OSMOREGULATION OF EARTHWORM IN DIFFERENT SALINITY LEVELS

Carandang | Cruz | Pasumbal | Salem | Tolentino

Institute of Biology, University of the Philippines Diliman, Diliman, Quezon City

ABSTRACT

This experiment aims to observe the regulatory functions of earthworms under varying saline conditions.
Six groups of seven earthworms, submerged in aged tap water 6 hours prior to the experiment, were
exposed to solutions of different saline levels (0.03 M, 0.06 M, 0.09 M, 0.12 M, 0.14 M and 0.15 M
NaCl). Their weights and volumes prior to saline exposure were recorded, with changes in weight
monitored at 20 minute intervals for 80 minutes. The final volume was recorded after the 80 minute total
immersion time. Experimental results showed a decrease in weight for all samples, 0.03 M, 0.06 M and
0.09 M concentration solutions displayed a linear trend, while 0.12 M, 0.14 M and 0.15 M concentrations
showed fluctuations in weight loss but still an overall decrease. The minimal change was not observed at
0.12 M which was the isotonic solution. Based on weight change over time, concentrations 0.03 M and
0.06 M showed the least change, while based on percent weight change over time, concentrations from
0.03-0.09 M showed the least percent weight change. The theoretical data was consistent with the data
gathered in hypertonic solutions where the earthworms would decrease in body weight per time due to
water loss, however it did not correspond to the actual data for hypotonic solutions.

INTRODUCTION environmental osmolarity. Osmoregulators, on

Osmoregulation is the ability of an organism to
maintain its fluid level. This is a major
determining factor for an animal’s geographic
distribution due to its role in the maintenance of
cell volume homeostasis (Randall et al., 2002).
Lacking true cell walls as found in bacteria,
plants, algae, and fungi, most cells of animals
become osmotically equalized with the
extracellular fluid (ECF) that bathes them. Thus,
the osmotic composition of the ECF directly
affects cell volume homeostasis. To equalize
pressure exerted by the ECF with that exerted by
the intracellular fluid (ICF), it is necessary to
equalize the amount of solutes in both
(Sherwood et al., 2013).
An animal’s osmoregulatory capabilities can be
classified under one of two types:
osmoconformers and osmoregulators.
Osmoconformers exhibit little to no
osmoregulation leading to their body fluids’
osmolarity changing in accordance to the
the other hand, maintain an osmolarity different
from the environment. The concentrations in
which the animal is capable of tolerating
determines the environments it can inhabit,
classifying them as either euryhaline or
stenohaline. The former are able to tolerate wide
variations in salinity while the latter only able
tolerate a narrow range (Randall et al., 2002).
Terrestrial animals are by necessity
osmoregulators to some extent because the
environment (air) has no solutes, the earthworm
however, is an exception. Due to the moist
conditions of the soil earthworms are found in,
their environment is effectively aquatic
(Sherwood et al., 2013). Their osmoregulatory
organs, nephridia, employ mechanisms of
filtration, reabsorption and secretion for fluid
level maintenance (Randall et al., 2002).
This experiment aimed to observe these
osmoregulatory organs of earthworms and their
efficacy under varying saline conditions by concentration increases for the three lowest
observing changes in mass and volume. concentrations, while the three highest
concentrations showed fluctuations.
MATERIALS AND METHODS
Observing the percent change in weight, all
The process of osmoregulation was observed in groups showed a significant decrease after 20
earthworms using saline solutions with varying minutes, and the percent change in weight
concentrations of sodium chloride. Prior to the increased and stabilized afterwards. A small
experiment, 21 earthworms were immersed in fluctuation was observed in setups immersed in
aged tap water for six hours. The earthworms 0.03 M, 0.06 M and 0.09 M NaCl showing
were separated into three groups containing minimal activity in osmoregulation - indicating
seven worms each. Each group of sample were that the cells of the earthworm are isotonic
weighed and reweighed after 15 minutes until no within these concentrations.
change in weight was observed. After weighing
each group, the volume was measured by
placing each group in graduated cylinder
containing a recorded amount of aged tap water,
and the volume displacement was obtained.
Each group of earthworms were immersed in
assigned concentrations of sodium chloride
(0.03 M, 0.06 M, 0.09 M, 0.12 M, 0.14 M and
0.15 M) and weighed in 20 minute intervals
until 100 minutes to observe the effect of the
varying salt concentrations to the samples. After
100 minutes of immersion, the volume of each
group was measured again. The change in
volume and weight for each group was
presented using a graph and the trend was
analyzed.

