Estuarine, Coastal and Shelf Science 83 (2009) 265–276
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Estuarine, Coastal and Shelf Science
journal homepage: www.elsevier.com/locate/ecss
Effect of a simulated oil spill on natural assemblages of marine phytoplankton
enclosed in microcosms
J. González a, *, F.G. Figueiras b, M. Aranguren-Gassis a, B.G. Crespo b, d, E. Fernández a, X.A.G. Morán c,
M. Nieto-Cid b, e
a
Laboratorio de Ecoloxı́a Mariña, Departamento de Ecoloxı́a e Bioloxı́a Animal, Facultade de Ciencias do Mar, Universidade de Vigo, Ctra. Colexio Universitario s/n, 36310 Vigo
(Pontevedra), Spain
b
Instituto de Investigaciones Marinas, CSIC, Eduardo Cabello 6, 36208 Vigo, Spain
c
Instituto Español de Oceanografı́a, Centro Oceanográfico de Xixón, Camı́n de L’Arbeyal, s/n, Xixón, Spain
d
Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA
e
Marine Chemistry and Geochemistry Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 January 2009
Accepted 2 April 2009
Available online 15 April 2009
Two microcosm experiments were carried out to simulate the effect of sporadic oil spills derived from
tanker accidents on oceanic and coastal marine phytoplankton assemblages. Treatments were designed
to reproduce the spill from the Prestige, which took place in Galician coastal waters (NW Iberia) in
November 2002. Two different concentrations of the water soluble fraction of oil were used: low
(8.6 0.7 mg l1 of chrysene equivalents) and high (23 5 mg l1 of chrysene equivalents l1). Photosynthetic activity and chlorophyll a concentration decreased in both assemblages after 24–72 h of
exposure to the two oil concentrations, even though the effect was more severe on the oceanic
assemblage. These variables progressively recovered up to values close or higher than those in the
controls, but the short-term negative effect of oil, which was generally stronger at the high concentration, also induced changes in the structure of the plankton community. While the biomass of nanoflagellates increased in both assemblages, oceanic picophytoplankton was drastically reduced by the
addition of oil. Effects on diatoms were also observed, particularly in the coastal assemblage. The
response of coastal diatoms to oil addition showed a clear dependence on size. Small diatoms (<20 mm)
were apparently stimulated by oil, whereas diatoms >20 mm were only negatively affected by the high oil
concentration. These differences, which could be partially due to indirect trophic interactions, might also
be related to different sensitivity of species to PAHs. These results, in agreement with previous observations, additionally show that the negative effect of the water soluble fraction of oil on oceanic
phytoplankton was stronger than on coastal phytoplankton.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
phytoplankton assemblages
oil spills
microcosms
Galicia
Prestige
1. Introduction
Among all adverse effects that large oil spills due to tanker
accidents cause in coastal zones, the most evident can be easily
observed on seashores and nearby sediments. At these places,
sessile organisms as well as other organisms with low mobility are
directly affected by the accumulation of the insoluble fraction of oil.
Nevertheless, planktonic life is also disturbed by this type of marine
oil pollution. Thus, oil slicks over the sea surface not only limit gas
exchange through the air–sea interface, they also reduce light
penetration into the water column affecting phytoplankton
* Corresponding author.
E-mail address: [email protected] (J. González).
0272-7714/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ecss.2009.04.001
photosynthesis. Furthermore, crude oil constitutes a large reservoir
of the highly toxic polycyclic aromatic hydrocarbons (PAHs), which
soluble fraction is rapidly released into the water column following
the spill. Since PAHs remain dissolved in seawater for an extended
period of time, even after the insoluble fraction has been removed
(e.g. González et al., 2006), and the structure and function of pelagic
marine food webs basically depend on the energy and matter
supplied by phytoplankton photosynthesis, understanding how
PAHs affect to phytoplankton populations is a relevant issue (e.g.
Ostgaard et al., 1984b; Kelly et al., 1999; Hjorth et al., 2007, 2008).
Current knowledge on the toxic effect that these contaminants
cause to natural assemblages of marine phytoplankton is still
unclear and sometimes contradictory. Several investigations have
addressed the effect of a single PAH on natural phytoplankton
assemblages (Kelly et al., 1999; Marwood et al., 1999; Hjorth et al.,
266
J. González et al. / Estuarine, Coastal and Shelf Science 83 (2009) 265–276
2007) or the influence of crude oil (Kusk, 1978; Ostgaard et al.,
1984a,b) and diesel oil on cultured phytoplankton species (Chan
and Chiu, 1985). However, few studies have addressed the response
of natural phytoplankton to sporadic oil inputs (Yamada et al.,
2003; Ohwada et al., 2003). While some investigations reported
negative effects on phytoplankton (Ostgaard et al., 1984a,b; Sargian
et al., 2005), others found stimulatory effects (Oviatt et al., 1982).
Differences in sensitivity of taxonomic groups have also been
reported. In some cases, diatoms were found to be more affected by
oil than other phytoplankton groups (Harrison et al., 1986),
whereas other investigations did not show such difference (Vargo
et al., 1982).
Several oil spills due to tanker accidents have occurred along the
Galician coast (NW Spain) over the last 30 years (Varela et al.,
2006), mainly during rough weather in winter. On 13 November
2002, the Prestige oil tanker started leaking oil 30 miles off the
Galician coast (Álvarez-Salgado et al., 2006; González et al., 2006;
Ruiz-Villarreal et al., 2006). Six days later, after an erratic course,
the tanker broke in two and sank 150 miles offshore, releasing the
main amount of oil. It is estimated that more than 60,000 metric
tons of fuel (Garcı́a-Soto, 2004) were released into the water. As the
knowledge required for predicting its impact on phytoplankton was
scarce at that time, we designed a set of experiments directed to
improve our understanding on the effect that an oil spill of this type
has on natural phytoplankton assemblages.
In this study we examined the effect of the water soluble fraction of oil at both the physiological and the community level by
measuring the photosynthetic efficiency of photosystem II (PSII),
primary production and phytoplankton biomass and composition.
The aim was to evaluate the short-term response (less than 5 days)
of two different natural phytoplankton assemblages to experimental oil spills.
