Directory UMM :Data Elmu:jurnal:J-a:Journal of Experimental Marine Biology and Ecology:Vol251.Issue2.Aug2000:

Journal of Experimental Marine Biology and Ecology
251 (2000) 161–183
www.elsevier.nl / locate / jembe

Breakdown of phytoplankton pigments in Baltic sediments:
effects of anoxia and loss of deposit-feeding macrofauna
Thomas S. Bianchi a , *, Birgitta Johansson b ,1 , Ragnar Elmgren c
a

Institute of Earth and Ecosystem Sciences, Department of Ecology and Evolution Biology,
Tulane University, New Orleans, LA 70118 -5698, USA
b
Department of Zoology, Stockholm University, SE-106 91 Stockholm, Sweden
c
Department of Systems Ecology, Stockholm University, SE-106 91 Stockholm, Sweden

Received 16 September 1998; received in revised form 15 December 1999; accepted 28 March 2000

Abstract
We examined the decay of chlorophyll a and the carotenoid fucoxanthin in oxic and anoxic
sediment microcosms, with and without the deposit-feeding benthic amphipod Monoporeia affinis,

over 57 days at 58C. Deep frozen phytoplankton from the Baltic Sea proper was added to all but a
few microcosms. The range of chlorophyll a and fucoxanthin decay rate constants observed in
microcosms with phytoplankton addition was 0.04–0.07 day 21 . The fastest pigment decay and
build-up of chlorophyll breakdown products after phytoplankton addition were found in oxic
treatments with amphipods. No effects of amphipods on pigment breakdown were found in anoxic
treatments, or in treatments without phytoplankton addition. Greater losses of chlorophyll a in oxic
(96%) than in anoxic (80%) treatments after 57 days indicates that preservation of sedimentary
organic matter will be enhanced during periods of anoxia. Due to slow recruitment and
recolonization in Baltic sediments, a single anoxic event may cause long-term (years) absence of
significant macrobenthos. Anoxic events will thus not only reduce decay of plant pigments, and
presumably other organic matter, while they last, but will also have longer-term effects, through
elimination of macrofauna, which when present enhance organic matter decomposition.  2000
Elsevier Science B.V. All rights reserved.
Keywords: Plant pigments; Decomposition; Baltic Sea; Carbon cycling; Macrobenthos; Bioturbation;
Monoporeia; Oxygen concentration; Anoxia; Hypoxia

*Corresponding author. Tel.: 11-504-862-8000; fax: 11-504-862-8706.
E-mail addresses: tbianch@mailhost.tcs.tulane.edu (T.S. Bianchi), ragnar.elmgren@ecology.su.se (R.
Elmgren).
1

¨ Pl. 32101, SE-459 93 Ljungskile, Sweden. E-mail: birgitta.johansson@home.se.
Present address: Restenas,
0022-0981 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved.
PII: S0022-0981( 00 )00212-4

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1. Introduction
In recent decades, periods of hypoxia and anoxia have markedly modified the
distribution and abundance of macro- and meiobenthos below the primary halocline of
the Baltic Sea proper (Elmgren, 1975: meiofauna; Andersin et al., 1978; Rumohr et al.,
1996: macrofauna). Increasing oxygen deficiency has been mainly caused by nutrient
enrichment of the Baltic (Larsson et al., 1985; Rosenberg et al., 1990). This has resulted
in increased phytoplankton production (Elmgren, 1989; Stigebrandt, 1991), and a
consequent increase in sedimentation of organic matter to sediments (Jonsson and
Carman, 1994). Anoxic conditions in bottom waters and sediments in deeper parts of the
Baltic proper have resulted in extensive areas of laminated surface sediments (Jonsson et
al., 1990). Little is known about the long-term effect of loss of macrofauna on

decomposition of sedimenting organic matter, both in the Baltic (Cederwall and
Elmgren, 1990; Rosenberg et al., 1990) and in general (Diaz and Rosenberg, 1995).
The direct effect of bioturbation by macrofauna on decomposition of organic matter is
well documented in coastal ecosystems (Rhoads, 1974; Berner, 1980; Aller, 1982; Rice,
1986; Lopez and Levinton, 1987; Andersen and Kristensen, 1992; Webb and Montagna,
1993; Alongi, 1995). Bioturbation generally stimulates diagenesis of organic matter by
increasing oxygen availability and through mechanical fragmentation by feeding
activities (Berner, 1980; Lopez and Levinton, 1987; Bianchi et al., 1989). During
prolonged anoxic events, most macrofauna are eliminated and anaerobic metabolism
becomes the dominant pathway for breakdown of organic matter. Many studies have
shown that anaerobic bacteria are generally less efficient than aerobic bacteria in
decomposing organic matter (Fenchel and Finlay, 1995). Kemp (1990) and Lee (1992)
proposed that by killing bacterial grazers, lack of oxygen and sulfide toxicity can depress
the activity of the sedimentary microbial loop and result in the sequestering of bacteria
and bacterial products within anoxic sediments. Thus, periods of anoxia may enhance
the preservation of organic matter due to the destruction of macrofauna, a less efficient
pathway of anaerobic metabolism, and the disruption of the microbial loop (via changes
in bacterial grazers).
Due to its complex composition, it is difficult to understand decay dynamics of bulk
organic matter, whether in oxic or anoxic sediments, particularly when the sources of

