Effects of high pH on a natural marine planktonic community
Effects of high pH on a natural marine planktonic community
Effects of high pH on a natural marine planktonic community
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MARINE ECOLOGY PROGRESS SERIES<br />
Vol. 260: 19–31, 2003 Published September 30<br />
Mar Ecol Prog Ser<br />
<str<strong>on</strong>g>Effects</str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g> <strong>on</strong> a <strong>natural</strong> <strong>marine</strong> plankt<strong>on</strong>ic<br />
<strong>community</strong><br />
INTRODUCTION<br />
Marine waters have traditi<strong>on</strong>ally been c<strong>on</strong>sidered a<br />
<str<strong>on</strong>g>pH</str<strong>on</strong>g>-stable envir<strong>on</strong>ment with a <str<strong>on</strong>g>pH</str<strong>on</strong>g> value <str<strong>on</strong>g>of</str<strong>on</strong>g> 8 ± 0.5, due<br />
to the <str<strong>on</strong>g>high</str<strong>on</strong>g> buffer capacity found here (e.g. Hinga<br />
1992, 2002). This point <str<strong>on</strong>g>of</str<strong>on</strong>g> view is exemplified by a<br />
quote from Barker (1935b) who wrote: ‘Din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates<br />
are by no means as sensitive to small changes in <str<strong>on</strong>g>pH</str<strong>on</strong>g> as<br />
expected for organisms accustomed to such c<strong>on</strong>stant<br />
an envir<strong>on</strong>ment as the ocean’. C<strong>on</strong>sequently, effects <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
<str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g> <strong>on</strong> <strong>marine</strong> plankt<strong>on</strong>ic protists are not well documented;<br />
especially studies <str<strong>on</strong>g>of</str<strong>on</strong>g> the effects <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g> <strong>on</strong><br />
heterotrophic protists are sparse. This is in c<strong>on</strong>trast<br />
to freshwater ecology where the influence <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g> as a<br />
*Corresp<strong>on</strong>ding author. Email: pjhansen@zi.ku.dk<br />
Maria Fenger Pedersen, Per Juel Hansen*<br />
Marine Biological Laboratory, University <str<strong>on</strong>g>of</str<strong>on</strong>g> Copenhagen, Strandpromenaden 5, 3000 Helsingør, Denmark<br />
ABSTRACT: A <strong>natural</strong> plankt<strong>on</strong>ic <strong>community</strong> was incubated for 2 wk to study its resp<strong>on</strong>se to different<br />
levels <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g>, ranging from 8 to 9.5. A general increase in phytoplankt<strong>on</strong> biomass was observed<br />
over time in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 to 9 incubati<strong>on</strong>s. In the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5 incubati<strong>on</strong>, the phytoplankt<strong>on</strong> biomass<br />
decreased close to detecti<strong>on</strong> limit during the first week; however, at the terminati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment,<br />
the initial biomass level was regained. In the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 and 8.5 incubati<strong>on</strong>s, the diatoms Cerataulina<br />
pelagica, Cylindrotheca closterium and Leptocylindrus minimus became numerous, whereas in the<br />
<str<strong>on</strong>g>pH</str<strong>on</strong>g> 9 and 9.5 incubati<strong>on</strong>s, C. closterium solely made up the diatom biomass at the terminati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the<br />
experiment. Photosynthetic din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates <str<strong>on</strong>g>of</str<strong>on</strong>g> the genus Ceratium, which were initially abundant, did<br />
not grow well in any <str<strong>on</strong>g>of</str<strong>on</strong>g> the incubati<strong>on</strong>s, probably due to the low nutrient c<strong>on</strong>centrati<strong>on</strong>s. The protozooplankt<strong>on</strong><br />
biomass increased over time in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 to 9 incubati<strong>on</strong>s. In the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5 incubati<strong>on</strong>, the<br />
protozooplankt<strong>on</strong> biomass decreased close to detecti<strong>on</strong> limit during the first 3 d <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment<br />
and stayed at that level until the terminati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment. The biomass increase found in the<br />
<str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 to 9 incubati<strong>on</strong>s was due to an increase in the number <str<strong>on</strong>g>of</str<strong>on</strong>g> ciliates, because the heterotrophic<br />
din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellate number remained almost c<strong>on</strong>stant. Most protozooplankt<strong>on</strong> species incubated at <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5<br />
died; however, the ciliate Myri<strong>on</strong>ecta rubra survived at almost the same cell number as in the lower<br />
<str<strong>on</strong>g>pH</str<strong>on</strong>g> incubati<strong>on</strong>s. Overall a species successi<strong>on</strong> occurred am<strong>on</strong>g both phototrophic and heterotrophic<br />
protists when <str<strong>on</strong>g>pH</str<strong>on</strong>g> approached 9. In the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5 incubati<strong>on</strong>, the number <str<strong>on</strong>g>of</str<strong>on</strong>g> different protist taxa was<br />
reduced from 34 at the start <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment to 10 at the terminati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment. In c<strong>on</strong>clusi<strong>on</strong>,<br />
our study indicates that elevated <str<strong>on</strong>g>pH</str<strong>on</strong>g> (>9) in nature will affect the entire plankt<strong>on</strong> <strong>community</strong><br />
mainly by reducing the species richness and by favouring algal blooms due to loss <str<strong>on</strong>g>of</str<strong>on</strong>g> grazing.<br />
KEY WORDS: High <str<strong>on</strong>g>pH</str<strong>on</strong>g> · Plankt<strong>on</strong>ic protists · Ciliates · Diatoms · Din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates · Cylindrotheca<br />
closterium · Copepods<br />
Resale or republicati<strong>on</strong> not permitted without written c<strong>on</strong>sent <str<strong>on</strong>g>of</str<strong>on</strong>g> the publisher<br />
forcing factor has been c<strong>on</strong>sidered for decades. Models<br />
for estimating lake <str<strong>on</strong>g>pH</str<strong>on</strong>g> <strong>on</strong> the basis <str<strong>on</strong>g>of</str<strong>on</strong>g> the <str<strong>on</strong>g>pH</str<strong>on</strong>g> optima <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
the diatom species present in the lake even have been<br />
made (ter Braak & van Dam 1989).<br />
The view <str<strong>on</strong>g>of</str<strong>on</strong>g> the <strong>marine</strong> envir<strong>on</strong>ment as c<strong>on</strong>stant with<br />
respect to <str<strong>on</strong>g>pH</str<strong>on</strong>g> fluctuati<strong>on</strong>s has changed recently. This is<br />
mainly due to the anthropogenic nutrient enrichment<br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> many coastal areas, causing phytoplankt<strong>on</strong> blooms,<br />
and thereby <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g> values in places like bays,<br />
lago<strong>on</strong>s, salt p<strong>on</strong>ds and tidal pools (e.g. Droop 1959,<br />
Santhanam 1994, Macedo et al. 2001). The bestinvestigated<br />
locati<strong>on</strong> in Denmark where <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g> is<br />
found is Mariager Fjord. During the last 10 yr, the average<br />
<str<strong>on</strong>g>pH</str<strong>on</strong>g> value during summers has been 8.8, but during<br />
© Inter-Research 2003 · www.int-res.com
20<br />
calm sunny periods, <str<strong>on</strong>g>pH</str<strong>on</strong>g> values up to 9.75 have been<br />
measured (Hansen 2002). However, equivalent values<br />
have been recorded in <strong>marine</strong> pools and smaller increases<br />
in <str<strong>on</strong>g>pH</str<strong>on</strong>g> have been measured in the surface<br />
waters <str<strong>on</strong>g>of</str<strong>on</strong>g> the North Sea, where during a Phaeocystis<br />
bloom <str<strong>on</strong>g>pH</str<strong>on</strong>g> increased from 7.9 to 8.7 (Brussaard et al.<br />
1996). The durati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> elevated <str<strong>on</strong>g>pH</str<strong>on</strong>g> is quite variable; in<br />
a Portuguese coastal lago<strong>on</strong>, <str<strong>on</strong>g>pH</str<strong>on</strong>g> values above 8.5 are<br />
found all year round, whereas in rock pools and sediments,<br />
the <str<strong>on</strong>g>pH</str<strong>on</strong>g> can increase to 10, but <strong>on</strong>ly lasts for days<br />
or hours (Gnaiger et al. 1978, Macedo et al. 2001).<br />
An increase in <str<strong>on</strong>g>pH</str<strong>on</strong>g> in the <strong>marine</strong> envir<strong>on</strong>ment, as in<br />
