Chinese Journal of Oceanology and Limnology

Vol.20, No.3, P.237 - 243, 2002

COMBINED EFFECTS OF TEMPERATURE, IRRADIANCE AND

SALINITY ON GROWTH OF DIATOM SKELETONEMA COSTATUM*
YAN T i a n ( ~ ) , ZHOU Mingjiang ( ~ : ~ I ~ )
( Key Laboratory of Marine Ecology and Environmental Science, Institute of Oceanology,
Chinese Academy of Sctenees , Qingdao , 266071, Ch/na)
QIAN Peiyuan ( ~ )
( Biology Department, Hong Kong University of Sciences and Technology, Hong Kong)
Received Nov. 15, 2000; revision accepted May 20, 2001

Abstract 3-factor experiment was used to study the combined effects of temperature, irradiance
and salinity on the growth of an HAB species diatom Skeletonema costatum ( Grey. ) Cleve. The results
showed that temperature (12, 19, 25, 32~ ) , irradiance ( ( 0 . 0 2 , 0 . 0 8 , 0 . 3 , 1.6) x 1016quanta/(s 9
cm2 )) and salinity (10, 18, 25, 30, 35) significantly influenced the growth of this species. There were
interactive effects between any two of and among all three physical factors on the growth. In the experi-
ment, the most optimal growth condition for S . costatum was temperature of 25~ salinity of 18 - 35
and irradiance of 1.6 x 1016 quanta/(s ~cm2). The results indicated S. costatum could divide at higher
rate and were more likely to bloom under high temperature and high illumination from spring to fall. It was
able to distribute widely in ocean and estuary due to its adaptation to a wide range of salinities.

Key words: Skeletonema costatum, harmful algal bloom, temperature, irradiance, salinity

INTRODUCTION

Various causative factors, such as increased scientific awareness, utilization of coastal waters
for aquaculture, eutrophication and cyst transportation (Hallegreaff et al., 1995) has led to extens-
ive studies on the global increase in HAB (Harmful Algal Bloom). Yet the HAB mechanism is still
poorly understood. Each HAB species adapts to certain environment and may bloom during its opti-
mal conditions. Changes in environmental factors may lead to a shift in dominating species within a
phytoplankton community. Therefore, it is crucial to study the monoculture growth of harmful algae
under different environmental factors, in order to understand the HAB mechanism and establish
mathematical models.
Diatom Skeletonema costatum ( G r e v . ) Cleve distributes widely from north to south along the
Chinese coast and is one of the most frequently occurring HAB causative organisms (Zou and Wu,
1992; Hong et al., 1994; AFD, 1997). It is apparently non-toxic but still potentially harmful to
marine organisms because of oxygen depletion during algal decomposition. For example, an extens-
ive HAB covering about 1000 km2 in the exterior sea area of the Changjiang Estuary during June 30
- J u l y 14, 1987, seriously affected the marine~_ecosystem (Hang et al., 1989). However, only a
few HAB events of S . costatum were investigated in situ during which environmental factors were
monitored during its whole process (Hong et al., 1989, 1994; Hao, 2000). Because of the signifi-
cant impacts of this species on local waters, it was chosen to be investigated in the present study fo-
cused on possible interactions of three main physical factors of temperature, irradiance and salinity.
In order to explore the potential interactions between/within biophysical elements, a multi-factor de-

* This study was supported by the PREPP and National Key Basic Research Project (2001CB4097) NNSFC No. 39950001,
20177023, 49576301, 49906007 and KZCX2- 206.
 238 CHIN. J. OCEANOL. LIMNOL., 20(3), 2002 Vol.20

sign was employed in the present investigation. The main purpose of this study was to explore the
environmental conditions under which HAB would be more likely formed by this species and to pro-
vide data for mathematical models for coastal areas.

MATERIALS AND METHODS

Alga cultivation

S. costatum (Greville) Cleve was obtained from a culture of CCMP1332 (Culture Center of
Marine Phytoplankton, Maine, U . S . A . ) . The experiment was performed in the Biology Depart-
ment, Hang Kong University of Sciences and Technology. Cultures were grown in enriched seawater
(Harrison et al. 1980). One part of natural seawater blended with two parts of artificial seawater
(Harrison et a l . , 1980) were used to keep stability and provide natural trace substances for algal
growth. To avoid precipitation during autoclaving, the medium was passed under gravity pressure
through O. 2 ttm filter for sterilization using 316 stainless steel sanitary filter holder (142 nun). Ster-
ilized 50 ml glass test tubes with caps were used for culturing. Incubators (Powers Scientific SD
33SE) were used at the four designated temperatures. Different salinity media were obtained by di-
luting with double distilled seawater or adding salt according to artificial seawater recipe when mixing
natural and artificial seawater before enrichment. ATAGO hand refractometer was used for salinity
measurement. Light at 14h: 10h (light: dark) cycle was provided by Ostrum Lumilux cool white
fluorescent tubes and was attenuated by distance or screening. Irradiance was measured by Licor
model LI 185 meter with 2B collector).

