Marine Pollution Bulletin 80 (2014) 8–23

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Marine Pollution Bulletin

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Review

Acclimation and toxicity of high ammonium concentrations

to unicellular algae
Yves Collos a,⇑, Paul J. Harrison b
a
Ecologie des Systèmes Marins Côtiers (UMR5119), Université Montpellier 2, CNRS, IRD, case 093, 34095 Montpellier Cedex 5, France
b
University of British Columbia, Department of Earth & Ocean Sciences, Vancouver, BC V6T 1Z4, Canada

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

Keywords: A literature review on the effects of high ammonium concentrations on the growth of 6 classes of mic-
Ammonia/ammonium roalgae suggests the following rankings. Mean optimal ammonium concentrations were 7600, 2500,
Toxicity 1400, 340, 260, 100 lM for Chlorophyceae, Cyanophyceae, Prymnesiophyceae, Diatomophyceae, Raphid-
Phytoplankton ophyceae, and Dinophyceae respectively and their tolerance to high toxic ammonium levels was 39,000,
Acclimation
13,000, 2300, 3600, 2500, 1200 lM respectively. Field ammonium concentrations <100 lM would not
EC50 for ammonia
pH
likely reduce the growth rate of most microalgae. Chlorophytes were significantly more tolerant to high
ammonium than diatoms, prymnesiophytes, dinoflagellates, and raphidophytes. Cyanophytes were sig-
nificantly more tolerant than dinoflagellates which were the least tolerant. A smaller but more complete
data set was used to estimate ammonium EC50 values, and the ranking was: Chlorophyceae > Cyanophy-
ceae, Dinophyceae, Diatomophyceae, and Raphidophyceae. Ammonia toxicity is mainly attributed to NH3
at pHs >9 and at pHs <8, toxicity is likely associated with the ammonium ion rather than ammonia.
Ó 2014 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.1. Influence of pH, temperature and salinity on ammonia/ammonium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.2. Nitrogen cycle: ammonium sources and sinks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.3. High ammonium levels in various environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.4. Generalized response of growth rate to dissolved inorganic nitrogen concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.5. Sources of data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2. Effect of ammonium on growth rates of unicellular algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.1. Acclimation and adaptation of growth rate to high ammonium in cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.2. Influence of high ammonium levels on phytoplankton communities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3. Acclimation time for ammonium uptake at high ammonium concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.1. Transition phases of uptake rates at high ammonium concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.2. Cellular transport of ammonium and energetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.3. Effects of ammonium inhibition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.3.1. Direct Effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.3.2. Indirect Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.4. Strategies to cope with high ammonium concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
4. Bioassays and water quality criteria for ammonium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
5. Other examples of NH4 toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
5.1. Fish ponds/farms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
5.2. Removal of ammonia in wastewater treatment ponds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
6. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

⇑ Corresponding author. Tel.: +33 4 6714 4744; fax: +33 6714 3719.
E-mail address: Yves.collos@univ-montp2.fr (Y. Collos).

http://dx.doi.org/10.1016/j.marpolbul.2014.01.006
0025-326X/Ó 2014 Elsevier Ltd. All rights reserved.
 Y. Collos, P.J. Harrison / Marine Pollution Bulletin 80 (2014) 8–23
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Appendix A. Supplementary material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
1. Introduction
Nitrogen (N) is the element that generally limits phytoplankton
growth in many coastal waters and oceans (Ryther and Dunstan,
1971; Boynton et al., 1982; Hecky and Kilham, 1988; Howarth
and Marino, 2006). The forms of inorganic N have been suggested
to structure phytoplankton communities in a variety of environ-
ments, i.e. nitrate leading to diatom blooms (Lomas and Glibert,
1999) and ammonium stimulating flagellates/dinoflagellates
growth (Boynton et al., 1982; Malone et al., 1983; Price et al.,
1985; Probyn, 1985; Robert et al., 1986; Semeneh et al., 1998). In
addition to the form of N, high concentrations of ammonium that
often occur near marine outfalls have also been shown to influence
phytoplankton blooms. For example, Keller et al. (1987, see their
Table 2) reported that 100 lM ammonium was toxic for 200 spe-
cies or clones of oceanic phytoplankton as determined by fluores-
cence. Recently, high ammonium was suggested to be detrimental
to normal coastal diatom development in urbanized estuaries
(Wilkerson et al., 2006; Dugdale et al., 2007; Yoshiyama and Sharp,
2006; Glibert, 2010; Parker et al., 2012a,b, but see Cloern et al.,
2012). More specifically, it was argued that ammonium inhibited
nitrate uptake and delayed diatom blooms (Dugdale et al., 2007),
depressed primary production and suppressed diatom spring
blooms (Parker et al., 2012a), or led to low assimilation numbers
(Yoshiyama and Sharp, 2006). In contrast, ammonium has been re-
ported to stimulate diatom growth relative to nitrate not only in
cultures (Harvey, 1953; Thompson et al., 1989; Livingston et al.,
2002; Bender et al., 2012), but also in the field (Harris, 1959;
Takahashi and Fukazawa, 1982; Suksomjit et al., 2009b; Tada
et al., 2009).
In order to try to reconcile these opposing lines of evidence, the
literature on the effects of ammonium concentrations on marine
phytoplankton growth in laboratory cultures is reviewed. Optimal,
inhibitory and toxic concentrations were identified among the dif-
ferent classes of unicellular algae and the mechanisms responsible
for ammonium toxicity are briefly discussed. Some acclimation
mechanisms such as lag phases and multi-phasic uptake systems
were also identified and their consequences are discussed in the
present context of eutrophication in coastal zones. Since ammo-
nium toxicity is also a problem in sewage oxidation ponds, and
pulp mill effluents, the removal of ammonium is necessary in
wastewater treatment and in high capacity fish/shrimp ponds.
These examples are briefly discussed as well.
1.1. Influence of pH, temperature and salinity on ammonia/ammonium
Ammonium toxicity in water can be due to the effects of both
the unionized ammonia (NH3) and the ionized ammonium
ðNHþ 4 Þ. Ammonia is considered to be the most toxic form because
it is uncharged and lipid soluble and easily diffuses across mem-
branes. Since it is a gas, it is volatile and can be lost to the atmo-
sphere, especially in actively aerated cultures. In contrast to
ammonia, the charge on the membrane hinders the passage of
the charged ammonium ion. There is no chemical method that
can measure these two forms separately. Present chemical meth-
ods measure both forms that are often termed ‘total ammonia’
(i.e. NH4 + NH3). The relative concentration of each form is strongly
dependent on pH and to a lesser extent on temperature, and salin-
ity has only a minor influence (Whitfield, 1974; Emerson et al.,
9
1975; Bower and Bidwell, 1978; Spotte and Adams, 1983). In gen-
eral, at the pH of seawater at 8.0 and 20oC, only about 10% of the
total ammonia is present as the more toxic form, ammonia. Since
90% is present as the ammonium ion, it is preferable to use the
term ammonium for natural seawater. As pH increases, the con-
centration of ammonia increases dramatically (Fig. 1). For example,
in freshwater fish ponds at 30oC, when the pH increases from 7.0 to
9.0, the ammonia concentration increases over 60-fold, but it is
still only 45% of the ammonia + ammonium concentration. In this
case, the term ammonia (actually total ammonia as determined
by the chemical method) is generally used in freshwater aquacul-
ture since the concern is on ammonia toxicity to fish. Emerson
et al. (1975; see their Table 2) and Spotte and Adams (1983; see
their Table 1) give the %NH3 for a range of pHs and temperatures.
The ratio of unionized ammonia to ammonium ion increases by 10-
fold for each unit increase in pH and only 2-fold for each 10 °C rise
in temperature over the 0–30 °C range (Erickson 1985). Bower and
Bidwell (1978) also included the influence of salinity as well as pH
and temperature in their four tables. An increase in the ionic
strength of the solution (i.e. an increase in salinity or water hard-
ness in freshwater) causes only a very small decrease in the
%NH3. For example, an increase in salinity from 20 to >34 causes
a small decrease in %NH3 from 3.41 to 2.98 (Bower and Bidwell,
1978). The dissociation constant (pKa) of the ammonia/ammonium
reaction is about 9.3 depending a salinity, temperature, etc. In
summary, ammonia toxicity is almost solely attributed to NH3 at
higher pHs of about 9 and at pHs <8, any toxicity effects are more
likely associated with the ammonium ion than ammonia (Erickson,
1985). At a pH between 8 and 9, Erickson (1985) found that toxicity
to fish was due to both ammonia and ammonium and similarly for
other organisms (USEPA, 1999). In seawater at 20 °C, pH 8 and a
salinity of 30, ammonia comprises 3.8% of the total ammonia
(ANZECC/ARMCANZ, 2000), with ammonium contributing <1% of
the total toxicity (USEPA, 1989; Batley and Simpson, 2009). For
consistency in this review, we use ammonium, even though both
ammonia and ammonium are present at most pHs discussed here,
and we have used the units of lM and converted mg L1, the unit
that is used in freshwater studies, to lM.
During growth of algae on ammonium, the pH decreases due to
the release of H+ ions to the medium. In contrast, growth on nitrate
Fig. 1. Un-ionized ammonia as a function of pH and temperature. Reproduced with
permission from Hargreaves and Tucker (2004).
10 Y. Collos, P.J. Harrison / Marine Pollution Bulletin 80 (2014) 8–23

