JOURNAL OF THE Vol. 22, No.

3
WORLD AQUACULTURE SOCIETY September, 1991

Effect of pH and Decapsulation on the Toxicity of Ammonia

to the Brine Shrimp Arfemiufrunciscunu
MATTHEW
LANDAU’AND VERONICA SANCHEZ’
Department of Marine Science, Stockton State ColIege, Pomona, New Jersey 08240 USA

Abstract
Artemiu frunciscanu were hatched and tested for tolerance to ammonia at pH 6.5 and 8.5 in
artificial seawater with a salinity of 17 %. Nauplii seemed to be less tolerant of ammonia at pH
6.5 despite the fact that more of the ammonia is in the ionized form (NH,+). It is suggested that
this greater sensitivity is a result of the NH,+ competing with the Na+ during gut transfer. Nauplii
hatched from decapulated cysts were more tolerant of ammonia than those hatched from whole
cysts, probably because of a greater energy reserve.

Brine shrimp of the genus Artemiu are
typically found in isolated saline environ-
ments, which in the United States include
such places as Mono Lake and San Fran-
cisco Bay in California, Big Soda Lake in
Nevada, Hot Lake in Washington, and Great
Salt Lake in Utah.Besides being of ecological
interest, brine shrimp are also needed in
solar saltworks to produce a high quality salt
(Davis 1980) and are also important feed
organisms in the aquaculture industry (Sor-
geloos 1980). Culture methods for Artemiu
are varied and include the rearing of these
organisms in environments where high lev-
els of ammonia may be encountered, such
as wastewater facilities (McShan et al. 1974)
or manure based culture systems (Landau
et al. 1986). Despite the importance of brine
shrimp to the aquaculture industry, little
was known about this crustacean’s ability
to withstand ammonia toxicity stress until
recently. Chen et al. (1989) found that Ar-
temzu could survive in relatively high con-
centrations of total ammonia, and that am-
monia and nitrite acted synergistically as
toxicants on brine shrimp nauplii.
Ammonia is the main excretory product
of many aquatic organisms. Under typical
environmental conditions, the relatively
’ Corresponding author.
Present address: Fisheries Research Laboratory, to the animal’s ability to be cultured in high
Southern Illinois University at Carbondale, Carbon- ammonia environments. That is, under what
dale, Illinois 6290 1 USA.
0 Copyri&t by the World Aquaculture Society I99 I
nonpolar un-ionized form of ammonia,
NH3, diffuses from the animal into the water
since the concentration of NH3 is much
greater in the organism’s body fluids than
in the surrounding medium. However, when
levels of NH, in the water are elevated, the
diffusion process is slowed or stopped and
NH3 levels in the organism rise. If the me-
dium contains very high levels of NH3, it
may actually diffuse from the water into the
organism (Armstrong 1979).
The relative amount of un-ionized am-
monia molecules in an aqueous solution is
related primarily to the pH, but it is also a
function of the salinity and temperature of
the medium (Emerson et al. 1975; Bower
and Bidwell 1978). Low pH values result in
high levels of the ionized form (NH,+) which
is supposedly less toxic to aquatic organisms
because this molecule is much less mem-
brane soluble than the more nonpolar NH,
(Milne et al. 1958). It is therefore generally
held that ammonia toxicity is largely a func-
tion of the pH of the medium. Since acidic
waters contain relatively little un-ionized
ammonia, for most organisms a higher total
ammonia (ionized plus un-ionized) concen-
tration can be tolerated in mildly acidic en-
vironments.
This study examined the effect of pH on
the toxicity of ammonia to Artemiu relative
conditions could the effects of the ammonia