freshwater, is expected to cause a change in the plankt<strong>on</strong>ic<br />
protist compositi<strong>on</strong>. The most comm<strong>on</strong> <strong>marine</strong><br />
phytoplankt<strong>on</strong> species found to co-occur with <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g><br />
in nature are the din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates Heterocapsa triquetra,<br />
Prorocentrum minimum and P. micans, and the<br />
diatom Skelet<strong>on</strong>ema costatum (Macedo et al. 2001,<br />
Hansen 2002). However, many different taxa <str<strong>on</strong>g>of</str<strong>on</strong>g> <strong>marine</strong><br />
phytoplankt<strong>on</strong> are found to grow at <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g>. Growth<br />
experiments with m<strong>on</strong>ocultures in the laboratory have<br />
shown that some cryptophytes, diatoms, din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates<br />
and prymnesiophyte species can grow at <str<strong>on</strong>g>pH</str<strong>on</strong>g><br />
above 9, and a few species even above <str<strong>on</strong>g>pH</str<strong>on</strong>g> 10 (Goldman<br />
et al. 1982, Nimer et al. 1994, Elzenga et al. 2000,<br />
Schmidt & Hansen 2001, Hansen 2002).<br />
The knowledge <str<strong>on</strong>g>of</str<strong>on</strong>g> how heterotrophic organisms<br />
resp<strong>on</strong>d to <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g> is very sparse. While nothing is<br />
known about how copepods are affected by <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g>,<br />
we have some knowledge <str<strong>on</strong>g>of</str<strong>on</strong>g> the resp<strong>on</strong>se <str<strong>on</strong>g>of</str<strong>on</strong>g> protozooplankt<strong>on</strong><br />
to <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g>. Droop (1959) found that the<br />
heterotrophic din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellate Oxyrrhis marina is <str<strong>on</strong>g>high</str<strong>on</strong>g>ly<br />
<str<strong>on</strong>g>pH</str<strong>on</strong>g>-tolerant. (Pedersen & Hansen 2003, this issue)<br />
studied the effect <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g> <strong>on</strong> 4 ciliates and 2 heterotrophic<br />
din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates in laboratory cultures, and<br />
found that while some species are <str<strong>on</strong>g>high</str<strong>on</strong>g>ly <str<strong>on</strong>g>pH</str<strong>on</strong>g>-tolerant,<br />
others are quite sensitive to elevated <str<strong>on</strong>g>pH</str<strong>on</strong>g> and cannot<br />
survive at <str<strong>on</strong>g>pH</str<strong>on</strong>g> exceeding 8.9.<br />
Due to this very limited knowledge <str<strong>on</strong>g>of</str<strong>on</strong>g> the effects <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
<str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g> <strong>on</strong> the <strong>marine</strong> plankt<strong>on</strong> organisms, the aim <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
this paper was to m<strong>on</strong>itor the resp<strong>on</strong>se <str<strong>on</strong>g>of</str<strong>on</strong>g> a <strong>natural</strong><br />
plankt<strong>on</strong>ic <strong>community</strong> (>15 µm) to different levels <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
<str<strong>on</strong>g>pH</str<strong>on</strong>g>, thereby evaluating the effect <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g> <strong>on</strong> the<br />
species successi<strong>on</strong> and diversity <str<strong>on</strong>g>of</str<strong>on</strong>g> both phototrophic<br />
and heterotrophic protists. Because nutrient limitati<strong>on</strong><br />
may affect the successi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> protists, this was also<br />
taken into account.<br />
MATERIALS AND METHODS<br />
Water samples were collected from the pycnocline<br />
(water depth <str<strong>on</strong>g>of</str<strong>on</strong>g> 20 m) at a stati<strong>on</strong> located in the Øresund,<br />
Denmark, <strong>on</strong> August 25, 2000. The water (salinity<br />
22 psu) was collected using a Niskin water sampler, im-<br />
Mar Ecol Prog Ser 260: 19–31, 2003<br />
mediately filtered through a 160 µm filter to remove<br />
large zooplankters and stored in a 25 l c<strong>on</strong>tainer. The<br />
c<strong>on</strong>tainer was brought back to the laboratory and the<br />
water was poured into 4 clear 2.8 l Nalgene ® bottles<br />
(#1 to 4). The <str<strong>on</strong>g>pH</str<strong>on</strong>g> was elevated from the original 7.95 to<br />
8.0 (#1), 8.5 (#2), 9.0 (#3) and 9.5 (#4) by additi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> 0.1<br />
and 1 M NaOH. For <str<strong>on</strong>g>pH</str<strong>on</strong>g> measurements, a Sentr<strong>on</strong> ®<br />
2001 <str<strong>on</strong>g>pH</str<strong>on</strong>g> meter with a Red Line electrode, sensitivity<br />
0.01, and a 2 point calibrati<strong>on</strong> was used. To minimise<br />
the shock effects <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g>, the <str<strong>on</strong>g>pH</str<strong>on</strong>g> was raised by levels<br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> 0.5 units at 12 h intervals until the final value was<br />
reached. The bottles were mounted <strong>on</strong> a plankt<strong>on</strong><br />
wheel (1 rpm) and incubated for 14 d at 15 ± 1°C <strong>on</strong> a<br />
16:8 h light:dark cycle at an irradiance <str<strong>on</strong>g>of</str<strong>on</strong>g> 50 µE m –2 s –1 .<br />
The first sampling was carried out after 24 h; subsequent<br />
samplings were d<strong>on</strong>e every sec<strong>on</strong>d or third day<br />
during the next 14 d.<br />
At each sampling occasi<strong>on</strong>, the temperature and <str<strong>on</strong>g>pH</str<strong>on</strong>g><br />
were m<strong>on</strong>itored and the <str<strong>on</strong>g>pH</str<strong>on</strong>g> adjusted. A total water<br />
volume <str<strong>on</strong>g>of</str<strong>on</strong>g> 300 ml was removed; the water was used for<br />
nutrient (3 × 30 ml) and chlorophyll a analyses (chl a;<br />
65 to 95 ml) as well as enumerati<strong>on</strong> and identificati<strong>on</strong><br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> protists and copepods (105 to 135 ml was fixed in<br />
acidic Lugol’s iodine [final c<strong>on</strong>centrati<strong>on</strong> 1%] and kept<br />
in the dark and cold until examinati<strong>on</strong>). After sampling,<br />
the bottles were refilled with GF/C-filtered<br />
seawater (taken at the same locati<strong>on</strong> and time as the<br />
experimental water) and adjusted to the given <str<strong>on</strong>g>pH</str<strong>on</strong>g>,<br />
within <str<strong>on</strong>g>pH</str<strong>on</strong>g> 0.01 <str<strong>on</strong>g>of</str<strong>on</strong>g> the given value, before they were<br />
remounted <strong>on</strong> the plankt<strong>on</strong> wheel.<br />
Inorganic nutrients (NO 3 – , NO2 – , NH4 + , PO4 2– , SiO4 – )<br />
were analysed at the Nati<strong>on</strong>al Envir<strong>on</strong>mental Research<br />
Institute, Roskilde, Denmark (Grassh<str<strong>on</strong>g>of</str<strong>on</strong>g>f 1976). Chl a<br />
was measured in triplicate by filtrati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> 20 to 30 ml<br />
samples <strong>on</strong>to GF/C filters immediately after sampling.<br />
Filters were then extracted in 96% ethanol in the<br />
freezer overnight, and the next day centrifuged at<br />
1000 × g for 5 min before the fluorescence <str<strong>on</strong>g>of</str<strong>on</strong>g> the<br />
supernatant was measured with a Turner‚ TD-700<br />
Laboratory Fluorometer.<br />
Lugol-fixed samples for enumerati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> protists<br />
were, depending <strong>on</strong> cell c<strong>on</strong>centrati<strong>on</strong>s, poured in<br />
triplicates into 10 or 25 ml sedimentati<strong>on</strong> chambers.<br />
The dominant taxa (species or groups <str<strong>on</strong>g>of</str<strong>on</strong>g> species)<br />
were identified and enumerated using an inverted<br />
Olympus ® microscope at a magnificati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> 100 to<br />
400×, thereby focussing <strong>on</strong> protists >15 µm. Diatoms,<br />
din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates and ciliates dominated the samples and<br />
were identified using Dodge (1985), Hansen & Larsen<br />
(1992), Tomas (1996) and S<strong>on</strong>g et al. (1999). Diatoms<br />
and din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates that have chloroplasts <str<strong>on</strong>g>of</str<strong>on</strong>g> their own<br />
were grouped as ‘phytoplankt<strong>on</strong>’. Ciliates and din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates<br />
that do not have their own chloroplasts<br />
were grouped as ‘protozooplankt<strong>on</strong>’, even though<br />
some <str<strong>on</strong>g>of</str<strong>on</strong>g> them may c<strong>on</strong>tain functi<strong>on</strong>al chloroplasts.
