Influences of metal concentration in phytoplankton and seawater on metal assimilation and elimination in marine copepods

1067

Environmental Toxicology and Chemistry, Vol. 20, No. 5, pp. 1067–1077, 2001q 2001 SETAC

Printed in the USA0730-7268/01 $9.00 1 .00

INFLUENCES OF METAL CONCENTRATION IN PHYTOPLANKTON AND SEAWATERON METAL ASSIMILATION AND ELIMINATION IN MARINE COPEPODS

YAN XU, WEN-XIONG WANG,* and DENNIS P.H. HSIEHDepartment of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

(Received 18 April 2000; Accepted 26 September 2000)

Abstract—Radiotracer experiments were conducted to examine the influence of the concentration of Cd, Se, and Zn in ingestedphytoplankton (dinoflagellate Prorocentrum minimum and diatom Thalassiosira weissflogii) and in ambient seawater on metalassimilation and elimination efficiencies of three marine copepods, Acartia spinicauda, Paracalanus aculeatus, and Calanus sinicus.The assimilation efficiencies (AEs) decreased by 1.7 to 2.0 times, 1.4 to 4.1 times, and 1.3 to 2.2 times in the copepods with anincrease in metal concentration in ingested algae by 16 to 84 times, 14 times, and 45 to 153 times, for Cd, Se, and Zn, respectively.However, the physiologic turnover rate constant was relatively independent of the metal concentration in copepods. No evidencewas found of any interaction between Cd and Zn in their assimilation by copepods. Assimilation efficiencies of Cd were higher incopepods feeding on the dinoflagellate P. minimum, whereas the AEs of Zn were higher in copepods feeding on the diatom T.weissflogii. Differences in metal distribution in algal cytoplasm at different ambient metal concentrations may be partially responsiblefor the observed influence of metal concentration in algal cells on metal assimilation in copepods. However, metal desorption withinthe gut of the copepod may have little influence on metal assimilation, as a result of the short gut residence time of food particlesand the neutral gut pH. Our study also indicated that the ingestion rate of copepods was reduced by a higher concentration of Cdand Se, but was not affected by Zn concentration in the food particles. Consequently, partial regulation of metal trophic transferin response to increasing metal contamination may be achieved by a change in metal assimilation efficiency and the ingestionactivity of the copepod, but not by changes in metal turnover rates from the animals.

Keywords—Copepods Assimilation efficiency Elimination Metals

INTRODUCTION

Copepods are the dominant marine zooplankton in pelagicsystems, but relatively few studies have been conducted onmetal accumulation and elimination in marine copepods. Thecycling and transport of metals in pelagic systems can be con-siderably impacted by metal interactions with marine planktonaffected by metal transfers between marine phytoplankton andzooplankton. For example, the biological uptake of metals bymarine phytoplankton has been shown to have a strong effecton concentration of metals in the water column [1,2]. Oncemetals associated with phytoplankton are ingested by zoo-plankton, the metals can either be retained in the animals,egested as packed fecal pellets, or excreted into the dissolvedphase, all of which can considerably affect the fate and dis-tribution of metals in marine systems. Circumstantial evidencealso indicates that copepods may be particularly sensitive tometal pollution in marine environments [3]. Copepods (e.g.,the calanoid Acartia tonsa) frequently have been used in tox-icity testing [4], and considerable interest exists in their ac-cumulation of metals from the ambient environments, eitherby uptake from solution or from food.

The assimilation efficiencies of metals from ingested phy-toplankton have been measured in a few marine copepods [5–12]. Using the kinetic modeling approach, Wang and Fisher[10] found that uptake from the ingested food source can bean important route for metal accumulated by copepods. Recentstudies have shown that excretion by marine copepods rep-resents a significant route by which particulate metals are elim-inated into the dissolved form [10,13]. The influences of foodquality and quantity on the bioaccumulation of metals in ma-

* To whom correspondence may be addressed ([email protected]).

rine copepods have been examined [9,13]. Metal assimilationefficiencies (AEs) have been shown to be related to the metaldistribution in the cytoplasm of algae and the physiologic con-ditions of the algal cells [7,8,12,14]. However, most previousstudies have focused on a few calanoid copepods (e.g., A. tonsaand Temora longicornis) [7,9,10], and no systematic com-parison has been made of interspecies differences in metalassimilation among different copepods. The effects of envi-ronmental and chemical factors, such as metal concentration,on the trophic transfer and the biogeochemical cycling of met-als in marine systems remain largely unknown.

To assess the quantitative significance of trophic transferin metal accumulation in marine zooplankton, rigorous testsare needed on the variability of metal assimilation and elim-ination under diverse ecological and biological conditions. Ac-cording to a simple bioenergetic-based kinetic model, metalconcentration in the animals under steady-state conditions re-sulting from only trophic transfer is a function of metal AEfrom ingested food, metal concentrations in the ingested food,the animal’s ingestion rate, and the metal efflux from the an-imals [10]. In this study, we examined the effects of metalconcentrations in food particles and in ambient seawater onthe bioavailability of metals to marine copepods. Three cal-anoid copepods, Acartia spinicauda, Paracalanus aculeatus,and Calanus sinicus, that are dominant in Hong Kong coastalwaters were chosen. We determined experimentally severalimportant physiologic parameters controlling the metal uptakefrom the ingested food source. These parameters included themetal AEs, the ingestion rates of the animals, and the elimi-nation rate constants after uptake from both food and watersources.