2. Materials and methods
2.1. Experimental setup
Two microcosm experiments were conducted in October and
December 2004, using natural assemblages of primary producers
collected from a coastal station off the Rı́a de Vigo, NW Iberia 41
540 N, 8 550 E. These dates were selected because this is the time
period when the last oil spills occurred in the region (Prestige, 13
November 2002; Aegean Sea, 3 December 1992), and when it is
expected to find different phytoplankton populations in coastal
waters. The seasonal transition from upwelling to downwelling
conditions habitually occurs in this region in October (Figueiras
et al., 2002), and during this transition surface oceanic waters
containing Prochloroccocus are advected to the coast (Calvo-Dı́az
et al., 2004; Rodrı́guez et al., 2006). Since later, in winter, Prochlorococcus disappears from coastal waters (Rodrı́guez et al.,
2006), its presence in water samples collected near the coast in
autumn can be used as a tracer indicating the advection of oceanic
waters (Calvo-Dı́az et al., 2004).
Nine 4 l borosilicate glass flasks were filled with surface water
collected with Niskin bottles. Three of these flasks were used as
controls and the other six were utilised to test two experimental
treatments: low (LC; 8.6 0.7 mg l1 of chrysene equivalents) and
high (HC; 23 5 mg l1 of chrysene equivalents) oil concentration.
The HC was slightly lower than the concentration obtained in the
water accommodated fraction (34 mg l1 of chrysene equivalents)
prepared according to Singer et al. (2000) with the oil from the
Prestige (González et al., 2006). Therefore, the HC that we used
should be considered as the concentration expected in the water
just after the spill. Furthermore, both LC and HC were well above of
the values (0.09–4.84 mg l1 of chrysene equivalents) recorded in
the region 1 month after the Prestige disaster (González et al.,
2006).
The flasks were maintained in a culture chamber at 15 C, an
irradiance of 70 mmol quanta m2 s1 and a 14:10 h light–dark
photoperiod. The addition of the accommodated fraction of oil to
the experimental treatments was done by suspending in the water
several glass tubes with their surface area covered with oil from the
Prestige. These tubes were removed from the culture flasks after
24 h, once the concentration of the accommodated fraction of oil
reached stable values. After removing the tubes with the solid
phase of oil, air was gently bubbled into the flasks to ensure stirring.
Samples were drawn from the flasks before the addition of the
accommodated fraction (0 h) and at 24, 72 and 120 h to determine
the photosynthetic efficiency of PSII, chlorophyll a (chl a) concentration, primary production and plankton composition. Additional
samples were also taken at 48 and 96 h to determine the photosynthetic efficiency of PSII.
2.2. Dissolved PAHs
The evolution of PAH concentrations in the microcosms was
estimated by direct fluorescence at excitation/emission wavelengths of 280/350 nm. This simple and low-volume consuming
procedure was calibrated against the MARPOLMON protocol
(UNESCO, 1984), which is based on the extraction and concentration of the water soluble PAHs in hexane followed by its determination by fluorescence at excitation/emission wavelengths of 310/
360 nm. The conversion factor of direct fluorescence into PAH
concentrations referred to a chrysene standard (Ehrhardt et al.,
1991) was 0.33 0.02 with a standard error of the estimation of
1.1 mg l1 chrysene equivalents. Measurements of chrysene standards against Prestige oil extracted with hexane provided a factor of
5.5 0.4 mg Prestige oil mg chry1. We are aware that the composition of the accommodated fraction is not elucidated with this
procedure, which was used to keep the final water volume in the
flasks above 50% of the initial volume. However, spatial and
temporal distributions of oil concentrations in the Galician coast
determined by this procedure paralleled those of total PAH
concentrations determined by gas chromatography-mass spectrometry (González et al., 2006). PAH composition of the water
accommodated fraction of the Prestige fuel-oil was dominated by
naphthalene and its alkylated derivates (methylnaphtalenes,
dimetylnaphtalenes, and trimethylnaphtalenes), which accounted
for 89% of the total PAH concentrations, and this PAH composition
remained in the region 1 month after the Prestige disaster (González et al., 2006). Therefore, it can be assumed that our experiments basically assessed the effects induced by naphthalene and its
alkylated derivates.
2.3. Photosynthetic efficiency of PSII
The photosynthetic efficiency of photosystem II (Fv/Fm) was
measured with a fast repetition rate fluorometer (FRRF) as
described in Pérez et al. (2006). Briefly, the FRRF, which operated
with a frequency of 100 saturating flashes and 20 relaxation flashes,
was configured to only allow flashing the light chamber, where the
samples were measured in darkness. Three 40 ml replicates from
each microcosm were sampled daily, which were kept in darkness
for 30 min before doing measurements to allow relaxation of nonphotochemical quenching. Blank values were obtained by filtering
water from the microcosms through Millipore glass fibre filters, and
then measuring the Fv/Fm values as for the rest of the samples. A
mean value for each treatment was obtained.
The ratio Fv/Fm is related to the potential photochemical efficiency of PSII (Falkowski and Raven, 1997). When all PSII reaction
J. González et al. / Estuarine, Coastal and Shelf Science 83 (2009) 265–276
Table 1
Initial conditions for each experiment. For total chl a concentration and primary
production values are means standard error (n ¼ 9). DIN, dissolved inorganic
nitrogen (nitrate þ ammonium þ nitrite).
Variable
October
December
Temperature (sC)
Salinity (psu)
DIN (mmol kg1)
1
HPO2
4 (mmol kg )
SiO4H4 (mmol kg1)
15.5
35.609
1.03
0.10
1.23
13.9
34.642
2.68
0.08
1.49
Chl a (mg m3)
0.60 0.03
2.55 0.27
<2 mm (%)
2–20 mm (%)
>20 mm (%)
31
33
36
17
42
41
Phytoplankton (mg C m3)
27.0
98.5
<2 mm (%)
2–20 mm (%)
>20 mm (%)
24
37
39
5
57
38
Primary production
(mg C m3 h1)
1.73 0.28
20.43 7.74
<2 mm (%)
2–20 mm (%)
>20 mm (%)
16
32
52
15
52
33
Carbon: chl a (weight: weight)
45
36
<2 mm
2–20 mm
>20 mm
31
66
38
11
53
32
Heterotrophic dinoflagellates
and ciliates (mg C m3)
6.4
49.5
Dinoflagellates (%)
Ciliates (%)
73
27
95
5
centres are open, fluorescence is minimal (Fo), because the
excitation energy is primarily used in photosynthesis. In contrast,
when PSII centres are closed the excitation energy cannot be used
in photosynthesis and, hence, fluorescence rises to a maximum
value (Fm). Variable fluorescence (Fv) is defined as Fm–Fo.