causes an increase in the pH due to the release of OH- ions and dur- (Underwood and Provot, 2000). In coastal waters, the highest val-
ing growth on urea, there is little change in the pH (Raven and ues are found near sewage outfalls: >25 lM in Victoria Harbour,
Smith, 1976; Goldman et al., 1982a,b,c; Raven, 1988; Britto et al., Hong Kong (Xu et al., 2008), 40 lM in Santa Monica Bay, California
2001b). (MacIsaac et al., 1979), 150 lM at Whites Point, California (Thomas
and Carsola, 1980), 3000 lM in the Ems-Dollard estuary (Admiraal,
1.2. Nitrogen cycle: ammonium sources and sinks 1977). However, high values can also be found at salinities over 30,
such as in Annaba Bay (Algeria) where 100 lM was recorded in
Generally, ammonium concentrations in surface waters range summer (Ounissi and Fréhi, 1999).
up to 3 lM. In contrast, anthropogenic inputs of ammonium from While temporal increases can be found in some marine environ-
atmospheric deposition, agricultural activities and sewage are re- ments such as in Osaka Bay, increasing from 30 lM in 1980 (Yamochi
garded as ‘new’ nitrogen (Dugdale and Goering, 1967). Examples and Abe, 1984) to 300 lM in 2007 (Yamamoto et al., 2010), other
of anthropogenically produced atmospheric sources are transpor- maxima seem to be more stable such as in Suisun Bay (USA):
tation emissions, and volatilization from manure produced in ani- 27 lM in 1974 (Glibert, 2010), 16 lM in 2000–2003 (Wilkerson
mal farming. Atmospheric deposition can range from 10 to over et al., 2006), 14 lM in 2006 (Parker et al., 2012a,b). High frequency
40% of the new N loading to estuaries that are downwind of sampling has revealed large diel changes in ammonium concentra-
anthropogenic emissions (Paerl et al., 2002). On the east coast of tions that could increase 50-fold (from 0.1 to 5 lM) during the
the USA, atmospheric deposition can account for 10–40% of the night possibly due to grazing (Litaker et al., 1988; Yamamuro
new nitrogen loading to estuaries and may exceed riverine input and Koike, 1994; Horner-Rosser and Thompson, 2001).
in many areas. The air shed may exceed the watershed by 10–
20-fold (Paerl et al., 2002). Agricultural activities, including an in-
1.4. Generalized response of growth rate to dissolved inorganic
crease in intensive animal farming (especially pigs and chickens)
nitrogen concentrations
and the liberal use of fertilizer, have increased ammonium loading
to the coastal zone via atmospheric wet and dry deposition and
Fig. 2 shows the response of an estuarine diatom to a range of
groundwater (Raven et al., 1992).
concentrations of nitrate, nitrite and ammonium (Rao and Sridharan,
In the N cycle in marine ecosystems, ammonium is usually re-
1980). Notice the log scale and the large difference in the toxic
garded as ‘regenerated’ N and is produced by excretion from ani-
concentration between nitrate and ammonium. It illustrates that
mals and by bacterial regeneration/recycling of organic N
all three inorganic N substrates can become inhibitory above a cer-
compounds in the sediments and water column (Galloway et al.,
tain concentration. A similar experiment was conducted with ten
2003, 2004). Another minor source of ammonium in sediments
species of estuarine benthic diatoms and good growth occurred
that is produced via nitrogen cycle processes is dissimilatory ni-
at 17,000 lM nitrate, 1000–10,000 lM nitrite, but only 500 lM
trate reduction (DNR). In sediments with high sulphide concentra-
ammonium was inhibitory (Rao and Sridharan, 1980). It is interest-
tions, nitrification and denitrification may be inhibited, but
ing to see how few studies have compared the toxicity of these
dissimilatory nitrate reduction to ammonium may be enhanced
three inorganic N species. Here we focus on ammonia/ammonium
because sulfide acts an electron donor (An and Gardner, 2002;
toxicity in particular, even though other inorganic N forms can
Brandes et al., 2007). Enhanced DNR and reduced denitrification
become toxic at high levels. At concentrations that are not toxic,
may preserve available nitrogen in estuarine sediments.
ammonium has frequently been reported to produce higher
In anoxic, high organic sediments, the N transformation reac-
growth rates compared to nitrate and urea for a wide variety of
tions stop at the conversion of organic N to ammonium. Since
species (Paasche, 1971; Thompson et al., 1989; Giordano, 1997;
ammonium is highly soluble, it is recycled via pore water to the
Suksomjit et al., 2009a; Tada et al., 2009; Hii et al., 2011).
water column. The primary sinks for ammonium include phyto-
plankton and possibly bacterial uptake and microbial nitrification
(NH4 ? NO3). Nitrate may be converted to N2 via denitrification 1.5. Sources of data
processes when oxygen is low. Under anoxic conditions, anaerobic
oxidation of ammonium can occur (NH4 + NO 2 ! N2 ; called the
Overall, laboratory culture data from 45 freshwater and 68 mar-
anammox reaction; Galloway et al., 2003; Brandes et al., 2007). ine studies were used. This review is not intended to be exhaustive,
but it should be fairly representative of available data on the effect
1.3. High ammonium levels in various environments of ammonium on phytoplankton growth in a wide variety of aqua-
tic environments. Unfortunately, many laboratory studies did not
The highest values of ammonium are generally found in fresh- measure pH of the culture medium at the end of the growth period
water environments. Maxima ranged from 100 lM in Wascana and therefore it was not always possible to estimate the%NH3 in
Lake, Canada (Donald et al., 2011), 200 lM in prairie lakes, Canada
(Murphy and Brownlee, 1981), 300 lM in Lake Taihu, China (Chen
et al., 2003) and up to 680 lM recorded in summer 1990 in Lake
Little Mere, UK (Carvalho, 1994). Maxima sometimes increased
over time such as in Lake Donghu, China from 10 lM in the
1950s to 60 lM in the 1980s and up to 100 lM in 2001 (Dai
et al., 2012). In lagoons, maxima ranged from 9 lM in Thau lagoon,
France (Collos et al., 2005), to 300 lM in Bolmon lagoon, France
(Chomérat et al., 2007) and Balearic islands lagoons (Lucena-Moya
et al., 2012) and up to 400 lM in Portuguese lagoons (Coutinho
et al., 2012).
In estuaries, ammonium concentrations are generally related to
salinity (S), with high values in the less saline part, such as 300 lM
in the Ems-Dollard estuary (Admiraal, 1977), >400 lM in Deep Bay, Fig. 2. Growth rate (in units of day1) of the diatom Pleurosigma aestuarii as a
Hong Kong (Xu et al., 2010) or 1000 lM in the Colne estuary (UK) function of nitrogen concentrations (data from Rao and Sridharan, 1980). Ammo-
at S = 10, decreasing to 100 lM at S = 20 and 20 lM at S = 30 nium (triangles), nitrite (squares), nitrate (diamonds).
 Y. Collos, P.J. Harrison / Marine Pollution Bulletin 80 (2014) 8–23
the medium and its potential toxicity. Some studies used pH buf-
fers to keep pH relatively constant during batch culture growth.
In addition, many studies did not use ecologically important spe-
cies. Data were also examined for indications of possible acclima-
tion of unicellular algae to high ammonium levels and the%NH3
at known pHs.
Names of microalgae were checked against the Algaebase
(http://algaebase.org/) or the WoRMS (World Register of Marine
Species) database and currently accepted names were used when-
ever possible.
We used three categories of ammonium concentrations (e.g.
optimal, inhibitory and toxic) in relation to phytoplankton growth
rates that were usually assessed by an increase/decrease in cell
counts and/or in vivo fluorescence (i.e. long term effects). In a
few studies, growth rate decreased, but not cell yield, or vice versa.
Short term effects of ammonium additions based on 14C uptake
(Azov and Goldman, 1982; Collos, 1986 and references therein;
Turpin, 1991; Huppe and Turpin, 1994) or oxygen production
(Belkin and Boussiba, 1991) are not included here. Fluorometric
estimates of photosynthetic efficiency are mentioned where useful.
‘‘Optimal’’ concentrations were defined as those leading to maxi-
mal growth and were experimentally determined by measuring
growth rates over a range of ammonium concentrations. If no
gradient was used, optimal concentration data were included
if authors indicated that they were optimal (Chu, 1942, 1943;
Guillard and Wangersky, 1958; Kapp et al., 1975; Kim et al., 2012;
Pintner and Provasoli, 1963; ZoBell, 1935), or if growth on
ammonium was greater than growth on nitrate (i.e. the control)
at equimolar concentrations (Ryther, 1954; Stewart, 1964; Moss,
1973; Fabregas et al., 1989; Thakur and Kumar, 1999; Giordano,
2001; Shi et al., 2000; Suksomjit et al., 2009a,b; Chen et al.,
2011; Hii et al., 2011). It was often observed that there was a lag
phase in growth that increased as the initial ammonium concen-
tration increased in batch cultures (Collos, 1986 and references
therein, Bates et al., 1993; Matsuda et al., 1999; Collos et al.,
2004; Nagasoe et al., 2010; Park et al., 2010), without affecting
the growth rate reached after the acclimation period. We noted
this lag in final maximal growth rates where applicable. Inhibitory
concentrations significantly reduced growth rate compared to
optimum concentrations and were represented by the EC50, (the
effective concentration where growth rate was reduced by 50%, a
common term used in ecotoxicology studies). The toxic concentra-
tion is the concentration at which no growth was observed.
2. Effect of ammonium on growth rates of unicellular algae
Table 1 summarizes optimum, inhibitory and toxic ammonium
concentrations (details in Supplementary tables) for growth rates
of six classes of unicellular algae. The ranking of the six algal clas-
ses in terms of their tolerance to high ammonium levels was as
follows:
Chlorophyceae > Cyanophyceae > Diatomo
phyceae > Raphidophyceae > Prymnesiophyceae > Dinophyceae.
Table 1
Optimal, inhibitory and toxic ammonium concentration (lM) for growth of unicellular algae in batch cultures. Optimal concentrations were determined from data for which at
least one adverse concentration was identified or when the authors specifically mentioned they were optimal. Number of studies is in brackets.
Class Mean ± SD
Optimal
Chlorophyceae 7572 ± 7619 (17)
Cyanophyceae 2486 ± 1570 (10)
Diatomophyceae 337 ± 409 (27)
Dinophyceae 110 ± 77 (23)
Prymnesiophyceae 1432 ± 1197 (12)
Raphidophyceae 263 ± 332 (8)
11
The Kruskal–Wallis test and Dunn’s multiple comparison test
revealed that for toxic concentrations, Chlorophytes were signifi-
cantly more tolerant to high ammonium than diatoms
(p < 0.001), dinoflagellates (p < 0.001), and raphidophytes
(p < 0.05). Cyanophytes were significantly more tolerant than dino-
flagellates (p < 0.01). In general, comparing the six classes, dino-
flagellates were the least tolerant to high ammonium levels. The
ranking for inhibitory and optimal levels were similar.
For the Chrysophyceae, Keller et al. (1987) were able to grow
Ochromonas sp. at 100 lM ammonium and Watson and McCauley
(2005) found optimal concentrations at 140 lM for Uroglenopsis
americana and Synura petersenii and at 14 lM for Dinobryon
cylindricum. The toxic level for the latter three species was identical
at 1500 lM.
For the Cryptophyceae, only three studies were found (Antia
and Chorney, 1968; Guillard and Wangersky, 1958; Lourenço
et al., 2002), with toxic ammonium levels of 5000, 1200 and
11,000 lM for Hemiselmis virescens, Hillea sp., and Rhodomomas
sp. respectively.
Concerning the Dictyochophyceae, optimal growth of Aureococ-
cus anophagefferens occurred at 50 lM ammonium, with inhibitory
and toxic ammonium levels at 200 and 500 lM respectively
(Fan et al., 2003; Taylor et al., 2006).
A smaller data set was used that included gradients of ammo-
nium concentrations and their effects on growth rates (Table 2).
EC50 values were provided by investigators or could be calculated
from raw data. The EC50 for growth ranged from 30 to
56,230 lM. The ranking of EC50 values was slightly different from
the one derived from Table 1: Chlorophyceae > Cyanophy-
ceae > Diatomophyceae > Dinophyceae > Raphidophyceae, but the
dinoflagellates, the diatoms and the raphidophytes were not signif-
icantly different. Only the Chlorophyceae were significantly more
tolerant to ammonium than diatoms (p < 0.001), dinoflagellates
(p < 0.01) and the Cyanophyceae were more tolerant than the
Dinophyceae. When pH and temperature data were provided, we
calculated the ammonia concentration (rather than ammonia +
ammonium values that were used above) and determined EC50
values for ammonia toxicity for the five algal groups. We found
the following ranking: Cyanophyceae > Chlorophyceae > Diatomo-
phyceae > Raphidophyceae > Dinophyceae, but the chlorophytes,
the dinoflagellates, the diatoms and the raphidophytes were not
significantly different. Only the Cyanophyceae were significantly
more tolerant to ammonia than diatoms (p < 0.05) and dinoflagel-
lates (p < 0.01).
Many of the effects of ammonia on growth rates were deter-
mined in cultures grown at temperatures of 18 °C or higher. As
temperature increases from 10 to 30 °C, the %NH3 also increases
by 20% at pH 9.0, but only slightly at a pH of 8.0 (Fig. 1). There-
fore, growth rates at lower temperatures should be less sensitive
to a certain total ammonium concentration than at higher temper-
atures since NH3 is more toxic than ðNHþ 4 Þ. As discussed above for
Table 2, if the temperature and pH of the cultures were given, then
we calculated the EC50 values for NH3 rather than total ammonia
Mean ± SD Mean ± SD
Inhibitory Toxic
23,758 ± 25,922 (14) 39,181 ± 60,025 (8)
6616 ± 6514 (10) 12,982 ± 13,226 (13)
725 ± 839 (27) 3575 ± 4478 (16)
324 ± 283 (25) 1139 ± 2494 (29)
958 ± 1241 (9) 2304 ± 2651 (14)
635 ± 624 (8) 2474 ± 3843 (8)
Table 2