organic matter are unknown. Skopintsev (1981) showed that algal organic matter does
not degrade at a uniform rate, but rather as a combination of several pools, with different
rates of decomposition. Multi-first-order decay rate equations have been used to describe
the breakdown of different fractions of algal carbon (G model of Westrich and Berner,
1984). Specific molecular biomarkers can be used to identify sources as well as ages of
the sedimentary carbon (Sun and Wakeham, 1994; Harvey et al., 1995; Canuel and
Martens, 1996; Eglington et al., 1996). Plant pigments have been used as biomarkers of
sources of organic matter in aquatic systems (Watts et al., 1977; Mantoura and
Llewellyn, 1983; Bidigare et al., 1985; Wright et al., 1991; Bianchi et al., 1996), and for
tracing food resources of benthic invertebrates (Bianchi et al., 1988, 1991). The
degradation products of chloropigments can give clues to the availability of different
source materials to macro-invertebrates. Different characteristic tetrapyrrole derivatives
of chloropigments (phaeopigments) are formed as a result of bacterial or autolytic cell

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163

lysis, and of metazoan grazing activities (Sanger and Gorham, 1970; Shuman and
Lorenzen, 1975; Welschmeyer and Lorenzen, 1985; Hawkins et al., 1986; Bianchi et al.,

1988, 1991). The phaeopigment, phaeophorbide, has been shown to be well correlated
with the grazing activities of metazoans (Shuman and Lorenzen, 1975; Bianchi et al.,
1991). In controlled laboratory feeding experiments, the highest concentrations of total
phaeophorbides usually reflect the most intense metazoan grazing activity — which in
turn depends upon resource ‘quality’ or availability (Millie et al., 1993).
In this study, we used laboratory microcosms containing sediments collected from the
Baltic proper to examine the effects of anoxia and macrobenthos on the degradation of
plant pigments in phytodetritus. The macrobenthic species used was the amphipod
¨
Monoporeia affinis (Lindstrom),
a dominant deposit-feeder in most of the Baltic Sea
˚ 1950, Rumohr et al., 1996). It lives in soft sediments, mainly below 15–20
(Segerstrale,
˚ 1950; Jarvekulg,
¨
¨ 1973), tolerates salinities from fresh water up to
m depth (Segerstrale,
˚ 1950, 1959), feeds on surface sediment (Lopez and Elmgren, 1989),
13–15 (Segerstrale,
and is an effective bioturbator. Most individuals are found in the upper five centimeters

of sediment (Hill and Elmgren, 1987). Recent studies have shown that pigments and
most other compounds are more stable under anoxic than oxic conditions (Sun et al.,
1991; Sun and Wakeham, 1994; Harvey et al., 1995). Little attention has, however, been
given the role of macrofauna and anoxic events in preservation of organic matter, as
traced by pigment biomarkers. Our experiments were designed to test for the double
effect anoxic events may have on pigment decay. First directly, during anoxia / hypoxia,
by restricting breakdown to anaerobic pathways, and then indirectly, even after the
return of oxygen to bottom waters, by eliminating bioturbation and grazing on bacteria
by macrobenthos.

2. Methods and materials

2.1. Sources of experimental animals, sediments, and phytoplankton
Sediments and subadult amphipods, Monoporeia affinis, of the age class hatched in
early spring of the previous year, were collected with a benthic dredge at 30–40 m near
the Asko¨ Laboratory, northern Baltic proper (588 499 N, 178 389 E), in spring 1994.
Sediments were sieved through a 0.5-mm net to remove all macrofauna. In mid-April,
spring-bloom phytoplankton, dominated by diatoms, was collected with a 45-mm
plankton net, and deep-frozen until used.

2.2. Laboratory microcosms
The experiment was conducted in summer, in recirculating water tanks at 58C and in
total darkness. Artificial seawater was made from aquarium salt (‘hw-Marinemix 1 Bioelements’, Wiegant Gmbh, Germany), to a salinity of 6.5‰, near that at which
amphipods and phytoplankton were collected. One experimental tank (2.5 3 0.4 3 0.2 m,
200 l) each was used for four oxygen concentrations: 0.4 (called anoxia in the
following), 1.7, 3.0 (both called low oxic), and 11.6 mg O 2 l 21 (continuously air-

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bubbled, called oxic). The water was recirculated at 440 l h 21 by an Eheim pump with
an attached bio-filter. An oxygen meter (AOWL2 / KOB / 421, Processtyrning AB,
Sweden), regularly calibrated against the Winkler method, monitored and stored oxygen
concentrations on a data logger. Oxygen concentration was regulated by a flow of
nitrogen gas, which was automatically switched off by a magnetic valve controlled by
the oxygen meter, once the desired oxygen level was reached. The water surface was
covered with a sheet of Plexiglas to obtain low, stable oxygen concentrations.
Ammonium concentrations were determined every 2 weeks in all treatments, using a
modification of the method of Solorzano (1969).