10
A
mg C m-3
8
6
Synechococcus
Proclorochoccus
Picoeukaryotes
Therefore, Fv/Fm indicates the fraction of the absorbed energy
channelled to photosynthesis by PSII reaction centres. Thus, low
values of Fv/Fm, which have been related to nutrient deficiency in
natural conditions (e.g. Kolber and Falkowski, 1993), have also been
ascribed to toxic effects on phytoplankton (e.g. Juneau et al., 2002;
Pérez et al., 2006).
2.4. Size-fractionated carbon fixation
Primary production was determined by the radiocarbon
method. Three 40 ml light and one dark acid-cleaned pyrex bottles
were filled with water from each culture flask. Each bottle was
inoculated with 10 mCi NaH14CO3 and incubated for 2 h in the
culture chamber where the microcosms were maintained. After
that, samples were filtered sequentially through 20, 2 and 0.2 mm
pore size polycarbonate filters, which were exposed to concentrate
HCl fumes for 24 h to eliminate unincorporated 14C. Radioactivity of
the samples was measured with a liquid scintillation counter using
the external standard and the channel ratio methods to correct for
quenching.
2.5. Size-fractionated chlorophyll a concentration
To determine size-fractionated chl a concentration two samples
of 100 ml taken from each culture flask were filtered sequentially
through 20, 2 and 0.2 mm pore size polycarbonate. Filters were
frozen (20 C) before pigment extraction in 90% acetone over 24 h
in the dark at 4 C. A Turner TD-700 fluorometer calibrated with
pure chl a (Sigma) was used to determine chl a concentrations.
2.6. Phytoplankton community composition
Picophytoplankton was determined from samples of 1.8 ml fixed
with 2% glutaraldehyde using a FACSCalibur flow cytometer as
described in Calvo-Dı́az and Morán (2006). Carbon biomass was
estimated assuming a spherical shape and using volume-to-carbon
conversion factors: 230 fg C mm3 for Synechococcus, 240 fg C mm3
for Prochlorococcus and 237 fg C mm3 for picoeukaryotes (Worden
et al., 2004).
100
Nano-microphytoplankton
D
Microphytoplankton
60
40
2
20
0
0
40
B
80
4
60
mg C m-3
Picophytoplankton
C
Nanophytoplankton
40
30
Nanoflagellates
Dinoflagellates
Diatoms
267
Nanoflagellates
Dinoflagellates
Diatoms
Other flagellates
Dinoflagellates
Diatoms
20
20
10
0
0
October
December
October
December
Fig. 1. Biomass composition of initial phytoplankton assemblages enclosed for the experiments conducted in October and December.
268
J. González et al. / Estuarine, Coastal and Shelf Science 83 (2009) 265–276
30
A
Control
LC
HC
µg l-1 chrysene eq.
25
nanoplankton was not possible with this technique. In addition, the
preservation procedure could cause losses in the nanoplankton
fraction, which is mainly composed of flagellates.
2.7. Statistical analyses
20
Analysis of variance (ANOVA) was applied to daily data to
determine the significance of differences between means. Post-hoc
multiple-comparison tests were used at each sampling time to
detect significant differences between treatments in the variables
measured. The Bonferroni post-hoc test was used when the variance was homogeneous, whereas the T3 Dunnett’s test was used
when variance was not homogeneous.
15
10
5
0
3. Results
30
3.1. Initial conditions
B
µg l-1 chrysene eq.
25
20
15
10
5
0
0
24
48
72
96
120
Time (h)
Fig. 2. Time course of PAH concentrations expressed in chrysene equivalents during
the experiment of October (A) and December (B). Error bars represent the standard
error.
Nano- and microplankton were determined in samples
preserved in Lugol’s idone solution 2% final concentration. To avoid
an excessive consumption of water and keep the flasks until the end
of the experiment with >50% of the initial volume, only one sample
of 25 ml was taken from each flask at each sampling time. The
samples were then pulled according to control and treatments to
obtain final volumes (75 ml) suitable for counting. Initial abundances were only determined in the original samples, previously to
their separation into treatment and control flasks. Unfortunately,
the samples collected at 24 h from LC and HC treatments in the
experiment of October were lost. The preserved samples were
allowed to settle for 48 h in composite sedimentation chambers
and the organisms were counted and identified to the species level,
when possible, using an inverted microscope. Several transects
were scanned at 400 and 250 to enumerate the small species.
The larger species, often less abundant, were counted from scanning the whole slide at 100 magnification. Dimensions were
taken to calculate cell biovolumes after approximation to the
nearest geometric shape (Hillebrand et al., 1999). Cell carbon
biomass was estimated following Strathmann (1967) for diatoms
and dinoflagellates, Verity et al. (1992) for flagellates and Putt and
Stoecker (1989) for ciliates. Pigmented and non-pigmented dinoflagellates were differentiated following Lessard and Swift (1986)
and also making use of our historical records obtained with epifluorescence microscopy. It must be noted that a complete
discrimination
between
autotrophic
and
heterotrophic
Initial conditions differed between the two experiments
(Table 1). The water sample collected in October was warmer,
saltier and contained a concentration of dissolved inorganic
nitrogen lower than the sample collected in December. However,
phytoplankton biomass, expressed as chl a concentration or carbon
biomass was four times higher in December. The difference in
primary production was even higher, because the value measured
in December was 12-fold the value determined in October. The size
structure of the phytoplankton community also differed between
experiments. While in October the contribution of nano- (2–20 mm)
and microphytoplankton (>20 mm) to total phytoplankton carbon
biomass was similar (w40%), nanophytoplankton represented
w60% in December. The contribution of picophytoplankton
(<2 mm) was particularly low in December (5%), but accounted for
24% of the total phytoplankton carbon biomass in October. These
differences were not so evident in size-fractionated chl a and
primary production, although primary production of nanophytoplankton represented >50% of the total primary production
in December and microplankton accounted for 52% of the total
primary production in October.