1068 Environ. Toxicol. Chem. 20, 2001 Y. Xu et al.

MATERIALS AND METHODS

Adult copepods (A. spinicauda, P. aculeatus, and C. sin-icus) were collected with a plankton net (150- to 500-mm meshsize) from Port Shelter, Clear Water Bay, Hong Kong. Thecopepods were maintained under laboratory condition for 1 dand fed with the algal food. All experiments were carried outat 188C in glass fiber–filtered 30‰ salinity seawater.

Radiolabeling phytoplankton

Two species of phytoplankton, Prorocentrum minimum(CCMP 696), and Thalassiosira weissflogii (CCMP 1048),were maintained in unialgal, clonal cultures in an f/2 medium[15] at 188C with a light illumination of 100 mE/m2/s on a 14:10 h light:dark cycle. Cells in the late log phase were collectedonto 3-mm polycarbonate membranes and resuspended in 0.2mm of filtered seawater enriched with f/2 levels of N, P, Si(except for P. minimum), and vitamins, and f/20 levels of tracemetals without addition of Cu, Zn, and ethylenediaminete-traacetic acid (EDTA). The initial cell concentration in themedia was generally 2 3 104 cells/ml. Algal cells were grownat three different metal concentrations (Cd: 4.6, 45.9, and 459nM; Se: 6.7, 66.7, and 667 nM; Zn: 15.4, 154, and 1,538 nM).Radioisotope additions produced concentrations of 4.6 nM for109Cd, 0.72 nM for 75Se, and 0.05 nM for 65Zn. After 7 to 8 dof growth for P. minimum, or 4 to 5 d of growth for T. weiss-flogii, the cell density reached 1 to 2 3 105 cells/ml (three orfour divisions) and they were considered to be uniformly la-beled. Cells were then collected by filtration onto polycar-bonate membranes and resuspended into 0.2 mm of filteredunlabeled seawater. The cell density was counted before thecells were fed to the copepods. The distribution of radioiso-topes in the phytoplankton cytoplasm was determined by cen-trifugation; details were described in Fisher et al. [16] andReinfelder and Fisher [7].

In another experiment, cells of P. minimum were grown atfour different combined concentrations of Cd and Zn: 4.6 nMCd and 15.4 nM Zn; 4.6 nM Cd and 1,538 nM Zn; 459 nMCd and 15.4 nM Zn; and 459 nM Cd and 1,538 nM Zn. Ra-dioisotope additions were similar to previous experiments. Thecells were then collected before being fed to the copepods.

Assimilation and elimination of metals in copepods

The radiolabeled algal cells were added into 200 ml of glassfiber–filtered seawater at a concentration of 104 cells/ml forP. minimum, and 2 3 104 cells/ml for T. weissflogii. Copepodswere added to three replicate beakers for each treatment atdensities of 0.2 to 0.5 individuals/ml, and were allowed to feedon the radiolabeled phytoplankton for 0.3 to 0.5 h in the dark.The copepods were then collected with a 250-mm nylon mesh,rinsed with filtered seawater, and placed in 5 ml of filteredseawater. The radioactivity of the copepods was immediatelycounted for 2 to 3 min, and the copepods were returned to 200ml of filtered seawater to depurate their ingested radiolabeledfood materials for 1 to 2 d, under the same conditions and inthe presence of unradiolabeled food (without addition of met-als). Any fecal pellets egested during the 0.3- to 0.5-h radio-active feeding period were also immediately collected onto a40-mm nylon mesh, rinsed with filtered seawater, and assayedfor radioactivity. The total amount of radioactivity ingestedby the copepods during the 0.3- to 0.5-h radioactive feedingperiod was calculated as the sum of radioactivity retained inthe copepods and in the feces produced during the radioactive

feeding period. The radioactivity of the copepods was mea-sured every 6 to 8 h over the 1- to 2-d depuration period. Theseawater and food were replaced each time the radioactivityof the copepods was counted.

In a previous study, AEs were calculated either as the per-centage of metals retained in the animals after complete di-gestion and assimilation (i.e., the mass balance method), or asthe y-intercept of the regression between the natural log of thepercentage of metals retained in the copepods and the time ofdepuration during the second phase of depuration (i.e., the y-intercept method) [17]. Both the mass balance method and they-intercept method were used in this study to calculate themetal AEs [17]. In the mass balance method, metal assimilationwas assumed to be completed within 6 h of depuration (seeresults). In the y-intercept method, the regression was per-formed during the second phase of depuration, that is, phys-iologic turnover between 6 and 24 h or 48 h. Metal eliminationrate constants were calculated as the slope of the linear re-gression between the natural log of the percentage of metalsretained in the copepods and the time of depuration between6 and 24 h or 48 h [10].

