Effects of Salinity Concentration on Procambarus clarkia
Team Nakaokisuda!
Ashlynn Aoki, Erin Nakamura, Kenton Nakamura, Zachary Masuda
Shimamoto/Papa Jack, Period 2
May 21, 2012
Abstract:
We caught crayfish in the Manoa Stream and tested their survival with regard to salinity
concentration. We decided to stop the experiment when the LC50 rule (the lethal concentration
that kills of half the population) was met. The LC50 occurred at a salinity of 14.6ppt (parts per
thousand) at which two of the three crayfish died. The crayfish turn blue rather than stay at their
normal brownish-red color when they are stressed by the environment. Additionally, the
crayfish may regenerate certain body parts by shedding its exoskeleton and gain nutrition and
minerals from eating its old shell.
Background:
In this experiment, we were interested in finding the salinity tolerance of crayfish found
in streams that feed into the Ala Wai Canal. Crayfish (Crustacea decapoda) are also known as
crawdads, crawfish, and freshwater lobsters. They are members of the crustacean, Astacoidea,
and Parastacoidea families. The appearance of a crayfish resembles that of a small lobster and is
commonly eaten by humans (Needon, 1971). There are over six hundred different species of
crayfish (Fattener, 2004). Although we are not entirely sure of the exact species of crayfish we
are experimenting with, we assume that it is the Red Swamp species (Procambarus clarkii),
commonly known as ‘Opae Pake (Bishop Museum, 2012). This species of crayfish was first
introduced to the Hawaiian Islands in 1923, in taro patches around the Ahuimanu Stream.
Because of tidal influences, these crayfish are now commonly found in the Manoa and Palolo
Streams, which feed into the Ala Wai Canal (Bishop Museum, 2012).
Crayfish belong to a group of organisms called osmoregulators. In biology, maintaining
homeostasis is an important aspect of life. For osmoregulators, their internal environment is
different than their external environment; as a result, they must constantly work against osmosis
and exert energy to maintain a concentration gradient between themselves and their environment.
In crayfish, this is done through antennal glands, which serve as the basic excretory system
(Saladin, 2012).
As with all creatures in the crustacean family, the crayfish has a hard exoskeleton
encasing its body. This exoskeleton is made of chitin, a calcium rich material. A crayfish’s body
is structured into two sections, the cephalothorax (head and thoratic region) and the abdomen.
Connected to the head region are the antennules, which the crayfish uses for balance, touch, and
taste. Crayfish also use mandibles, or jaws, to break down their food. In addition, the crayfish
also have two pairs of maxillae, which are used to hold, tear, and transfer food as well as to draw
water over the gills. Connected to the thoratic region are maxillipeds, which hold food while the
crayfish is eating, and chelipeds, large claws used for hunting and protection. Also, crayfish have
four pairs of walking legs as well as five pairs of swimmerets, or pleopods. Each of the
crayfish’s legs has an attached gill so that water circulates over the gills as the legs move. The
first pair of swimmerets can be used to determine the gender of the crayfish. The males use this
pair of swimmerets to deposit their sperm into the oviducts of the female crayfish, thus these
swimmerets are much larger and more durable than the other pairs. On the other hand, female
swimmerets are all soft in order to carry the fertilized eggs and newly hatched babies. Crayfish
utilize their tail fans, made of a modified pair of uropods, to force water forward so that they are
able to move in a backwards direction (Massengale, 2012).
Crayfish have a unique organization of internal organs. Their gonads are located directly
above the heart. The crayfish also have a two-part stomach made of the “cardiac” stomach and
the “pyloric” stomach. The “cardiac” stomach is the larger or the two sections and stores most of
the food. The “pyloric” stomach serves as the digestive site and contains digestive glands that
produce digestive enzymes. In addition, crayfish have green glands which function as kidneys.
Crayfish utilize an open circulatory system consisting of the abdominal aorta and anterior dorsal
aorta arteries. Blood travels from the arteries to capillaries and finally to sinuses, tissue spaces
that serve as veins. Also, crayfish have a ventral nervous system. A crayfish’s brain is a mass of
nerve ganglion (small bundle of nerves) located near the esophagus. The brain is connected to
the crayfish’s eyes, antennae, and antennules through very thin nerves. A ventral nerve cord raps
around the esophagus and runs to the end of the abdomen (Crescent, 2012).
Crayfish also periodically undergo a process called molting. Because their constrictive
carapace (shell) makes it difficult for them to grow, they must change them occasionally. Before
molting, the crayfish withdraws a majority of the calcium from its shell and stores the calcium,
which allows the shell to harden, in two white “tablets” on the sides of its head. After a crayfish
sheds its previous shell, it emerges with a new, flexible shell which hardens within a short period
of time (University of California, 2011).
