Aquatic Toxicology 181 (2016) 22–28

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Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox

The influence of urea and nitrate nutrients on the bioavailability and

toxicity of nickel to Prorocentrum donghaiense (Dinophyta) and
Skeletonema costatum (Bacillariophyta)
Xu-Guang Huang a,b,∗ , Xie-Chang Lin a,c , Shun-xing Li a,b,d , Song-Li Xu e , Feng-Jiao Liu d
a
College of Chemistry and Environmental Science, Minnan Normal University, Zhangzhou, 363000, China
b
Fujian Province Key Laboratory of Modern Analytical Science and Separation Technology, Minnan Normal University, Zhangzhou, 363000, China
c
School of Environmental and Chemical Engineering, Nanchang Hangkong University, Nanchang, 330063, China
d
Key Laboratory of Coastal and Wetland Ecosystems, Ministry of Education, Xiamen University, Xiamen, 361005, China
e
Environmental Monitoring Station of Longwen, Zhangzhou, 363000, China

a r t i c l e i n f o a b s t r a c t

Article history: Nitrogen nutrients and nickel(Ni) are ubiquitous in aquatic environments, and they are important for
Received 23 July 2016 primary production of ocean ecosystem. This study examined the interaction of nitrogen nutrients (specif-
Received in revised form 24 October 2016 ically urea and nitrate) and Ni on chlorophyll (Chl a) concentration and photosynthesis parameters values
Accepted 26 October 2016
of Prorocentrum donghaiense and Skeletonema costatum. The data presented here indicate that low concen-
Available online 27 October 2016
tration of Ni for P. donghaiense and S. costatum can enhance both Chl a concentration and photosynthesis
parameters values when grown in urea containing environment. Despite this increase there was also an
Keywords:
observed depression in both species tested when incubated in high concentration of Ni for P. donghaiense
Nickel
Urea
and S. costatum regardless of incubating in urea or nitrate. Additionally, EC50 values of Chl a and Fv/Fm
Bioavailability for Ni at different time intervals were calculated in this study. These observations indicated that the Ni
Toxicity tolerance was higher in P. donghaiense as compared to S. costatum. The Ni tolerance of P. donghaiense
Prorocentrum donghaiense incubated in urea was higher than that incubating in nitrate. The same phenomenon was not observed in
Skeletonema costatum S. costatum, which indicated that the influence of urea was dependent on the species investigated. Thus,
urea input could impact Ni bioavailability and toxicity, and then affect the biodynamics thereafter.
© 2016 Published by Elsevier B.V.

1. Introduction urea are often the predominant components of dissolved nitrogen

Eutrophication is common in many estuarine and coastal
waters. It is mainly due to an increase in agricultural devel-
opment in recent years. This increase has become a serious
environmental problem (De Jong, 2006). It has been reported that
nutrient enrichment, accompanied by a variation in nutrient ratio
(due to disproportionate inputs of nutrients) has been shown to
profoundly affect the phytoplankton species composition and pro-
duction (Smith et al., 1999). Nutrient enrichment mainly refers to
an increase in nitrogen and phosphorus concentrations. Nitrogen
nutrients include dissolved inorganic nitrogen (i.e., nitrite, nitrate,
and ammonia) and dissolved organic nitrogen (DON) (i.e. urea,
amino acid and so on) in the ocean (Peers et al., 2000). Nitrate and
∗ Corresponding author. Present/permanent address: College of Chemistry and
Environmental Science, Minnan Normal University, Zhangzhou, 363000, China.
E-mail addresses: huangxuguang@munu.edu.cn, hxg226@sina.com
(X.-G. Huang).
in estuarine and coastal waters due to riverine input (Dyhrman
and Anderson, 2003; Liu et al., 2011). Phytoplankton use inor-
ganic nitrogen and can also take up and assimilate urea. It has
been demonstrated that the bioavailability of urea to dinoflagellate
enhances the intensity of dinoflagellate bloom (Bronk et al., 2007;
Dyhrman and Anderson, 2003). There are numerous lines of evi-
dences that indicate most phytoplankton (Antia et al., 1991), except
chlorophytes, use the Ni-containing enzyme urease to hydrolyze
urea to ammonium and carbon dioxide (Worms et al., 2007).
Consequently, these phytoplankton require Ni for growth in urea
containing environment, an important nitrogen source that can
support 5–50% of oceanic primary production (Dupont et al., 2010).
Thus, the potential interactions between phytoplankton commu-
nity composition, nitrogen and Ni can significantly influence the
ocean ecosystems (Dupont et al., 2010).
Ni is a necessary metal for organisms, but it is toxic at ele-
vated concentrations. It is important in the functioning of urease;
an important enzyme in the hydrolysis of urea to produce ammonia
and carbamate (Smyj, 1997). Because Ni plays such an important