RESULTS
Figure 1. Weights of earthworms in different
All setups containing earthworms immersed in salt solutions measured at 20 minute time
different concentrations showed a decrease in intervals.
weight as shown in Figure 1 and volume. The
volume change was two mL for the three lowest
concentrations, while only one mL change in
volume was observed in the three highest
concentrations. Since the least gradation of the
instrument was one mL, the volume change was
not very accurate. All groups did not have
identical initial weights therefore, the slope of
the line was used to compare each setup. There
was an increase in the magnitude of slope as the

Figure 2. Percent weight change of earthworms
in different salt concentrations over time.
DISCUSSION
A soil solution is composed of different salts
such as sodium chloride (NaCl), sodium sulfate
(Na2SO4), magnesium sulfate (MgSO4), calcium
sulfate (CaSO4), magnesium chloride (MgCl2),
potassium chloride (KCl), and sodium carbonate
(Na2CO3). Each of these salts contribute to the
salinity of soil, but NaCl is the most prevalent
and contributes the most impact on the salinity
stress of soil (Tavakkoli, Rengasamy, &
McDonald, 2010). The salt content of the soil is
caused by water runoffs, rainfall, and farming
activities. (Deng, 2012). The effect of rainfall on
the soil is that it dilutes the solutes, redistributes
nutrients and promotes migration of earthworms
because it affects soil nitrification process
thereby affecting the texture of the soil (Sun et
al., 2009).
In order for the earthworm to survive, it has
behavioral, physiological and structural
mechanisms. Behaviorally, it undergoes a
dormant state when the temperature becomes
much too hot called estivation by which it coils
itself into a ring to lower surface area to volume
ratio. This minimizes metabolic activity and
lessens water loss. Second, it migrates to a more
favorable area such as crawling to a surface at
night, to avoid predators and to lessen water loss
due to warm temperature, aided by light
sensitive photoreceptors because they do not
have eyes (Brusca, 2016).
A physiological mechanism of the earthworm is
by secreting mucus as a smooth protective layer
that protects from abrasion as well provides
protection from poisonous soils. It has
developed structural mechanisms in its
metanephridia, skin, and intestine. The
metanephridia is a network of connecting
tubules that absorbs nitrogenous wastes, excess
solutes, and water to stores them for excretion or
reabsorption. This regulates the movement of
solutes. (Reece et al., 2014)
Experimentally, all setups showed a decreasing
trend line in the body weight of the earthworms
the longer they are submerged in varying salt
solutions. Water, in hypotonic solutions, would
enter osmotically from the cell membrane of the
test samples due to the higher concentration
gradient of sodium chloride in the internal
environment. Sodium chloride would also exit
from the cell membrane of the earthworms since
the ions would move from a point of high
concentration to a site of low concentration
through facilitated diffusion. In order to
maintain osmolarity and homeostasis the
earthworm would excrete hypotonic urine into
the environment. Hypotonic urine is composed
of high amounts of water and low concentration
of solutes. Water is readily excreted in large
amount due to its continuous uptake while the
solutes were reabsorbed into the bloodstream to
maintain its concentration gradient. This
decrease in overall body weight over time is due
to the lack of nutrients which is necessary for the
production of ATP that is needed to maintain the
osmoregulatory processes of the organism in
which it would start to have difficulty in
maintaining homeostasis. Theoretically, the
overall body weight should increase over time in
hypotonic solutions, since the water would flow
osmotically into the cell membrane of the
earthworms but this does not coincide with the
experimental data which decreased over time
(Ramsay, 1948).
Water in hypertonic solutions would passively
flow from the inside of the cell to the
extracellular environment. This would lead to
shriveling of the cells and thus the overall body
weight of the earthworms since the cells are
mostly made up of water. Sodium chloride