Crayfish thrive in freshwater environments. Most prefer running water (such as streams
and rivers) over stagnant waters because many of them are not tolerable to polluted waters. If
they are to reside in stagnant waters, the lack of water flow will cause their environment to build
up in pollution (Shukla, 2011). In addition, crayfish also prefer dark, cool environments and thus
are often hidden under rocks and vegetation. Crayfish are unable to survive in extremely cold
temperatures as well as environments that are low in oxygen (Crayfishfacts.net, 2008).
The environments which crayfish live in need to maintain certain temperatures, pH
levels, salinity levels, and oxygen content. Temperatures of water ideal for crayfish survival
range from 18-25 C (room temperature) (Ball, 2001). The average pH level which crayfish prefer
is 7.5-8.5. If the pH level is too low, crayfish have difficulties molting their shells. The water
must also maintain an appropriate level of calcium or difficulties in molting will also occur (Ball,
2001). The amount of oxygen needs to be at an appropriate level. Crayfish use gills and therefore
breathe oxygen through water. If there is an inefficient amount of oxygen in the water, crayfish
attempt to gather oxygen from the air above their environment (Planeta Acocil). Crayfish are
normally found in freshwater but are sometimes said to survive in water with higher salinity
contents. Their survival in waters of higher salinity is directly proportional to the size of crayfish
(TheFishSite, 2010).
Crayfish are most active during night and are also scavengers. They are nocturnal and
normally hunt for food during night. They prefer hiding and burrowing under objects such as
rocks, leaves, tree roots, and other objects. When startled, crayfish quickly move in a backwards
direction using their tails. But normally, a crayfish will slowly move in a forward direction, using
its legs (Nale).
Because crayfish are omnivorous, they do not have a specific diet whatsoever and will eat
just about anything. Examples of plant and animal organisms that they will eat are fish, shrimp,
worms, elodea, plankton, insects, and snails. In addition, they can also be cannibalistic if they are
not provided with a sufficient amount of food or shelter in their habitat (Ball, 2007).
The LC50 rule is a method used to determine the conclusion of an experiment. The LC50
rule states that when 50% or more of the population dies at a specific salinity, then that salinity is
determined to be the lethal concentration (Canadian Centre, 2005)
Materials and Methods:
The materials we used are listed as follows: one Sper Scientific Water Quality Pen, a
wooden tank with the dimensions 33” x 33” x 5”, two plastic barriers, two plastic buckets, one
Penguin BiO-Wheel Mini water filter, a Stellar air pump, three spherical air stones, TetraMin
Tropical flakes, a turkey baster, leaves, a small fishing net, a screen, Hawaiian salt, measuring
spoons, and a turkey baster.
Our control group is composed of two crayfish in the same plastic bucket. Our
experimental group is composed of four crayfish in the wooden tank. The experimental tank is
divided into four quadrants by the two plastic dividers. Our control group has one air stone, and
the experimental tank has one Penguin BiO-Wheel Mini water filter, with two air stones. Each
day, the control crayfish are transferred to the other plastic bucket filled with clean tap water.
The crayfish in both the tank and the control bucket are then fed an abundant supply of fish
flakes. Here is a picture of our setup:
The wooden boards, cones, and racks are only there to secure the screen to the wooden tank.
The objects also serve as protection from the stray cat which wanders around the neighborhood.
We added leaves to the control bucket and tank to try to create a somewhat stream-like
environment.
When leftover fish flakes and crayfish waste pollute and turn the water murky, cleaning
must be done. Since the water in the control buckets are changed daily, there are no concerns
about unsanitary conditions in our control group. The water in our experimental group, however,
gradually turns murky due to fish flakes (see the Analysis of Data Collected portion below).
There are two types of cleaning of the tank that we performed during our experiment: a quick
and a thorough cleaning. The quick cleaning, which usually takes about 10 minutes, involves the
caretaker scooping and removing excess fish flakes and crayfish waste from each quadrant of the
tank by using the small fishing net. The thorough cleaning, which usually takes anywhere
between 30 minutes to an hour, involves packaging the crayfish into Ziploc bags, replacing all of
the water in the tank, eliminating all waste, and rinsing all components of the experimental tank
with care. These components include the fragile plastic barriers, the water filter, and the tank
itself. While the tank is being cleaned, the crayfish wait in plastic bags filled with plain tap
water.