http://dx.doi.org/10.1016/j.aquatox.2016.10.027
0166-445X/© 2016 Published by Elsevier B.V.
 X.-G. Huang et al. / Aquatic Toxicology 181 (2016) 22–28
role in metabolism of urea, it is essential to study the function and
fate of Ni in the context of marine biogeochemical cycles. Numerous
studies have been conducted to investigate this role of Ni. However,
current reported data focused on Ni accumulation in, or uptake
from water, the dissolved phase by marine biota and its transfer
in food chain (Mehta et al., 2000; Singh et al., 1992; Wang et al.,
2007; Wang and Dei, 2001). The major contribution to net primary
production in coastal and estuarine waters could be impacted by
metal pollution including Ni. Despite these studies there has been
a lack of study, especially in urea incubation, on the influence of
Ni on growth and photosynthesis parameters. Therefore the data
presented here addresses this important issue.
Prorocentrum donghaiense and Skeletonema costatum are 2 key
species that contribute to harmful algal blooms in the coastal
waters of China (Ou et al., 2008). In the past few years, they had
formed extensive blooms in East China Sea (Ou et al., 2008); a region
that coexisted with an abundance of urea. These observations sug-
gest urea might affect the bioavailability of Ni in coastal waters, thus
influencing the growth and photosynthesis parameters of the phy-
toplankton that was examined. However, no work has been done to
investigate this possibility. This study investigates the influence of
different nitrogen resource (specifically nitrate and urea) and Ni on
the growth and photosynthesis parameters of P. donghaiense and
S. costatum. The data reported here aims to determine the impacts
and mechanisms of urea on Ni bioavailability and risk assessment
in the phytoplankton.
2. Materials and methods
2.1. Culture conditions
Seawater was collected from the Zhangzhou Bay (23.62◦ N,
117.61◦ E), which is in the Fujian province in China. Samples were
stored at 20 ◦ C for 6 months, and filtered through acid-washed Pall
Acropak Supor capsule 0.22 ␮m filters before use. A flow injection
analyzer (FIA) was used to determine the background nutrient con-
centration in the seawater. The background concentration of total
dissolve nitrogen (TDN) was measured using a Shimadzu TOC-TN
analyzer (Shimadzu Corp., Kyoto, Japan). Urea concentration was
measured using the diacetylmonoxime reagent method for seawa-
ter at room temperature (Goeyens et al., 1998). The seawater was
added to a closed vessel with mixed acid (HNO3 :H2 O2 , v:v = 2:1). It
was then microwave digested for 10 min at 10 atm, then used for
determining the background concentrations of Ni in the seawater
using inductively coupled plasma mass spectrometry (ICP-MS).The
amount of Ni was 1.23 ␮mol L−1 , and the relative standard devi-
ation was 1.3%. This coastal seawater, with both Ni and nutrient
enrichment, could be used for Ni bioavailability and risk assessment
by phytoplankton experiments.
Unialgal cultures of P. donghaiense and S. costatum were obtained
from the State Key Laboratory for Marine Environmental Science
(Xiamen University), the samples were collected from Yongtze
River estuary in 2003. They were maintained in seawater, f/10 lev-
els of phosphate and Si for S. costatum only, at 140 ␮mol photons
m−2 s−1 by a light: dark cycle as 14:10 h. Additionally, the samples
were fortified with vitamins, but without trace metals. Different
N (added as NaNO3 , urea) concentrations at 20 ◦ C.The cells were
transferred to a new medium every 3–6 days, to ensure that the
cells were acclimated to these nutrient conditions.
2.2. Algal growth assays
During the growth experiments, the algal cells were cultured in
the filtered seawater in a media containing f/10 levels of phosphate,
silicate (only for S. costatum) and vitamins without trace metals.
23
Concentrations of 172 ␮mol L−1 N (f/10 levels, added as NaNO3 ,
urea) were also added. Incubation was at 20 ◦ C. Stock solutions
of Ni were added to the algal medium for treatments at different