Pedersen & Hansen: <str<strong>on</strong>g>Effects</str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g> <strong>on</strong> a plankt<strong>on</strong>ic <strong>community</strong><br />
The dimensi<strong>on</strong>s <str<strong>on</strong>g>of</str<strong>on</strong>g> 10 cells <str<strong>on</strong>g>of</str<strong>on</strong>g> each taxa were measured<br />
and cell volumes were estimated from linear<br />
dimensi<strong>on</strong>s using simple volumetric formulae. Cellular<br />
carb<strong>on</strong> c<strong>on</strong>tent was then calculated using the following<br />
equati<strong>on</strong>s (Menden-Deuer & Lessard 2000) where V is<br />
the cell volume:<br />
Din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates: pgC cell –1 = 0.760 × V 0.819<br />
Diatoms >3000 µm 3 : pgC cell –1 = 0.288 × V 0.811<br />
Other protists: pgC cell –1 = 0.216 × V 0.939<br />
Although the water was pre-screened through a<br />
plankt<strong>on</strong> net (mesh size 160 µm), some developmental<br />
stages <str<strong>on</strong>g>of</str<strong>on</strong>g> small copepods passed the filter. To evaluate<br />
their grazing impact <strong>on</strong> the protist <strong>community</strong>, the<br />
copepods were identified to species and developmental<br />
stage to estimate the clearance rate <strong>on</strong> potential<br />
prey examined in this paper. Enumerati<strong>on</strong> and identificati<strong>on</strong><br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> copepods in some <str<strong>on</strong>g>of</str<strong>on</strong>g> the Lugol-fixed samples<br />
was c<strong>on</strong>ducted using a dissecti<strong>on</strong> microscope. The<br />
prosome length <str<strong>on</strong>g>of</str<strong>on</strong>g> each species was used for identificati<strong>on</strong><br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> the developmental stages. The maximum clearance<br />
was estimated for copepods using the equati<strong>on</strong>s<br />
given in Hansen et al. 1997 and taking size into c<strong>on</strong>siderati<strong>on</strong>.<br />
RESULTS<br />
<str<strong>on</strong>g>pH</str<strong>on</strong>g> and inorganic nutrients<br />
The <str<strong>on</strong>g>pH</str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g> <strong>natural</strong> seawater samples was adjusted to<br />
<str<strong>on</strong>g>pH</str<strong>on</strong>g> 8, 8.5, 9 and 9.5 respectively, and kept stable by<br />
adjustments at each sampling occasi<strong>on</strong> (Fig. 1). The<br />
incubati<strong>on</strong>s with <str<strong>on</strong>g>pH</str<strong>on</strong>g> levels between 8 and 9 showed a<br />
maximum reducti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> 0.1 <str<strong>on</strong>g>pH</str<strong>on</strong>g> units between sampling<br />
days. In the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5 treatment, however, a reducti<strong>on</strong><br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> 0.2 <str<strong>on</strong>g>pH</str<strong>on</strong>g> units occurred between samplings, with<br />
1 excepti<strong>on</strong> at Day 7, where the reducti<strong>on</strong> was 0.5 <str<strong>on</strong>g>pH</str<strong>on</strong>g><br />
units. The nutrient c<strong>on</strong>centrati<strong>on</strong>s <str<strong>on</strong>g>of</str<strong>on</strong>g> NH 4 + , NO3 – ,<br />
NO 2 – and PO4 2– (Fig. 2) remained at the initial level in<br />
the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 to 9 incubati<strong>on</strong>s, with a slight decrease<br />
towards the end <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment. However, while<br />
the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5 incubati<strong>on</strong> experienced an increase in<br />
inorganic nutrients from the start <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment to<br />
the end, an excepti<strong>on</strong> was found for PO 4 2– , which<br />
decreased to the detecti<strong>on</strong> limit after Day 3. The c<strong>on</strong>centrati<strong>on</strong><br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> SiO 4 – in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 and 8.5 incubati<strong>on</strong>s<br />
remained at a c<strong>on</strong>centrati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> 2 to 6 µM throughout<br />
the durati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment (Fig. 2). In the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9<br />
incubati<strong>on</strong>, the SiO 4 – c<strong>on</strong>centrati<strong>on</strong> increased to a<br />
maximum <str<strong>on</strong>g>of</str<strong>on</strong>g> 12.5 µM at Day 7, whereafter it<br />
decreased to reach 9 µM at Day 14. In the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5<br />
incubati<strong>on</strong>, the SiO 4 – kept increasing throughout the<br />
experiment to reach a maximum c<strong>on</strong>centrati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
31 µM at Day 14.<br />
Fig. 1. <str<strong>on</strong>g>pH</str<strong>on</strong>g> fluctuati<strong>on</strong>s in the experimental bottles during the<br />
2 wk incubati<strong>on</strong>s. (y) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.0; (j) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.5; (S) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.0; (m) <str<strong>on</strong>g>pH</str<strong>on</strong>g><br />
9.5. <str<strong>on</strong>g>pH</str<strong>on</strong>g> was adjusted at each sampling date to keep the<br />
<str<strong>on</strong>g>pH</str<strong>on</strong>g> c<strong>on</strong>stant<br />
The plankt<strong>on</strong>ic <strong>community</strong><br />
Phytoplankt<strong>on</strong><br />
The phytoplankt<strong>on</strong> <strong>community</strong> was quantified in<br />
bulk using both chl a measurements and direct cell<br />
counts. The initial chl a c<strong>on</strong>centrati<strong>on</strong> was 2.4 µg l –1 in<br />
all treatments (Fig. 3A). In the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 to 9 incubati<strong>on</strong>s,<br />
the chl a c<strong>on</strong>centrati<strong>on</strong> increased 2- to 3-fold during<br />
the first 10 d. During the next 4 d, the chl a c<strong>on</strong>centrati<strong>on</strong><br />
stabilised in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 and 8.5 incubati<strong>on</strong>s, whereas<br />
the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9 incubati<strong>on</strong> experienced a further 3-fold<br />
increase. In the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5 incubati<strong>on</strong>, the chl a c<strong>on</strong>centrati<strong>on</strong><br />
declined more than 90% from Day 0 to 7. However,<br />
at the terminati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment, the initial<br />
chl a c<strong>on</strong>centrati<strong>on</strong> was almost regained.<br />
The phytoplankt<strong>on</strong> biomass (estimated from direct<br />
counts) was initially 53 µg C l –1 (Fig. 3B). A general<br />
increase in biomass was observed in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 and<br />
8.5 incubati<strong>on</strong>s, increasing 3- and 14-fold, respectively.<br />
In the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9 incubati<strong>on</strong>, no increase was observed<br />
during the first week; however, during the last<br />
week, the biomass increased to reach the same level as<br />
found in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.5 incubati<strong>on</strong>. Unlike the other incubati<strong>on</strong>s,<br />
a significant decrease was observed in the<br />
<str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5 incubati<strong>on</strong> during the first week. However, during<br />
the following week an increase to almost the initial<br />
biomass occurred.<br />
The focus <str<strong>on</strong>g>of</str<strong>on</strong>g> the phytoplankt<strong>on</strong> group was <strong>on</strong><br />
diatoms and din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates because they were the<br />
most dominant classes in the present experiment<br />