Each tank housed 14 boxes (18 3 18 cm), with holes on the sides covered with 45-mm
net and filled with 3 cm of sieved sediment (organic content 6%) (Fig. 1). Twelve boxes
received phytoplankton additions corresponding to 0.80 g dry weight per box or about
10 g C m 22 (assuming 40% of dry weight is carbon, based on normal values for spring
¨
bloom material in the area, A. Sjosten,
personal communication). This material settled
quickly on the sediment surface, but was not mixed down into the sediment when added.
Six boxes were left without animals, and six received 40 amphipods each, corresponding
to 1250 m 22 , which is within the normal range of field densities (Ankar and Elmgren,
1976). Two boxes were left without phytoplankton addition, one without and the other
with 40 amphipods. The experiment lasted 57 days. Soda-straw (5 mm I.D.) sediment
samples were taken weekly from the anoxic and high oxygen treatments (0.4 and 11.6
mg O 2 l 21 ). Low oxic treatments (1.7 and 3.0 mg O 2 l 21 ) were sampled only at the end
of the experiment. Samples were stored at 2 808C before analysis. There was no
noticeable loss of sediment from boxes in the tank during the experiment. On ending the

Fig. 1. The experimental system. All four tanks were the same, except that the highest oxygen tank was
bubbled with air, not N 2 . All oxygen meters used the same data logger. Twelve boxes (grey) received a
phytoplankton addition; the amphipod indicates boxes to which 40 Monoporeia affinis were added. Two boxes

(white) received no phytoplankton, but one had amphipods added.

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165

experiment, amphipods were sieved out, transferred to well-oxygenated water and living
and dead individuals counted after 30 min. The carbon input to our experimental
treatments was similar to that from a coastal spring phytoplankton bloom in the Baltic
proper. Larsson et al. (1986) reported a sedimentation of 4.9 g C m 22 during a spring
bloom event near the Asko¨ Laboratory, while Graf et al. (1982) recorded 11.5 g C m 22
over a 4-week spring bloom period in Kiel Bight, southwestern Baltic Sea.

2.3. Pigment analyses
Plant pigments in sediment and spring-bloom material were extracted using 100%
acetone according to Bianchi et al. (1995), followed by reversed-phase high-performance liquid chromatography (HPLC) after Wright et al. (1991), as modified by
Bianchi et al. (1995, 1996). A Waters model 996 photodiode array detector set at 438 nm
for absorbance, and a Shimadzu fluorescence detector with excitation at 406 nm and
emission at 660 nm were used. The injector was connected to a reversed-phase C 18
Alltech absorbosphere column (5 mm particle size; 250 3 4.6 mm I.D.) via a guard

column. After injection (100 ml), a gradient program (1 ml min 21 ) began isocratically
with mobile phase A (80:20 v / v methanol / 0.5 M ammonium acetate, aq.; pH 7.2 v / v),
ramped to 100% mobile phase B in 8 min (90:10 v / v acetonitrile / HPLC grade water),
and then changed to 20% B and 80% mobile phase C (100% ethyl acetate) in 24 min. A
return to 100% B in 10 min followed, with a final ramping to 100% A in 4 min.
High purity HPLC standards of chlorophylls a and b were obtained from Sigma Co.
Hoffman LaRoche kindly provided a fucoxanthin standard. Phaeophytins a and b were
produced by acidification (30 ml of 2 N HCl added to 1 ml of pigment solution)
followed by a clean-up separation using Alltech prep-cartridges to remove the acid.
Chlorophyllide a was prepared by activation of chlorophyllase in a diatom culture
(Thalassiosira sp.) by overnight incubation in the dark with 50% aqueous acetone.
Phaeophorbide a was then prepared by acidification (as above) of this chlorophyllide
solution. Although a number of phaeophorbide a-like components have been reported in
the literature (Mantoura and Llewellyn, 1983; Bianchi et al., 1988), phaeophorbide a
produced like this appears as a single peak. Replicate (n 5 3) and standard precision for
plant pigments ranged from 3 to 6%.