concentrations (0.17, 1.7, 17 ␮mol L−1 and 0.17, 0.85, 1.7 mmol L−1 ).
The algal biomass measurements were performed from 0 to 96 h,
and all treatments were performed in triplicate. Chl a was used as
an indicator for the concentration of P. donghaiense and S. costatum
during the experiments, using the method of Juneau P et al. (Juneau
et al., 2002).
2.3. Photosynthesis parameters
Photosynthetic parameters were measured daily up to 4 days,
treated samples using a pulse amplitude modulated fluorometer
(PHYTO-PAM, Heinz Walz, Germany). The measurement principle
of PAMF is based on changes in the Chl a fluorescence level after
application of a saturated light pulse from which the photosyn-
thetic yield as well as quenching was calculated. These parameters
were used to reflect the impact of certain stress factors on photo-
synthetic pathway. The maximum photochemical efficiency of PSII
was calculated as Fv/Fm. Chl a content was estimated by photosyn-
thesis parameters (Chl a) and were also performed to account for
phytoplankton biomass changes (Ahmed and Häder, 2010).
Light-response curves were determined for all treated samples
each day. Cells were first dark-adapted for 15 min before measure-
ment. Cells were then exposed to increasing illumination intensity
in 15 steps from 0 to 3500 ␮mol m−2 s−1 . After 20 s of each illu-
mination, a saturating pulse was applied, photosynthetic yield
and rETR (electron transport rate) were measured automatically.
Then, the maximum relative electron transport rate (rETRmax) was
calculated from the light-response curves. A minimum of four inde-
pendent samples for all photosynthesis parameters were measured
for the same treatment (Ahmed and Häder, 2010).
2.4. Data analysis and statistical analysis
Principal response curve (PRC) analyses were performed with
in vivo Chl a fluorescence parameters (rETRmax and Fv/Fm) and Chl
a in order to obtain a comprehensive overview of different nitrogen
nutrients on Ni toxicity during the experiment.
Dose response curves were constructed for photosynthetic
yields (Fv/Fm) and Chl a using a 3-parameter log-logistic model fol-
lowing the equation (Eq. (1)). The EC curve has a sigmoidal shape,
from which many toxicity values such as EC50 were determined.
d
y=
b
1 + (c/EC50 )
Where y is the response variable (inhibition percentage), c is Ni
concentration, d is the response when the concentration tends to
infinity, EC50 of Ni concentration that results in 50% inhibition of
parameters and b is a scale factor.
For all parameters, the effects of exposure time, different
nitrogen nutrients and Ni concentration were tested by repeated
measure ANOVA. The effects of different nitrogen nutrient on Ni
exposure were then tested at each exposure time by one-way
ANOVA. The ANOVA was followed by a Tukey-HSD test by SPSS
statistics 19.0. A p value of 0.05 levels were considered significant,
3. Results
3.1. Effects of Ni and nitrogen nutrients on the Chl a content of P.
donghaiense and S. costatum
During the 96 h exposure, compared to the control treatment,
the Chl a of P. donghaiense content at 0.17 ␮mol L−1 , 1.7 ␮mol L−1 ,
24 X.-G. Huang et al. / Aquatic Toxicology 181 (2016) 22–28
Fig. 1. The influence of Ni on the Chl a content of P. donghaiense (A was nitrate, B was urea) and S. costatum (C was nitrate, D was urea) in different nitrogen conditions.
(䊉) control, (䊏) 0.17 ␮mol L−1 , () 1.7 ␮mol L−1 , () 17 ␮mol L−1 , () 0.17 mmol L−1 , () 0.85 mmol L−1 , (䊐) 1.7 mmol L−1 .
17 ␮mol L−1 and 0.17 mmol L−1 of Ni treatment decreased 7.79%,
0.63%, 19.32%, 35.13%, respectively (Fig. 1A), when incubated with
nitrate. During the 48 h exposure, the Chl a content decreased
significantly at 0.17 ␮mol L−1 of Ni treatment (p < 0.05), and then
increased slowly (Fig. 1A). After 24 h exposure, the Chl a con-
tent was barely detectable at 0.85 mmol L−1 and 1.7 mmol L−1 of Ni
treatments.
The Chl a content of P. donghaiense in treatments incubated with
urea and Ni (from 0 to 100 mg L−1 ) are shown in Fig. 1B. During
