Salt and Salmon: the Effects of Hard Water Ions on Fertilization

1
Salt and Salmon the Effects of Hard Water Ions
on Fertilization Pamela Brannock1, Michael S.
Stekoll2, 3, Barbi Failor2, Ivan Wang2.
1Department of Biology and Marine Biology, Roger
Williams University, Bristol, RI, 02809 2Juneau
Center School of Fisheries and Science,
University of Alaska Fairbanks, 3Department of
Natural Science, University of Alaska Southeast,
Juneau, AK 99801
RESULTS
ABSTRACT Mine effluents contain a variety of
ionic species that may be harmful to important
organisms living in the discharge area. In this
study the industrial effluent from the Red Dog
Mine (near Kotzebue, AK) was modeled in the
laboratory in order to determine the response of
developing salmon to this specific effluent.
Previous experiments demonstrated that an
increase in total dissolved solids (TDS) in the
simulated mine effluent caused a decrease in the
fertilization rate of exposed salmon eggs. The
current study attempted to determine which
specific ionic species were responsible for this
decrease in fertilization. Concentrations of K,
Ca2, SO4, and Mg2 typical of their presence in
a 2500 ppm mine simulation effluent were tested
in a salmon egg fertilization experiment. Since
previous experiments demonstrated that NaCl at
the same osmotic equivalent as the 2500 ppm
solution has no effect on salmon fertilization,
counter ions were selected to be Na and Cl-.
Compounds utilized in this experiment were
Na2SO4, CaCl2, MgCl2, and KCl. A set of 24-hour
assays was preformed on both king and pink salmon
provided by Douglas Island Pink and Chum, Inc.
(DIPAC) Macaulary Hatchery in Juneau, AK.
Treatments consisted of the molar equivalents of
the chemical species in the 2500 ppm TDS
concentration, four times that amount, and ¼ that
amount. A 2500ppm solution and process water from
Salmon Creek (the water supply that feeds the
hatchery) were run as controls. Assays
illustrated that both CaCl2 and Na2SO4, had the
greatest detrimental effect on egg fertilization.
This result suggests that Ca2 and SO4 are
responsible for the main effect in the simulated
mine effluent. But caution is advised due to the
confounding effect of Naand Cl- ions at such
high concentrations.
The results of the bioassays illustrated that
both CaCl2 and Na2SO4, had the greatest
detrimental effect on egg fertilization.
(Figures 8 and 9) The water control and the
2500ppm solution in both assays demonstrated
results similar to those obtained in previous
studies. For the king salmon assay the control
and 2500ppm average fertilization rates were
96.25 and 5, respectively. For the pink salmon
assay the average fertilizations for the water
and 2500ppm were 90 and 6.25, respectively.
In the king salmon assay the KCl treatment
caused the least effect on the fertilization of
the eggs (Figure 8). The MgCl2 treatment
appeared to have some effect on the fertilization
of the eggs. Both Na2SO4 and CaCl2 had the most
detrimental effect on the survival of the eggs.
Eggs exposed to the highest concentrations in
both treatments produced no discernable cell
mass. The pink salmon assay produced similar
results to the king salmon assay. (Figure 9) KCl
had the least effect on the fertilization of the
eggs. MgCl2 appeared to have a more negative
effect on the fertilization of the pink salmon
eggs compared to king salmon. As in the king
salmon assay, both Na2SO4 and CaCl2 had the
greatest detrimental effect on the survival of
the eggs. Eggs exposed to CaCl2 had
fertilization rates that decreased dramatically
as the concentrations were increased. All eggs
were unfertilized at the highest Na2SO4
concentration.
Figure 8. The fertilization averages for the king
salmon eggs at the different treatments
Figure 9. The fertilization averages for the pink
salmon eggs at the different treatments
INTRODUCTION Mine effluents contain a variety of
ionic species that may be harmful to important
organisms living in the discharge area. For
example, the Red Dog Mine near Kotzebue, Alaska
(Figure 1) discharges an effluent that is very
high in total dissolved solids (TDS). It is
unknown what effect these high concentrations
have on the ecosystem surrounding the discharge
area. Recently studies were initiated to begin
to examine the effects of such effluents more
closely. A study performed by Stekoll et al.
used a simulated Red Dog Mine effluent in order
to determine the response of developing salmon to
this effluent. Results showed that eggs exposed
to high concentrations of TDS during
fertilization had a significant decline in
fertilization success and subsequent survival
(Figure 2). The current study utilized the
stimulated effluent to determine which specific
ionic species were responsible for the decrease
in fertilization and survival rate of pacific
salmon eggs. Utilizing the chemical composition
for the simulated effluent, we calculated the
molar equivalents of K, Ca2, SO4, and Mg2 as
they exist in the 2500ppm TDS. (Tables 1 2)
Since the previous studies indicated that an NaCl
concentration with the same osmotic equivalent as
the 2500ppm TDS solution had no effect on the
fertilization, Na and Cl- were used as counter
ions. The salts used in this experiment were
Na2SO4, CaCl2, MgCl2, and KCl. Two acute assays
were performed to determine whether one or all
the ionic species were responsible for the
decrease in fertilization rates. Determining
which ionic species is responsible for the low
fertilization in pacific salmon eggs will help
understand the mechanism of the TDS effect and
will aid in determining whether a discharge limit
for that specific ion needs to be set.