Experiment design
Temperature, irradiance and salinity were chosen to cover the range which HAB species could
likely experience in Chinese coastal waters. The setup followed 3-factor design: combinations of 4
temperature levels (12, 19, 25, 32 ~C), 5 salinity levels (10, 18, 25, 30, 35) and 4 irradiance
levels ( ( 0 . 0 2 , 0.08, 0 . 3 , 1 . 6 x 1016quanta/(s-cm2) ). Treatments (4 x 5 x 4) were conducted in
triplicate. Culture at mid-exponential phase was transferred to 40 ml fresh medium in each test tube
to initiate the experiment. Dilution rate was about 5 % . Determinations of biomass ( in vivo fluores-
cence) were made at 24 h intervals by inserting the whole tube into a Turner Designs Model 10 flu-
orometer after mixing by about six inversions of the tube. The growth rate was calculated as tt = In
( F z / F I ) ( t 2 - t l ) , where F2 is fluorescence at time tz and F1 is fluorescence at time t~. The
growth rate of each transfer was obtained by averaging daily growth rates during exponential phase.

Statistical analysis

3-way ANOVA (Zar, 1996) was applied in analyzing the effects of temperature, salinity and
irradiance on the growth rate. Tukey multiple comparison test was used to compare treatment means
when'factors did not significantly interact with each other. When there was any significant interac-
tion between (among) factors (P < 0 . 0 5 ) , the effect of a single factor on the treatment was tested,
with other factor(s) fixed.

RESULTS

The respective effects of temperature, salinity and irradiance on growth of S . costatum

The respective effects of temperature, irradiance and salinity on growth of S. costatum are
shown in Table 1 after 3-way ANOVA analysis. The results showed that temperature, irradiance and
No.3 YAN et a l . : COMBINED E F F E C T S O F 3 - F A C T O R ON DIATOM G R O W T H 239

salinity all had significant effects on the growth of S . costatum (P < 0 . 0 0 1 ) . The growth rate in-
creased with temperature from 12 - 25~ but dropped at higher temperature of 32~ increased
with salinity from 10 - 35, increased with irradiance from 0 . 0 2 x 10 ~6 to 0 . 3 x 10 ~6 quanta/s"
cm 2 ) ) , but did not increase anymore at higher irradiance of 1.6 x 10 ~ quanta/(s" c ~ ) ) .

Table 1 The respective effects of temperature, s a l i n i t y a n d i r r a d i a n c e o n growth of S . costa.

turn. I n t h e s a m e g r o w t h r a t e column, values that do not differ significantly at 0 . 0 5 level i n
Tukey tests are indicated by the same letters (a or b)
Temperature Growth rate Growth rate Irradianee Growth rate
(%) (/d) Salinity (/d) ( x 1016 quanta/(s" cm2 ) ) (/d)

12 0.4753 10 - 0.2222" 0.02 - 0.0283"

19 0.5757 18 0.0724 ~ 0.08 0.0278 ~
25 0. 8622 25 0.246 2a 0.3 0.3087 b
32 - 1.3019 30 0.3204b 1.6 0.3031 b
35 0.3474b

1.5
1
LI 1.51.2
0.5
(
-0.~ -0.
-1
-l.5 -1
~ 3 5
-2
,,-,, -2. fi
-1.5
-2 35
T
25 32

:52i /' 35 1:52I

12 19 ~ 25 32 10 12 19 ~/ 10

F i g . 1 The c o m b i n e d effects of t e m p e r a t u r e a n d salinity on the growth of S . costatum

Table 2 Summary of 3-way ANOVA results for v a r i o u s e f f e c t s o f d i f f e r e n t t e m p e r .

a t u r e s , s a l i n i t i e s a n d i r r a d i a n c e s o n t h e growth of S . costatum
Treatments df F P
Temperature ( ~C ) 12 19 25 32 3 208. 839 * *
Salinity 10 18 25 30 35 4 398. 894 * *
Irradianee (1016 q u a n t a / ( s ' e m 2 ) ) 0.02 0.08 0.3 1.6 3 4130.487 * *
Temperature x Irradianee 9 135.704 * *
Temperature x Salinity 12 189.390 * *
Irradianee Salinity 12 124. 883 * *
Temperature x Irradlanee x Salinity 36 128.477 * *
* * . P<O.O01
 240 CHIN. J. OCEANOL. LIMNOL., 20(3), 2002 Vol.20