12
Estimates of EC50 values (for growth, except where noted) for NH4 + NH3 (total ammonia) and for NH3 only calculated from final pH values for cultures growing at various PAR values, temperature and at a range of total ammonia
concentrations. In some studies growth on NO3 was the control. Al: Alexandrium; Am: Amphiprora; As: Asterionella; Ch: Chaetoceros; Chla: Chlamydomonas; Chlo: Chlorella; Cyc: Cyclotella; Cyl: Cylindrotheca; D: Dunaliella; FW:
freshwater; Gym: Gymnodinium; Gyr: Gyrodinium; H: Heterosigma; K: Karenia; L: Lingulodinium; M: Microcystis; N: Nephroselmis; Na: Navicula; NA: not available; NS: not significant; P: Prorocentrum; Pl: Pleurosigma; S: Skeletonema; Sce:
Scenedesmus; Stim: stimulation of growth by ammonium; Syn: Synechocystis. T: Trentepolia; PAR: photosynthetically available radiation in lmol photons m2 s1.

Class Genus/species PAR Temp. (°C) pH NH4 + NH3 (lM) NH3 (lM) n EC50 EC50 Control References
Initial Final NH4 + NH3 (lM) NH3 (lM)
Chloro phyceae Chla. reinhardtii 100 25 NA NA 100–10,000 NA 3 Stim Stim Giordano et al. (2003)
Chlorella sp. 40–100 23–29 7.2 NA 5000–30,000 60–360 3 Stim Stim Kim et al. (2012)
Chlo. protothecoides Dark 28 6.1 4.0 80,000–725000 80–725 6 NS NS Shi et al. (2000)
Chlo. vulgaris 250 18–22 5.9 5.7 2400–12,500 1.2–6.3 5 8046 3 NO3 Pratt and Fong (1940)
Chlo. vulgaris 250 18–22 5.2 3.7 2400–12,500 1.2–6.3 5 3805 3 NO3 Pratt and Fong (1940)
Chlo. vulgaris 250 18–22 4.5 3.0 2400–12,500 1.2–6.3 5 6333 3 NO3 Pratt and Fong (1940)
Chlo. vulgaris 250 18–22 3.8 3.2 2400–12,500 1.2–6.3 5 5622 3 NO3 Pratt and Fong (1940)
Chlo. vulgaris A23 100 28 ± 2 7.0 4.0 10,000–71,428 70–500 8 31,622 210 Przytocka-Jusiak et al. (1977)
Chlo. vulgaris AA 100 28 ± 2 7.0 8.0 10,000–143000 70–1250 8 56,234 562 Przytocka-Jusiak et al. (1977)
Chlo. vulgaris 70 20 ± 2 7.0 7.0 714–71,428 3–286 10 NS NS NO3 Tam and Wong (1996)
Dunaliella sp. 200–340 20 NA NA 25–200 NA 8 NS NS NO3 Thomas et al. (1980)