2.4. Statistics
Differences in chlorophyll a breakdown rates could not be analyzed with linear
regression, since variances differed too much between treatments. Instead, the time at

which 70% of the initial pigment concentration had been lost was calculated by linear
interpolation, and differences between treatments were tested using the Kruskal–Wallis
test. The breakdown of fucoxanthin and the build-up of breakdown products of
chlorophylls were mostly analyzed using simple linear regression, logarithmically
transformed when needed to homogenize variances. Resulting slopes were tested for
significant differences, using Bonferroni’s correction for multiple comparisons where
appropriate.
Pigment concentrations and amphipod survival at the end of the experiment (including

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low oxic treatments) were compared using analysis of variance (ANOVA) followed by
the Student–Newman–Keuls (SNK) multiple comparison test.. Homogeneity of variances was tested with Cochran’s C-test. Previous experiments have not shown
differences between tanks with the same oxygen concentration, and therefore only one
tank was used for each oxygen concentration.

3. Results

3.1. Amphipod survival
No amphipods survived in the two lowest oxygen concentrations in treatments with
phytoplankton addition and survival was low also at the highest oxygen concentration
(Fig. 2). The number of amphipods alive at the end of the experiment was significantly
greater at 11.6 mg O 2 l 21 than at 3.0 mg O 2 l 21 (ANOVA, P , 0.001, SNK). Also,
significantly more dead amphipods were found at 11.6 mg O 2 l 21 (ANOVA, P , 0.001,
SNK). Ammonium concentration showed no significant difference among oxygen
treatments (data not shown), and was always below 0.9 mg NH 3 -N l 21 , far below the
LD 50 values (50–150 mg NH 3 -N l 21 ) typically reported for amphipods (Kohn et al.,
1994).

3.2. Effects of macrobenthos and anoxia on pigment decay
Pigment concentrations for day 0 should be the same in all treatments with additions,
and were calculated from analyses of the added spring bloom material and of the bulk
sediment without additions (see figures), taking respective weights into account. This
means that no value for between-replicate variability is available for day 0.
The time taken for chlorophyll a concentrations to decrease by 70% differed
significantly among treatments (Wilcoxon rank sum test, P 5 0.0002), and took about 18
(with animals) to 22 days (without animals) in oxic treatments, and significantly longer,
about 30 days, in anoxic treatments (Kruskal–Wallis test). The addition of animals made
a significant difference in oxic, but not in anoxic treatments. A further noticeable
difference was that concentrations were uniformly low at the end of the experiments in
all oxic treatments, that is no significant effect of animal presence remained after 57
days. At the end, almost all ( ¯ 96%) chlorophyll a was lost in the oxic treatments, also
from those without phytoplankton addition (Fig. 3), while only about 80% was lost in
the anoxic treatments. All anoxic treatments had similar concentrations at day 57, with
little of the chlorophyll a added with the spring-bloom material remaining. Judging from
concentrations in the controls without additions of spring-bloom material (Fig. 3),a
much larger fraction of the initial sediment chlorophyll a was left.. There were no
apparent effects of amphipods in treatments without organic matter additions (Fig. 3).
On day 57, remaining chlorophyll a concentrations could be compared at four different
oxygen concentrations, with and without amphipods, using ANOVA (Table 1). There
were significant differences among treatments, the SNK-test showing that chlorophyll a
concentrations were lower, the higher the oxygen concentration, with all oxygen levels

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167

Fig. 2. Percent living and dead amphipods at the end of the experiment in the four oxygen treatments with
phytoplankton addition (%). Animals recovered dead likely died within a week of the end of the 57-day
experiment. Error bars denote standard deviation, based on six replicates.

significantly different (Fig. 9). There were no significant effects on chlorophyll a
concentration at day 57 due to the presence or absence of amphipods (Table 1, Fig. 3).
The chlorophyll a breakdown products chlorophyllide a and phaeophytin a, as well as
total phaeophorbide and phaeopigments, all increased in concentration with time (Figs.
4–7). Concentrations of chlorophyllide a increased much more rapidly in the oxic
treatment than in the anoxic treatment (Fig. 4). The unreplicated day 0 value and has
been disregarded in the statistical treatment. In the oxic treatment, the chlorophyllide a
concentrations increased linearly from day 9, with slopes (6standard error) of
0.012760.0010 and 0.013660.0012 with and without amphipods, respectively (not
significantly different). In the anoxic treatments, no increase could be discerned until
after about day 30. Between days 30 and 57, the slope was higher than in the oxic
treatment, 0.017660.0018 with amphipods and 0.018060.0008 without (not signifi-

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T.S. Bianchi et al. / J. Exp. Mar. Biol. Ecol. 251 (2000) 161 – 183

Fig. 3. Concentrations (nmol g dry sediment 21 ) of chlorophyll a in oxic and anoxic treatments with and
without amphipods (closed symbols). Treatments without phytoplankton addition are signified by open
symbols. Error bars show standard deviation, based on six replicates; there were no replicates for treatments
without phytoplankton additions.