,.5
1
0.5 J
i
0
-0.5
-1 I
-1.5 I

-2. t
t

10 1 8 ~ 4
"~ 25 30
35-~L1 L2 L3
35 L1

1.5

0.5
1
15,f
C
-0. ~
-1
-1.~ -1_~
-,~
-2. ~ -2. 5 "

-- 25 ,m~-~_~ / L3
S 35L1 L2 30 35L1 L2 ~

Fig. 2 The combined effects of salinity and irradiance on the growth of S . costatum

o.+~ oit _
-~ i + +~
t. -":t a +
32 LI L2 L3 < ' ~ /~. ~ 32 L1
L3 --...~ ~ ~" 25
i L4 12 L4 12 19 L412 19

o
9 1-1 5 I:Ircadiance
m05-1
-o:~I --~~/.o+i ---~,.-~ [] 0-0 5
El -0 5-0
(quanta.s-1.cm-2)
LI: 0.02X lO16
[]-1--0 5 L2: O.08X1016
9 5--1
-~: ~ ~o~
L1 ,'~,-..~ ~ 32
-~:~ :------~ B-2-1 5
1.3:0.3 10z6
9 -2 5 - 2 I..4: 0.16X 1016
L2 ~ 2 5 ~" L1 ~ j - ~ 5 3 2
9 -3--2 5

Fig. 3 The combined effects of temperature and irradiance on the growth of S. costatum

Figs 1 - 3 and Table 2 show the combind effects of temperature, irradiance and salinity on the
growth of S . costatum and interactions between any two of and among the three factors. Fig. 1
shows the effect of temperature and salinity on the growth of S . costatum at different irradiance lev-
els. At low irradiance of 0 . 0 2 x 10 t6 q u a n t a / ( s . c m 2 ) , there were no significant differences among
the growth rates at 12,19 and 25% ( P > 0 . 0 5 ) ; while at higher irradiances of 0 . 3 x 1016 and 1 . 6
No.3 YAN et al. : COMBINED EFFECTS OF 3-FACTOR ON DIATOM GROWTH 241

x 1016 q u a n t a / ( s ' c m 2 ) , the growth rate increased with temperature from 1 2 - 25~ Under each
irradiance level, the growth rate dropped significantly with decreased salinity at 32 ~ Fig. 2 shows
the effect of irradiance and salinity on the growth of S . costatum at different temperature levels. At
temperature of 12, 19 and 25~ the growth rate increased significantly with irradiance. Even at
lowest irradiance 0 . 0 2 x 1016 q u a n t a / ( s ' c m z) and lowest temperature 12~ S . costatum could
grow, although very slowly. But at high temperature of 32~ the cell density declined very rapidly,
indicating algal cells could not survive at 32 ~ The growth rate dropped significantly with decreased
salinity and decreased irradiance, showing the conditions of high temperature, low irradiance and
low salinity were most difficult for survival of this species. Fig. 3 shows the effect of temperature and
irradiance on growth of S . costatum at different salinity levels. At each salinity level, growth rate
increased with increased irradiance, and temperature from 1 2 - 25~ under each irradiance.
The results showed there were significant effects of temperature, irradiance and salinity on the
growth of S . costatum and interactions between any two of and among the three factors ( P < 0.001).
This experiment showed that the most optimum growth conditions for S . costatum were temperature of
25~C, salinity of 18 - 3 5 and irmdiance of 1.6 x 1016 quanta/(s'cm 2 ). The growth rate under these con-
dition was 1.5/d.