Y. Collos, P.J. Harrison / Marine Pollution Bulletin 80 (2014) 8–23

D. salina 60 30 7.6 NA 10–5000 0.3–1.5 5 Stim Stim Norici et al. (2002)
D. tertiolecta CCAP 65 18 7.0 7.0 250–16,000 1–48 7 NS NS Fabregas et al. (1989)
D. tertiolecta UTEX NA 23 7.8 NA 1000–50,000 40–2000 4 4536 225 NO3 Chen et al. (2011)
N. pyriformis 35 20 ± 1 8.7 8.7 1–40 0.2–8 7 224 2 NO3 Källqvist and Svenson (2003)
Sce. acuminatus 200 NA 7.5 NA 7000–70,000 NA 6 NA NA Park et al. (2010)
Sce. obliquus 10 30 9.8 9.2 900–4000 730–3240 6 3162 1258 NO3 Abeliovich and Azov (1976)
Cyano phyceae Anabaena azotica 70 25 8.3 ± 0.2 1000–50,000 100–5000 6 2448 388 NO3 Dai et al. (2008)
Arthrospira platensis 72 30 8.0 10.4 400–6400 30–480 3 Stim Stim Carvalho et al. (2004)
M. aeruginosa 315 40 25 8.3 ± 0.2 500–10,000 50–1000 7 3258 316 NO3 Dai et al. (2008)
M. aeruginosa 905 40 25 8.3 ± 0.2 500–10,000 50–1000 6 3735 251 NO3 Dai et al. (2008)
Nostoc sp. 70 25 8.3 ± 0.2 500–10,000 50–1000 6 1105 466 NO3 Dai et al. (2008)
Synechococcus sp. 40 25 8.3 ± 0.2 500–50,000 50–5000 6 8961 1995 NO3 Dai et al. (2008)
T. odorata 48–122 28 7.0 7.0 350–35,000 3–245 3 Stim Stim Tan et al. (1993)
Diatomo phyceae Am. hyalina 100 22 7.5 NA 1000–3000 14–42 4 1224 14 NO3 Tadros and Johansen (1968)
As. japonica 200 20 NA NA 50–200 NA 8 NS NA NO3 Thomas et al. (1980)
Chaetoceros sp. NA NA 8.1–8.2 NA 1–4000 NA 6 300 NA Zgurovskaya and Kustenko (1968)
Chaetoceros sp. 100 21 8.0 NA 50–1500 2–62 10 662 21 NO3 Suksomjit et al. (2009a)
Ch. affinis 200 20 NA NA 50–200 NA 8 NS NA NO3 Thomas et al. (1980)
Cy. cryptica 100 22 7.5 NA 5–3000 0.1–42 6 NS NS NO3 Tadros and Johansen (1968)
Cy. cryptica Dark 25 7.5 7.7 1700–20,100 31–362 8 243 126 NO3 Pahl et al. (2012)
Gyrosigma spencerii 85 12 8.0 (Tris) 100–5000 2–100 5 1200 25 Admiraal (1977)
Na. acceptata 6 100 22 7.5 NA 1000–3000 14–42 4 NS NS NO3 Tadros and Johansen (1968)
Na. acceptata 8 100 22 7.5 NA 5–3000 0.1–42 3 Stim NA NO3 Tadros and Johansen (1968)
Na. arenaria 85 12 8.0 (Tris) 100–5000 2–100 5 1256 13 Admiraal (1977)
Na. cryptocephala 85 12 8.0 (Tris) 100–5000 2–100 5 2901 61 Admiraal (1977)
Na. perminuta 40 25 8.0 NA 75–4000 4–216 10 302 16 Underwood and Provot (2000)
Na. salinarum 85 12 8.0 8.4 100–5000 2–100 5 64 1 Admiraal (1977)
Na. saprophila 100 22 7.5 NA 5–3000 0.1–42 5 Stim Stim NO3 Tadros and Johansen 1968)
Nitzschia closterium 150 21 8.1 9.0 286–1143 12–49 18 714 NA Adams et al. (2008)
Nitzschia dissipata 100 22 7.5 NA 10–3000 0.1–42 4 982 14 NO3 Tadros and Johansen (1968)
Nitzschia dissipata 85 12 8.0 NA 100–5000 2–100 5 1609 34 Admiraal (1977)
Nitzschia dubiformis 85 12 8.0 NA 100–5000 2–100 5 1025 21 Admiraal (1977)
Nitzschia pungens 100 17 NA NA 220–880 NA 4 NS NA Bates et al. (1993)
Pl. aestuarii 25 21 NA NA 1000–20,000 NA 5 4116 NA NO3 Rao and Sridharan (1980))
S. costatum NA NA 8.1 7.7 1–4000 NA 6 316 NA Zgurovskaya and Kustenko (1968)
S. costatum 200 20 NA NA 50–200 NA 5 126 NA NO3 Thomas et al. (1980)
S. costatum 100 17 NA NA 220–880 NA 4 NS NA NO3 Bates et al. (1993)
S. sp. (Dokai Bay) 100 21 8.0 9.4 10–1500 0.4–62 10 423 14 NO3 Suksomjit et al. (2009a)
S. sp. (Harima Nada) 100 21 8.0 NA 10–1500 0.4–62 10 135 5 NO3 Suksomjit et al. (2009a)
 Y. Collos, P.J. Harrison / Marine Pollution Bulletin 80 (2014) 8–23 13

and therefore the temperature effect has been taken into account

Zgurovskaya and Kustenko (1968)

in these calculations.
For data in Table 2, the incubation duration ranged between 1
Chang and McClean (1997)
Chang and McClean (1997)
Chang and McClean (1997)

and 21 days. For those data sets where initial and final pH was doc-
Suksomjit et al. (2009a)

Suksomjit et al. (2009a)

Suksomjit et al. (2009a)

umented, there were 8 experiments in which the pH decreased (6

Chang and Page (1995)

Chang and Page (1995)
Chang and Page (1995)
Nagasoe et al. (2010)
Thomas et al. (1980)

Thomas et al. (1980)

in freshwater and 2 in seawater media) and 9 in which the pH in-

creased (3 in freshwater and 6 in seawater). There was no pH
Barker (1935)
Barker (1935)

change in 8 experiments, generally due to the use of Tris (Fabregas

et al., 1989) or TAPS buffers (Dai et al., 2008), or very careful mon-
itoring of pH (Tan et al., 1993; Tam and Wong, 1996; Källqvist and
Svenson, 2003). The largest pH decrease was a 3 unit drop over
24 h during growth of Chlorella vulgaris under continuous light
(Przytocka-Jusiak et al., 1977) and a 2.1 unit drop during growth
NO3
NO3

NO3

NO3

of Chlorella protothecoides in darkness (Shi et al., 2000). The largest

increase was 2.4 units during growth of Arthrospira platensis
(Carvalho et al., 2004). In the later case, the final pH of 10.4,
compounded by a temperature of 30 °C, led to an estimate of 90%
NO3

ammonia in the medium. Otherwise, except for the studies of

NA
NA
NA

NA

NA

NA

NA
NA
NA
19

28
9

1
1

Abeliovich and Azov (1976), Adams et al. (2008), Källqvist and

Svenson (2003) and Suksomjit et al. (2009a), the final pH values
did not exceed 8.5, and remained in a range where the concentra-
753(lag)NA

tion of ammonia was very low and nearly negligible.

A recent study (Dai et al., 2012) investigated the influence of
2264
2674
234
126

753
489
379
183

556
101

ammonium on the Photosystem II (PSII) quantum yield of 18 fresh-

NS
96
77
30

water algal species and ranked them according to their EC50 values
in relation to the trophic state ranging from oligotrophic to hyper-
10
5
5
5
8
8
8
5
4
4
6
7
6
6
6

trophic. Overall, EC50 ranged from 260 to 500 lM for diatoms in so-
called ‘‘oligo-’’ and ‘‘mesotrophic’’ regimes, from 560 to 1500 lM
0.2–62

0.2–62

0.4–62

for cyanobacteria in eutrophic regimes, and from 1500 to

0–3
0–3
NA
NA
NA
NA
NA

NA

NA

NA
NA
NA

18,500 lM for chlorophytes in hypertrophic regimes. Quantum

yield assesses short-term effects, but nevertheless, it reflects the
longer term effects depicted in Table 2.
Other variables, such as the Photosynthetically Available Radia-
10–10,000
10–10,000

tion (PAR) could modulate the toxic effects of high ammonium lev-
10–1500
27–875
27–875
27–875
50–200

7–4000
5–250
6–200
6–200
6–200

5–500

5–500

els and generally toxicity was higher at higher irradiances. Early

work by Guillard (1963) indicated that ammonium inhibition of
growth was higher in high light than in dim light. Similarly, Admi-
25–200

raal (1977) reported that inhibition of photosynthesis of estuarine

benthic diatoms growing on 500 lM ammonium was enhanced at
7.2
7.2
7.7
NA
NA
NA

NA
NA
NA

NA
NA
NA
NA
NA

high PAR. This was confirmed by Hillebrand and Sommer (1996)

who found 300 lM to be toxic to Pseudo-nitzschia multiseries at
230 lmol photons m2 s1, but not at 25 lmol photons m2 s1.
8.1
NA
NA
NA
NA
NA
8.0
NA
6.0
6.0

8.0
NA
NA
NA
8.0

On shorter time scales (minutes to hours), Drath et al. (2008) re-

ported more inhibition of PS II activity in Synechocystis at 40 than
at 10 lmol photons m2 s1. The sensitivity of Microcystis aerugin-
osa to ammonium increased with PAR in the range 50–1000 lmol
photons m2 s1 (Dai et al., 2012). In contrast to these five studies
NA

NA
21

18
18

21
15
15
15
21
20
20
20

20

20

above, two studies reported greater ammonium inhibition of

growth at low PAR for A. minutum (Chang and Page, 1995) and
200–340 20

Heterosigma akashiwo (Chang and McClean, 1997).

150

160
100

100
200
100
100

100

100
NA
25
50

80
40

2.1. Acclimation and adaptation of growth rate to high ammonium in

cultures
Chattonella antiqua
Gym. splendens
Gyr. instriatum

Peridinium sp.
L. polyedrum

Many of the laboratory experiments use species that have been

K. mikimotoi

H. akashiwo
A. minutum
A. minutum
A. minutum

H. carterae
H. carterae
H. carterae
P. micans
P. micans

in culture collections for many years and there is always concern

about acclimation/adaptations that could have occurred in the cul-
ture collection. Berge et al. (2012) tested species that were main-
tained in culture collections for many years on enriched medium
Raphido phyceae

and it is likely that they experienced nutrient exhaustion and high

Dino phyceae

pH frequently before they were transferred to new medium. They

found that these long term strains tended to have lower growth
rates and increased tolerance to high pH. Therefore, it is ideal to
use recently isolated ecologically important species and simulated
environmental factors to ensure applicability to field observations.
14 Y. Collos, P.J. Harrison / Marine Pollution Bulletin 80 (2014) 8–23

Table 3
Induction of ammonium uptake by unicellular algae in laboratory cultures at various physiological N states and growing on various N sources. Lag = time to reach Vmax for
ammonium uptake. PAR in lmol photons m2 s1. NA = not available. Nat: natural. Lim: limited; starv: starved; suff: sufficient.