(Fig. 3C,D). At the start <str<strong>on</strong>g>of</str<strong>on</strong>g> the incubati<strong>on</strong>s, the din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates<br />
dominated the phytoplankt<strong>on</strong> <strong>community</strong>.<br />
However, during the 2 wk incubati<strong>on</strong>, the relative<br />
importance <str<strong>on</strong>g>of</str<strong>on</strong>g> diatoms increased at all <str<strong>on</strong>g>pH</str<strong>on</strong>g> levels and<br />
they became dominant. At <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 and 8.5, the diatoms<br />
21
22<br />
Mar Ecol Prog Ser 260: 19–31, 2003<br />
Fig. 2. Fluctuati<strong>on</strong>s in nutrient<br />
c<strong>on</strong>centrati<strong>on</strong>s measured during<br />
the 2 wk experimental period.<br />
(A) NH 4 + , (B) SiO4 – , (C) NO3 – +<br />
NO 2 – , (D) PO4 2– . (y) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.0; (j)<str<strong>on</strong>g>pH</str<strong>on</strong>g><br />
8.5; (S) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.0; (m) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5.<br />
Symbols represent means <str<strong>on</strong>g>of</str<strong>on</strong>g> triplicates<br />
± SE<br />
Fig. 3. Phytoplankt<strong>on</strong> c<strong>on</strong>centrati<strong>on</strong>s<br />
in the 4 incubati<strong>on</strong>s<br />
during the 2 wk experimental<br />
period. (A) Chlorophyll a,<br />
(B) phytoplankt<strong>on</strong> biomass<br />
(µg C l –1 ), (C) diatom biomass,<br />
(D) phototrophic din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellate<br />
biomass. (y) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.0;<br />
(j) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.5; (S) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.0; (m) <str<strong>on</strong>g>pH</str<strong>on</strong>g><br />
9.5. Symbols represent means<br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> triplicates ± SE
Pedersen & Hansen: <str<strong>on</strong>g>Effects</str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g> <strong>on</strong> a plankt<strong>on</strong>ic <strong>community</strong><br />
grew throughout the durati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment,<br />
whereas the din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates <strong>on</strong>ly grew for the first 3 d.<br />
At <str<strong>on</strong>g>high</str<strong>on</strong>g>er <str<strong>on</strong>g>pH</str<strong>on</strong>g>, the biomass <str<strong>on</strong>g>of</str<strong>on</strong>g> both diatoms and din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates<br />
either remained c<strong>on</strong>stant or declined during<br />
the first week <str<strong>on</strong>g>of</str<strong>on</strong>g> the incubati<strong>on</strong>, and <strong>on</strong>ly growth <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
diatoms was observed during the sec<strong>on</strong>d week <str<strong>on</strong>g>of</str<strong>on</strong>g> the<br />
incubati<strong>on</strong>s.<br />
The successi<strong>on</strong> am<strong>on</strong>g the species within the studied<br />
phytoplankt<strong>on</strong> groups varied according to the <str<strong>on</strong>g>pH</str<strong>on</strong>g> level<br />
(Figs. 4 & 5). The 2 lowest <str<strong>on</strong>g>pH</str<strong>on</strong>g> incubati<strong>on</strong>s, <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 and<br />
8.5, experienced almost no successi<strong>on</strong> am<strong>on</strong>g species.<br />
Here, all the identified species were present throughout<br />
the experimental period.<br />
In the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5 incubati<strong>on</strong>, a pr<strong>on</strong>ounced successi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
species occurred during the incubati<strong>on</strong> period. At the<br />
end <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment, Cylindrotheca closterium was<br />
the <strong>on</strong>ly species am<strong>on</strong>g the diatoms that thrived. Its<br />
growth rate at <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5 was similar to the growth rates<br />
obtained in the lower <str<strong>on</strong>g>pH</str<strong>on</strong>g> incubati<strong>on</strong>s (Fig. 4, Table 1).<br />
Am<strong>on</strong>g the din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates, Prorocentrum micans, P.<br />
minimum and Heterocapsa triquetra all survived at<br />
<str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5, whereas the initial dominant species, Ceratium<br />
furca, C. fusus and C. tripos, died out (Fig. 5).<br />
In the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9 incubati<strong>on</strong>, the successi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> species was<br />
less pr<strong>on</strong>ounced, and <strong>on</strong>ly a few species died out. It<br />
was interesting to note however that some species<br />
apparently grew faster in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9 incubati<strong>on</strong> compared<br />
to in the incubati<strong>on</strong>s at lower <str<strong>on</strong>g>pH</str<strong>on</strong>g> (Figs. 4 & 5).<br />
Protozooplankt<strong>on</strong><br />
At the start <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment, the protozooplankt<strong>on</strong><br />
biomass was 20 µg C l –1 in all incubati<strong>on</strong>s (Fig. 6A). In<br />
the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8, 8.5 and 9 incubati<strong>on</strong>s, a general increase in<br />
biomass was found over time. An 8-fold increase in<br />
biomass was observed at the terminati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment<br />
in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 and 9 incubati<strong>on</strong>s, whereas <strong>on</strong>ly a<br />
3-fold increase in biomass was found in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.5<br />
incubati<strong>on</strong>. In the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5 incubati<strong>on</strong>, the biomass<br />
decreased about 5-fold during the first 3 d and stayed<br />
at that level until the terminati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment.<br />
The increase in biomass in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8, 8.5 and 9 incubati<strong>on</strong>s<br />
was mainly caused by ciliates, because the<br />
heterotrophic din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates were found to be relatively<br />
c<strong>on</strong>stant throughout the experiment. In the<br />
<str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5 incubati<strong>on</strong>, both ciliates and heterotrophic<br />
din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates declined in biomass throughout the<br />
durati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment (Fig. 6B,C).<br />
The successi<strong>on</strong> am<strong>on</strong>g the species within the studied<br />
protozooplankt<strong>on</strong> groups varied according to the <str<strong>on</strong>g>pH</str<strong>on</strong>g><br />
level (Figs. 7 & 8). The 2 lowest <str<strong>on</strong>g>pH</str<strong>on</strong>g> incubati<strong>on</strong>s, <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8<br />
and 8.5, experienced almost no successi<strong>on</strong> am<strong>on</strong>g<br />
species. Here, all the identified species were present<br />
throughout the experimental period. In the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5<br />
Fig. 4. Cell c<strong>on</strong>centrati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> some selected diatoms in the 4 incubati<strong>on</strong>s<br />
during the 2 wk experimental period. (A) Cylindrotheca<br />
closterium, (B) Cerataulina pelagica, (C) Leptocylindrus<br />
minimus. (y) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.0; (j) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.5; (S) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.0; (m) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5.<br />
Symbols represent means <str<strong>on</strong>g>of</str<strong>on</strong>g> triplicates ± SE<br />