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169

Table 1
Two-factorial ANOVA comparing chlorophyll a and total phaeophoride concentrations (day 57) at oxygen
concentrations 0.4, 1.7, 3.0, and 11.6 mg O 2 l 21 , with or without amphipods (ns5not significant)
Effect

Oxygen
With / without amphipods
Interaction

Chlorophyll a

Phaeophorbides

df

F value

P value

F value

P value

3
1
3

221.9
3.0
1.8

,0.0001
ns
ns

62.2
34.2
2.9

,0.0001
,0.0001
,0.046

cantly different), but because it acted for a shorter time, it still gave lower final
concentrations.
The concentration of phaeophytin a increased more in the oxic treatment with
amphipods than in the oxic treatment without amphipods and in both anoxic treatments
(Fig. 5). Excluding the unreplicated day 0 value, the phaeophytin a concentration in the
oxic treatment with amphipods seems to have increased in a step fashion between days
23 and 30, with little change before or after. The difference between those two periods is
highly significant (two-sample t-test, P 5 0.0001). The other treatments are obviously
different, with more of a linear increase from day 9 to day 57 (except that day 57 tends
to have lower concentrations than day 50). The slope estimates, oxic without amphipods
0.017160.0009, anoxic with amphipods 0.014760.0009, anoxic without amphipods
0.012660.0014, are not significantly different among treatments.
Total phaeophorbide concentrations increased over time, particularly after day 30.
Excluding the unreplicated day 0 value, a slope for days 9–57 can be calculated for all
treatments with added organic matter, except oxic with amphipods, where values after
day 30 were clearly higher than in the other three groups (Fig. 6). The slope is
0.034760.0030 for the oxic treatment without amphipods, which is significantly greater
than in both anoxic treatments: with amphipods 0.014060.013, without 0.013960.0017
(no significant difference).
The total phaeopigment concentrations (Fig. 7) showed a development similar to that
of phaeophytin a, with both oxic treatments diverging very clearly from the anoxic ones
from day 30, and oxic with amphipods clearly higher than without. For the anoxic
treatments, a regression for days 9–57 gives identical slopes with amphipods
(0.037160.0022) and without (0.037260.0042). Total phaeopigments in oxic and
anoxic treatments without added organic matter appeared not to differ between
treatments with and without amphipods (Fig. 7). On day 57, total phaeopigment
concentrations could be compared for four different oxygen concentrations, with and
without amphipods, using ANOVA (Table 1, Fig. 9). For total phaeopigment concentrations, a significant interaction term was found. The SNK showed significantly
lower phaeopigment concentrations at levels of 0.4 and 1.7 mg O 2 l 21 for treatments
with and without amphipods and at 3 mg O 2 l 21 without amphipods. Intermediate
concentrations of phaeopigments were found in treatments with amphipods at 3 mg O 2
l 21 , and at 11.6 mg O 2 l 21 without amphipods. The highest phaeopigment concentrations were found at 11.6 mg O 2 l 21 with amphipods (Fig. 9).
Concentrations of fucoxanthin, a carotenoid marker for diatoms, showed a decrease

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T.S. Bianchi et al. / J. Exp. Mar. Biol. Ecol. 251 (2000) 161 – 183

Fig. 4. Concentrations (nmol g dry sediment 21 ) of chlorophyllide a in oxic and anoxic treatments with and
without amphipods. Treatments without phytoplankton addition are signified by open symbols. Error bars show
standard deviation, based on six replicates; there were no replicates for treatments without phytoplankton
addition.

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171

Fig. 5. Concentrations (nmol g dry sediment 21 ) of phaeophytin a in oxic and anoxic treatments with and
without amphipods. Treatments without phytoplankton addition are signified by open symbols. Error bars show
standard deviation, based on six replicates; there were no replicates for treatments without phytoplankton
addition.

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Fig. 6. Concentrations (nmol g dry sediment 21 ) of total phaeophorbides in oxic and anoxic treatments with
and without amphipods. Treatments without phytoplankton addition are signified by open symbols. Error bars
show standard deviation, based on six replicates; there were no replicates for treatments without phytoplankton
addition.

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173

Fig. 7. Concentrations (nmol g dry sediment 21 ) of total phaeopigments in oxic and anoxic treatments with and
without amphipods. Treatments without phytoplankton addition are signified by open symbols. Error bars show
standard deviation, based on six replicates; there were no replicates for treatments without phytoplankton
addition.