Species PAR T (°C) pH Previous N source Physiological NH4 addition (lmol N/l) Lag (h) References
status
Chlamydomonas reinhardii 65 25 7.0 NO3 N starv. 16 h 900 0 Thacker and Syrett (1972)
Chlamydomonas reinhardii 65 25 7.0 NH4 N suff. 200 0 Cullimore and Sims (1980)
Chlamydomonas reinhardii 500 25 7.0 NH4 N suff. 200 0 Florencio and Vega (1983)
Chlorella fusca 70 25 7.4 NH4NO3 N starv.16 h 9000 48 Syrett and Morris (1963)
Chlorella fusca 140 25 7.4 NH4NO3 N starv. 16 h 9000 24 ‘‘ ‘‘ ‘‘
Chlorella vulgaris Dark 25 6.0 NH4NO3 N starv. 16 h 10,000 0 Syrett and Fowden (1952)
Ditylum brightwellii 175 15 NA NO3 N.A. 1 75 Eppley et al. (1969)
Emiliana huxleyi 150 13 NA NO3 N suff. 5 85 Page et al. (1999)
Emiliana huxleyi 150 13 NA NO3 N suff. 10 60 ‘‘ ‘‘ ‘‘
Emiliana huxleyi 150 13 NA NO3 N suff. 17 72 ‘‘ ‘‘ ‘‘
Lingulodinium polyedrum 175 15 NA NO3 NA 3 24 Eppley et al. (1969)
Lingulodinium polyedrum 100 18 NA NO3 N starv. 24 h 10 0 Harrison (1976)
Haslea ostrearia 100 NA 7.8 NO3 N suff. 30 48 Robert and Maestrini (1986)
Nitzschia ovalis 100 NA 7.8 NO3 N suff. 30 48 Robert and Maestrini (1986)
Nitzschia ovalis nat PAR NA NO3 N starv. 24 h 40 24 Maestrini et al. (1986)
Phaeodactylum NA NA 7.6– NO3 N suff. 200 96 ZoBell (1935)
tricornutum 8.0
Phaeodactylum 200 20 8.0 NH4 N suff. 300 0 Cresswell and Syrett (1982)
tricornutum
Platymonas striata 90 20 NA NH4 N starv. 1 h 1200 1 Ricketts (1988)
Skeletonema costatum 310 17 NA NA N suff. 5 0 Conway (1977)
Skeletonema costatum 490 17 NA NH4 N lim. 8 0 Conway (1977)
Skeletonema costatum NA 13 NA NO3 N starv. <24 h 8 24 DeManche et al. (1979)
Thalassiosira antarctica 50 0 NA NO3 N suff. 25 0 Döhler (1991)
Thalassiosira antarctica 50 0 NA NO3 N suff. 50 2 Döhler (1991)

Przytocka-Jusiak et al. (1977) acclimated an ammonium sensi-
tive strain of Chlorella vulgaris originally maintained at 9860 lM,
by culturing in medium containing inhibitory (53,570 lM), but
not toxic, ammonium concentrations. The cells did not divide,
but there was no lethal effect on cell survival. Four transfers at
7-d intervals were necessary to acclimate the cells to these exces-
sively high ammonium concentrations. It was speculated that
some cellular mechanism regulated the external pH so that it did
not vary as much after acclimation (one unit or less over 24 h).
In contrast, for the non-acclimated strain, the pH in the culture
medium dropped by four units (8–4) over 24 h. Once Chlorella
was acclimated, the C/N ratio exhibited values typical of N-limited
cells at lower ammonium concentrations (12, 14.5 and
17.6 mol C mol1 N at 35,710, 17,860 and 9860 lM respectively
and 6.3–7.9 at higher ammonium concentrations between 54,570
and 142,860 lM).
The reverse acclimation (called de-adaptation by Przytocka-
Jusiak et al. (1977)) was also carried out in the same study using
6 successive transfers in a medium with 9860 lM of either ammo-
nium, nitrate or urea. The acquired tolerance to high ammonium
was found to be stable for at least 30 days, with a slight (624 h)
time lag for cells pre-conditioned on nitrate or urea. Since 30 days
could represent about 50 cell generations, adaptation rather than
acclimation might have been involved.
2.2. Influence of high ammonium levels on phytoplankton
communities
Ammonium concentrations up to 150 lM in coastal or estuarine
waters (Admiraal, 1977; Thomas et al., 1980; Han et al., 1992; Suksomjit
et al., 2009a,b; Yamamoto et al., 2010) may be toxic to many diatom spe-
cies such as Chaetoceros (Suksomjit et al., 2009b), Coscinodiscus (Guillard,

Table 4
Phase transitions in the ammonium uptake system of unicellular algae. Vmax2/Vmax1: ratio of low affinity uptake rate to high affinity uptake rate; A: Alexandrium; C: Chlorella;
D: Dunaliella; N: Nostoc; Pr: Prorocentrum; Ps: Pseudo-nitzschia; R: Rhizosolenia; T: Thalassiosira.