Marked differences also occurred in phytoplankton composition (Fig. 1). Concerning picophytoplankton (Fig. 1A), with similar
biomasses in October and December (6.0 and 5.5 mg C m3,
respectively); the most obvious difference referred to Prochlorococcus, which accounted for 50% of the total picophytoplankton biomass in October but was absent in December (Fig. 1A).
Synechococcus was present in the initial phytoplankton populations of the two experiments, though accounting for a larger
fraction of the total picophytoplankton biomass in October than in
December (18 and 5%, respectively). Picoeukaryotes dominated in
December (95%) and only represented 31% of the picophytoplankton biomass in October. Differences were also evident within
the nano-microphytoplankton community (Fig. 1B). Thus, the total
nano-microphytoplankton biomass, which was almost equally
distributed among diatoms, dinoflagellates and nanoflagellates in
October, was however dominated by nanoflagellates in December
(54%), when autotrophic dinoflagellates represented only 2% and
diatoms accounted for the remaining 44%. Although nanoflagellates dominated in the 2–20 mm size-fraction in the two
cases (Fig. 1C), its contribution was more important in December
(94% of nanophytoplankton biomass) than in October (68%). In
October the contribution of small diatoms (22%) and small dinoflagellates (10%) within the 2–20 mm size-fraction was also relatively important. Dinoflagellates (53%) and diatoms (45%)
co-dominated within the microphytoplankton fraction in October,
while in December this fraction was almost exclusively composed
of diatoms (92%) (Fig. 1D). Total phytoplankton carbon:chl a ratios
J. González et al. / Estuarine, Coastal and Shelf Science 83 (2009) 265–276
0.5
Fv/Fm
0.4
October
0.5
A
269
December
B
0.3
0.3
0.2
**
*
*
*
0.4
*
0.2
0.1
0.1
0.0
*** ***
7
mg C m-3 h-1
6
0.0
60
C
Control
LC
HC
5
50
D
40
30
3
2
**
20
*
***
10
** *
***
0
1.5
Control
LC
HC
*
4
1
mg chl a m-3
*
*
0
8
E
F
6
1.0
4
*
0.5
*
0.0
0
24
*
48
72
**
2
0
96
120
Time (h)
0
24
48
72
96
120
Time (h)
Fig. 3. Time course of the photosynthetic efficiency of PSII (A, B), primary production (C, D) and chlorophyll a concentration (E, F) in the controls, low (LC) and high (HC) oil
treatments during the experiments conducted in October and December. Error bars represent the standard error. Asterisks indicate the level of significance of the differences
between the treatments and the control (* ¼ p < 0.05; ** ¼ p < 0.01; *** ¼ p < 0.001).
(45 in October and 36 in December) were within the range
habitually reported for healthy phytoplankton growing without
nutrient limitation.
Heterotrophic nano-microplankton biomass was noticeably
higher in December (Table 1), being almost exclusively
composed of dinoflagellates (95%). Heterotrophic dinoflagellates
and ciliates <20 mm were relatively more abundant in October
(67 and 90% of the biomass of heterotrophic dinoflagellates and
ciliates respectively) than in December, when large dinoflagellates accounted for 68% of the biomass of heterotrophic dinoflagellates. Heterotrophic dinoflagellates and ciliates represented
19 and 33% of the total plankton biomass in October and
December, respectively.
3.2. PAH concentrations
The initial values and the evolution of PAH concentrations were
similar in the two experiments (Fig. 2). Mean initial values in the HC
treatments (23 5 mg l1 of chrysene eq.) were approximately
three times higher than those in the LC treatment (8.6 0.7 mg l1
chrysene eq.). Then, as previously described (Yamada et al., 2003),
PAH concentrations gradually diminished, showing a more
pronounced decrease within the first 48 h because of the rapid
degradation of low molecular weight PAHs.
3.3. Photosynthetic efficiency of PSII
The evolution of the Fv/Fm ratio in the control flasks showed
contrasting differences between experiments (Figs. 3A,B). Thus,
the Fv/Fm ratio, after an initial decrease from 0.43 to 0.36,
remained constant at 0.35 0.02 in the control until the end of
the experiment of October. In contrast, the Fv/Fm ratio remained
constant at 0.45 during the first 24 h in the experiment of
December, and then showed a constant decrease at a rate of
0.04 0.004 d1 (r2 ¼ 0.96; p < 0.001) to reach a value of
0.30 0.02 at 120 h. The negative effect of oil addition was
particularly evident in October, and it was more pronounced in
the HC treatment (Fig. 3A), where the Fv/Fm ratio fell near zero
values at 24 and 48 h. In this experiment a recovery occurred
after 72 h, leading to Fv/Fm values in the two treatments which
were not significantly different from those measured in the
control during the last 2 days (Fig. 3A). The negative effect of
PAHs in the experiment of December was only perceptible in the
HC treatment at 48 h (Fig. 3B). For this experiment significant
higher values of Fv/Fm (40% higher than in the control) were
observed in the HC treatment during the last 2 days (Fig. 3B).
The Fv/Fm values in the LC treatment of December did not differ
from the values in the control, and therefore they declined at
a similar rate.
270
J. González et al. / Estuarine, Coastal and Shelf Science 83 (2009) 265–276
mg C m-3 h-1
1.2
October
<2µm
A
10
8
December
<2µm
B
6
0.8
4
0.4
2
*
0
0.0
**
*
4
C
mg C m-3 h-1
25
Control
LC
HC
3
2
2-20µm
*
10
***
***
**
*
*
5
***
0
24
>20µm
E
Control
LC
HC
20
1
3
D
15
0
mg C m-3 h-1
30
2-20µm
20
2
**
>20µm
F
16
*
12
1
***
8
**
0
4
*
**
**
0
0
24
48
72
96
120
0
24
48
72
96
120
Time (h)
Time (h)
Fig. 4. Time course of primary production in the controls, low (LC) and high (HC) oil treatments for the three size-fractions tested during the experiments conducted in October and
December: picophytoplankton (A, B), nanophytoplankton (C, D) and microphytoplankton (E, F). Error bars represent the standard error. Asterisks indicate the level of significance of
the differences between the treatments and the control (* ¼ p < 0.05; ** ¼ p < 0.01; *** ¼ p < 0.001).