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174

over time (Fig. 8), which can be described by linear regression of logarithmic
concentration values. Taking into account that day 0 is unreplicated, the following slopes
and degrees of explanation (r 2 ) were estimated:
Oxic with amphipods:
Oxic without amphipods:
Anoxic with amphipods:
Anoxic without amphipods:

slope
slope
slope
slope

5
5
5
5

2 0.0561,
2 0.0497,
2 0.0455,
2 0.0446,

r 2 5 0.968
r 2 5 0.975
r 2 5 0.927
r 2 5 0.973

Using Bonferroni’s method, the calculated slopes must differ by at least 0.004 to be
significantly different, and all but the two anoxic treatments are thus significantly
different. Fucoxanthin concentration thus decreased most rapidly in the oxic treatment
with amphipods, slower in the oxic treatment without amphipods and most slowly in the
two anoxic treatments (Fig. 8). By day 57, total loss of fucoxanthin was over 90% in all
treatments with added organic matter. As for chlorophyll a, more fucoxanthin had been
lost in oxic than in anoxic treatments without added spring-bloom material, but the
presence on animals seemed to make little difference in any of theses treatments (Fig.
8).

3.3. Pigment decay constants
Apparent first-order decay rate constants were calculated from the relationship:
Gt 5 Gi e 2kt
where Gt 5concentration of pigment at time t (nmol g dry sed 21 ), Gi 5initial concentration of pigment (nmol g dry sed 21 ), t5time (days), and k5decay rate constant
(day 21 ). Changes in chlorophyll a and fucoxanthin were fitted using least squares
non-linear regression of concentration versus time to yield the rate constant (k, given in
Table 2) of this simple first-order equation.

4. Discussion

4.1. Pigment decay constants
The range of chlorophyll a decay rate constants observed in both anoxic and oxic
sediments, with and without amphipods (0.04–0.07 day 21 ), was within the range
observed for pigments in previous studies (Leavitt and Carpenter, 1990; Bianchi and
Findlay, 1991; Sun et al., 1991, 1993a,b; Sun and Wakeham, 1994). Decay rate
constants in oxic treatments were faster than in anoxic treatments, which agrees with the
general paradigm of slower decay kinetics under anoxic conditions (Fenchel and Finlay,
1995). Fucoxanthin decay rate constants in this study were also similar to chlorophyll a,
as were decay constants found in decomposing freshwater epiphytic algae (Bianchi and
Findlay, 1991). Decay rate constants under anoxic and oxic conditions found for both
fucoxanthin and chlorophyll a in this study (0.04–0.07 day 21 or 15–26 year 21 , Table

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175

Fig. 8. Concentrations (nmol g dry sediment 21 ) of fucoxanthin in oxic and anoxic treatments with and without
amphipods. Treatments without phytoplankton addition are signified by open symbols. Error bars show
standard deviation, based on six replicates; there were no replicates for treatments without phytoplankton
addition.

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Table 2
First-order decay constants (k) (day 21 ) of chlorophylls and carotenoids from phytoplankton detritus in eight
treatments of a laboratory experiment a
Treatment

Chlorophyll a
(k)
(9–57 days)

Fucoxanthin
(k)
(9–57 days)

Oxic a (1)
Oxic a (2)
Anoxic a (1)
Anoxic a (2)

0.0760.02
0.0560.02
0.0460.01
0.0460.01

0.0760.02
0.0660.02
0.0560.01
0.0560.01

a

Decay constants of pigments were calculated for all treatments during the time periods of days 9–57.
(6S.D.) Indicates 95% confidence limits.

1), are within range of that found for lipids, proteins, and carbohydrates in phytodetritus
decay experiments (Harvey et al., 1995; Canuel and Martens, 1996). This consistency is
somewhat surprising, since past studies have shown oxygen-poor, hydrolysis-resistant
molecules such as lignins, lipids, and plant pigments to be more sensitive to oxygen
effects than more labile substrates such as carbohydrates and amino acids (Hedges and
Keil, 1995).
Pigments ‘bound’ to structural compounds such as lignins or surface waxes in higher
plants have slower decay rate constants than similar pigments from non-vascular sources
(Webster and Benfield, 1986; Bianchi and Findlay, 1991). Thus, despite significant
differences in the molecular weight constituents of organic matter in higher plants versus
algae, many of these compounds (i.e. lipids, plant pigments) appear to decay at
comparable rates under similar redox conditions. These results indicate that much of the
decomposition of these compounds is occurring in the ‘free’ state, similar to chlorophyll
a under anoxic conditions (Sun et al., 1991).

4.2. Effects of macrobenthos on pigment decay
Although amphipod survival was low even in the high oxygen (11.6 mg O 2 l 21 )
treatment, some survived the experiment, and those recovered dead must have died
recently, because dead amphipods decompose totally in a week or less (Elmgren et al.,
1983; Johansson, unpublished). The sediment surface turned black soon after organic
matter addition, and local anoxic conditions within the sediment, with possible sulfide
production, may have contributed to the high amphipod mortality. The amount of
organic material added by death of amphipods, 30–40 mg dry weight, was small in
comparison with the 800 mg dry weight of algal material added (assumes an amphipod
dry weight of 1 mg, half of which was lost in those recovered dead). The addition of
organic material from dead amphipods in some treatments is thus unlikely to have
influenced pigment decay rates. The enhanced pigment decay rates found in the presence
of amphipods are thus a conservative result, in the sense that the same initial amphipod
density would likely have stimulated pigment breakdown even more, had mortality been
lower. Pigments in boxes without organic matter addition had lower decay rates,
presumably because they were homogeneously mixed into sediments, and thus largely