DISCUSSION

This experiment showed that S . costatum had high growth rate at temperature of 19 - 25 ~
salinity of 10 - 35 and irradiance of 0.3 x 1016 - 1.6 x 1016 q u a n t a / ( s ' c m 2 ) , and so is more likely
to bloom under high temperature and the high illumination from spring to fall. It could distribute
widely in ocean and estuary due to its adaptation to relatively wide salinity ranges. Growth rate of
S . costatum could reach 1.5/d at the most optimum condition in the experiment and could be about
1/d even at slightly less than optimum conditions, such as temperature of 19~ irradiance of 1.6
1016 quanta/( s" crn2 ) or temperature of 25 ~ irradiance of 1.6 1016 quanta/( s" cm2 ), salinity of
10. As one of the most common HAB species, it was found growing optimally at temperature of 20 -
26~ and salinity of 25 - 30 in Bohai Bay (Zou, 1983), which coincided with our results. It usual-
ly peaks in early spring or late summer and could reach density of 1.4 107 cell/m 3 in the Yellow
River Estuary, 4 . 5 x 109 cell/m 3 in Jiaozhou Bay, 7 1 0 6 - - 2 . 6 101~ in the Changjiang
River Estuary and 4.48 x 109 in Dapeng Bay(Zou, 1992). The high growth rate and wide adapta-
tion of this species could probably explain why it is one of the most common HAB species in coastal
waters all over the world.
In a common HAB occurrence area, the northern part of Jiaozhou Bay, an HAB of S . costa-
turn occurred during July 3 - 8, 1998. The changes of chl-a and environmental factors, including
temperature, salinity, pH, DO, PO4, SiO3, NH4, NO3 and NO2, were monitored during this
HAB' s formation and decay. For main physical factors, the investigation results showed the temper-
ature was 2 2 . 7 ~ before the HAB, rose to 2 6 . 5 ~ and remained at 2 5 . 6 - 28.0~ during the
bloom. Salinity decreased from 31.24 to 30.59 before the HAB due to rainfall on June 30 - July 1,
and remained at 30.31 - 30.78 during the bloom (Hao, 2000). From our results, we can see that
salinity of around 30, irradiance of 1.6 x 1016 quanta/( s" cm2 ) (light usually reaches this level in
summer in this environment), increased temperature from 19 to 25 ~ which was near the range of
22 - 27 ~ in situ of the boom, the growth rate of S . costatum could rise from 1.0/d to 1 . 4 / d .
Therefore, this increased growth of S . costatum due to temperature shift may be one of the causative
factors for the dramatic increase of algal cells from late June to early July during the HAB forming
process, aside from the additional nutrient supply brought by rain caused runoff to the bloom area.
 242 CHIN. J. OCEANOL. LIMNOL., 20(3), 2002 Vol.20

In J i n ' s study ( 1 9 6 5 ) , S . costatum growth was satisfactory at temperature of 8 - 32~ and

salinity of 7 - 50 and optimized at temperature of 20 - 26 ~ salinity of 25 - 30. Our results were
basically in agreement with his results, although our strain of the species was not able to grow well
at 32~ This may be caused by differences between strains. In a study of biological characteristics
of Genus Skeletonema, Shi found two biomass peaks of the same species, S . costatum in Jiaozhou
Bay, one was in August and one in Jan - Feb. However, the two strains showed different adapta-
tions to temperature under the same nutrient level, at 15 ~ and 20 ~ the growth rate of the summer
strain was 0 . 9 8 9 and 1. 559/d, respectively, while that of the winter strain was 1. 260/d and 1.
452/d (Shi, 1990). But for the Genus Skeletonema in China, it is divided into two species: S.
costatum and S . tropicum. The former species, with 1 - 2 chromoplasts, is eurythermal and eury-
haline, while the latter one, with 5 - 7 chromoplasts, is polythermal and oligohaline (Zou, 1992).
In a study of intraspecific variation in the temperature niche component of the diatom Skeletonema
costatum from Korean coastal waters (Yih, 1995), it was found there were intraspecific variations of
the mean growth rate and mean final biomass yield under three different light intensities. However,
the coefficients of variation were not greater than 10% in any of the 7 S . costatum clones. Their
findings indicated the different strains may be acclimated to their environment and differ in environ-
mental adaptation. This may be the second reason for the wide distribution and common occurrence
of S . costatum HAB in coastal waters.
In our experiments, we applied fully enriched medium in monoculture and focused on physical
factors. However, in the natural environment, there are competitions among algal species and possi-
ble interactions between and among species in the environment, by metabolic excretions from algae
(which may inhibit or stimulate the growth of other species) (Honjo, 1993), as well as other im-
portant HAB formation influencing factors, such as nutrients (Hong et a l . , 1994; Cannon, 1996;
Davidson et a l . , 1999; Hao, 2000). Therefore, the growth behavior of each species was not the
same in the environment as that in monocuhure under fixed chemical and physical conditions. For
example, S . costatum sometimes reached its density peak at different conditions other than opti-
mum, such as in early spring at the Yellow River Estuary. For better understanding of the HAB
mechanism, the effect of nutrients and inter-specific competition on S . costatum should be studied
further.

ACKNOWLEDGEMENT

The authors wish to thank Dr. Qiu J. for statistics consultation.

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