3.4. Primary production
Differences between experiments also occurred in the evolution
of primary production. While total primary production in the
control of October increased continually from 1.55 0.19 to
5.05 0.86 mg C m3 h1 (Fig. 3C), in the control of December
showed a high increase from 10.21 4.68 to 51.03 5.05 mg C m3
h1 within the first 24 h (Fig. 3D), to then decline to 6.42 1.96 mg
C m3 h1 at 120 h. Primary production at 24 and 72 h in the LC and
HC treatments of October was significantly lower than the values
measured in the control, with the reduction being higher at 24 h
(92% in the two treatments) than at 72 h (70 and 89% in LC and HC,
respectively). Later, primary production in both treatments picked
up to values not significantly different from values in the control
(Fig. 3C). The reduction in primary production was only significant
at 24 h in the experiment of December, being the decrease in the LC
treatment (30%) a half of that observed in the HC treatment (62%)
(Fig. 3D). In contrast, primary production at 120 h in the HC treatment of December was nearly three times higher than primary
production in the control (Fig. 3D).
Regarding size-fractionated primary production, the three sizefractions in the LC and HC treatments of October showed an initial
decrease at 24 h (Figs. 4A,C,E). Although differences between
treatments and control for the 2–20 mm size-fraction were not
significant at 72 h (Fig. 4C), the other two size-fractions showed
significant lower values in the HC treatment at that time (Figs. 4A,E).
At the end of this experiment, significant lower values were only
found for the >20 mm size-fraction in the HC treatment (Fig. 4E).
Primary production of the 2–20 mm size–fraction in the LC and
HC treatments of December clearly decreased at 24 and 72 h
(Fig. 4D), with the decrease being higher in the HC treatment,
particularly at 24 h. For the fraction >20 mm values significantly
lower than in the control were only measured at 24 h in the HC
treatment, whereas higher values were found at the end of the
experiment (Fig. 4F). Primary production of the <2 mm fraction
followed a similar pattern to that of the 2–20 mm fraction (Fig. 4F),
though differences were not significant.
3.5. Chlorophyll a
After a first slight decrease, total chl a concentration in the control
flasks of October (Fig. 3E) increased linearly (r2 ¼ 0.998; p < 0.05) to
a value of 0.99 0.31 mg chl a m3 at 120 h (Fig. 3E). On the
contrary, chl a concentration in the control flasks of December
(Fig. 3F) increased from 2.56 0.20 to 3.94 0.27 mg chl a m3
within the first 24 h, then remained constant, and finally showed
J. González et al. / Estuarine, Coastal and Shelf Science 83 (2009) 265–276
mg chl a m-3
0.3
A
1.2
0.2
0.8
0.1
0.4
0.0
0.0
1.0
0.8
mg chl a m-3
October
<2µm
C
4.0
2-20µm
0.6
B
*
D
2-20µm
Control
LC
HC
2.0
*
0.4
1.0
0.2
0.0
0.0
0.6
mg chl a m-3
December
<2µm
3.0
Control
LC
HC
271
E
3.0
>20µm
0.4
2.0
0.2
1.0
*
0.0
0
24
48
72
96
120
Time (h)
F
>20µm
0.0
0
24
48
72
96
120
Time (h)
Fig. 5. Time course of chlorophyll a concentration in the controls, low (LC) and high (HC) oil treatments for the three-size-fractions tested during the experiments conducted in
October and December: picophytoplankton (A, B), nanophytoplankton (C, D) and microphytoplankton (E, F). Error bars represent the standard error. Asterisks indicate the level of
significance of the differences between the treatments and the control (* ¼ p < 0.05).
a slight decline at the end of the experiment (3.04 1.86 mg chl
a m3 at 120 h). Chl a concentration in both LC and HC treatments
was significantly lower than in the control at 72 h in the experiment
of October. Concentrations significantly lower were also recorded at
24 h in the LC treatment. For the experiment of December concentrations significantly lower than in the control were only measured
at 24 h in the HC treatment (Fig. 3F).
Size-fractionation shows that the evolution of total chl a in the
control and treatments of the two experiments was largely
controlled by changes occurring in the two largest fractions
(Fig. 5). Although lower chl a values were generally measured in
oiled flasks in October, significant differences were only found for
the >20 mm fraction in the HC treatment at 120 h (Fig. 5E).
Significant lower chl a concentrations were also measured for the
size-fraction <2 mm in the HC treatment of December (Fig. 5B).
Higher chl a concentrations were however recorded in December
for >20 and 2–20 mm size-fractions in the HC treatment (Figs.
5D,F), though differences were only significant for the 2–20 mm
fraction (Fig. 5D).
3.6. Size structure of the phytoplankton community
The addition of the water accommodated fraction of oil caused
a significant reduction in the biomass of picophytoplankton in
October (Fig. 6A), with the decrease being more pronounced in the
HC treatment. Although this diminution occurred in the three
components (Prochlorococcus, Synechococcus and picoeukaryotes),
it was especially severe for Prochlorococcus and Synechococcus,
which disappeared from both LC and HC treatments at 24 h
(Table 2). Thus, the slight recovery observed at 120 h in the two
treatments was due to picoeukaryotes (Table 2). Prochlorococcus
and Synechococcus also declined in the control flasks, from where
they disappeared at 72 h (Table 2). In contrast, picoeukaryotes
increased in the control at a rate of 2.0 0.43 mg C m3 d1
(r2 ¼ 0.91; p < 0.05). The final C:chl a ratios of this phytoplankton
fraction in the control (77), the LC treatment (15) and the HC
treatment (30) was higher, lower and equal than the initial ratio
(Table 1).