the 96 h exposure, compared to the control treatment, the con-
centration of Chl a at 17 ␮mol L−1 and 0.17mmol L−1 treatments
decreased by 21.82% and 38.15%, respectively. However, increased
Chl a content (4.07% and 4.44%) was observed to be significant
(p < 0.05) at treatments of 0.17 ␮mol L−1 and 1.7 ␮mol L−1 of Ni. Fur-
thermore, at the treatment of 0.85 mmol L−1 and 1.7 mmol L−1 of Ni,
the Chl a content fell significantly (p < 0.05) after 24 h exposure.
During the 96 h exposure, compared with the control treat-
ment, the Chl a content of S. costatum in nitrate (Fig. 1C) and
at 0.17 ␮mol L−1 , 1.7 ␮mol L−1 , 17 ␮mol L−1 or 0.17 mmol L−1 of Ni
decreased 6.96%, 31.7%, 37.8% and 61.8%, respectively. Also, the
Chl a content of S. costatum in urea (Fig. 1D) at 0.17 ␮mol L−1 ,
1.7 ␮mol L−1 and 17 ␮mol L−1 of Ni increased 20.3%, 6.2% and 3.0%,
respectively; and it decreased 46.1% at 0.17 mmol L−1 of Ni. Mean-
while, the Chl a levels decreased to an undetectable threshold at
0.85 mmol L−1 and 1.7 mmol L−1 of Ni regardless of incubating in
nitrate or urea after 24 h exposure.
Table 1 Test statistics on the effects of Ni concentration and
nitrogen sources on Chl a of algae during a 96 h culture period. This
data indicated that incubating in different nitrogen sources, the val-
ues of Chl a were significantly different (p < 0.05) in P.donghaiense,
but not significantly different (p > 0.05) in S. costatum.
3.2. Effects of Ni and nitrogen nutrients on the photosynthesis
parameters (Fv/Fm and rETRmax) of P. donghaiense and S.
costatum
While P. donghaiense and S. costatum incubated in Ni and nitro-
gen nutrients (urea and nitrate) for different times (24, 48, 72
and 96 h), the key indicators, photosynthesis parameters including
maximal photochemical efficiency of PsiI (Fv /Fm ), and maximum
relative electron transport rate (rETRmax ) were measured. The data
indicated the effects of Ni on the Fv/Fm and rETRmax of algal cells
within the 0–96 h exposure (Figs. 2 and 3). While P. donghaiense
incubated in nitrate culture after 96 h, compared with control
treatment, Fv /Fm and rETRmax at 1.7 ␮mol L−1 , 17 ␮mol L−1 and
0.17 mmol L−1 of Ni treatment were depressed 5.5%, 11.7%, 13.5%
and 36.6%, 80.3%, 83.5%, respectively (Figs. 2A and 3A). However,
while P. donghaiense exposure to urea within 0–96 h, the Fv /Fm
and rETRmax at 0.17 ␮mol L−1 –1.7 ␮mol L−1 of Ni were significantly
higher than that of control treatment (p < 0.05). However, after
a 96 h incubation period, compared with control treatment, the
Fv /Fm and rETRmax of P. donghaiense at 17 ␮mol L−1 , 0.17 mmol L−1 ,
0.85 mmol L−1 and 1.7 mmol L−1 of Ni treatment were depressed
21.7%, 48.2%, 78.7%, 99.3% and 32.1%, 40.3%, 94.5%, 94.2%, respec-
tively. After 24 h exposure, the treatments of 0.85 mmol L−1 and
1.7 mmol L−1 , photosynthesis parameters of P. donghaiense regard-
less of nitrogen nutrients were decreased, to the point that none
were detectable.
The Fv /Fm and rETRmax of S. costatum incubated in urea and
nitrate within the 0–96 h exposure showed the similar trend as
that of P. donghaiense (Figs. 2C, D, 3C and D). While S. costatum
incubated in nitrate and urea culture after 96 h, compared with
control treatment, the values of Fv /Fm and rETEmax decreased with
the increased Ni concentration (from 17 ␮mol L−1 to 1.7 mmol L−1 ).
However, while S. costatum exposure to nitrate and urea within
0–96 h, the Fv /Fm and rETRmax at 0.17 ␮mol L−1 –1.7 ␮mol L−1 of
Ni were not significantly higher than that of control treatment
(p > 0.05). Notably, this observed phenomenon is different than that
of P. donghaiense.
Meanwhile, Table 1 presents data that indicate incubating in
different nitrogen sources, the values of Fv/Fm were significantly
different (p < 0.05) in P. donghaiense, but were not significantly
different (p > 0.05) in S. costatum. Statistical results support a signif-
icant interaction (p < 0.05) between Ni concentration and nitrogen
sources for P.donghaiense (Table 1).
 X.-G. Huang et al. / Aquatic Toxicology 181 (2016) 22–28 25