178
 TOXICITY OF AMMONIA TO ARTEMIA FRANCISCANA
be minimized and would decapsulation im-
prove or hinder the hatching rate in such
an environment?
Materials and Methods
Artemia franczscana (Utah strain) were
donated by Sander’s Brine Shrimp Co., Og-
den, Utah, USA. Unless otherwise stated,
cysts were decapsulated according to the
method of Sorgeloos et al. (1 977) and stored
until needed in a saturated NaCl solution
at 4 C. All cultures and stock solutions were
made up in an artificial seawater (Bower and
Holm-Hansen 1980), made from reagent
quality chemicals, which was diluted with
distilled/deionized water to 17Yb0. The dis-
tilled/deionized water was tested by the sa-
licylate-hypochlorite method (Bower and
Holm-Hansen 1980) and was determined
to contain no detectable amounts of am-
monia. Ammonia solutions were made from
reagent grade NH,CI; test dilutions were
made from a concentrated stock solution
and the pH was adjusted just prior to each
bioassay.
Approximately 36 hours before a bioas-
say was to begin, decapsulated cysts were
placed in flasks of well-aerated artificial sea-
*
water kept at 23 2 C. Newly hatched stage
I, and I, (D’Agostino 1965) brine shrimp
nauplii were collected with a pipet and dis-
tributed into 2 ml depressions on spot plates
( 12 depressions per plate). Each depression
held only one nauplius in the ammonia so-
lution to be tested.
In experiments designed to test the effect
of pH on ammonia toxicity, pH values of
6.5 and 8.5 were used with ammonia con-
centrations ofO, 0.5,0.75, 1.0, and 1.5 g N /
liter. Two plates (24 animals) were used for
each pH-ammonia test solution. Spot plates
were placed in large covered dishes which
included wet paper towels to maintain the
humidity near 100% in the dish. Only
healthy, actively swimming nauplii were
used. After 24 hours in the test solution the
nauplii were examined under a dissecting
microscope and their general condition was
recorded as either healthy, weak (swimming
179
slowly), very weak (unable to move off the
bottom of the depression, irregular move-
ments), or dead (did not move even when
probed).
In experiments to test the effect of decap-
sulation on ammonia toxicity, undecapsu-
lated cysts were placed in aerated artificial
seawater to hatch, and freshly decapsulated
cysts (not stored at 4 C) were similarly treat-
ed 12 hours later. The result was a nearly
simultaneous hatching of the cysts in both
flasks. The nauplii were distributed into de-
pressions in a medium of pH 7.0 with 0.75
g N/liter. Four plates of nauplii (48 animals)
from both decapsulated and non-decapsu-
lated cysts were used in the bioassay.
The percent of ionized (NH,+) and un-
ionized (NH,) ammonia present was cal-
culated using the equations of Bower and
Bidwell ( 1 978). A pkaSof 0.28 was assumed
for the 17Yb0 artificial seawater.
Results
The LD5o values and their 95% confi-
dence limits (CL) were calculated using a
probit regression and weighted coefficients
(Wardlaw 1985). At pH 6.5 and 8.5 the LD5o
values were 0.73 g total N/liter (95% CL =
0.66 g to 0.85 g total N/liter) and 0.99 g
total N/liter (95% CL = 0.80 g to 1.36 g
total N/liter), respectively. The amount of
un-ionized ammonia at the LD5o concen-
trations was 1.05 mg N as NH3/liter at pH
6.5 and 125.04 mg N as NH3/liter at pH
8.5.
A more sensitive estimate of the toxicity
of the ammonia was generated, taking into
account sub-lethal effects, using the follow-
ing original toxicity index (T.I.) for each
pH-ammonia test solution:
+
(Ndead x 3) (Nvery weak x 2)
T.I. =
+
(Nweak x 1)
total N tested at pH, and
ammonia concentration,
The T.I. values were regressed against the
total N concentrations. The slope of the line
at pH 6.5 (b = 3.0) was not significantly
180 LANDAU AND SANCHEZ

greater than the slope at pH 8.5 (b = 2.1)

(comparison of reduced major axis between
samples, Beerbower 1968), although it is
suggestive of a greater sensitivity to total
ammonia at the lower pH value (Fig. 1).
The mortality percentages for decapsu-
lated (29.2 f 14.4) and non-decapsulated
(45.7 f 19.9) cysts were compared. Nauplii
which hatched from decapsulated cysts were
better able to withstand the ammonia (0.05
< P < 0.10, one-tailed Student’s t-test).
Discussion
Brine shrimp, Artemia, appear to be more
tolerant of ammonia than most other aquat-
ic organisms. For example, for fourth stage
larval lobsters, Hornarus arnericanus, the
LDso has been reported to be about 1.4 mg I I I I I
N as un-ionized ammonidliter (Delistraty 025 05 075 10 125 I50

et al. 1977), and for penaeid shrimp a 48

hour LD50 of 1.29 mg N as un-ionized am- -05 4 Tom1 Ammonia (&‘I)