incubati<strong>on</strong>, some species died out, whereas the remaining<br />
species were alive at the terminati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the<br />
experiment. However, unlike in the case <str<strong>on</strong>g>of</str<strong>on</strong>g> the phytoplankt<strong>on</strong>,<br />
n<strong>on</strong>e <str<strong>on</strong>g>of</str<strong>on</strong>g> the protozooplankt<strong>on</strong> species took<br />
over.<br />
In the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9 incubati<strong>on</strong>, the successi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> species was<br />
less pr<strong>on</strong>ounced, and <strong>on</strong>ly a few species died out<br />
(Figs. 7 & 8). It was interesting to note however that<br />
some ciliate species apparently grew faster in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9<br />
incubati<strong>on</strong> compared to in the incubati<strong>on</strong>s at lower <str<strong>on</strong>g>pH</str<strong>on</strong>g>.<br />
23
24<br />
Copepods<br />
In terms <str<strong>on</strong>g>of</str<strong>on</strong>g> numbers, the copepod <strong>community</strong> in the<br />
incubati<strong>on</strong>s c<strong>on</strong>sisted mainly <str<strong>on</strong>g>of</str<strong>on</strong>g> small Oith<strong>on</strong>a spp.<br />
and <strong>on</strong>ly a few much larger copepods <str<strong>on</strong>g>of</str<strong>on</strong>g> the genera<br />
Pseudo-/Paracalanus spp. were found in the sample<br />
volumes studied. At the initiati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment, a<br />
c<strong>on</strong>centrati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> 84 copepods per litre were found,<br />
including all developmental stages. In the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 incubati<strong>on</strong>,<br />
the number <str<strong>on</strong>g>of</str<strong>on</strong>g> copepods increased slightly<br />
during the experimental period, whereas a slight<br />
decrease was found at <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.5. No copepods were<br />
found in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9 and 9.5 incubati<strong>on</strong>s after Days 5<br />
and 0, respectively (data not shown). Thus, the aver-<br />
Mar Ecol Prog Ser 260: 19–31, 2003<br />
Fig. 5. Cell c<strong>on</strong>centrati<strong>on</strong>s <str<strong>on</strong>g>of</str<strong>on</strong>g> selected phototrophic din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates in the 4 incubati<strong>on</strong>s during the 2 wk experimental period.<br />
(A) Prorocentrum micans, (B) P. minimum, (C) Heterocapsa triquetra, (D) Ceratium tripos, (E) Ceratium furca, (F) C. fusus.<br />
(y) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.0; (j) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.5; (S) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.0; (m) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5. Symbols represent means <str<strong>on</strong>g>of</str<strong>on</strong>g> triplicates ± SE<br />
Table 1. Cylindrotheca closterium. Growth rate (d –1 ) for the<br />
incubati<strong>on</strong>s <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.0, 8.5, 9.0 and 9.5<br />
<str<strong>on</strong>g>pH</str<strong>on</strong>g> Growth rate (d –1 )<br />
Days 0 to 7 Days 7 to 14 Days 0 to 14<br />
8.0 0.54 ± 0.07 0.53 ± 0.05 0.54 ± 0.05<br />
8.5 0.62 ± 0.09 0.56 ± 0.003 0.56 ± 0.04<br />
9.0 0.65 ± 0.07 0.97 ± 0.04 0.81 ± 0.02<br />
9.5 0.48 ± 0.06 0.93 ± 0.04 0.70 ± 0.05<br />
age length and total calculated biomass increased<br />
over time <strong>on</strong>ly in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 and 8.5 incubati<strong>on</strong>s (Figs. 9<br />
& 10).<br />
The calculated maximum clearance for the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 and<br />
8.5 incubati<strong>on</strong>s revealed that about 6% <str<strong>on</strong>g>of</str<strong>on</strong>g> the water<br />
was cleared daily at the start <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment. C<strong>on</strong>siderable<br />
variati<strong>on</strong> in calculated clearance was found<br />
and no c<strong>on</strong>sistent increase was observed during the<br />
experiment, probably due to the small sample sizes<br />
used. However, taking all data <strong>on</strong> clearance from the<br />
<str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 and 8.5 incubati<strong>on</strong>s into c<strong>on</strong>siderati<strong>on</strong>, the maximum<br />
and the average rate corresp<strong>on</strong>ded to a removal<br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> 38 and 10% <str<strong>on</strong>g>of</str<strong>on</strong>g> the prey populati<strong>on</strong>s per day,<br />
respectively (Table 2).<br />
DISCUSSION<br />
How did elevated <str<strong>on</strong>g>pH</str<strong>on</strong>g> affect the phytoplankt<strong>on</strong><br />
<strong>community</strong>?<br />
The phytoplankt<strong>on</strong> communities incubated at <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9<br />
and 9.5 clearly developed differently from those<br />
incubated at <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 and 8.5, with the most pr<strong>on</strong>ounced<br />
differences found at <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5 (Fig. 3). In the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5 incu-
Pedersen & Hansen: <str<strong>on</strong>g>Effects</str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g> <strong>on</strong> a plankt<strong>on</strong>ic <strong>community</strong><br />
bati<strong>on</strong>, a 75% decline in total phytoplankt<strong>on</strong> biomass<br />
occurred during the first week. However, the effect<br />
was <strong>on</strong>ly transient, because the phytoplankt<strong>on</strong> biomass<br />
increased again during the sec<strong>on</strong>d week <str<strong>on</strong>g>of</str<strong>on</strong>g> incubati<strong>on</strong><br />
to 2 /3 <str<strong>on</strong>g>of</str<strong>on</strong>g> the initial phytoplankt<strong>on</strong> biomass. The<br />
reas<strong>on</strong> for this reducti<strong>on</strong> in biomass was that some species<br />
declined in numbers or totally disappeared (Figs. 4<br />
& 5). This resulted in a decline in the total number <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
algal taxa from 21 at the start <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment to a<br />
total <str<strong>on</strong>g>of</str<strong>on</strong>g> <strong>on</strong>ly 5 at the terminati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment,<br />
with <strong>on</strong>ly 1 species making up for 70% <str<strong>on</strong>g>of</str<strong>on</strong>g> the algal<br />
biomass (Figs. 3, 4 & 11).<br />
How do these results compare with laboratory data<br />
<strong>on</strong> single species? In a recent review, Hansen (2002)<br />
found that in laboratory cultures, <strong>on</strong>ly 7 out <str<strong>on</strong>g>of</str<strong>on</strong>g> a total <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
35 phytoplankt<strong>on</strong> species were able to grow at <str<strong>on</strong>g>pH</str<strong>on</strong>g><br />
exceeding 9.5. Am<strong>on</strong>g species that had the capability<br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> growing at <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g> were the din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates Prorocentrum<br />
minimum and P. micans, which also were<br />
some <str<strong>on</strong>g>of</str<strong>on</strong>g> the <strong>on</strong>es growing in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5 incubati<strong>on</strong> in<br />
the present experiment. Likewise, some <str<strong>on</strong>g>of</str<strong>on</strong>g> the species<br />
which during the incubati<strong>on</strong> period disappeared or<br />
decreased in numbers in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9 and 9.5 incubati<strong>on</strong>s<br />
have been reported to be unable to grow at this <str<strong>on</strong>g>high</str<strong>on</strong>g><br />