Metal depuration in copepods after uptake from thedissolved phase

Experiments were conducted to examine the metal uptakeand depuration in copepods (A. spinicauda) exposed to variousdissolved metal concentrations. Radioisotopes and stable met-als were added to 0.2 mm of filtered seawater and equilibratedfor 24 h before the addition of the copepods. The stable metalconcentrations were: 4.6, 45.9, and 459 nM for Cd; 133.3,6,667, and 13,333 nM for Se; and 15.4, 769, and 1,538 nMfor Zn. Radioisotopes were added to achieve concentrationsof 4.6 nM for 109Cd, 1.4 nM for 75Se, and 0.05 nM for 65Zn.

The copepods (500 individuals/L) were exposed to the dis-solved metals for 24 h in three replicate beakers for eachtreatment. The copepods then were collected with a 250-mmmesh, and the radioactivity was measured. Fifty individualsfrom each replicate were allowed to depurate in 200 ml offiltered seawater supplied with unlabeled algal cells (P. min-imum). The radioactivity retained in the copepods was mea-sured every 6 to 8 h, and the seawater and food were replacedeach time the radioactivity was counted. Depuration of metalsfrom the copepods was followed for 3 d, during which timelittle mortality occurred.

The distribution of metals in the copepods after 1 d ofdissolved uptake and after 3 d of depuration was determinedas described in Reinfelder and Fisher [14] and Wang and Fisher[10]. Briefly, copepods were collected onto 8-mm polycarbon-ate membranes and rinsed with filtered seawater. The weaklysurface-bound metals were removed by 10 ml of 0.1 mMEDTA. The copepods were then extracted with 3 ml of 0.2NaOH at 658C for 3 h. This was necessary for a completeextraction of soft tissue [10,18]. The tissues were then filteredthrough a 8-mm polycarbonate membrane and rinsed twicewith 2 ml of 0.2 N NaOH. The radioactivity of different frac-tions (EDTA washing, exoskeleton, and soluble tissue filtrate)was determined. We pooled the copepods from all three rep-licates because of the low radioactivity retained by the animalsafter 3 d of depuration.

Release of Cd and Zn from intact algal cells at pH 8 and 6

The effect of pH on releases of Cd and Zn from the intactdiatom T. weissflogii were examined as described in Wang et

Metal concentration effect on trophic transfer in copepods Environ. Toxicol. Chem. 20, 2001 1069

Table 1. Metal concentration and distribution in two phytoplankton (Prorocentrum minimum and Thalassiosira weissflogii) after exposure todifferent metal concentrations. The uptake of Se in the diatom T. weissflogii was not measured. All metals increased concurrently among treatments

so that only three levels of metal treatments were used

Metal concentration (nM)

Cd Se Zn

% Metal on cells afterradiolabeling

Cd Se Zn

Metal concentration (fg/cell)

Cd Se Zn

% Metal in algal cytoplasm

Cd Se Zn

Prorocentrum minimum (fed to copepod Acartia spinicauda)4.6

45.9459

6.766.7

667

15.4154

1,538

100100

54

1381.4

757578

2.932.9

218

0.42.55.5

4.246.8

632

28.526.320.2

66.164.957.5

23.721.417.6

Prorocentrum minimum (fed to copepod Paracalanus aculeatus)4.6

45.9459

6.766.7

667

15.4154

1,538

858444

850.8

788986

3.340.3

237

0.32.24.5

3.042.1

458

33.327.018.3

47.341.028.9

22.918.015.1

Thalassiosira weissflogii (fed to copepod Paracalanus aculeatus)4.6

45.9459

15.4154

1,538

967932

887584

3.318.552.1

6.034.9

272

54.031.427.1

67.949.025.6

Thalassiosira weissflogii (fed to copepod Calanus sinicus)4.6

45.9459

15.4154

1,538

666443

676771

2.323.3

194

4.648.5

633

22.817.914.8

49.245.020.4

al. [19] and Wang and Fisher [20]. Diatom cells were radio-labeled as described above and were resuspended into 150 mlof 0.2-mm–filtered seawater at a concentration of 1.5 3 104

cells/ml. The retention of metals in the cells was measured atpH 8 (control) and pH 6. Seawater of pH 6 was prepared byaddition of Ultrex 0.5 N HCl (Merck, Damstadt, Germany).Two replicates were used for each treatment. Periodically, a10-ml sample was filtered onto a polycarbonate membrane andthe radioactivity was measured.

Ingestion rate of copepods

To examine the ingestion rate of the copepods feeding onalgal cells containing different metal concentrations, algal cells(P. minimum) were grown at different concentrations of Cd(4.6, 45.9, and 459 nM), Se (6.7, 66.7, and 667 nM), or Zn(15.4, 154, and 1,538 nM). After 7 to 8 d of growth, the cellswere collected onto 3-mm polycarbonate membranes and re-suspended into filtered seawater. The algal diets were thenadded into 200 ml of glass fiber–filtered seawater at a con-centration of 104 cells/ml. Copepods (A. spinicauda) were add-ed at a density of 0.4 to 0.5 individuals/ml and allowed to feedon the cells for 2 h in the dark. Three replicates were used foreach treatment. The cell density before and after the feedingwas counted by a Coulter counter. The ingestion rate (IR; mgcell/copepod/h) was calculated by the following equation [21]:

IR 5 W·V·(C 2 C )/(N·t)0 t

where W is the dry weight of the cell (mg); V is the volumeof seawater (ml); C0 and Ct are the concentrations of algalcells before and after feeding (cells/ml), respectively; N is thenumber of copepods used in the experiment; and t is the feed-ing time (h).