Organism Phase transitions (lmol N L1) Vmax2/Vmax1 References

Cultures A. catenella 60–100 2.5 Collos et al. (2004)
Chlorella sp. 4000 3 Kim et al. (2012)
C. vulgaris 8000–10,000 3 Syrett (1956)
C. vulgaris 500–1000 3 Schlee and Komor (1986)
D. tertiolecta 500–800 4 Hellio and Le Gal (1998)
D. tertiolecta 1200–1900 6 Hellio and Le Gal (1998)
Nannochloropsis sp. 450–550 6 Hii et al. (2011)
N. muscorum 100–200 16 Srivastava and Amla (1993)
Ps. delicatissima 4–10 1.7 Loureiro et al. (2009)
R. truncata 50–10 4 Döhler (1991)
T. weissflogii 30–40 1.4 Sivasubramanian and Rao (1988)
T. antarctica 50–100 5 Döhler (1991)
Field samples
Prairie lakes Microcystis 20–70 40 Murphy and Brownlee (1981)
Atlantic coast Diatoms 35–40 3 Collos et al. (1989)
Choptank River Pr. minimum 7–12 2.1 Glibert et al. (2001)
Thau lagoon
October 2001 A. catenella 20–30 6 Collos et al. (2007)
November 2001 A. catenella 5–10 1.4 Collos et al. (2004)
 Y. Collos, P.J. Harrison / Marine Pollution Bulletin 80 (2014) 8–23
1963), Cyclotella (Guillard, 1963; Tadros and Johansen 1968), Nitzschia
seriata (Guillard, 1963), Pseudo-nitzschia multiseries (Hillebrand and
Sommer, 1996), some species of Skeletonema (Guillard, 1963), coastal
clones of Thalassiosira pseudonana (Guillard, 1963). Skoglund and Jensen
(1976) found that S. costatum, Thalassiosira pseudonana and Phaeodacty-
lum tolerated ammonium concentrations to >450 lM. In contrast, higher
ammonium may actually stimulate growth of other phytoplankters such
as Synechococcus (Birdsey and Lynch, 1962; Kapp et al., 1975; Neilson
and Larsson, 1980; Dai et al., 2008).
Suksomjit et al. (2009b) compared the ammonium tolerance of
Skeletonema isolated from the Seto Inland Sea (low ambient ammo-
nium) with an isolate from Dokai Bay (ammonium 100 lM) and
found that growth rate decreased at 500 lM in the former, but the
Dokai Bay isolate tolerated ammonium up to 1500 lM. This
suggests that Skeletonema from Dokai has adapted to the continu-
ally high ammonium concentrations of 100 lM in Dokai Bay.
This is somewhat similar to the study of Han et al. (1992) who
found that the volume specific photosynthetic rate of S. costatum in
Tokyo Bay reached maximal values at around 140 lM ammonium
and decreased at higher concentrations. Takahashi and Fukazawa
(1982) using natural communities from Osaka Bay (Japan), re-
ported that 50 lM enrichment stimulated the growth of Eutreptiel-
la sp. (0.5 vs. 0.4 d1), Skeletonema costatum (1.0 vs. 0.7 d1) and
Thalassiosira sp. (0.4 vs. 0.2 d1) relative to 10 lM, while the
reverse was true for Gymnodinium sp. (0.15 vs. 0.45 d1) and
Heterosigma sp. (0.5 vs. 0.3 d1).
Domingues et al. (2011) reported specific net growth rates for several
classes of phytoplankton in a tidal estuary following differential enrich-
ments with ammonium (from 1 to 100 lM) and nitrate (100 lM) over
several seasons. Overall, green algae growth was consistently stimulated
by ammonium additions (up to 100 fold)). Cyanobacteria (reported in
summer only) growth rates were strongly enhanced by high ammonium
(up to 8-fold increase). Growth rates of diatoms decreased significantly
in spring and summer with ammonium additions >50 lM, but this could
be due to silicate limitation (estimated from data in Domingues et al.,
2010). Dinoflagellates were clearly inhibited by 100 lM ammonium in
the spring–summer transition and in summer (even reaching negative
growth rates in summer).
Ammonium appears to favor cyanobacterial dominance in
lakes, possibly because of their superior uptake kinetics (Table 4),
whereas nitrate enrichment may selectively stimulate growth of
eukaryotes whose nitrate reductase can be more easily induced
than that of cyanobacteria (Blomqvist et al., 1994). Similarly, Don-
ald et al. (2011) found that ammonium and urea additions favored
non-heterocystis cyanobacteria and chlorophytes at the expense of
diazotrophic taxa.
The total ammonia concentration from a sulphite pulp mill in
northern Florida may reach >100 lM which was shown to be toxic
to phytoplankton assemblages with reductions in algal biomass
and species richness (Livingston et al., 2002). Microcosm experi-
ments with the dominant diatom Skeletonema showed inhibition
between 7 and 20 lM (i.e. surprisingly low compared to other
studies) and major effects at >30 lM. For this system in Florida,
it was recommended that total ammonia should not exceed
10 lM.
High ammonium concentration was a selective factor in the dis-
tribution of benthic diatoms on an estuarine mudflat (Admiraal
and Peletier, 1980). Navicula salinarum and Gyrosigma fasciola tol-
erated ammonium concentrations of 7000–10,000 lM, while four
other species were less tolerant at 2000–7000 lM.
3. Acclimation time for ammonium uptake at high ammonium
concentrations
In the previous section, we reviewed the effect of ammonium
on growth rates which are relatively long term and integrate short
15
term physiological processes. Here we review initial short-term
physiological responses of algal cells suddenly exposed to a pulse
of ammonium that can occur near effluent sources. These short-
term transient responses over a few hours may or may not trans-
late into long term effects on community composition since a brief
lag/induction/acclimation period, for example, may not influence
the outcome of species competition in the field that is expected
to occur over several days of growth.
In several studies, there appeared to be an induction or acclima-
tion period for ammonium uptake at high ammonium concentra-
tions (i.e. ammonium uptake does not reach Vmax immediately
after the addition of ammonium). Table 3 summarizes the lag
phase (or lack of it) that could be identified in several time series
studies. Some lag phases were very long and up to 75–85 h in
the early study by Syrett and Morris (1963) on Chlorella fusca (Mor-
ris, 1974). Possibly, the large amount of unbuffered ammonium
chloride added (9000 lM) led to an immediate decrease in the
ambient pH (Collos et al., 1992) that was deleterious to the initial
uptake of ammonium or to growth in general. This time lag prob-
ably reflects an acclimation to high ammonium levels, with a very
high interspecies variability, because overall, it was surprising that
this time lag was not related to ammonium concentrations. For
example, small ammonium additions (1–8 lM) still led to 24 h
time lags for D. brightwellii (Eppley et al., 1969) and S. costatum
(DeManche et al., 1979). Possibly, those long time lags were due
to previous growth on nitrate. Conway (1977) showed that precon-
ditioning on ammonium led to much smaller lags in ammonium
uptake for S. costatum and in agreement with ZoBell (1935) and
Cresswell and Syrett (1982) who worked on P. tricornutum. For spe-
cies such as Chlorella that are very tolerant of high (10,000 lM)
ammonium, there is still a significant acclimation period for the
ammonium uptake system to reach its maximum capacity even
when the cells were previously grown on ammonium (Syrett and
Morris, 1963). This shows that there are detrimental effects of high
ammonium levels on the ammonium uptake system. Page et al.
(1999) mentioned the lack of ‘‘surge uptake’’ that is often observed
and speculated that an ‘‘end-product regulator’’ might have shut
down surge uptake.
3.1. Transition phases of uptake rates at high ammonium
concentrations
Table 4 summarizes the data on phase transitions (i.e. imple-
mentation of a second higher rate uptake system at higher
substrate concentrations). This phenomenon of bi-phasic or multi-
phasic uptake systems is well known in higher plants (Clarkson
and Lüttge, 1991), but less so in microalgae. The ratios of
maximum uptake rates for high affinity systems (operating at
low concentrations) to low affinity systems (at high concentra-
tions) have been calculated from available published data. Such
ratios in uptake are variable, but in some cases they can reach very
high values, particularly among the Cyanophyceae, less so among
the diatoms. Phytoplankton seem to exhibit such phase transitions
across a wide variety of classes (cyanobacteria, diatoms, dinoflag-
ellates) and habitats, ranging from prairie lakes to coastal lagoons
(Table 4). In natural populations, these phase transitions led to a
2- to 6-fold increase in ammonium uptake over a rather narrow
concentration range, and even more for Microcystis. This appears
to be a nutrient acquisition strategy rather than a lack of cell
control on uptake. Apart from P. delicatissima for which there are
no data on growth rates, the highest ammonium concentrations
in Table 4 have not been found to be toxic to growth of Alexandrium
catenella (Collos et al., 2004), Chlorella vulgaris (Urhan, 1932), T.
weissflogii (Conover, 1975), Microcystis (Dai et al., 2008) and Nanno-
chloropsis (Hii et al., 2011). This enhanced uptake at high ammo-
nium concentrations enables some species to acquire the limiting
16 Y. Collos, P.J. Harrison / Marine Pollution Bulletin 80 (2014) 8–23
nutrient faster than other species and enables them to exploit tran-
sient elevated concentrations (Conway et al., 1976). This confers an
ecological advantage over species that do not have a multi-phasic
uptake system, as often observed in higher plants (Clarkson and
Lüttge, 1991).
3.2. Cellular transport of ammonium and energetics
The undissociated and uncharged ammonia molecule is lipid
soluble and therefore it easily enters through the membrane and
high intracellular concentrations can depolarize the membrane
and possibly inhibit anion transport and affect the cell’s metabo-
lism. In contrast, membranes are relatively impermeable to the
charged ammonium ion.
Internal pools of ammonium can reach up to 30% of the cell N
content for A. catenella (Collos et al., 2006) and up to 66% of the cel-
lular N content for T. weissflogii (Conover, 1975) without any
apparent detrimental effect on cell growth. At typical cytoplasmic
and vacuolar pHs of around 7–8 (Altenburger et al., 1991 and ref-
erences therein; Taylor et al., 2012), ammonium would be the
dominant form rather than ammonia.
From an energetic point of view, ammonium should enhance
growth relative to nitrate, especially at low light. However, con-
trary to expectations, Thompson et al. (1989) showed that higher
growth rate only occurred at high light where energy is not limit-
ing. As suggested by Collier et al. (2012), ‘‘it is possible that the pre-
dicted lower metabolic cost of ammonium compared to nitrate in
terms of only N assimilation is offset by higher costs in terms of re-
pair of photodamaged PSII and processes such as detoxification of
oxygen radicals" (Bendixen et al., 2001).
3.3. Effects of ammonium inhibition
Ammonium appears to be the ideal N source since its oxidation
state eliminates the need for its reduction in the cell and thus it can
be utilized immediately for the synthesis of amino acids. However,
if the ammonium concentration is too high, it can be toxic and re-
sult in reduced growth. As observed in higher plants, the threshold
of ammonium toxicity varies widely and thus there are sensitive
and insensitive species. In the next section, some of the reasons
for the occurrence of ammonium toxicity and its alleviation are ex-
plored for microalgae and some higher plants.
3.3.1. Direct Effects
The most spectacular effect of high ammonium is cell lysis
where algal cells burst immediately (Provasoli, 1958; Nagasoe
et al., 2010), or within a few hours (Zgurovskaya and Kustenko,
1968) after ammonium addition. Early work on Chlorella vulgaris
(Syrett, 1953; Hattori, 1957) indicated large increases in respira-
tion upon addition of ammonium to nitrogen starved cells, and
the oxygen absorbed was correlated with the ammonium taken
up. Very elegant experiments using an isotope dilution method