Table 1
Test statistics on the effects of Ni concentration and nitrogen sources on Chl a and Fv/Fm of algae during 96 h culture. ANOVA (F) value for repeated measures and significance
(p).

P. donghaiense S. costatum

F P F P

Chl a Time(T) 157.2 <0.001 622.9 <0.001

Ni concentration(Nc) 276.5 <0.001 185.0 <0.001
Nitrogen sources(Ns) 81.1 <0.001 0.006 0.938
Nc×Ns 5.6 0.01 6.7 <0.001
Fv /Fm Time(T) 160.5 <0.001 87.5 <0.001
Ni concentration(Nc) 126.3 <0.001 451.3 <0.001
Nitrogen sources(Ns) 9.9 0.04 1.2 0.276
Nc×Ns 3.1 0.018 1.1 0.396

Fig. 2. The influence of Ni on the Fv/Fm of P. donghaiense (A was nitrate, B was urea) and S. costatum (C was nitrate, D was urea) in different nitrogen conditions.
(䊉) control, (䊏) 0.17 ␮mol L−1 , () 1.7 ␮mol L−1 , () 17 ␮mol L−1 , () 0.17 mmol L−1 , () 0.85 mmol L−1 , (䊐) 1.7 mmol L−1 .

3.3. Determination of EC50
Tab.2 showed the growth inhibition of P. donghaiense and S.
costatum caused by Ni in the nitrate and urea. A concentration-
response relationship was observed, EC50 values of Chl a content
of P. donghaiense was 0.188 mmol L−1 , 0.185 mmol L−1 (nitrate) and
0.389 mmol L−1 , 0.353 mmol L−1 (urea) after 24 h and 96 h incuba-
tion. The data suggest the same trend on EC50 values of Fv/Fm of
P. donghaiense in nitrate and urea after 24 h incubation. However,
the EC50 values of rETRmax of P. donghaiense and S. costatum were
more difficult to determine regardless of incubating in urea and
nitrate. Knowable statistics showed only EC50 values of Chl a were
derived after 48 h incubation. Meanwhile, it was determined that
the EC50 values of Chl a content of S. costatum are lower than that
of P. donghaiense both in nitrate and urea (Table 2).
4. Discussion
Results presented here demonstrate that Chl a content of P.
donghaiense and S. costatum in low Ni treatment were higher than
that in the control treatment (Fig. 1). And then, Chl a concentration
dropped dramatically with the increasing of stress concentration
and increased exposure to higher concentration of Ni. The results
indicated that low concentration Ni can promote the growth of P.
donghaiense and S. costatum while incubated in urea. Previously
published studies indicate that phytoplankton can accumulate Ni,
a cofactor in the urease enzyme, which hydrolyzes urea into ammo-
nium nitrogen (Egleston and Morel, 2008; Price and Morel, 1991).
Ni is a necessary component for the composition of urease as, with-
out it, no urease can be synthesized or functional (Dupont et al.,
2010). Previous research suggested that the growth rate of phy-
toplankton was influenced by urease activity, while it was grown
on urea (Solomon and Glibert, 2008). Results from Dyhrman’s study
and previous studies suggested that Alexandrium sp., which became
the dominant species in marine waters, has a bearing on the higher
urease activity (Dyhrman and Anderson, 2003). While Ni is ubiq-
uitously distributed throughout nature; chemical affinities and
physical size affect its bioavailability (Wen et al., 2006).
It has been reported that high concentration of Ni is toxic to
algal cells. The toxicity is thought to be due to the limited abil-
ity of the cells to regulate and protect themselves (Mulrooney and
Hausinger, 2003). The data suggested extra Ni was not part of ure-
ase prosthetic group and can be harmful to algal growth when the
Chl a content and Fv/Fm value declined (Figs. 1 and 2). As indicated
previously, Ni is an essential element in living organisms, how-
ever, higher concentrations are toxic for humans, animals, plants,
26 X.-G. Huang et al. / Aquatic Toxicology 181 (2016) 22–28