METHODS   Fertilization trails were performed on
king salmon (Oncorhynchus tshawytscha) and pink
salmon (Oncorhynchus gorbuscha). Douglas Island
Pink and Chum (DIPAC), Inc. Macaulay Hatchery in
Juneau, Alaska (Figure 3) provided fertile male
and female salmon. On July 26, 2001, two female
and three male king salmon were collected from
the raceways of the Macaulay Hatchery. A second
assay was performed on August 9, 2001 using three
female and three male pink salmon. Female
gametes were obtained by making an incision on
the ventral portion allowing the eggs to fall
into a 1-gallon Ziploc plastic bag (Figure 4A).
Males were spawned into a 4 oz (100ml) wax-paper
cup by gently squeezing the ventral portion of
the fish (Figure 4B). Gametes were kept at 4ºC
and transported to the lab area where all female
gametes and all male gametes were combined in
separate containers. For the bioassays
four chemical species were being tested K,
Ca2, SO4, and Mg2. These common ions were in
the compound forms of Na2SO4, CaCl2, MgCl2, and
KCl.. Three different concentrations of each
salt were utilized in this experiment the molar
equivalent of the species as it occurs in the
2500ppm TDS solution, four times (4X) that
amount, and ¼ that amount (¼X). Two controls
were utilized water from Salmon Creek (the water
supply that feeds Macaulay Hatchery) and the
simulated 2500ppm solution. All solutions were
prepared using Salmon Creek water. Four
replicates of each of the fourteen treatments
were used. Exposure chambers consisted of
one-liter plastic tri-pour beakers set in a water
bath to maintain constant temperature (Figure 5).
The beakers were equipped with an air stone and a
removable lid. Replicates of all fourteen
treatments were assigned randomly to each water
bath. A plastic ½ tablespoon was used to measure
approximately 50 eggs. All eggs were exposed to
the solutions for five seconds prior to
fertilization by filling a 100 mL (4oz) wax-paper
cups halfway full of the test solution and then
adding the eggs. After allowing the eggs to sit
in the test solution for five seconds, a syringe
was used to add milt to the test solution plus
eggs (Figure 6). Five drops of milt from a 0.3
cc syringe and two drops from a 1 cc syringe were
added in the king and pink assays, respectively.
The cup was then filled with the appropriate test
solution. Two-minutes after the addition of the
sperm the eggs were rinsed with the same test
solution to remove the remaining milt. Rinsing
consisted of carefully pouring off the test
solution in the cup and then refilling the cup
with same test solution. After the eggs were
rinsed, they were placed in approximately 900 mL
of exposure solution in the tri-pour beakers.
Temperature, pH, dissolved oxygen (DO), and
TDS/conductivity were measured prior to the
exposure bioassay. At the end of the king salmon
bioassay the pH and TDS/conductivity were
measured. At the beginning of the pink salmon
bioassay, before eggs were added the pH was
adjusted between 7.3 and 7.4.An aeration device
was placed in the solution for the duration of
the assay. After 24-hours, the eggs were removed
from the tri-pour beaker and placed in
Whirl-packsTM where they were cleared and
preserved in Stockard solution. Samples of 20
eggs per Whirl-pack were then later examined
under a microscope to determine whether
fertilization had occurred and the cellular stage
reached by the eggs (Figure7).
CONCLUSIONS The data illustrate that Ca2 and
SO4 reduce the success of fertilization of both
pink and king salmon eggs. The finding of Ca
affecting salmon eggs in a negative manner
supports the findings of Ketola and others in
1988. Their research indicated water rich in
calcium solutions detrimentally affected eggs of
three species of fish when they were placed in
the solution prior to fertilization. It is
thought that excess calcium affects the
water-hardening phase of the salmon eggs. It
still remains unclear what role SO4 plays in the
reduction of fertilization success in the pink
and king salmon eggs. In addition the Mg2
results are intriguing. What role Mg2 plays in
the reduced fertilization rate is also not
understood. Future studies examining the effect
these ions have on the fertilization success of
pacific salmon at equivalent molar values are
needed to be performed to understand the true
effect each ion has on fertilization. These
results are important information that will
assist state agencies to set upper limits for the
discharge of industrial effluents.
REFERENCES Ketola, H. G., Longacre, D., Greulich,
A., Phetterplace, L., Lashomb, R. (1988). Hogh
Calcium Concentration in Water Increases
Mortality of Salmon and Trout Eggs. Progressive
Fish-Culturist, 50, 129-135.  Stekoll, M.,
Smoker, W., Wang, I., Failor, B. (2000). Salmon
as a Bioassay Model of Effects of Total Dissolved
Solids. 2000 Annual Report for ASTF Grant
98-1-012.
ACKNOWLEDGEMENTS   This project was supported by
the National Science Foundations (NSF) Research
Experience for Undergraduate (REU) program at the
University of Alaska, Southeast. Dr, Michael
Stekoll allowed me to participate in this
opportunity and supervised me throughout the
project. Barbi Failor assisted in conducting the
research, analyzing and data. Ivan Wang assisted
with the data analysis. I wish to thank NSF, the
American Geophysical Union (AGU) Ocean Sciences,
and the American Society of Limnology and
Oceanography (ASLO) for inviting me to
participate in presenting at this conference
Figure 4. A) Female Spawning B) Male Spawning