(Syrett, 1956) revealed that not only was more CO2 produced dur-
ing ammonium assimilation than during nitrate assimilation, but
CO2 was also fixed in darkness, in much greater proportions than
that following nitrate addition. This was probably the first evi-
dence for the anaplerotic pathway of carbon assimilation in
microalgae.
In the light, ammonium additions to ammonium limited or
starved cultures often lead to a transient photosynthetic suppres-
sion for a few hours (Collos and Slawyk, 1979, 1984; Turpin,
1983; Elrifi and Turpin, 1985; Collos, 1986 and references therein).
These suppressions are generally observed on time scales of 15 min
(Turpin, 1983) to 6 h (Collos and Slawyk, 1979, 1984) and this is
then followed by a stimulation of carbon fixation (Turpin, 1983;
Collos and Slawyk, 1984) relative to a control without ammonium
addition.
The time scale of ammonium perturbations is of utmost impor-
tance in the interpretation of such changes in carbon fixation re-
sults but remains totally unknown. Studies using high frequency
(hourly) sampling revealed very large (50-fold, from 0.1 to 5 lM)
and reproducible variations in ammonium concentrations between
day and night over several diel cycles in the Newport River estuary
(Litaker et al., 1988). The enclosures used by Parker et al. (2012b)
led to the development of S. costatum with an initial ammonium le-
vel of about 10 lM, and this is consistent with the relatively high
EC50 values for this species in Table 2. The apparent inhibition of
ammonium uptake in the same study (p. 582, their Fig. 6) is similar
to the ‘‘induction’’ phenomenon described in Table 3 when data are
plotted as rates vs. concentrations, but the mechanism involved re-
mains unexplained so far. Thus, it is unlikely that the 24 h incuba-
tions used by Parker et al., (2012a) could have led to such an
apparent inhibition of primary production based on the results of
the studies discussed above.
Concerning the effect of ammonium on pigments, while the
chlorophyll a (chla) content of Prorocentrum micans did not change
between 1 and 71 lM NH4, that of S. costatum decreased by a factor
of 2.5 between 1 and 71 lM and by a factor of 9 between 1 and
714 lM (Zgurovskaya and Kustenko, 1968), in parallel with de-
creases in photosynthesis (oxygen evolution). This was similar to
the response of Nostoc sp. where a 10-fold decrease in chla content
was reported between 1000 and 10,000 lM (Dai et al., 2008).
In Dunaliella tertiolecta, the chla content first increased from 3
to 6 pg/cell between 250 and 1000 lM, then decreased from 6 to
3 pg/cell as ammonium further increased from 1000 to
32,000 lM, without effect on growth (Fabregas et al., 1989). This
indicates that there are compensatory mechanisms to counteract
detrimental effects on pigments. In Chlorella vulgaris, the chla con-
tent increased continuously from 0.1 to 2.1 pg/cell) between 714
and 71,428 lM (Tam and Wong, 1996), and also in Chlamydomonas
reinhardtii (from 1.1 to 3.9 pg/cell) between 100 and 10,000 lM
(Giordano et al., 2003). Thus, there are clear detrimental effects
of high ammonium levels on the chla content of diatoms and cya-
nophyceae, that are also reflected in reduced growth rates (Table 2),
but none in Chlorophyceae. In contrast, Leong and Taguchi (2004)
reported an increase (4-fold) in chla /cell between 6 and 100 lM
ammonium, and they attribute this to ‘‘low growth and larger cells
due to the detrimental effect’’ of ammonium on growth.
The free internal ammonium resulting from ammonium uptake
is considered as source of stress (Giordano et al., 2003) by causing
an intracellular pH disturbance (Britto and Kronzucker, 2002).
Ammonium toxicity has long been thought to be due to uncoupling
of photophosphorylation (Zhu et al., 2000; Britto and Kronzucker,
2006), but new evidence points to direct PS II photodamage due
to ammonia binding to the Mn complex (Drath et al., 2008).
3.3.2. Indirect Effects
The paradigm of pH changes brought about by ammonium up-
take (Brewer and Goldman, 1976; Goldman and Brewer, 1980) is
not really borne out by this review where a great variety of changes
were observed (Table 2). The causes of pH changes in culture med-
ia during microalgal growth are probably complex and possibly
also due to CO2/HCO3 uptake (Admiraal, 1977; Carvalho et al.,
2004; Waser et al., 1998).
Ammonium toxicity in higher plants has received extensive
investigation and review relative to microalgae (Britto et al.,
2001a,b; Britto and Kronzucker, 2002; Kronzucker et al., 2001).
Ammonium in soils can range up to 2000–4000 lM and large scale
forest decline has been linked to anthropogenic ammonium inputs
and soil acidification (Pearson and Stewart, 1993; Britto and Kron-
zucker, 2002). Reasons for ammonium toxicity include, proton
 Y. Collos, P.J. Harrison / Marine Pollution Bulletin 80 (2014) 8–23
extrusion, cytosolic pH disturbances displacement of crucial cat-
ions, and shifts in carbohydrate status. Sensitive plants such as bar-
ley show chlorosis of the leaves, significantly reduced growth, a
decrease in cellular cations and a marked acidification in the
hydroponic medium at 10,000 lM vs. 100 lM. Britto et al.
(2001a,b) showed that at high ammonium concentrations, barley
roots experience a breakdown in the regulation of ammonium in-
flux, leading to excessive amounts of ammonium in the cytosol.
This high ammonium concentration is pumped out of the cells at
a high energetic cost (e.g. a 40% increase in root respiration) which
may be responsible for the reduction in growth. This ammonium
efflux may be up to 80% of the influx and has been viewed as futile
transmembrane ammonium cycling, a new hypothesis to explain
ammonium toxicity (Britto et al., 2001a,b; Kronzucker et al.,
2001). In contrast, rice is insensitive to high ammonium because
ammonium depolarizes the plasma membrane, whereas the poten-
tial difference in barley appears to be ammonium insensitive
which leads to a high influx of ammonium. Ammonium toxicity
has been shown to occur even in pH-buffered medium, suggesting
that toxicity is not related only to changes in external pH and
ammonium-induced cytosolic pH disturbance (Bligny et al.,
1997). Recent work (Britto and Kronzucker, 2006) indicates that
it is the energy-intensive nature of futile cycling of NH4 that leads
to toxicity (high rate of ion cycling for low affinity transport
systems).
3.4. Strategies to cope with high ammonium concentrations
Cells have several strategies to tolerate high ammonium con-
centrations. In higher plants, it is not known why ammonium tox-
icity does not occur if both nitrate and ammonium are in the
growth medium (Britto et al., 2001b). Ammonia and ammonium
may be toxic to the cell since intracellular pools may be in the mil-
limolar range (Britto et al., 2001a). Thus, ammonium toxicity may
be alleviated by converting it quickly to amino acids (e.g. gluta-
mine/glutamate) via glutamine synthetase that has a high affinity
for ammonium (low Km) and glutamate dehydrogenase activities.
One suggestion for the higher tolerance of green algae to high
ammonium is that they have higher GS/GDH activities and hence
the ammonium is converted quickly into amino acids, rather than
accumulating in the cell (Klochenko et al., 2003).
In the anaplerotic pathway, ammonium stimulates PEPCase that
leads to rapid incorporation of ammonium into organic com-
pounds to avoid toxicity (Giordano et al., 2003). In addition to
the reduction in cytosolic ammonium by various synthesis path-
ways, ammonium may be transported to the vacuole where acid
trapping of ammonium may occur. Ammonium may also be re-
moved from the cytosol by ammonium extrusion/efflux to the
medium (Britto et al., 2001a).
In areas receiving high anthropogenic nitrogen loads, there is
concern over the loss of seagrass beds and in some cases, the mass
mortality of seagrasses has been suggested to be related to high
ammonium (van Katwijk et al., 1997; Van der Heide et al., 2008).
There are suggestions that ammonium may prevent an annual
population from adopting a perennial reproduction strategy (van
Katwijk et al., 1997). Ammonium concentrations may reach
>200 lM with pHs of >9 in shallow estuaries due to anthropogenic
inputs and algal biomass decomposition. Brun et al. (2002) as-
sessed the toxicity of ammonium pulses on the survival and
growth of Zostera noltii and found that the toxic effect depended
on the internal carbon balance between photosynthesis and su-
crose reserves that are needed to sustain nitrogen assimilation
and the conversion into amino acids. Normally, there is an above
and below ground mobilization of sucrose to meet increased car-
bon demands arising from ammonium assimilation. Brun et al.
(2002) found that a repeated large pulse of ammonium quickly in-
17
creased nitrogen transport and assimilation, and carbon demands
and drained the carbon pool, resulting in intracellular accumula-
tion of ammonium and hence toxic effects to photosynthesis and
growth. At pH 8, Zostera marina showed no effects of high ammo-
nium concentrations up to 150 lM, but at pH 9, it became necrotic
in a few days (van der Heide et al., 2008). Remarkably, with high
shoot densities at pH 9, there was no necrosis and toxic effects ap-
peared to be alleviated through ‘joint’ ammonium uptake occur-
ring under high shoot densities. Thus, ammonium toxicity was
dependent on shoot density. In another study with Z. marina, van
Katwijk et al. (1997) observed ammonium toxicity at 125 lM
and even down to 25 lM. They found that low light enhanced
ammonium toxicity because photosynthesis was reduced along
with carbon stores that are needed for the assimilation of ammo-
nium into amino acids. Similarly, Villazán et al. (2013) found that
elevated ammonium and low light formed a deadly combination
for Z. marina that explained why eelgrass and other seagrasses
deteriorate under nitrogen-rich, low light conditions. Higher tem-
peratures (15 vs. 20 °C) increased toxicity due to higher uptake
rates of ammonium and higher carbon-consuming respiration
rates. Eelgrass leaves are more susceptible to toxicity than roots,
possibly because leaves have a higher uptake rate and the sur-
rounding seawater pH is 8.2, compared to pH of 7.5 in sediments,
despite the much higher ammonium concentrations in sediments
of between 100–1000 lM.
In a comparative study of two duckweeds, Monselise and Kost
(1993) found that Lemna gibba from a high ammonia site was more
efficient at detoxifying excessive ammonia by simply accelerating
the usual GS-GOGAT nitrogen assimilation system (i.e. an ‘‘ammo-
nium trapping’’ process), producing glutamine and c-aminobuty-
rate and leaving little intracellular free ammonia/ammonium. In
contrast, Wolffia arrhiza from a low ammonium site had a less effi-
cient ammonium assimilation system, probably due to a relative
shortage of glutamate. This suggestion was supported by the addi-
tion of a-ketoglutarate which resulted in a significant decrease in
intracellular ammonia. Thus, a-ketoglutarate is a precursor for
the ammonium trapping mechanism. Substantial evidence indi-
cates that d-amides are key detoxification products when plants
are exposed to high levels of total ammonia and they act as storage
reservoirs or sinks for intracellular ammonia/ammonium which
may be toxic for cells.
4. Bioassays and water quality criteria for ammonium
Direct toxicity assessment or whole effluent toxicity testing is
an important part of the regulatory framework in many countries
and is used for compliance monitoring of various effluents and
contaminated waters (USEPA, 1989, 1999). Guidelines for toxicity
testing recommend that a range of organisms be used, including
micro and macroalgae, echinoderms, bivalves, a gastropod, crusta-
ceans and fish. It is important that pH be kept constant during the