The negative effect of oil on picophytoplankton was not so
marked in the experiment of December (Fig. 6B), where it was only
slightly perceptible in the HC treatment. Synechococcus, which only
represented 5% of the total picophytoplankton biomass in the
original sample of December (Table 1), decreased in the control and
treatments (Table 2), though the initial biomass was recovered in
the control and LC treatment at 120 h (Table 2). The evolution of
picophytoplankton biomass in December (Fig. 6B) followed that of
picoeukaryotes (Table 2), characterised by a continuous increase
that resulted in a final biomass 5–6-fold the initial one. This
272
J. González et al. / Estuarine, Coastal and Shelf Science 83 (2009) 265–276
A
Control
LC
HC
12
mg C m-3
Table 3
Biomass (mg C m3) evolution of the main autotrophic plankton groups identified in
the control (C), low (LC) and high oil (HC) treatments during the experiment conducted in October. ND, not determined.
Group
Time (h)
C
LC
HC
Nanoflagellates
0
24
72
120
8.7
8.7
40.1
27.5
8.7
ND
37.2
51.5
8.7
ND
23.1
30.6
Diatoms <20 mm
0
24
72
120
2.8
10.4
14.2
13.6
2.8
ND
1.6
15.9
2.8
ND
0.6
1.6
Diatoms >20 mm
0
24
72
120
3.7
2.3
18.8
13.2
3.7
ND
7.3
16.1
3.7
ND
3.4
0.6
8
*
4
**
***
0
40
**
**
***
B
mg C m-3
30
20
10
*
0
0
24
48
72
96
120
Time (h)
Fig. 6. Time course of picophytoplankton biomass in the controls, low (LC) and high
(HC) oil treatments during the experiments conducted in October (A) and December
(B). Error bars represent the standard error. Asterisks indicate the level of significance
of the differences between the treatments and the control (* ¼ p < 0.05; ** ¼ p < 0.01;
*** ¼ p < 0.001).
contrasts with the evolution of chl a concentration in the <2 mm
size-fraction (Fig. 5B) which declined following the increase at 24 h.
Therefore, the final C:chl a ratios of the picophytoplankton fraction
in the control and LC and HC treatments of December (50, 61 and
300, respectively) were appreciably higher than the initial ratios
(Table 1).
Treatment
Although the lack of replicates for nano- and microphytoplankton does not allow a statistical analysis, differences
between both experiments and treatments apparently occurred
(Table 3; Fig. 7). The biomass of nanoflagellates, which showed an
initial decrease in the two treatments of both experiments,
however recovered at the end, when values in the treatments were
similar or even higher than in the controls (Table 3; Fig. 7A). In all
cases (treatments and controls) the final biomass of nanoflagellates
was higher than at the beginning of the experiment, w4 times in
October (Table 3) and w9 times December (Fig. 7A).
The evolution of diatoms was different and showed dependency
on size and experiment. In October, the biomass of the two sizefractions of diatoms in both LC and HC treatments was lower than
in the control at 72 h (Table 3). Later, at the end of the experiment,
there was a recovery in the biomass of both size-fractions in the LC
treatment, whereas in the HC treatment continued declining until
values lower than the initial ones (Table 3). The final biomasses of
the two size-fractions of diatoms in the control and LC treatment
were four to six higher than the initial biomasses. These two sizefractions of diatoms depicted a different evolution in December
(Figs. 7B,C). Thus, the biomass of diatoms >20 mm in the HC
treatment was always lower than in the control and LC treatment
(Fig. 7C), while the biomass of diatoms <20 mm in the HC treatment
was 5-fold the biomass in the control at the end of the experiment
(Fig. 7B). This fraction of diatoms had also higher biomass in the LC
treatment than in the control (Fig. 7B). The two fractions of diatoms
Table 2
Mean values standard error of picophytoplankton biomass (mg C m3) in the control (C), low (LC) and high oil (HC) treatments during the two experiments conducted in
October and December. Prochlorococcus was not present in December.
Group
Time (h)
October
December
C
LC
HC
Synechococcus
0
24
72
120
1.10 0.04
0.63 0.09
0.0
0.0
1.27 0.02
0.0
0.0
0.0
1.26 0.19
0.0
0.0
0.0
Prochlorococcus
0
24
72
120
3.02 0.19
1.57 0.14
0.0
0.0
4.06 0.11
0.0
0.0
0.0
3.56 1.07
0.0
0.0
0.0
Picoeukaryotes
0
24
72
120
1.86 0.20
2.61 0.31
5.12 0.36
12.04 1.60
1.92 0.12
0.73 0.29
1.29 0.87
2.74 1.91
2.19 0.59
0.0
0.18 0.03
1.19 0.67
C
LC
0.27 0.02
0.16 0.01
0.18 0.04
0.24 0.02
HC
0.22 0.01
0.17 0.01
0.09 0.02
0.22 0.05
0.47 0.21
0.24 0.02
0.02 0.01
0.04 0.01
–
–
–
–
–
–
–
–
–
–
–
–
5.22 0.66
13.56 1.02
25.38 4.11
23.46 3.03
4.56 0.63
11.12 1.41
23.62 1.99
27.31 3.99
4.41 0.14
6.28 0.89
14.35 8.78
28.82 3.50
J. González et al. / Estuarine, Coastal and Shelf Science 83 (2009) 265–276
600
A
Autotrophic dinoflagellates did not show any clear pattern of
evolution, either in the controls or treatments (data not shown),
probably because their low abundance led to low counting accuracy
and so to an erratic evolution. However, final biomasses in the
control and treatments of the experiment of October were lower
than the initial ones, while in December were slightly higher.
The increase in carbon biomass observed in the experiment of
December resulted in high final C:chl a ratios (265 143 and
200 138 for nanophytoplankton and microphytoplankton,
respectively) which, however, were not extremely high in the
experiment of October (86 28 and 43 16).
Nanoflagellates
500
mg C m-3
400
300
200
100
3.7. Heterotrophic dinoflagellates
0
100
B
The low abundance of heterotrophic dinoflagellates and ciliates
(Table 1) did not permit to obtain conclusive information about
their evolution in the control and treatments during the experiment of October. Nevertheless, the biomass of heterotrophic
dinoflagellates in December showed a sudden increase in the
control and LC treatment at the end of the experiment (Fig. 9A).
This increase, which was not so evident in the HC treatment, was
due to small dinoflagellates (Fig. 9B), because large heterotrophic
dinoflagellates declined (Fig. 9C). The decrease of large heterotrophic dinoflagellates was considerable in the LC and HC treatments
at 24 h. Later, large heterotrophic dinoflagellates continued
declining in the control and in the HC treatment, but manifested
a slight recovery in the LC treatment.