After moving all of our materials to AP Biology room at school on day 29 of our
experiment, we still continued quick and thorough cleaning with the same materials; however,
instead of using Ziploc bags to keep the crayfish separated from the tank as the water was
cleaned, the crayfish were put in plastic containers. Using plastic tubes from the bio room, we
were also able to thorough clean the tanks via siphoning the water. We used the turkey baster to
suck up and expel leftover food and crayfish waste as well. Our Penguin BiO-Wheel Mini water
filter stopped working sometime after winter break (actual date was not recorded). As a result,
the water in the experimental tank was not filtered and circulated.
We concluded the experiment according to the LC50 rule, stopping when we discovered
that one of two remaining crayfish died.
Data: The following table shows the daily salinity level in parts per thousand (ppt).
Day:
1
2
3
4
5
6
7
8
9
10
Control Salinity Concentration
(ppt)
0.21
0.22
0.22
0.22
0.22
0.22
0.22
0.22
0.22
Experimental Salinity
Concentration (ppt)
0.21
0.21
0.22
0.22
0.22
0.23/
0.33
(QC)*
0.33
0.34
0.35
(QC)
0.36
0.37
Day:
11
12
13
14
15
16
17
18
19
20
Control Salinity Concentration
(ppt)
0.22
0.23
0.22
0.22
0.22
0.21
0.22
0.22
0.22
0.22
Experimental Salinity
Concentration (ppt)
0.39/0.31
(TC)*
0.32
0.33
0.33
0.34
0.35
0.36/0.34
(QC)
0.35
0.36
0.37
(QC)
Day:
Control Salinity Concentration
(ppt)
Experimental Salinity
Concentration (ppt)
21
22
23
24
25
26
27
28
0.22
0.22
0.22
0.22
0.22
0.21
0.21
0.37
0.38/0.50
(TC)*
0.51
0.51
0.52
0.53
0.53
29
32
0.22
0.22
0.25
0.54
0.54/
0.61
0.63
(QC)
* - indicates an addition of Hawaii salt into the water.
/ - indicates the salinity differences. The value on the left of the slash mark is the salinity before,
and the value on the right of the slash mark is the salinity after.
(QC) - indicates a quick cleaning. Only excess fish flakes and crayfish waste were removed.
(TC) – indicates a thorough cleaning. The entire experimental tank was cleaned.
Note that the only time Hawaiian salt was added was whenever we decided to increase
the salinity of the experimental tank (days 5 and 22) and performed a thorough cleaning (days 11
and 22) as the old water was dumped out and the new water’s salinity concentration had to be
matched. We altered the salinity concentration twice. On day 5, we added a total of one
teaspoon of Hawaiian salt to the tank water. On day 22, we added three more teaspoons of
Hawaiian salt so that the total salt in the water measures at four teaspoons. The new salinity
concentrations were always measured an hour after the salt was added to ensure that the salt
dissolved. The addition of Hawaii salt and the increase in salinity did not seem to have any
noticeable effects on the crayfish.
We moved the crayfish to the AP Biology on day 29, January 14, and continued the
experiment. The data for days 32 to 91 can be found attached at the end of the lab report. Note
that we added enough salt to the tank every three to five days to increase the salinity of the water
by approximately 1 ppt. We quick or thorough cleaned the tank every day other than days 88
through 91. Thorough cleaning was only performed when the water was too murky.
Analysis:
After conducting the lab for 26 consecutive days, we noticed many similar behavioral
features of the crayfish in our experimental and control groups. The crayfish in the experimental
and control tanks both love to hide either under the leaves, in corners, or near the air stones. The
crayfish rarely venture out into the open unless there is food present. When the crayfish spot the
TetraMin fish flakes floating on the surface of the water, they arch their back and reach toward
the surface of the water. When fish flakes are within reach, we noticed that the crayfish use their
giant claws, chelipeds, and the first two pairs of walking legs to gather food from its
surroundings and bring the flakes to its mouth. The two back pairs of walking legs are used for
support and stability while the crayfish eats. When performing thorough and quick cleanings of
the experimental and control groups, we noticed that the crayfish were somewhat frightened in
the presence of our small fish net. As a result, the crayfish propelled themselves backward, not
forward, into the furthest corner from the net and curled their abdomen underneath the rest of
their body. Another notable observation concerning the crayfish is that they shed and may
sometimes eat their exoskeletons as a source of nutrients. When crayfish eat their exoskeletons,
their excrements appear a pinkish-orange color rather than the normal brownish-black color. We
also noticed the leaves in each group appeared smaller each day. After watching the crayfish for
an extended amount of time, we witnessed the crayfish nibbling and eating parts of the leaf.
A side (left) and top (right) view of the crayfish in quadrant IV climbing on the plastic
bindings in the experimental tank.