Radioactivity measurements and statistical analysis

Radioactivity was measured with a Wallac 1480 NaI(Tl)gamma detector (Wallac, Turku, Finland). All measurementswere related to appropriate standards and calibrated with spill-over. The gamma emission of 109Cd was determined at 22 keV,

of 75Se at 264 keV, and of 65Zn at 1,115 keV. Counting timeswere adjusted to yield a propagated counting error , 5%.Statistical analysis was performed by one-way or two-wayanalysis of variance (ANOVA) tests.

RESULTS

Effects of metal concentration on metal assimilation andelimination in copepods

A major difference was found in metal concentration inalgal cells exposed to different ambient concentrations (Table1). Concentrations varied by 16 to 84 times for Cd, 14 to 15times for Se, and 45 to 153 times for Zn over a 100 timesdifference in ambient metal concentration. A much lower up-take of Cd and Se was found at the highest ambient metalconcentration, whereas the percentage of Zn associated withthe algal cells was similar among the Zn concentrations. Forall metals, an increase in ambient concentration resulted in adecline in metal distributions in the algal cytoplasm, especiallyin the diatom T. weissflogii. Differences also were found inmetal distribution in algal cytoplasm for the same algal speciesused in two different experiments.

Generally, the pattern of metal depuration was characterizedby a rapid loss within the first 3 h, followed by a more gradualloss (Figs. 1 and 2). The initial rapid loss was probably dom-inated by egestion of unassimilated materials. No major dif-ference in the depuration pattern was observed among differentmetals, although in some cases Cd appeared to be depuratedat a slower rate between 6 and 24 h than between 24 and 48 h.

The AEs estimated by the mass balance method were gen-erally lower than those estimated by the y-intercept method(Table 2). With the exception of the AE of Se in A. spinicauda,the AEs for all three metals decreased significantly with in-creasing metal concentrations in the algal cells (p , 0.05, one-way ANOVA). For Cd and Zn, no differences in AEs wereobserved between the two lowest metal concentrations, but theAEs decreased with further increase in algal metal concentra-tions. The AEs of Zn were similar when the two copepodspecies were fed with the same algal diet, but were greater in


Fig. 1. The retention of Cd, Se, and Zn in two copepods (Paracalanusaculeatus and Acartia spinicauda) after a pulse ingestion of radio-labeled dinoflagellates (Prorocentrum minimum). Algal cells main-tained at 4.6 nM of Cd, 6.7 nM of Se, or 15.4 nM of Zn (m); algalcells maintained at 45.9 nM of Cd, 66.7 nM of Se, or 154 nM of Zn(.); algal cells maintained at 459 nM of Cd, 667 nM of Se, or 1,538nM of Zn (v). Data are mean 6 standard deviation (n 5 3).

Fig. 2. The retention of Cd and Zn in two copepods (Paracalanusaculeatus and Calanus sinicus) after a pulse ingestion of radiola-beled diatoms (Thalassiosira weissflogii). Algal cells maintained at4.6 nM of Cd or 15.4 nM of Zn (m); algal cells maintained at 45.9nM of Cd or 154 nM of Zn (.); algal cells maintained at 459 nMof Cd or 1,538 nM of Zn (v). Data are mean 6 standard deviation(n 5 3).

copepods fed with the diatom T. weissflogii than with thedinoflagellate P. minimum. Acartia spinicauda assimilatedmore Cd and Se than did P. aculeatus, and C. sinicus had thelowest Cd AE among the three species.

The physiologic turnover rate constants were similar foreach metal in both P. aculeatus and C. sinicus, which suggests

that metals were lost at a similar rate. However, Cd and Znwere eliminated at a slower rate in A. spinicauda than in theother two copepods. The physiologic turnover rate constantswere unaffected by the metal concentrations in the algal cells(p . 0.05, one-way ANOVA). In our experiments, fecal pelletegestion represented only a small fraction of the total metalloss from the copepods during the physiologic turnover period,suggesting that excretion was the major pathway for metal lossfrom the copepods into the ambient environment.

When all experiments were considered, a significant cor-relation was found between the metal AEs and the metal dis-tribution in the algal cytoplasm for Se and Zn (Fig. 3). Nosignificant relationship was found for Cd. However, the CdAE generally increased with increasing metal distribution inthe algal cytoplasm for each copepod feeding on a single algaldiet. Considering all three metals, no relationship was foundfor copepods fed on the dinoflagellate, whereas a significantrelationship was found when copepods fed on the diatom (Fig.4).