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177

protected from oxygen, unlike phytoplankton additions, which were added on top of the
sediment. That the added phytoplankton had been frozen may also have made its
pigment easier to break down.
Based on changes in chlorophyll a concentrations with time, it appears that Baltic
macrofauna (amphipods) can significantly increase the decomposition rate of phytoplankton pigments in sediments. The highest decay rate for chlorophyll a was found in
the oxic treatment with amphipods. Similarly, the highest phaeophorbides and lowest
chlorophyll a concentrations were found at the end of the experiment in this treatment
(Fig. 9). These results agree with previous studies, which showed that macrofauna
enhance the decomposition rate of bulk organic matter (Rhoads, 1974; Aller, 1982; Rice,
1986). More specifically, it has been shown that metazoans are primarily responsible for
the conversion of chlorophylls to phaeophorbides (Shuman and Lorenzen, 1975;
Welschmeyer and Lorenzen, 1985; Bianchi et al., 1988). Recent work also has shown
that pyrolised phaeopigments such as pyrophaeophorbide a and pyrophaeophytin a
accounted for more than 70% of the breakdown pigments formed during grazing on
phytoplankton by copepods (Head and Harris, 1992, 1994, 1996). Pyrophaeophorbide a
has the same structure as phaeophorbide a except that the methylated carboxyl group has
been eliminated from the isocyclic ring from the C-13 proprionic acid group. Grazing
activity of meiofauna may also have contributed to increased production of phaeophorbides in the oxic treatment without amphipods during the last weeks of the experiment.
In another study, it was shown that pigment decay rates in experimental sediments with
very few meiofauna were lower than in those with abundant meiofauna (Sun et al.,
1993a). We did not measure meiofauna in the experimental sediments. Natural
meiofaunal biomass in the sediments of this region average 1.2 g m 22 shell-free dry
weight (Ankar and Elmgren, 1976), but previous experimental work has shown that
meiofaunal content in sediment treated as in our experiment tends to be about half the
field density. A field study in the Baltic showed that 1–2 months were required for
meiobenthic populations to increase markedly after the settling out of the spring
´
phytoplankton bloom (Olafsson
and Elmgren, 1997).
Estimates of sediment processing by M. affinis support the idea that meiofauna and
bacteria played a significant role in the breakdown of pigments. Based on previous
experimental measurements of M. affinis ingestion rates (same age- and size class, at
78C, Elmgren et al., 1986; Lopez and Elmgren, 1989), we calculated that the 40
amphipods in our treatments could only have ingested 400–800 mm 3 of surface
sediment day 21 . Lopez and Elmgren (1989) found that M. affinis feeds primarily on
surface sediment. If only the top mm of sediment was eaten it would take amphipods
40–80 days to ingest the top millimeter of sediment in an experimental box. Since there
was considerable amphipod mortality, and added algal material presumably was
gradually worked down into the sediment, the amphipods could not have ingested all
added algal material during the 57-day experiment, and could therefore not alone explain
the breakdown of almost all added chlorophyll.
While recent studies have shown pyrolised phaeopigments to be the dominant
products of chlorophyll a decay during grazing by copepods, we saw no evidence for
production of such decay products. This difference in decay products might be explained
by differences in grazing efficiency between benthic and pelagic food webs (Bianchi et

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Fig. 9. Concentrations (nmol g dry sediment 21 ) of total chlorophyll a (A) and phaeopigments (B) on the last
day of the experiment (day 57) in the four different oxygen treatments (mg l 21 ) that had received an addition
of spring bloom phytoplankton, with and without amphipods. Error bars denote standard deviation, based on
six replicates.

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179

al., 1991). Chlorophyllide and phaeophytin are usually produced by processes other than
metazoan grazing (i.e. cell lysis and senescence, microbial decay), and did not increase
as dramatically as did total phaeophorbides in oxic treatments. There appeared to be a
delay in production of total phaeopigments in relation to loss of chlorophyll a. A similar
time lag was observed in Long Island Sound sediments, and was attributed to timedependent growth patterns of water column phytoplankton followed by later inputs of
zooplankton feces to the sediments (Sun and Wakeham, 1994; Sun et al., 1994). While
most of the chlorophyll a was lost by day 23 in both oxic and anoxic treatments, the
most rapid increase in total phaeopigments in oxic treatments occurred after day 23 or
30 (Figs. 3 and 7). The breakdown of chlorophyll a to colorless products (Klein et al.,
1986) is not likely to explain this delay, since the total amount of phaeopigments
produced agreed with total chlorophyll a loss during the entire experiment. There are,
however, numerous unidentified breakdown products from chlorophyll a digestion via
copepod grazers (Head and Harris, 1992). The delayed appearance of phaeopigments
may therefore have been due to intermediate pigment decay products, not resolved with
the HPLC method used.