Diatoms <20µm
80
Control
mg C m-3
273
60
LC
HC
40
20
4. Discussion
0
500
C
Diatoms >20µm
mg C m-3
400
300
200
100
0
0
24
48
72
96
120
Time (h)
Fig. 7. Time course of the biomass of nanoflagellates (A), small diatoms (B) and large
diatoms (C) in the control, low (LC) and high (HC) oil treatments during the experiment of December.
increased over time in the treatments and control, being the
increase especially important in small diatoms in the HC treatment,
where biomass at 120 h was 25-fold the initial biomass (Fig. 7B).
These changes largely reflected those occurring in the initially more
abundant species within each fraction: Thalassionema nitzschioides
and Guinardia striata in the fraction >20 mm (Figs. 8A,B), and
Chaetoceros socialis and Ch. gracilis in the fraction <20 mm
(Figs. 8C, D). Other large diatoms, illustrated here by Nitzschia
longissima (Fig. 8E) and Lauderia annulata (Fig. 8F), showed
different responses. Despite the negative effect of the high PAH
concentration, almost all species were apparently favoured by the
low oil concentration.
According to these results, it can be asserted that the objective of
this research (i.e. to assess the effect of the water soluble fraction of
oil on two different phytoplankton assemblages) was achieved. The
assemblage enclosed in October, characterised by low phytoplankton abundance and high importance of the picophytoplankton fraction in which Prochlorococcus was abundant (Table 1,
Fig. 1), points to their oceanic origin. Prochlorococcus is found in the
coastal waters of Galicia only when warm and high saline oceanic
waters are advected to the region (Calvo-Dı́az et al., 2004). Typically, Prochlorococcus acquires major importance at the time of
seasonal upwelling–downwelling transition (Rodrı́guez et al.,
2006), which occurs in late summer–beginning of autumn (Figueiras et al., 2002). At this time, surface water of subtropical origin
reaches the Galician shelf forced by downwelling (Rodrı́guez et al.,
2006; Crespo et al., 2007). On the other hand, the assemblage
enclosed in December, with dominance of nanophytoplankton and
much less importance of picophytoplankton without presence of
Prochlorococcus (Table 1, Fig. 1) is commonly found in coastal waters
of Galicia during winter (Figueiras et al., 2002; Rodrı́guez et al.,
2003; Cermeño et al., 2006; Arbones et al., 2008).
The evolution of the controls indicates that, when enclosed, the
response of the two assemblages was different. The phytoplankton
assemblage of October, which should be close to the steady state
fuelled by recycling, experienced an initial reduction in biomass
and physiological ability that was followed by a recovery. In this
oceanic assemblage, probably well adapted to rather stable conditions, manipulation and enclosing would have stressed the phytoplankton populations causing the observed initial reduction. The
disappearance of the thinner phytoplankton (Prochlorococcus and
Synechococcus) from the control flasks (Table 2) points to this
circumstance. The following increase in chl a concentration
(Fig. 3E) and primary production (Fig. 3C) and specifically no
further decreases in the Fv/Fm ratio (Fig. 3A) suggest that phytoplankton was not severely limited by nutrients during this experiment (Kolber and Falkowski, 1993). This inference is also
274
J. González et al. / Estuarine, Coastal and Shelf Science 83 (2009) 265–276
mg C m-3
160
160
Th. nitzschioides
120
120
80
80
40
40
0
0
30
mg C m-3
A
C
60
Ch. socialis
20
20
0
0
E
G. striata
D
Ch. gracilis
40
Control
LC
HC
10
20
B
3
N. longissima
Control
LC
HC
F
L. annulata
mg C m-3
16
2
12
8
1
4
0
0
0
24
48
72
96
120
Time (h)
0
24
48
72
96
120
Time (h)
Fig. 8. Time course of the biomass of the diatoms Thalassionema nitzschioides (A), Guinardia striata (B), Chaetoceros socialis (C), Ch. gracilis (D), Nitzschia longissima (E) and Lauderia
annulata (F) in the control, low (LC) and high (HC) oil treatments during the experiment of December.
supported by the relatively low final C:chl a ratio (68) recorded in
the control. In contrast, the high increase observed at 24 h in total
(Fig. 3D) and fractionated primary production (Figs. 3B,D,F) and
also in chl a concentration (Figs. 3F and 5B,D,F) during the experiment of December, suggest that this coastal assemblage of phytoplankton was favoured when it was enclosed. During winter, losses
due to vertical mixing of the water column are of great importance
at temperate latitudes. Therefore, any process reducing these losses, such as when phytoplankton is enclosed, will cause the accumulation of cells that, under suitable nutrient conditions (Table 1),
would increase primary production. However, in the absence of
further nutrient supply, the evolution of an assemblage of this type,
far of the equilibrium, will lead to nutrient limitation and, consequently, to the decline in primary production. The continuous
decrease observed in the Fv/Fm ratio after the first 24 h (Fig. 3B)
supports this deduction, as well as the extremely high C:chl a ratio
(261) measured at the end of this experiment. The continuous
accumulation of phytoplankton biomass, clearly perceptible in this
experiment of December (Fig. 7), could be in part due to low grazing
pressure, since large heterotrophic dinoflagellates, which are major
predators of diatoms (Sherr and Sherr, 2007), declined continuously in the control (Fig. 9C), and small dinoflagellates only
increased at the end of the experiment (Fig. 9B).
Apart from this different evolution of the two assemblages in
the controls, the negative effect of the water soluble fraction of oil
was evident in the two experiments. Primary producers exposed to
simulated oil spill experienced a significant reduction in the
photosynthetic efficiency of PSII within the first 48–72 h, being
stronger the impact on the oceanic assemblage of October (Figs.
3A,B). The reduction was also more pronounced in the HC treatments, suggesting a positive relationship dose-damage, at least
within the range of concentrations assessed. This negative effect on
photosynthetic efficiency is related to the accumulation of the
relatively hydrophobic PAHs in the hydrophobic thylakoid
membranes of the cells (Marwood et al., 1999; Sargian et al., 2005),
where PSII is located, interfering with electron transport and
photosynthesis (Duxbury et al., 1997).