Interactive effects of Cd and Zn on their assimilation incopepods

Metal concentrations in the dinoflagellate P. minimum in-creased with increasing metal concentration in ambient sea-water (Table 3). A high Zn concentration (1,538 nM) depressedCd accumulation and distribution into the algal cytoplasm.Conversely, no major effect of Cd concentration was foundon Zn accumulation in the algal cells. The metal depurationpattern was similar for animals fed algae with different com-bined metal concentrations (Fig. 5). The calculated AEs werecomparable at the same metal concentrations. Statistical anal-ysis indicated that no interaction occurred between Cd and Znin their assimilation in copepods at the experimental concen-trations examined. However, metal concentration in the algaldiets did have an effect on their assimilation. The AEs of Cdand Zn were significantly higher (p , 0.05) at a lower metalconcentration. The AEs of Zn in the copepods were also muchlower than the AEs of Cd. In this experiment, no correlationwas found between the metal AEs in copepods and the metaldistribution in the algal cytoplasm. The physiologic turnoverrate constants of Cd and Zn were also comparable among thedifferent treatments (p . 0.05), suggesting that metal concen-tration has no notable effect on the turnover rate of metals incopepods.

Release of Cd and Zn from intact diatom cells

Metal release behavior was similar in intact algal cells con-taining different metal concentrations (Fig. 6). At pH 8 (con-trol), little Cd was lost from the cells within the first hour ofresuspension, and afterwards the desorption exhibited a linearpattern over time. In contrast, a large fraction of Zn was de-sorbed into the dissolved phase within the first hour, and after-wards very little loss occurred from the intact cells. When thealgal cells were transferred to pH 6.0, a rapid metal desorptionoccurred within the first hour, especially for Zn. However, verylittle Cd was lost at the lowest Cd concentration (4.6 nM).More metals were desorped from the intact cells with increas-ing metal load in the algae.

Metal turnover rate in copepods after dissolved uptake

After 1 d of uptake from the dissolved phase, metal dep-uration in A. spinicauda was characterized by two distinctphases, an initial rapid loss within the first 6 h, followed by


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com

pari

son)

.


Fig. 3. Relationship of metal assimilation efficiency in copepods withmetal distribution in algal cytoplasm. Acartia spinicauda fed withProrocentrum minimum (v); Paracalanus aculeatus fed with P. min-imum (V); P. aculeatus fed with Thalassiosira weissflogii (.); Cal-anus sinicus fed with T. weissflogii (,). Data are mean 6 standarddeviation (n 5 3).

Tab

le3.

Met

alco

ncen

trat

ion

and

dist

ribu

tion

inth

edi

nofl

agel

late

Pro

roce

ntru

mm

inim

um(u

sed

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od)

atdi

ffer

ent

com

bine

dco

ncen

trat

ions

ofC

dan

dZ

n,an

dth

eas

sim

ilat

ion

effi

cien

cy(A

E)

and

phys

iolo

gic

turn

over

rate

cons

tant

(k)

ofm

etal

sin

the

cope

pod

Aca

rtia

spin

icau

da.

See

text

for

calc

ulat

ion

ofA

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dk.

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ues

are

mea

ns6

stan

dard

devi

atio

n(n

53)

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and

Bin

dica

tesi

gnifi

cant

diff

eren

cebe

twee

nth

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ents

( p,

0.05

),w

here

asA

and

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indi

cate

nosi

gnifi

cant

diff

eren

cebe

twee

nth

etw

otr

eatm

ents

Met

alco

ncen

trat

ion

(nM

)

Cd

Zn

Met

alin

cell

s(f

g/ce

ll)

Cd

Zn

%M

etal

inal

gal

cyto

plas

m

Cd

Zn

AE

(%)

Mas

sba

lanc

em

etho

d

Cd

Zn

y-In

terc

ept

met

hod

Cd

Zn

k(d

21 )

Cd

Zn

4.6

4.6

459

459

15.4

1,53

8 15.4

1,53

8

3.4

3.1

265

241

6.5

629 8.

91,

007

38.5

23.3

18.7

16.3

22.8

13.0

13.8

21.7

55.8

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63.

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26.1

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31.0

63.

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7.9

61.

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3.8

60.

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8.5

61.

0A

4.6

60.

6B

67.8

65.

7A

70.5

64.

5A

31.6

62.

4B

38.1

65.

1B

7.8

60.

2A

4.4

60.

2B

9.8

61.

1A

5.4

60.

5B

0.64

60.

070.

626

0.04

0.62

60.

110.

656

0.09

0.42

60.

150.

456

0.05

0.52

60.

050.

496

0.14

Fig. 4. Relationship of metal assimilation efficiency in the copepodswith metal distribution in the algal cytoplasm. Cd (v); Zn (V); Se(.). Pro 5 Prorocentrum minimum; Tw 5 Thalassiosira weissflogii.Data are mean 6 standard deviation (n 5 3).

a second slower phase of depuration (12–58 h; Fig. 7). A muchgreater fraction of metals was lost from the copepods withinthe first 6 h of depuration with increasing metal concentration(p , 0.05, one-way ANOVA). The physiologic turnover rateconstant was calculated as the slope of the linear regressionbetween the natural log of the percentage of metals retainedin the copepods and the time of depuration (during the secondphase of loss; Table 4). The calculated turnover rate constants


Fig. 5. The retention of Cd and Zn in the copepod Paracalanus acu-leatus after a pulse ingestion of the radiolabeled dinoflagellate Pro-rocentrum minimum with different combined metal concentrations.Algal cells maintained at 4.6 nM of Cd and 15.4 nM of Zn (v); algalcells maintained at 4.6 nM of Cd and 1,538 nM of Zn (V); algal cellsmaintained at 459 nM of Cd and 15.4 nM of Zn (.); algal cellsmaintained at 459 nM of Cd and 1,538 nM of Zn (,). Data are mean6 standard deviation (n 5 3).