<str<strong>on</strong>g>pH</str<strong>on</strong>g>. This is for instance the case for Ceratium tripos<br />
and C. lineatum, which are known for their sensitivity<br />
to <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g>, being unable to grow at <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.4 and 8.7,<br />
respectively. The diatom Cylindrotheca closterium was<br />
the <strong>on</strong>ly species found to obtain a <str<strong>on</strong>g>high</str<strong>on</strong>g> growth rate in<br />
the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5 incubati<strong>on</strong> (Fig. 4, Table 1). This diatom is<br />
known as a <str<strong>on</strong>g>high</str<strong>on</strong>g>ly <str<strong>on</strong>g>pH</str<strong>on</strong>g>-tolerant species, which is able to<br />
sustain maximum growth up to <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.2 in laboratory<br />
cultures (Barker 1935a, Grant et al. 1967, Humphrey &<br />
Subba Rao 1967). Thus, in c<strong>on</strong>clusi<strong>on</strong>, there is good<br />
accordance between the observati<strong>on</strong>s made in the present<br />
study and the literature found <strong>on</strong> laboratory experiments<br />
c<strong>on</strong>cerning <str<strong>on</strong>g>pH</str<strong>on</strong>g> tolerances <str<strong>on</strong>g>of</str<strong>on</strong>g> phytoplankt<strong>on</strong>.<br />
How did elevated <str<strong>on</strong>g>pH</str<strong>on</strong>g> affect the heterotrophic<br />
<strong>community</strong>?<br />
Elevated <str<strong>on</strong>g>pH</str<strong>on</strong>g> had a marked effect <strong>on</strong> both the protozooplankt<strong>on</strong><br />
and the copepod <strong>community</strong>. In the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9<br />
and 9.5 incubati<strong>on</strong>s, the development in terms <str<strong>on</strong>g>of</str<strong>on</strong>g> both<br />
biomass and species compositi<strong>on</strong> differed from the<br />
lower <str<strong>on</strong>g>pH</str<strong>on</strong>g> incubati<strong>on</strong>s. Many protozooplankt<strong>on</strong> survived<br />
and even grew in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9 incubati<strong>on</strong>, whereas <strong>on</strong>ly a<br />
few species survived the 2 wk exposure to <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5<br />
(Figs. 6 & 9). N<strong>on</strong>e <str<strong>on</strong>g>of</str<strong>on</strong>g> the copepod species was found to<br />
survive in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9 and 9.5 incubati<strong>on</strong>s for more than<br />
5 and 1 d, respectively, indicating that copepods are<br />
more sensitive to <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g> than protozooplankt<strong>on</strong>.<br />
Our knowledge <str<strong>on</strong>g>of</str<strong>on</strong>g> how elevated <str<strong>on</strong>g>pH</str<strong>on</strong>g> affects the<br />
growth and survival <str<strong>on</strong>g>of</str<strong>on</strong>g> <strong>marine</strong> protozooplankt<strong>on</strong> and<br />
Fig. 6. C<strong>on</strong>centrati<strong>on</strong>s <str<strong>on</strong>g>of</str<strong>on</strong>g> protozooplankt<strong>on</strong> in the 4 incubati<strong>on</strong>s<br />
during the 2 wk experimental period. (A) Total protozooplankt<strong>on</strong><br />
biomass, (B) ciliate biomass, (C) heterotrophic<br />
din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates biomass. (y) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.0; (j) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.5; (S) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.0;<br />
(m) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5. Symbols represent means <str<strong>on</strong>g>of</str<strong>on</strong>g> triplicates ± SE<br />
copepods is limited to a couple <str<strong>on</strong>g>of</str<strong>on</strong>g> laboratory studies <strong>on</strong><br />
heterotrophic din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates and ciliates (Droop 1959,<br />
Pedersen & Hansen 2003). In these studies, it was<br />
found that the heterotrophic din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellate Oxyrrhis<br />
marina and the prostomatid ciliate Balani<strong>on</strong> comatum<br />
both were able to grow quite fast at a <str<strong>on</strong>g>pH</str<strong>on</strong>g> above 9.5,<br />
whereas the din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates Gyrodinium dominans and<br />
the ciliates Rimostrombidium caudatum, R. veniliae<br />
and Favella ehrenbergii had <str<strong>on</strong>g>pH</str<strong>on</strong>g> growth limits ranging<br />
25
26<br />
from 8.8 to 9.3. When these organisms were exposed to<br />
<str<strong>on</strong>g>high</str<strong>on</strong>g>er <str<strong>on</strong>g>pH</str<strong>on</strong>g>, they died out. Thus, the data we obtained<br />
from the incubati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> field samples are not in c<strong>on</strong>flict<br />
with the laboratory results and they point to the fact<br />
that elevated <str<strong>on</strong>g>pH</str<strong>on</strong>g> may cause species successi<strong>on</strong> am<strong>on</strong>g<br />
heterotrophic organisms. The fact that we did not find<br />
any protozooplankt<strong>on</strong> or copepod species that could<br />
resp<strong>on</strong>d to the increase in algal biomass in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5<br />
incubati<strong>on</strong> suggests that <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g> may result in a<br />
reducti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the total grazing pressure <strong>on</strong> the <strong>natural</strong><br />
phytoplankt<strong>on</strong> <strong>community</strong>.<br />
Nutrient limitati<strong>on</strong> and grazing effects<br />
The growth <str<strong>on</strong>g>of</str<strong>on</strong>g> the large-celled phototrophic din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates<br />
(e.g. Ceratium spp.) stopped after 3 d <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
incubati<strong>on</strong> in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 and 8.5 experiments, whereas<br />
small-celled phototrophic din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates and diatoms<br />
were able to grow throughout the experiment (Fig. 5).<br />
Thus, these large din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates were either subjected<br />
to a heavy grazing pressure or became nutrientlimited.<br />
Substantial grazing by the dominating copepods<br />
(cyclopoids) and ciliates <strong>on</strong> Ceratium spp. seems<br />
unlikely, simply because they cannot handle them<br />
(Hansen et al. 1994, Nielsen & Kiørboe 1994). In the<br />
present study, the <strong>on</strong>ly potential grazers <strong>on</strong> the Ceratium<br />
spp. were the heterotrophic din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates,<br />
Mar Ecol Prog Ser 260: 19–31, 2003<br />
especially the Protoperidinium spp. (Hansen 1991,<br />
Buskey 1997, Naustvoll 2000), but their growth also<br />
stopped in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 and 8.5 experiments after 3 d <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
incubati<strong>on</strong>, indicating a very limited grazing pressure<br />
(Fig. 8).<br />
The initial c<strong>on</strong>centrati<strong>on</strong>s <str<strong>on</strong>g>of</str<strong>on</strong>g> inorganic nitrogen and<br />
phosphorus in the water in the present experiments<br />
were quite low, but close to Redfield’s ratio <str<strong>on</strong>g>of</str<strong>on</strong>g> N:P 16:1.<br />
At the end <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiments, this ratio had changed<br />
towards a ratio <str<strong>on</strong>g>of</str<strong>on</strong>g> 5:1 in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 to 9 incubati<strong>on</strong>s, suggesting<br />
an inorganic nitrogen limitati<strong>on</strong>. It is well<br />
known that smaller cells have lower half-saturati<strong>on</strong><br />