would passively enter into the cell due to its
concentration gradient wherein there is lower
concentration inside. In order to regulate
osmolarity, the earthworms would secrete
hypertonic urine that is composed of high
concentration of solutes and low amount of
water. This theoretical data coincides to that of
the gathered data where it was shown that the
overall body weight of the earthworms decreases
over time due to loss of water (Ramsay, 1948).
Isotonic solutions having a concentration of 0.12
M would only show minimal change in body
weight over time. The minimal change was not
observed at 0.12 M in the experiment. Based on
weight change over time (Figure 1),
concentrations 0.03 M and 0.06 M showed the
least change. Based on percent weight change
over time (Figure 2), concentrations from 0.03-
0.09 M showed the least percent weight change.
Volume displacement is another method used in
determining the weights of the samples.
Theoretically, the worms submerged in
hypotonic solutions would have larger volume
displacements compared to isotonic and
hypertonic ones since they took up water which
added to their overall body weight. Only a
volume displacement of 2 mL in aged tap water
was done due to the relatively large scale of the
50 mL graduated cylinder used.
By definition, ionic regulation is the
maintenance of the concentration of ions in a
compartment while volume regulation is the
maintenance of solvent or water concentration.
(McNab, 2002). Osmoregulation is the general
term for the processes by which animals control
solute concentrations and balance water gain and
loss. (Reece et al, 2014) These processes are
inextricably associated to each other for
biological process to continue. (Ramsay &
Kelley, 2014).
CONCLUSION
Earthworms are organisms that have
semipermeable membrane where water and
solutes can be transported into. It regulates its
osmolarity through its metanephridia which also
function as its excretory organs. They are
usually found in fresh water-logged areas that
are hypotonic to its internal body fluids. In order
to maintain homeostasis it would secrete
hypotonic that is composed of low concentration
of solutes and large amounts of water. However,
if put in saltwater environments, it would then
secrete hypertonic urine composed of low
amounts of water and high concentration of
solutes.
The theoretical data coincides with the data
gathered in hypertonic solutions where the
earthworms would decrease in body weight per
time due to water loss, however it did not
correspond to the actual data for hypotonic
solutions where the earthworms should have
increased in body weight due to continuous
uptake of water.
REFERENCES
Brusca, R. C., Moore, W., & Shuster, S. M.
(2016). Invertebrates. 399.
Deng Q, Hui D, Zhang D, et al. (2012) Effects
of Precipitation Increase on Soil Respiration: A
Three-Year Field Experiment in Subtropical
Forests in China. Bond-Lamberty B, ed. PLoS
ONE, 7(7). doi:10.1371/journal.pone.0041493.
McNab, B. K. (2002). The physiological
ecology of vertebrates: a view from energetics.
London: Cornell University Press. 134.
Ramsay, J. A. (1948). THE OSMOTIC
RELATIONS OF THE EARTHWORM.
Retrieved from
http://jeb.biologists.org/content/jexbio/
26/1/46.full.pdf.
Ramsay, J. A., & Kelley, F. C. (2014).
Regulation of water and salt balance. Retrieved
from
https://www.britannica.com/science/excretion/R
egulation-of-water-and-salt-balance
Randall, D., Burggren, W., & French, K. (2002).
Eckert Animal Physiology: 5th ed. New York:
W. H. Freeman and Company.
Reece, J. B., Urry, L. A., Cain, M. L.,
Wasserman, S. A., Minorsky, P. V., Jackson, R.
B. (2014). Campbell biology: 10th Ed. 698. 971.
Sherwood, L., Klandorf, H., & Yancey, P. H.
(2013). Animal physiology: From genes to
organisms. Belmont, CA: Brooks/Cole.
Sun, B., Zheng, X., Hu, F., Hong, B., Wang, L.,
& Sui, Y. (2009). Effect of temperature, rainfall
and soil properties on farmland soil nitrification.
Huan Jing Ke Xue, 30(1), 206-213. Retrieved
from
https://www.ncbi.nlm.nih.gov/pubmed/1935388
2.
Tavakkoli, E., Rengasamy, P., and McDonald,
G.K. (2010). High concentrations of Na+ and
Cl- ions in soil solution have simultaneous
detrimental effect on growth of faba bean under
salinity stress. Journal of Experimental Botany,
61(15), 4449-4459. doi: 10.1093/jxb/erg251.