Fig. 7. The retention of Cd, Se, and Zn in the copepod Acartia spin-icauda after 1 d of exposure to metals in the dissolved phase atdifferent concentrations. Copepods exposed to 4.6 nM of Cd, 133.3nM of Se, or 15.4 nM of Zn (m); copepods exposed to 45.9 nM ofCd, 6,667 nM of Se, or 769 nM of Zn (.); copepods exposed to 459nM of Cd, 13,333 nM of Se, or 1,538 nM of Zn (v). Data are mean6 standard deviation (n 5 3).

Fig. 6. The percentages of Cd and Zn desorbed from the intact diatomThalassiosira weissflogii inoculated at different metal concentrationsand resuspended at pH 8.0 (control) and pH 6.0. Algal cells resus-pended at pH 8.0 (m); algal cells resuspended at pH 6.0 (v). Dataare mean 6 standard deviation (n 5 2).

were comparable for each metal among different metal con-centration treatments (p . 0.05, one-way ANOVA), suggestingthat metal concentration in copepods had no major influenceon the physiologic turnover rate.

After 1 d of uptake, Cd concentration in the copepods wasdirectly proportional to its concentration in the ambient sea-water, whereas only a 55 times increase occurred in Zn con-centration in copepods with an increase in Zn concentrationin the ambient seawater by 100 times (Table 4). A greaterfraction of Cd was found in the soft tissue (72–77%) than inthe exoskeleton (23–28%), whereas more Zn was found in theexoskeleton (74–79%) than in the soft tissue (21–26%; Fig.8). Selenium was equally distributed in the exoskeleton andsoft tissue of the copepod. No major influence of metal con-centration on metal distributions was found in the copepodsafter the 1-d uptake or after the 3-d depuration. By the end of3 d of depuration, the distribution of Cd in the soft tissue andexoskeleton remained rather constant, whereas Se and Zn dis-tributions in the exoskeleton decreased, suggesting that theirlosses were faster than the losses from the soft tissue, espe-cially for Se.

Ingestion rates of copepods at different metalconcentrations

The ingestion rates of the copepod A. spinicauda wereinversely related to Cd and Se concentration in the dinofla-gellate P. minimum (Fig. 9), but ingestion rate was unaffectedby the Zn concentration. For example, at the highest Cd andSe concentration, the ingestion rate decreased by 39 to 40%compared to the ingestion rate measured at the lowest metal


Tab

le4.

Met

alup

take

and

depu

rati

onra

teco

nsta

ntin

the

cope

pod

Aca

rtia

spin

icau

daaf

ter

upta

kefr

omth

edi

ssol

ved

phas

efo

r1

dat

diff

eren

tm

etal

conc

entr

atio

ns.

Val

ues

are

mea

ns6

stan

dard

devi

atio

na

Met

alco

ncen

trat

ion

Wat

er(n

M)

Cd

Se

Zn

Cop

epod

afte

r1

dof

upta

ke(m

g/g

dry

wt)

Cd

Se

Zn

k 1(d

21 )

Cd

Se

Zn

k 2(d

21 )

Cd

Se

Zn

4.6

45.9

459

133

6,66

713

,333

15.4

769

1,53

8

1.6

11.9

96.9

1.6

234

728

8.5

385

489

0.43

60.

39b

1.33

60.

12b

2.46

60.

25b

1.47

60.

99b

8.29

60.

71b

8.97

60.

61b

0.62

60.

34c

1.69

60.

13c

1.59

60.

44c

0.59

60.

150.

556

0.03

0.60

60.

08

0.89

60.

110.

676

0.25

0.71

60.

16

0.62

60.

070.

586

0.05

0.61

60.

06

ak 1

5de

pura

tion

rate

cons

tant

betw

een

0an

d6

h;k 2

5de

pura

tion

rate

cons

tant

(phy

siol

ogic

turn

over

)be

twee

n12

and

58h.

bS

tati

stic

ally

sign

ifica

ntef

fect

ofm

etal

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entr

atio

nat

p,

0.00

1(o

ne-w

ayA

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).c

Sta

tist

ical

lysi

gnifi

cant

effe

ctof

met

alco

ncen

trat

ion

atp

,0.

05(o

ne-w

ayA

NO

VA

).