The crayfish uses its two back pairs of walking legs to
hold itself to the plastic binding while the two front pairs of walking legs and chilepeds to
gather the floating fish flakes.
After transferring the crayfish to the bio room, we noticed a few more oddities. The
crayfish escaped many times, even with the screen, wooden boards and rack on top of the tank.
We did not witness the crayfish escape, but we believe that the crayfish would climb on the
plastic bindings (as seen in the pictures in the previous paragraph) and eventually on the plastic
barriers and out of the tank. There were four incidences that occurred throughout the experiment
involving crayfish outside the tank or control bucket.
In addition, when we were adding salt to the tank at one point, salt was accidentally
poured on the crayfish. As a result, the crayfish attempted to brush the salt crystals off by using
its chelipeds and front two pairs of walking legs. Furthermore, after several weeks, we also
noticed that the control crayfish readily climbed into the net during the daily cleaning of the
control bucket, demonstrating habituation in its behavior. Also, during the experiment, we
noticed a white mold-like growth on the exoskeleton of all crayfish, possibly due to improper
cleaning or overfeeding; however, this growth did not appear to have any effect on the health or
behavior of the crayfish (based on the survival of the control crayfish).
Another observation that our group made was that the crayfish did not eat their
exoskeletons at higher salinities, therefore, we removed the exoskeletons from the tank starting
at day 61 (7.75 ppt). We also noticed that the crayfish demonstrated the ability to regenerate
their chelipeds, based on the crayfish in quadrant II.
Several characteristics were noticeable in dead or dying crayfish. For example, one of
the most noticeable changes in the crayfish was the change in color of dying crayfish.
Originally, the exoskeleton was colored reddish brown. However, as the salinity increased, the
majority of the crayfish, with the exception of the control, began to turn blue. Then, a day or two
before each crayfish died, our group noticed that the crayfish would continually flip upside
down, exposing its ventral side to the air, and struggle to reorient itself.
Compare the blue coloring of the dead crayfish from quadrant II (top left) to
the red hue of the surviving crayfish (top right) and control crayfish (bottom).
Following the LC50 rule, we stopped our experiment at day 87, when the salinity was 14.6
ppt and when one of the remaining two crayfish died. Then, we gradually diluted the salinity
over the course of five days before releasing the crayfish back into Manoa stream. In this five
day interval, we noticed that the exoskeleton of the crayfish in quadrant 1 returned to its original
red-brown hue.
Although we made many observations, we had several errors while conducting our
experiment. Our control and experimental groups are not in similar environments; the control
bucket is a cylindrical, plastic bucket while the tank is a wooden square. Additionally, the
control crayfish was subject to daily cleaning since the control bucket lacked a water filter. Our
crayfish are not all the same size, shape, and age, meaning that each crayfish is affected
differently by the changes in salinity. Because of the environmental differences, the crayfish in
the experimental tank can climb on the plastic bindings inside the tank to reach for floating fish
flakes. When the crayfish in the experimental tank do this, their chilepeds and antennules
sometime come out of the water. Our experiment should be conducted a couple more times for
the sake of accuracy and good data.
Conclusion:
After experimenting with salt concentrations on a group of crayfish, the following can be
concluded from the lab:
-Salinity levels of the surrounding water greatly affects a crayfish’s ability to survive. Not only
do the crayfish die at high salinity levels of approximately 10 ppt and higher, but the
crayfish are affected by the salt at lower concentrations as well.
-A blue body color in Procambarus clarkii is an indication that the crayfish is stressed under
current environmental conditions and will act abnormally and possibly die.
-Crayfish have the ability to eat their own exoskeleton, allowing the crayfish to extract nutrients
and minerals, turning their waste pink in color in the process.
-Crayfish have the ability to regenerate certain body parts, such as their large claws, chelipeds,
via shedding their exoskeleton.
Although we humans are not crustaceans like Procambarus clarkia, we did evolve from
aquatic creatures which eventually transitioned on to land. Studying crayfish is a significant
topic not only because we’re discovering the differences between freshwater and saltwater
marine creatures, but also because it is important to know how organisms interact with their
environment. Like crayfish, humans have to constantly adapt to changes in environment
between home, school, work and other places.
For further research concerning the Ala Wai, we suggest trying to test why crayfish
cannot survive in the Ala Wai – is the salinity concentration too high, or is their survival
dependent on another factor, such as food source, temperature and pH? Maybe predation is an
issue, and if so, which organisms in the murky Ala Wai prey on Procambarus clarkia? By
determining which environmental conditions are suitable for crayfish to thrive in, humans can
breed crayfish to serve as food for other aquatic animals and humans as well.
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