c<strong>on</strong>stants for nutrient uptake <str<strong>on</strong>g>of</str<strong>on</strong>g> inorganic nitrogen<br />
than large cells (e.g. Hein et al. 1995). This indicates<br />
that the most likely explanati<strong>on</strong> for the lack <str<strong>on</strong>g>of</str<strong>on</strong>g> growth<br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> Ceratium spp. was nutrient limitati<strong>on</strong>, and that<br />
this apparently also affected their predators (see Lynn<br />
et al. 2000).<br />
The <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5 incubati<strong>on</strong> differed in respect to inorganic<br />
nutrient c<strong>on</strong>centrati<strong>on</strong> from the other incubati<strong>on</strong>s<br />
(Fig. 2). At the end <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment, the NH 4 + c<strong>on</strong>centrati<strong>on</strong><br />
had increased 2- to 3-fold, whereas the phosphate<br />
c<strong>on</strong>centrati<strong>on</strong> had decreased to almost detecti<strong>on</strong><br />
level. The reas<strong>on</strong> for the rapid increase in NH 4 + is<br />
undoubtedly due to a quick remineralisati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> dead<br />
organisms, whereas the decrease in inorganic phosphorus<br />
is due to the much lower solubility <str<strong>on</strong>g>of</str<strong>on</strong>g> inorganic<br />
phosphorus at very <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g> (Otsuki & Wetzel 1972,<br />
Fig. 7. Cell c<strong>on</strong>centrati<strong>on</strong>s <str<strong>on</strong>g>of</str<strong>on</strong>g> selected<br />
ciliates in the 4 incubati<strong>on</strong>s during the<br />
2 wk experimental period. (A) Strombidium/Strobilidium<br />
spp. 50 µm, (C) Strombidium/Strobilidium<br />
spp. 25 to 50 µm, (D) Mesodinium<br />
pulex, (E) Myri<strong>on</strong>ecta rubra.<br />
(y) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.0; (j) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.5; (S) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.0;<br />
(m) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5. Symbols represent means<br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> triplicates ± SE
Pedersen & Hansen: <str<strong>on</strong>g>Effects</str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g> <strong>on</strong> a plankt<strong>on</strong>ic <strong>community</strong><br />
Kümmel 1981). This resulted in a potential phosphorus<br />
limitati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the phototrophic organisms, which may<br />
have affected the species compositi<strong>on</strong>. However, it is<br />
noteworthy that the growth rate <str<strong>on</strong>g>of</str<strong>on</strong>g> Cylindrotheca<br />
closterium was similar or even <str<strong>on</strong>g>high</str<strong>on</strong>g>er in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5 incubati<strong>on</strong><br />
compared to the rates found in lower <str<strong>on</strong>g>pH</str<strong>on</strong>g> incubati<strong>on</strong>s<br />
(Table 1). In fact, the <str<strong>on</strong>g>high</str<strong>on</strong>g>est growth rate <str<strong>on</strong>g>of</str<strong>on</strong>g> this<br />
diatom was found in the sec<strong>on</strong>d week in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5<br />
Fig. 8. Cell c<strong>on</strong>centrati<strong>on</strong>s <str<strong>on</strong>g>of</str<strong>on</strong>g> selected heterotrophic din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates<br />
in the 4 incubati<strong>on</strong>s during the 2 wk experimental<br />
period. (A) Protoperidinium divergens, (B) P. pellucidum,<br />
(C) Katodinium glaucum. (y) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.0; (j) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.5; (S) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.0;<br />
(m) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5. Symbols represent means <str<strong>on</strong>g>of</str<strong>on</strong>g> triplicates ± SE<br />
Fig. 9. Biomass <str<strong>on</strong>g>of</str<strong>on</strong>g> copepods (Oith<strong>on</strong>a and Pseudo-/Paracalanus)<br />
in the 4 incubati<strong>on</strong>s during the 2 wk experimental<br />
period. (A) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.0, (B) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.5, (C) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.0, (D) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5<br />
27
28<br />
Fig. 10. Average size <str<strong>on</strong>g>of</str<strong>on</strong>g> the copepod Oith<strong>on</strong>a in the 4 incubati<strong>on</strong>s<br />
during the 2 wk experimental period. (A) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.0,<br />
(B) <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.5. Symbols represent means ± SE<br />
incubati<strong>on</strong>, where the phosphorus limitati<strong>on</strong> should<br />
also have been the str<strong>on</strong>gest (Fig. 2).<br />
The silicate c<strong>on</strong>centrati<strong>on</strong> was c<strong>on</strong>stant in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8<br />
and 8.5 incubati<strong>on</strong>s throughout the durati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the experiment,<br />
and thus silicate does not seem to be limiting<br />
in these incubati<strong>on</strong>s (Fig. 2B). Dramatic increases in<br />
silicate c<strong>on</strong>centrati<strong>on</strong> were however observed in the<br />
<str<strong>on</strong>g>pH</str<strong>on</strong>g> 9 and 9.5 incubati<strong>on</strong>s mainly during the first 3 d <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
the experiment. The largest increase in silicate c<strong>on</strong>centrati<strong>on</strong><br />
was found in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5 incubati<strong>on</strong>, which<br />
reached a c<strong>on</strong>centrati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> ~30 µM silicate at the terminati<strong>on</strong><br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> the incubati<strong>on</strong>. This c<strong>on</strong>centrati<strong>on</strong> is ~3 times<br />
<str<strong>on</strong>g>high</str<strong>on</strong>g>er than the maximum c<strong>on</strong>centrati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> silicate typ-<br />
Table 2. Estimated copepod clearance in percent <str<strong>on</strong>g>of</str<strong>on</strong>g> the total<br />
water volume per day for the incubati<strong>on</strong>s <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8.0, 8.5, 9.0<br />
and 9.5<br />
<str<strong>on</strong>g>pH</str<strong>on</strong>g> Water volume cleared by the copepod <strong>community</strong><br />
Day 0 Day 5 Day 10 Day 12<br />
8.0 5.8 1.2 6.1 37.9<br />
8.5 5.8 9.6 13.0 2.3<br />
9.0 5.8 0.5 0.0 0.0<br />
9.5 5.8 0.0 0.0 0.0<br />
Mar Ecol Prog Ser 260: 19–31, 2003<br />
ically found in the spring before the diatom bloom has<br />
started (Richards<strong>on</strong> & Christ<str<strong>on</strong>g>of</str<strong>on</strong>g>fersen 1991). The largest<br />
<str<strong>on</strong>g>pH</str<strong>on</strong>g> adjustments were d<strong>on</strong>e in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9 and 9.5 incubati<strong>on</strong>s,<br />
both at the initiati<strong>on</strong> and during the experiment.<br />
It is therefore likely that we have added silicate al<strong>on</strong>g<br />
with the additi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> NaOH, because the NaOH soluti<strong>on</strong><br />
was stored in glass bottles and it is well known that<br />
silicate is <str<strong>on</strong>g>high</str<strong>on</strong>g>ly soluble at <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g>. The silicate c<strong>on</strong>centrati<strong>on</strong><br />
in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9.5 incubati<strong>on</strong> was, however, still<br />
much lower than what typically is added to ordinary<br />
algal growth media, like the f/2 medium (final c<strong>on</strong>centrati<strong>on</strong><br />
100 to 200 µM; Guillard 1972). Thus, it does not<br />