monialliter was determined by Wickins

(1 976). The values reported here, however,
closely approximate the LDsovalues of Chen
et al. (1 989) of 14.17 mg N as un-ionized
ammonialliter, and 839.5 mg total N as am-
monidliter, for brine shrimp (San Francis-
co Bay strain).
Thurston et al. (1 98 1) found that in fat-
head minnows and rainbow trout the LD5o
values for total ammonia decreased as ex-
pected when the pH increased, but the ac-
tual amount of un-ionized ammonia needed
to reach the LD5o increased with the pH.
For example, for trout the LD50 at pH 6.5 1
was 161 mg N as total ammonialliter and
0.13 mg N as un-ionized ammonidliter,
while at pH 8.29 the LD5O was 13.0 mg N
as total ammonidliter and 0.658 mg N as
un-ionized ammonidliter. Their explana-
tion of this phenomenon was that either NH3
ions were more toxic at lower pH values
than at higher pH values, or that NH4+itself
exerts a toxic effect, although much less so
than NH3.
Similar results were observed in larval
prawns, Macrobrachiurn rosenbergii, kept
in water with a salinity of 1 2 7 ~
(Armstrong
et al. 1978). They also suggested that NH4+
FIGUREI . The effect of p H on the toxicity of total
ammonia to Artemia. The open circles represent p H
8.5 and the triangles represent p H 6.5.
exerted a toxic effect and proposed a mech-
anism to explain the role of both the ionized
and un-ionized form’s effects: At high pH
values it is the NH3 molecule which causes
most of the observed toxicity, because it is
able to enter the organism from the medium
by simple diffusion through the lipid rich
membranes; at lower pH values it is the
NH4+molecule that is responsible for a large
portion of the toxicity because it competes
with Na+ which is actively transported by
the organism in an effort to osmoregulate.
The effects of NH4+on Na+ transport have
been discussed by Mangum et al. (1 976) and
Weiland and Mangum (1975).
In the case of Arternia the problem seems
more complex. Results reported here are
supported by many unpublished trials in the
laboratory, and seem to indicate that for
brine shrimp raised in a salinity of 177m,
total ammonia seems to be more important
than un-ionized ammonia, and in fact the
ionized form may actually be slightly more
 TOXICITY OF AMMONIA T O ARTEMIA FRANCISCANA
toxic. These results may, in part, be ex-
plained by the low salinity and the direct
effects of the pH on the organism. Although
they can survive at salinities of 59b to 2057m
(D’Agostino 1965), brine shrimp are nor-
mally found in isolated hypersaline envi-
ronments where few other organisms are able
to live and compete with Artemia. For ex-
ample, Great Salt Lake water has about
223.6 grams [sodium plus chloride] per liter
(Whitehead and Feth 196 l), while seawater
typically has about 30.9 grams [sodium plus
chloride] per liter (Riley and Chester 197 1).
It is the sodium and chloride ions in the
medium that are used by the brine shrimp
to control osmotic pressure of the body flu-
ids (Croghan 1958a) by regulating the
movement of water from the gut into the
hemolymph. These ions are actively ex-
changed between the medium and the or-
ganism (Croghan 1958b, 1958~).The pres-
ent study suggests that it is especially
important for Artemia to efficiently take up
Na+ effectively in a medium of 17o/oo since
the ion is relatively scarce. At higher salin-
ities there would be more Na+ available and
the competition with the NH,+ ions would
be less severe. Therefore, it is probable that
at a higher salinity the NH4+would be less
stressful.
In addition to the inhibition ofNa+trans-
port caused by ionized ammonia in the low
pH medium, there are also direct effects of
the higher H+ concentration. It has been
shown that a pH of 6.5 is stressful for newly
hatched brine shrimp of the Great Salt Lake
strain (D’Agostino 1965; McShan et al.
1974). It is likely that ammonia plus ele-
vated H+ combine to give higher mortality
rates at the lower pH.
Cysts which have been decapsulated pro-
duce nauplii which are more resistant to the
effects of ammonia than those nauplii which
hatched from whole cysts. Decapsulation is
the removal of the chorion, the hard outer
layer of the cyst. Bruggeman et al. (1980)
have suggested that decapsulation allows
nauplii to break out of the cysts more easily;
therefore, nauplii use up less of their caloric
181
reserves in the hatching process and are ini-
tially stronger and healthier organisms. This
study suggested that nauplii which hatched
from decapsulated cysts were better able to
withstand the stress of the high ammonia
environment because they had greater en-
ergy reserves that could be used in osmo-
regulation.
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