The reduction in the photosynthetic efficiency of PSII caused by
the addition of PAHs derived in a parallel decrease in primary
production (Fig. 3), which again was more severe in the oceanic
assemblage, where the three size-fractions were affected by the
two PAH concentrations used (Fig. 4). In contrast, the largest fraction of the coastal assemblage, mainly composed of diatoms, was
more resistant to this type of contamination, since it was only
affected by the highest PAH concentration. The initial decrease
observed in chl a concentration (Fig. 5) and primary production
(Fig. 4) agrees with previous results found in mesocosm experiments (Siron et al., 1996; Sargian et al., 2005). Although in these
preceding investigations no recovery was reported for the oiled
treatments, here we found that primary producers recovered from
the initial negative effect 72 h after the experimental oil spill.
Increases in phytoplankton abundance and primary production
J. González et al. / Estuarine, Coastal and Shelf Science 83 (2009) 265–276
275
predation pressure and not to the stimulatory effect of oil on
phytoplankton. Our results also suggest that, after the first direct
and negative effect of PAHs on the photosynthetic mechanism,
indirect effects occurred later, probably operating through the food
web, which could be responsible for the recovery observed in
primary producers. Thus, the pronounced decrease recorded in
large heterotrophic dinoflagellates during the experiment of
December (Fig. 9C) could release predation on large (>20 mm)
diatoms (Sherr and Sherr, 2007) and so allow their increase
(Fig. 7C). Similarly, the low increase experienced by small (<20 mm)
heterotrophic dinoflagellates in the HC treatment (Fig. 9B) could
partially relax predation on small diatoms allowing their sudden
increase observed at the end of the experiment (Fig. 7B). It must be
mentioned here that Chaetoceros socialis (Fig. 8C) is a small chainforming diatom that in size-fractionation is easily recovered in the
fraction >20 mm, and it could explain the increase in production
(Fig. 4F) and chl a concentration (Fig. 5F) recorded for this fraction
at the end of the experiment of December.
In addition to the effects induced by PAHs on the photosynthetic
mechanism of phytoplankton and trophic relationships within the
microbial community, our results also point to the existence of
taxon and species-specific responses (Table 3; Figs. 6–8). All
phytoplankton groups and size-fractions showed an initial decrease
after oil addition. However, the final biomass of the groups and also
the final chl a concentration of the size-classes differed according to
the communities enclosed. Oceanic picophytoplankton was clearly
more sensitive than coastal picophytoplankton to the addition of
PAHs (Table 2; Fig. 6). Nanoflagellates, however, increased at the
end of the two experiments, showing in some cases higher values
than in the control (Table 3; Fig. 7A), and they could in part
contribute to the final recovery observed in chl a in both experiments (Figs. 3E,F). However, large diatoms, which were affected by
the highest concentration of PAHs in the two experiments (Table 3,
Fig. 7C), were apparently stimulated by the low PAH concentration
in December. To what extent this is a direct or indirect stimulatory
effect of PAHs could not be discerned, but on occasions they were
attributed to regenerated nutrients resulting from the breakdown
of plankton exposed to PAHs (Hjorth et al., 2007, 2008), which
could favour the growth of some phytoplankton species. Different
responses in phytoplankton sensitivity to PAHs have also been
described in other studies (Siron et al., 1996; Kelly et al., 1999) and
they were ascribed to community composition and seasonal
factors, such as seawater temperature and nutrient concentration,
as well as type and quantity of the PAHs added. For example, the
diatom Skeletonema costatum, which showed high sensitivity to oil
in cold waters (Ostgaard et al., 1984b), was however recognized as
one of the most oil tolerant species in experiments carried out in
temperate waters (Vargo et al., 1982).
Fig. 9. Time course of the biomass of total (A), small (B) and large heterotrophic
dinoflagellates (C) in the control, low (LC) and high (HC) oil treatments during the
experiment conducted in December.
following the oil addition were reported on other occasions (Vargo
et al., 1982; Carman et al., 1997; Kelly et al., 1999). In some of these
studies, where repeated additions of oil were made (Vargo et al.,
1982; Carman et al., 1997), an increase in phytoplankton abundance
was found in spite of a decrease in photosynthetic rates. This
different effect on phytoplankton abundance and primary
production was attributed to changes in predation due to the
negative effect of oil on the abundance of heterotrophs. Kelly et al.
(1999) investigated the response of primary producers to the
addition of a single PAH (phenanthrene) and also reported an
increase in phytoplankton abundance attributed to the decrease in
5. Conclusion
These results demonstrate that the input of the water soluble
fraction of oil into the ocean causes a transitory, short-term,
negative effect on phytoplankton that later derived to changes in
the structure of the plankton community. The temporal evolution
of primary production and biomass and the final composition of the
community depended on the initial community. Although these
results confirm previous observations, here we additionally show
that the negative effect of PAHs on oceanic phytoplankton was
stronger than on coastal phytoplankton. Oceanic picophytoplankton was greatly affected by the addition of PAHs, while coastal
phytoplankton, specifically diatoms, was apparently more resistant
to this type of pollution. The observed increase in diatom abundance in coastal phytoplankton exposed to PAHs might be related
to indirect trophic interactions resulting from the release of
276
J. González et al. / Estuarine, Coastal and Shelf Science 83 (2009) 265–276
predation, although differences in sensitivity to PAHs might also be
important. However, these results should be taken with caution,
because the confinement of the organisms could induce changes in
the populations enclosed and so modify the natural prey-predator
interactions. Nevertheless, this experimental approach allowed us
to study the effect of PAHs on natural assemblages of phytoplankton, avoiding the influence of water mixing and transport that
would mask the response during in situ research.
Acknowledgements
We thank T. Ulla and P. Pérez for their helpful assistance. This
research was funded by the project IMPRESION (VEM2003-20021)
of the Spanish Ministerio de Educación y Ciencia. J. G. was supported by a FPU fellowship and M. A. G. by a FPI fellowship, both of
the Spanish Ministerio de Educación y Ciencia. B. G. C. and M. N. C.
were supported by CSIC-ESF I3P predoctoral fellowships.
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