Fig. 3. The influence of Ni on the ETRmax of P. donghaiense (A was nitrate, B was urea) and S. costatum (C was nitrate, D was urea) in different nitrogen conditions.
(䊉) control, (䊏) 0.17 ␮mol L−1 , () 1.7 ␮mol L−1 , () 17 ␮mol L−1 , () 0.17 mmol L−1 , () 0.85 mmol L−1 , (䊐) 1.7 mmol L−1 .

Table 2
The EC50 of growth parameters (Chl a and Fv/Fm) for algae incubated in nitrogen nutrients (nitrate and urea, mmol L−1 ).

Algae N sources Parameters Stress time/h

24 48 72 96

P. donghaiense Nitrate Chl a 0.188 0.181 0.204 0.185

Fv /Fm 0.204 0.342 Nr Nr
Urea Chl a 0.389 0.353 0.337 0.353
Fv /Fm 0.455 0.382 Nr Nr
S. costatum Nitrate Chl a 0.171 0.176 0.196 0.154
Fv /Fm 0.180 Nr Nr Nr
Urea Chl a 0.182 0.174 0.177 0.175
Fv /Fm 0.261 Nr Nr Nr

Note: Nr indicates the data is not elicited.

etc. (Twining et al., 2012). The accumulation of metals in aquatic
macrophytes often causes a variety of physiological changes, some
of which directly contributed to tolerance capacity of the macro-
phytes (Ding et al., 2007; Prasad et al., 2001). Physiological toxicity
effects were observed at 1 mg L−1 –100 mg L−1 (Ni) for P. dong-
haiense and 10–100 mg L−1 (Ni) for S. costatum; the data presented
here indicates excess Ni caused phytoplankton necrosis or death
(Figs. 1, 2 and 3).
Photosynthesis is an important metabolic process for phyto-
plankton, as such, it has been used as a stress indicator. In fact,
reduced photosynthesis activity is a parameter that is measured
and commonly documented in phytoplankton exposed to heavy
metal (Hou et al., 2007). In the experiments presented here, all
photosynthesis parameters (Fv /Fm , rETRmax ) were reduced at high
concentration of Ni (above 0.85 mmol L−1 ) in P. donghaiense and S.
costatum (Figs. 2 and 3). This observation presents a possible role
for previous data indicating that the functional groups on the cell
wall of algae (negative charges, hydroxide radical and amidogen)
have a greater affinity for compounds containing positively charged
metal ions (Dalla Vecchia et al., 2005; Ding et al., 2007; Mehta et al.,
2000; Wen et al., 2006). Additionally, as the concentration of metal
ions increases, more metals can also bind to metabolically inactive
regions of algae, for example the cell wall, which will result in the
disruption of cellular processes and ultimately death (Webster and
Gadd, 1996). Furthermore, excessive amounts of metal destroy the
endomembrane system of the chloroplast and inhibit the synthe-
sis of Chl a, which results in a decline of chlorophyll content and
photosynthesis activity (Dalla Vecchia et al., 2005).
The observed decrease in photosynthesis efficiency in both S.
costatum and P. donghaiense after exposure to Ni may be a result
of decreased effectiveness while utilizing light energy. This might
have resulted from damage to reaction center molecules and a
decrease in Chl a concentration (Chen et al., 2008). As described in
previous works (Ahmed and Häder, 2010; Lu et al., 2000) analysis of
photosynthesis parameters can be used as a tool to assess changes
in the photosynthetic performance of algae upon heavy metal pol-
lution. Based on the results reported here, EC50 values of Chl a and
photosynthesis parameters of P. donghaiense after 96 h incubation
were slightly higher than that of S. costatum in the same nitrogen
environment (Table 2), which indicated that Ni tolerance of P. dong-
haiense was higher than that of S. costatum. A recent study showeds
that interspecific difference can express very different heavy-metal
resistance, as in view of the functional groups containing the type
and quantity in the algal cells surface were disparate (Huang et al.,