4.3. Anoxia and macrofaunal bioturbation effects on organic matter preservation in
the Baltic
Field studies have demonstrated increased preservation of sedimentary organic matter
in laminated sediments during anoxic periods in the Baltic (Jonsson et al., 1990). The
role of productivity and oxygen in preservation of sedimentary organic matter has been
intensely debated in recent years, but no consensus has been reached (Henrichs and
Reeburgh, 1987; Canfield, 1989; Fenchel and Finlay, 1995). Different decay rate
constants of individual molecular biomarkers and bulk organic matter may explain some
dissimilarity between oxic and anoxic treatments. In a recent study by Harvey et al.
(1995), decay rates and overall turnover rates of different biochemical fractions in
decomposing phytoplankton organic matter were significantly higher in oxic than in
anoxic conditions. Harvey et al. (1995) also demonstrated that despite greater losses of
organic matter in oxic treatments, bacterial abundance and metabolism were similar
under anoxic and oxic conditions, indicating that oxygen increased rates of organic
matter decomposition. Conversely, decomposition of individual carbon compounds in a
stratified water column was not affected by oxygen in another study (Lee, 1992).
Numerous studies have shown that there is little or no effect of oxygen on decay of
some easily decomposed substrates derived from phytoplankton, such as sugars and
amino acids (see discussion paper by Hedges and Keil, 1995). Hydrolysis-resistant,
usually oxygen-poor substrates such as lignin, lipids, and carotenoid pigments are major
exceptions (Hedges and Keil, 1995). The greater loss of chlorophyll a in higher oxygen
treatments by day 57 shows that oxygen increased pigment decay. Thus, slower rate of
pigment decay observed at low oxygen concentrations in our experiment supports the
idea that preservation of sedimentary organic matter is likely to be enhanced during
periods of anoxia, at least in the short-term (months to years).
Temporal effects of recruitment may obscure the relationship between macrofauna
and oxygenated sediments in the Baltic. For example, sediments with no macrofauna

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may have oxygen levels where macrofauna can survive but recruitment has not yet
occurred. Our experiments demonstrate that hypoxic and anoxic events, severe enough
to eliminate or greatly reduce macrofauna, may have effects on pigment decay well
beyond those caused by elimination of oxic break-down pathways during the period of
acute oxygen deficiency. Even after high oxygen levels return to bottom water, the
macrobenthos is gone, along with its stimulatory effect on pigment decay. Pigments will
be less exposed to decay agents, and anaerobic breakdown pathways will continue to
prevail in all but the thinnest surface layer. When oxygen was introduced by switching
from anoxic to oxic conditions in experimental sediments, chlorophyll a concentrations
remained constant, indicating that oxygen alone is not sufficient for pigment decay (Sun
et al., 1993a). In deep Baltic sediments, macrofaunal recolonization is slow, and it may
take 1–2 years before significant macrofauna activity returns. In recent years, hypoxia
and anoxia have been frequent enough in the Baltic proper to keep very large areas free
from macrobenthos, either continuously, or for very long periods (Andersin et al., 1978;
Rumohr et al., 1996). Our experiments support the idea that, at least in the short term,
this will enhance organic matter preservation in general, and that of plant pigments in
particular. The latter observation is important for efforts to use plant pigments in
sediment cores for reconstructing paleoecological conditions in the Baltic Sea (Bianchi
et al., 2000).

5. Conclusions
On the basis of chlorophyll a and fucoxanthin decay rates, and rates of production of
chlorophyll breakdown products, in experimental oxic and anoxic sediments, with and
without deposit-feeding amphipods, we conclude that:
1. Plant pigments decay more rapidly in oxic than anoxic sediments
2. The highest decay rates were found in oxic treatments with amphipods
3. Macrofauna appear to enhance (directly and indirectly) the breakdown of phytoplankton inputs to Baltic sediments
4. Anoxic events reduce plant pigment decay rates while they last, and have a further
long-term effect through their elimination of benthic macrofauna, which if present
stimulate pigment decay

Acknowledgements
We thank M. Argyrou, A. Bennett, E. Engelhaupt, and C. Lambert for assistance with
¨
graphics and HPLC analyses. B. Soderlund
helped collect animals and start experiments.
¨
¨ of the Department of Mathematics, Stockholm University, gave
Anders Bjorkstrom
´ and an anonymous
excellent statistical help. We thank G. Ejdung, S. Hansson, L. Byren
referee for helpful comments. The chemistry laboratory at the Department of Systems
Ecology performed the ammonium analyses. This work was supported by grants from
the National Science Foundation (INT-9500018) to TSB and the Swedish Natural

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181

Science Research Council and the Swedish Environment Protection Agency to
RE. [SS]

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