tests because variations in pH can influence ammonium and metal
toxicity and other components of the effluent (Hogan et al., 2005).
In Australia, effluent discharge from Melbourne’s sewage treat-
ment plant caused a decrease in brown macroalgae and an increase
in opportunistic green macroalgae near the discharge site. Subse-
quent chronic toxicity testing using the microalgal growth rate of
Nitzschia closterium, macroalgal germination and cell division of
Hormosira banksii, larval development of the scallop Chlamys
asperrima, confirmed that ammonium was the major cause of efflu-
ent toxicity (Hogan et al., 2005; Adams et al., 2008). Macroalgal cell
division and germination was the most sensitive test (EC50 = 100 -
lM), followed by scallop larval development (EC50 = 200 lM) and
finally the microalgal growth inhibition test (EC50 = 700 lM). Com-
parisons of toxicity using calculated unionized ammonia (NH3) and
18 Y. Collos, P.J. Harrison / Marine Pollution Bulletin 80 (2014) 8–23
total ammonia gave similar results. High ammonium toxicity val-
ues for four marine amphipods ranged from a EC50 of 3000 to
10,000 lM, probably because amphipods are exposed to higher
ammonium concentrations associated with the sediments as com-
pared to the water column (Kohn et al., 1994).
In a very carefully designed bioassay, Källqvist and Svenson
(2003) used the chlorophyte Nephroselmis pyriformis since it was
the most sensitive of the nine species that they tested. They used
3 mM HEPES buffer to control pH and removed NH3 by raising
the pH to >11 with vigorous bubbling for >2 h. This is one of the
very few studies that has evaluated the toxicity of ammonium
and ammonia separately. Most other studies have assessed the
combined effect of ammonia and ammonium (i.e. total ammonia)
and since toxicity increased with increasing pH, these studies in-
ferred that the toxicity was mainly due to ammonia (e.g. Hogan
et al., 2005; Adams et al., 2008). Källqvist and Svenson (2003)
found that ammonia had an EC50 of 2.4 lM, while the EC50 for
ammonium was 100 times higher at 224 lM and the EC50 for to-
tal ammonia at pH = 8.0 was 71 lM. They concluded that there is a
joint toxicity effect (i.e. both ammonia and ammonium), but that
ammonium is much less toxic, possibly because transport of this
charged ion through the membrane is restricted compared to the
passive entry of the uncharged ammonia molecule.
There are no guidelines for ammonium in sediments. The Aus-
tralian and New Zealand guidelines (termed ‘trigger values’) for
marine and estuarine waters are as follows : 65 lM total ammonia
for 95% species protection (at 20 °C and pH 8), derived from
chronic no observable effects (ANZECC/ARMCANZ, 2000). Canada
has no recommended guideline for ammonium in marine waters
(CCME, 2000). The EU directive for freshwater has guidance and
imperative values of 14.3 and 71.5 lM, respectively. The USEPA
has a chronic marine criterion of 54.3 lM total ammonia (USEPA,
1989). Batley and Simpson (2009) revised the trigger value (i.e.
no further action is required) for ammonia using a greatly ex-
panded toxicity data set and their new 95% species protection va-
lue was 32.9 lM, about half the previous value of 65 lM. For
sediment pore waters, a trigger value of 300 lM was recom-
mended. They stated that a guideline concentration of 565 lM
(i.e. the concentration below the EC50 for 95% of the species), rep-
resents a major risk of acute toxicity to sensitive species.
5. Other examples of NH4 toxicity
5.1. Fish ponds/farms
In fish aquaculture, ammonia toxicity is a common problem for
the fish. It is a by-product of protein metabolism from their high
protein diet and it is excreted from the gills. This production of
ammonia from the fish is taken up by phytoplankton, but when
the algal blooms crash, ammonia increases and toxicity for the fish
may occur. There is interest in finding phytoplankton species that
can tolerate high ammonium concentrations and reduce ammo-
nium concentrations for the more sensitive fish. Nannochloropsis
sp. appears to be an ideal species since it can tolerate up to
900 lM ammonium and grows significantly faster on ammonium
than nitrate (Hii et al., 2011). In fish ponds, there is a daily pH cycle
due to algal photosynthesis and the removal of CO2. The pH may
increase to >9 in the late afternoon, causing a >50-fold increase
in ammonia (Wurtz, 2003). Since the %NH3 is pH dependent, the
fish must endure toxic ammonium levels for a few hours and the
pond may heat up in the late afternoon, causing a further small in-
crease in the ammonia concentration. Ammonia may be produced
in/near the sediments due to the decomposition of the algae and
the uneaten fish food and depending on the aeration system, it
may diffuse from the sediments or be mixed up into the water
column.
Another ammonia sink besides algal uptake is nitrification, the
bacterial oxidation of NH3 to NO 
2 and NO3 . Ammonia concentra-
tions tend to be higher in the winter (180 to 285 lM) in the fish
ponds and lowest in summer (35 lM) due to the highest algal
growth in summer (Hargreaves and Tucker, 2004). The complex
nitrogen biogeochemistry of aquaculture ponds has been well re-
viewed by Hargreaves (1998).
A common problem in many ponds is the occurrence of the tox-
in-producer Prymnesium parvum. For some fish, ammonium sul-
phate can be added to an ammonia concentration of 10–20 lM
that will kill P. parvum, but minimize mortality of several species
of bass (Barkoh et al., 2004). Of course, the safe level for fish varies
greatly with species, size and life stage. The average of the mean
acute toxicity values for 32 freshwater fish is 200 lM compared
to 130 lM for marine fish, indicating that marine species are
slightly more sensitive to ammonia toxicity that freshwater species
(Randall and Tsui, 2002).
5.2. Removal of ammonia in wastewater treatment ponds
Ammonia is one of the major constituents of domestic waste-
water and concentrations range from 10–200 mg L1 (1000–
20,000 lM) (Konig et al., 1987; Thomas et al., 1980). There has
been considerable interest in using high rate oxidation ponds to
treat wastewater (Abeliovich and Azov, 1976). One alga that is par-
ticularly tolerant to ammonium is Chlorella vulgaris and hence it
has a potential role in the removal of ammonium from wastewater
effluent since it grows well at concentrations ranging up to
20,000 lM (Przytocka-Jusiak et al., 1984; Tam and Wong, 1996;
Kim et al., 2010). The freshwater alga, Scenedesmus obliquus, has
a high growth rate in laboratory cultures (5–8 h doubling time),
but in the sewage ponds, a retention time of >100 h was necessary
to maintain a low ammonium concentration because growth was
slow when ammonium concentrations were >2000 lM and pH
was >8 (Abeliovich and Azov, 1976). In follow-up experiments,
Azov and Goldman (1982) observed that when the pH rises to
9.5 at 25 °C, 2000–3000 lM total ammonia led to a 50–90% reduc-
tion in photosynthesis. At the higher temperature of 25 °C, only
one-third the total ammonia is required to produce the same free
ammonia as at 10 °C and 15 times less total ammonia is required
at pH 10 as at pH 8. Therefore, it is essential to know the pH and
temperature of the pond from which the concentration of ammo-
nia can be calculated and not just the total ammonia concentration.
When algal systems are used for wastewater treatment and the
source of N cannot be regulated, pH control is necessary to avoid
algal ammonia toxicity (Azov and Goldman, 1982). Goldman
et al. (1982c) compared some freshwater and marine species and
concluded that marine species cannot tolerate pH > 9.5, except
for Phaeodactylum tricornutum which grew at a pH of 10.3. The un-
ique ability of P. tricornutum to grow at high pHs is a major factor
that explains its frequent dominance in large-scale outdoor cul-
tures (Goldman et al., 1982c). Therefore, P. tricornutum would ap-
pear to be an ideal/unique species for further studies on the
interaction of pH and ammonium toxicity. Of the freshwater spe-
cies, Scenedesmus obliquus grew better at a pH of 10.6 than Chlorella
vulgaris.
Duckweed systems are another option for sustainable wastewa-
ter treatment because they have high growth rates and high nutri-
ent content that can be used for animal feeds (Körner et al., 2001).
At low total ammonia concentrations of <70 lM, toxicity to Lemna
gibba was due to both ammonia and ammonium and the relative
contribution of each N form was pH dependent. However at
higher concentrations (up to 500 lM), the toxicity was due
mainly to ammonia. Using another species of duckweed
 Y. Collos, P.J. Harrison / Marine Pollution Bulletin 80 (2014) 8–23
(Spirodela polyrrhiza), Caicedo et al. (2000) found that both N forms
caused growth inhibition, but ammonia inhibition occurs at much
lower concentrations than ammonium inhibition. An increase in
pH can produce other cellular effects besides increasing the ammo-
nia/ammonium ratio and the use of buffers was recommended in
order to separate the pH effects from the total ammonia concentra-
tion effect (Caicedo et al., 2000).
6. Conclusions
The effects of ammonium on microalgae can occur on two dif-
ferent time scales: long term growth rates (days) and short term
physiological processes such as uptake rates, photosynthetic rates,
and enzyme activities that occur over minutes to hours. Growth
rates generally integrate these short term transient physiological
responses to changes in ammonium concentrations that occur over
a few hours. These short term responses may or may not translate
into changes in long term community composition since a tran-
sient lag/induction/acclimation period may not influence the out-
come of species competition in the field that is expected to occur
over several days of growth.
In spite of large intra- and inter-specific variability, there are
significant differences between classes of unicellular algae with re-
spect to the effect of high ammonium levels on growth rates. Lab-
oratory experiments indicate that growth rates of chlorophytes
followed by diatoms were the most tolerant and dinoflagellates
were the most sensitive to high ammonium. With EC50 values
ranging from 30 to 2700 lM for dinoflagellates, 60–4000 lM for
diatoms and up to 3800–56,000 lM for chlorophytes, and most
field ammonium concentrations well below 100 lM, ammonium
toxicity effects on growth rates are not likely to occur in the field.
In addition, toxicity occurs more frequently at high pHs >9 where
ammonia is more abundant, and these high pHs are seldom ob-
served in the field. The ranking of algal groups observed in the cul-
tures is somewhat reproduced in the field, although such
comparisons are fraught with difficulties such as long-term accli-
mation of strains in cultures (Berge et al., 2012) and problems with
identification of species such as ‘‘Chlorella-like’’ cells in the lower
size classes in field samples. There is a need for more well designed
laboratory experiments, similar to the excellent study by Källqvist
and Svenson (2003) to assess the toxicity of ammonium separately
from ammonia over a range of pHs, temperatures, irradiances and
over a range of ecologically relevant ammonium concentrations.
More studies are needed for recently isolated ecologically impor-
tant species and an assessment of environmental factors, especially
pH and any multiplicative effects with temperature, light, and
salinity.
Acknowledgements
We thank Patricia Glibert, Richard Dugdale, Francis Wilkerson,
Jim Cloern and an anonymous reviewer for their comments that
improved the manuscript.
Y.C. acknowledges support from CNRS.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.marpolbul.201
4.01.006.
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