Fig. 8. The distribution of Cd, Se, and Zn in the copepod Acartiaspinicauda after 1 d of exposure to metals in the dissolved phase atdifferent concentrations (before depuration), and after 3 d of depu-ration in nonradioactive water. Exoske 5 exoskeleton; ST 5 softtissue. Low metal 5 copepods exposed to 4.6 nM of Cd, 133.3 nMof Se, or 15.4 nM of Zn; Medium metal 5 copepods exposed to 45.9nM of Cd, 6,667 nM of Se, or 769 nM of Zn; High metal 5 copepodsexposed to 459 nM of Cd, 13,333 nM of Se, or 1,538 nM of Zn.

Fig. 9. The ingestion rate of the copepod Acartia spinicauda feedingon the dinoflagellate Prorocentrum minimum with different metalconcentrations. Low 5 Cd concentration of 2.9 fg/cell, Se concen-tration of 0.4 fg/cell, or Zn concentration of 4.2 fg/cell; Medium 5Cd concentration of 32.9 fg/cell, Se concentration of 2.5 fg/cell, orZn concentration of 46.8 fg/cell; High 5 Cd concentration of 218 fg/cell, Se concentration of 5.5 fg/cell, or Zn concentration of 632 fg/cell. Values are means 6 standard deviation (n 5 3).


concentration. However, statistical analysis indicated that theinfluence of metal concentration on the ingestion rate of acopepod was not significant (p . 0.05, one-way ANOVA).

DISCUSSION

Assimilation of metal in marine copepods

Metal transfer in marine planktonic food webs is controlledby several important physiologic and geochemical parameters,including metal assimilation, efflux, and metal concentrationsin ingested food particles [22]. Our study suggested that metalAEs in different copepod species were inversely related to themetal concentration in algal diets. Wang and Fisher [20] dem-onstrated that marine mussels (Mytilus edulis) responded to achange in metal concentration in ingested food particles in anelement-specific way. Assimilation of Se was not affected byits concentrations in ingested diatoms, whereas Cd assimilationincreased and Zn assimilation decreased with increasing metalconcentration in food particles. In a recent study, Schmidt etal. [12] measured Fe assimilation in marine copepods (Acartiasp.) fed with Fe-limited and Fe-replete diatoms (T. weissflogii),and found that the Fe AEs in copepods were 1.7 times higherfrom Fe-limited than from Fe-replete diatom cells.

A decrease in metal AE with increasing metal contami-nation in ingested food suggests that the copepods partiallymay be able to regulate their internal metal concentrations, asfound in other crustaceans [23–27]. Generally, the animalswere able to regulate their total body burden of metals up toa threshold level. A mechanism is maintenance of body metalconcentrations by efficient excretion with little control of metaluptake. Nugegoda and Rainbow [28] demonstrated that thedecapod Palaemon elegans regulated its body Zn to a constantlevel when exposed to elevated concentrations by balancingZn excretion with Zn uptake. In contrast, Chan and Rainbow[29] found that the crab Carcinus meanas maintained a con-stant Zn body burden by maintaining low Zn uptake rate ratherthan a high elimination rate. In these previous studies, trophictransfer was not considered as contributing to metal regulation.Our study of marine copepods indicated that metal assimilationmay be an important process for copepods in regulating theirmetal uptake from the ingested food source.

The metal uptake rate from ingested food is a function ofthe metal AE, the metal concentration in food, and the inges-tion rate of the copepod [10]. A further regulation of the metalinflux rate can also be achieved by a reduction in the ingestionrate of the copepod. Our study demonstrated that the ingestionactivity of copepods was responsive to a change in Cd and Seloads but not to Zn loads in the food particles. A reduction inthe animal’s ingestion activity may lead to a decline in theinflux of metals from the ingested food source. However, reg-ulation of the metal body burden by variation in metal AE andthe ingestion rate of the copepod may be inadequate, becauseonly a 2 times difference in AEs and a 1.3 times differencein feeding rates were found when metal concentration in theingested food particles varied by one to two orders of mag-nitude.

Several mechanisms may account for the observed variationof metal AEs in response to the metal concentration in foodparticles. Reinfelder and Fisher [7] suggested the existence ofa liquid digestive strategy for marine copepods, in which theanimals obtained nearly all their metals from the cytoplasmof prey cells. In this model, metals associated with the cellsurface were passed through the guts and packaged into fecalpellets rapidly, whereas metals in the algal cytoplasm were

assimilated and followed the fate of carbon in marine foodwebs [30]. However, only Hutchins et al. [8] demonstrated thatthe cytoplasmic distribution critically affected metal assimi-lation in marine copepods for a specific metal such as Fe. Inour study, we also found a positive relationship between theAEs and the metal distribution in the algal cytoplasm, furthersuggesting that the partitioning of these metals in the algalcytoplasm was crucial in determining the assimilation of met-als from food particles with different metal loads. Furthermore,a significant relationship between metal AEs and their distri-bution in algal cytoplasm for Se and Zn suggested that thelatter can partially account for the difference of metal AEsobserved for different species of copepods feeding on the sameor different algal diets.