seem likely that these <str<strong>on</strong>g>high</str<strong>on</strong>g> silicate c<strong>on</strong>centrati<strong>on</strong>s<br />
should have had any negative impact <strong>on</strong> the algal<br />
species compositi<strong>on</strong> in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9 and 9.5 incubati<strong>on</strong>s.<br />
The <str<strong>on</strong>g>pH</str<strong>on</strong>g>-tolerant species (Prorocentrum micans, P.<br />
minimum, Heterocapsa triquetra and Cylindrotheca<br />
closterium) did better in terms <str<strong>on</strong>g>of</str<strong>on</strong>g> growth in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9<br />
incubati<strong>on</strong> compared to the lower <str<strong>on</strong>g>pH</str<strong>on</strong>g> incubati<strong>on</strong>s<br />
(Figs. 4A & 5A,B,C). This is in c<strong>on</strong>trast to laboratory<br />
studies, which indicate that the growth rates <str<strong>on</strong>g>of</str<strong>on</strong>g> these<br />
species are reduced at <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9 compared to at <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8<br />
(Schmidt & Hansen 2001, Hansen 2002). Thus, the reas<strong>on</strong><br />
for the better growth <str<strong>on</strong>g>of</str<strong>on</strong>g> these species in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9<br />
could instead be reduced grazing.<br />
Am<strong>on</strong>g the potential grazers, <strong>on</strong>ly ciliates and copepods<br />
occur in such numbers that they potentially may<br />
play a role as grazers <strong>on</strong> the 4 <str<strong>on</strong>g>pH</str<strong>on</strong>g>-tolerant species. Significant<br />
predati<strong>on</strong> due to the ciliates can however be<br />
ruled out. First, n<strong>on</strong>e <str<strong>on</strong>g>of</str<strong>on</strong>g> them can ingest the l<strong>on</strong>g<br />
diatom Cylindrotheca closterium, and <strong>on</strong>ly the large<br />
(>50 µm) ciliates can ingest din<str<strong>on</strong>g>of</str<strong>on</strong>g>lagellates as large<br />
as Heterocapsa triquetra and Prorocentrum minimum<br />
(e.g. Hansen et al. 1994). Sec<strong>on</strong>d, the number <str<strong>on</strong>g>of</str<strong>on</strong>g> the<br />
Fig. 11. Number <str<strong>on</strong>g>of</str<strong>on</strong>g> taxa (species or size groups) found at<br />
Day 14 in c<strong>on</strong>centrati<strong>on</strong>s >0.1 cells ml –1
Pedersen & Hansen: <str<strong>on</strong>g>Effects</str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>high</str<strong>on</strong>g> <str<strong>on</strong>g>pH</str<strong>on</strong>g> <strong>on</strong> a plankt<strong>on</strong>ic <strong>community</strong><br />
large ciliates was actually <str<strong>on</strong>g>high</str<strong>on</strong>g>est in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9 incubati<strong>on</strong>,<br />
compared to the incubati<strong>on</strong>s at lower <str<strong>on</strong>g>pH</str<strong>on</strong>g>, and<br />
thus the grazing loss due to these ciliates should have<br />
been the largest in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9 incubati<strong>on</strong> (Fig. 7).<br />
The dominant copepods (cyclopids) found in the<br />
incubati<strong>on</strong>s have the potential to feed <strong>on</strong> all the <str<strong>on</strong>g>pH</str<strong>on</strong>g>tolerant<br />
phytoplankt<strong>on</strong> (Hansen et al. 1994, Nielsen &<br />
Kiørboe 1994). Therefore, a significant grazing impact<br />
by the copepods <strong>on</strong> these algal populati<strong>on</strong>s may have<br />
occurred in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 and 8.5 incubati<strong>on</strong>s. Our estimates<br />
suggest that the copepods may have been able<br />
to remove about 10% <str<strong>on</strong>g>of</str<strong>on</strong>g> the standing phytoplankt<strong>on</strong><br />
stock per day (Table 2). Thus, the loss <str<strong>on</strong>g>of</str<strong>on</strong>g> grazing by<br />
copepods in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9 incubati<strong>on</strong> is a likely explanati<strong>on</strong><br />
for the observed better net growth <str<strong>on</strong>g>of</str<strong>on</strong>g> the <str<strong>on</strong>g>pH</str<strong>on</strong>g>-dominant<br />
phytoplankt<strong>on</strong>, but the remineralisati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> dying <str<strong>on</strong>g>pH</str<strong>on</strong>g>sensitive<br />
phytoplankt<strong>on</strong> has probably also c<strong>on</strong>tributed.<br />
Copepods including cyclopoids can also easily feed<br />
<strong>on</strong> ciliates (Stoecker & Capuzzo 1990, Nielsen &<br />
Sabatini 1996, Nakamura & Turner 1997), and thus it<br />
is <str<strong>on</strong>g>high</str<strong>on</strong>g>ly likely that the better net growth <str<strong>on</strong>g>of</str<strong>on</strong>g> ciliates<br />
at <str<strong>on</strong>g>pH</str<strong>on</strong>g> 9 can be explained, at least partly, by copepod<br />
grazing <strong>on</strong> the ciliates in the <str<strong>on</strong>g>pH</str<strong>on</strong>g> 8 and 8.5 incubati<strong>on</strong>s<br />
(Table 2). However, we did not count the small-sized<br />
protists (
30<br />
mum, the diatom Skelet<strong>on</strong>ema costatum or the cleptoplastidic<br />
ciliate Myri<strong>on</strong>ecta rubra (Fenchel et al. 1995).<br />
During the rest <str<strong>on</strong>g>of</str<strong>on</strong>g> the year, the phytoplankt<strong>on</strong> <strong>community</strong><br />
is much more diverse.<br />
In c<strong>on</strong>clusi<strong>on</strong>, the present experiments with the<br />
exposure <str<strong>on</strong>g>of</str<strong>on</strong>g> a plankt<strong>on</strong>ic <strong>community</strong> to a fixed <str<strong>on</strong>g>pH</str<strong>on</strong>g><br />
show that an increase in <str<strong>on</strong>g>pH</str<strong>on</strong>g> kills <str<strong>on</strong>g>pH</str<strong>on</strong>g>-sensitive organisms<br />
and causes a decrease in the species richness <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
both phototrophic and heterotrophic organisms. Our<br />
results indicate that the resp<strong>on</strong>se <str<strong>on</strong>g>of</str<strong>on</strong>g> an ecosystem to an<br />
elevated <str<strong>on</strong>g>pH</str<strong>on</strong>g> very much depends up<strong>on</strong> the durati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
the exposure. Our results also suggest that copepods<br />
are quite sensitive to elevated <str<strong>on</strong>g>pH</str<strong>on</strong>g> compared to protozooplankt<strong>on</strong>.<br />
In their absence, phytoplankt<strong>on</strong> is <strong>on</strong>ly<br />
subjected to grazing from the other protists. This will<br />
favour phytoplankt<strong>on</strong> species that have developed<br />
grazing-resistant mechanisms such as toxin producti<strong>on</strong>,<br />
poor food quality or shape.<br />
Acknowledgements. We are indebted to Christian Marc<br />
Andersen for analyzing the metazooplankt<strong>on</strong> samples and to<br />
Torkel Gissel Nielsen for valuable comments and suggesti<strong>on</strong>s<br />
to the paper. We also thank Benni Winding Hansen for the<br />
kind use <str<strong>on</strong>g>of</str<strong>on</strong>g> his fluorometer. The work was funded by both the<br />
Danish Natural Research Council (project #9801391 & #21-01-<br />
0539) and the European Commissi<strong>on</strong>’s Envir<strong>on</strong>ment & Sustainable<br />
Development (ESD) (FP-V, research into the development<br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> Sustainable Marine Ecosystems, Key acti<strong>on</strong> 3)<br />
under c<strong>on</strong>tract EVK3-CT-1999-00015 BIOHAB (Biological<br />
C<strong>on</strong>trol <str<strong>on</strong>g>of</str<strong>on</strong>g> Harmful Algal Blooms in European coastal waters).<br />
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Submitted: July 9, 2002; Accepted: June 17, 2003<br />
Pro<str<strong>on</strong>g>of</str<strong>on</strong>g>s received from author(s): September 15, 2003<br />
31