2015). This suggest that Ni, which is either a nutritant or toxic ele-
ment to algae, is not only dependent on the concentration of metal
 X.-G. Huang et al. / Aquatic Toxicology 181 (2016) 22–28
ionbut is also regulated by affinity to a specific functional group.
Several studies have shown that Ni adsorption capacity, suscepti-
bility and tolerance varied with different species of algae (Huang
et al., 2015). Based on the results presented here, we can presume
that different Ni tolerance between S. costatum and P. donghaiense
was dependent on the incorporable functional groups on cell walls
(Huang et al., 2015).
Additionally, it has been reported that higher rates of urea
uptake and urease activity were often associated with the pres-
ence of dinoflagellate in both the Chesapeake Bay and the Choptank
River (Solomon, 2006). Importantly, other studies confirm that
dinoflagellates utilize urease (Dyhrman and Anderson, 2003), and
the urease activity of dinoflagellates is normally higher than that
of diatoms. These data imply that dinoflagellates may require more
Ni for metabolism, which may present a potential explanation of
the observed higher Ni tolerance in P. donghaiense than that of S.
costatum.
In addition, it should be pointed out that the EC50 values of P.
donghaiense incubated in urea for 96 h were higher than the cul-
tures in nitrate medium. Those results indicate urea can slow down
the toxicity of Ni in P. donghaiense. However, the same phenomenon
was not observed in S. costatum. Although dissolved organic nitro-
gen, such as urea, can benefit Ni uptake in P. donghaiense while it is
at a low concentration (less than 0.17 ␮mol L−1 ) (Fig. 1B). However
the data suggests that this phenomenon is concentration specific
because there was no observed change in high Ni concentration
samples. Huang et al. (2015) reported that Ni content in cells is
correlated with the ambient Ni and nitrogen nutrient levels, and
the main Ni distribution is protein-Ni content. Nitrogen nutrients
stimulated protein synthesis, altering the concentration of specific
proteins, such as urease and glutathione. Thus facilitating Ni accu-
mulation and detoxify cells (Wang et al., 2007). The data presented
here is complementary to previous work by Hong et al. (2009)
that suggested urea enrichment might increase the percentage of
protein-Ni than that of nitrate, which also indicated the ability to
detoxify of urea was stronger than that of nitrate.
5. Conclusions
The main conclusions from our study are as follows:
(1) Low Ni concentration (0.17–1.7 ␮mol L−1 ) promoted the
growth of P. donghaiense and S. costatum incubated in urea.
(2) The Ni resistance of P. donghaiense was significantly higher than
that of S. costatum regardless of incubating in urea or nitrate.
(3) Urea can alleviate the toxicity of Ni in P. donghaiense, but the
same phenomenon was not observed in S. costatum.
Thus, urea input could affect the Ni bioavailability and toxicity
for phytoplankton, and then affect the phytoplankton community
thereof.
Acknowledgements
We thank Mr Yue Gao for providing culture strains of P. dong-
haiense and S. costatumn. Assistance with English was graciously
provided bu Dr. Ms.Yu wen. This work was supported by the
National Natural Science Foundation of China (41206096 and
40506020), the Program for New Century Excellent Talents in Uni-
versity (NCET-11 0904), Outstanding Youth Science Foundation of
Fujian Province, China (2010J06005), and the Science & Technology
Committee of Fujian Province, China (2012Y0065).
27
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