Interspecies differences in metal AEs seem to be mainlydue to the difference of metal penetration into the cytoplasmof algal cells, which is consistent with the study by Reinfelderand Fisher [7]. For example, a significant relationship wasdocumented for Se between its AEs in two copepods (A. spin-icauda and P. aculeatus) and its distribution in the dinofla-gellate P. minimum. However, this relationship was less ob-vious for a single copepod species (P. aculeatus) feeding ondifferent algal diets (dinoflagellate and diatom), especially forCd. Thus, difference of Cd AEs observed between two algaldiets was not due to the difference of partitioning of Cd in thecytoplasm of these two algae. In our study, we also consistentlyfound that the AEs of Zn from P. minimum were much lowerthan the AEs from the diatom T. weissflogii, suggesting thatdifferent algal species can considerably affect Zn uptake incopepods. In contrast, Wang et al. [9] found no major differ-ence in metal AEs in copepods (A. tonsa and T. longicornis)feeding on different phytoplankton diets. The Zn AEs were ashigh as 83% in copepod fed P. minimum in that study.

Metal desorption within an animal’s gut may affect metaluptake in marine invertebrates [19,31,32]. In marine copepods,the gut pH was nearly neutral [33]; thus, metal desorption dueto the influence of lowered pH may be insignificant for thecopepods. However, our study suggested that desorption canbe significant for Zn in intact cells at normal seawater pH,presumably because a great fraction of Zn was in the extra-cellular pool. Very little desorption of Cd from the diatomcells occurred within the first hour because most Cd was par-titioned in the intracellular compartment (Dei and Wang, un-published data). After 1 h of resuspension, the loss of Cd maybe due to the efflux from the intact cells [34]. In our study,desorption seemed to be greater with increasing metal con-centrations in the algae as a result of greater partitioning ofmetals in the extracelluar pool. Given the decreased assimi-lation with increasing metal loads in the cells, desorption with-in the copepod’s gut may not control metal assimilation incopepods, presumably because of the short gut residence timeof food particles (4–20 min; Y. Xu, unpublished data).

Metal elimination in marine copepods

Our study provides a quantitative measure and comparisonof metal elimination by marine copepods after uptake from thefood source and from the dissolved phase. Elimination can bean important route by which metals are released from copepodsinto the dissolved phase. Previous studies demonstrated thatmetal efflux is dominated by excretion into the dissolved phase[8–10,13]. In these studies, most depurated metals were de-tected in the dissolved phase with little association with thefecal pellets during the physiologic turnover period. However,


egestion via fecal pellet production dominated metal loss dur-ing the initial digestive period [10]. In an earlier study, Wanget al. [9] measured a physiologic turnover rate constant of 0.3to 2.6/d for different metals after a pulse ingestion of radio-labeled food particles. Our present measurements on the metalphysiologic turnover rate were consistent with these previousstudies [9,10].

Our study showed that metal physiologic turnover rate wasindependent of the metal concentration in the food particlesand in the ambient seawater. One possibility was that littledifference occurred in metal body burden in copepods whenthe physiologic turnover rate was measured (during the secondphase of depuration). Similarly, Hudson et al. [35] found thatthe mean elimination rate of phosphorous did not vary withthe total phosphorus concentration in freshwater lakes, con-sistent with other measurements of nutrient turnover in plank-tonic systems [36,37].

The physiologic turnover rate was found to be related tometal species, duration and route of metal uptake, amount offood ingested, and copepod species [9,10,13]. Dietary Cd andSe were generally turned over at a faster rate than Zn in thethree species of copepods examined in our study. However,the turnover rates of these metals were comparable after uptakefrom the aqueous phase, despite the considerable differencein their distributions in the soft tissue and exoskeleton of thecopepods. Furthermore, we found that the physiologic turnoverrates of Cd and Zn were slower in the smaller copepod (A.spinicauda) than in the two larger copepods (P. aculeatus andC. sinicus) under the same experimental conditions. Thus, themetabolic process may have little control on the physiologicturnover of metals in copepods. Wang and Fisher [10] foundthat metals were turned over at a greater rate after uptake fromfood than after uptake from the water. They speculated that agreater fraction of metals was distributed in copepod soft tissueafter uptake from food, and desorption from copepod exo-skeletons was slower than excretion from the soft tissue. Inour study, we did not observe a major difference in metalturnover rate after uptake from the dissolved phase for 1 dand after a pulse ingestion of food particles.

Our measured physiologic turnover rates in copepods weremuch higher than those measured in other marine invertebratessuch as bivalves (0.01–0.03/d) [38,39]. Such an exceedinglyhigh physiologic turnover rate may have prevented the accu-mulation of metals in copepods to a high concentration level.According to a simple model, the trophic transfer factor ofmetals is inversely related to the physiologic turnover rateconstant of the metal [22]. Thus, a high physiologic turnoverrate may reduce the trophic transfer factor and minimize thebiomagnification of metals during their trophic transfer, as hasbeen observed in many field studies [40].

Acknowledgement—We thank the two anonymous reviewers for theirdetailed comments on this paper. This work was supported by a Com-petitive Earmarked Research Grant from the Hong Kong ResearchGrant Council (Hong Kong University of Science and Technology6137/99M) to W.-X. Wang.

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Documents

Influences of metal concentration in phytoplankton and seawater on metal assimilation and elimination in marine copepods