Auburn University

Uca pugilator Project

Data Analysis and Discussion

Undergraduate groups data:

Closures 1 and 2

Ana Gabriela Itokazu

Camryn Swain
Danyang Wang

BSEN 6510 - Ecological Engineering

Supervised by Dr. David Blersch

November 23, 2019

 Abstract

The microcosmological approach is beneficial for both academic learning as well as re-
search purposes. The data extracted from a microcosm is usually more reliable and precise,
therefore the knowledge derived from it can serve better as the basis for a more holistic
method. The use of microcosms in college classes results in substantial advantage for both
mentor and mentees, whereas the latter can apply and test their abilities, the first have a
convenient method to transfer the knowledge: a pocket-size ecosystem. This is the third
document of a series for the course, being the first a guidance on habitat and abiotic factors
for the design, the second a specific review on the ecological aspect of the respiration rate
in crabs. This document aims to analyze the undergraduate groups’ data and interpreting it
within the context of the conceptual models for the aquatic ecosystem. The data provided
includes pH, temperature, alkalinity, salinity, nitrogen, phosphate, hardness, dissolved oxy-
gen and humidity. The parameters were derived from two different experimental closures,
of 1 and 2 days. The values and fluctuations are discussed within the microcosm design
predicament, offering more guidance and suggestions to be implemented before the final
closure experiment.
Contents
1 Prologue 3

2 Introduction 3

3 Discussion 4
3.1 pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.2 Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.3 Salinity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.4 Humidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.5 Alkalinty and Hardness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.6 Dissolved Oxygen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.7 Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.8 Nitrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

4 Final comments 8

Bibliography 8

5 Supplementary Material 10

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1 Prologue
This document is part of a series produced during the course BSEN 6510 - Ecological
Engineering, at Auburn University (Fall 2019). The core project for this course is a hands-on
design and operation of a closure microcosm for a fiddler crab (Uca pugilator Bosc 1802). In
nature, these crabs inhabit intertidal sand-flats and feed from the sediment, including algae,
bloodworms and detritus. The microcosms that are in operation during this course have the
goal of supporting the crab’s survival through a 7-day closure experiment. The system can be
open energetically e.g. light and electricity, but has to be materially closed e.g. water, oxygen.
Before the experiment, the groups have the opportunity of testing their design and assumptions,
first as a 1-day closure trial, and after improvements, on a 2-day closure trial, which can be
followed by more changes before the final experiment.
While the project design and execution is conducted by undergraduate students groups,
with the supervision of Professor Dr. David Blersch, the graduate students act as a scientific
committee, gathering information and analyzing the data provided by the undergraduate groups.
This document is a compilation and analysis of the data the undergraduate groups provide for
the first trials.

2 Introduction
The academic purpose of allowing a shorter trial for these microcosms is to test and select
theoretical assumptions. Considering that the four groups in the class made different designs
and had separate tests, it is hard to treat them all as replicates, or even formally compare them.
Not only because of the differences between the systems per se, but also because of the lack of
standardization and periodicity of sampling.
For this report, it will be presented short discussions for the variables, relating two or more
of them whenever necessary, including those that were not common across all reports. This
will be useful for the undergraduate groups to decide if it is needed to add more variables to the
measurements for the final closure experiment. Presumably, no big changes in the microcosms
will be made until the final experiment, due to time constraints. Considering this, the present
report aims to provide support for the current designs, listing good practices for sampling and
data documentation, in addition to considerations on the initial aspects of the systems before
closure, providing a guide for the undergraduate groups and anticipating normalization of future

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data.
The data provided by the undergraduate groups and discussed in this document is partially
summarized in Figures (Figure S1a to Figure S1d), and detailed in the Supplementary Material,
in Table S1. For some data points, there was an uncertainty about the timeline of the closure as
described in the reports, and vagueness for methodological descriptors, therefore the analysis
discussed in this report will take into consideration the specific organized data provided in Table
S1 in this document.

3 Discussion

3.1 pH
According to the pH data of the four groups, both pre and post-closure data are lower than the
ideal range of Uca pugilator (Figure S1a). Even though this can be counter-intuitive, the relative
low initial pH can be a smart choice due to alkalinity, hardness and ammonia interactions, as
described in section 3.5. During closure, pH will tend to increase gradually, and if it increases
too much, it can reach the dangerous turnpoint for ammonia to ammonium ratio (Figure S2, pH
around 8.4) which is one of the main concerns for crab’s health.
It is important to note that a low pH can be as harmful to the overall health of the animal as a
high pH. Lower pH levels can affect gill function and active ion transport. Some predictions on
pH fluctuations can be made based on the amount of biomass and its metabolic rates in the tank
and values for other parameters, i.e. if algal biomass is abundant, the pH tends to rise slowly,
and if the alkalinity (see Section 3.5) is relatively high, there is less concern on the acidic side
of the range.

3.2 Temperature
All of the four microcosms temperature are within the optimal range (Figure S1b). Some
of the groups are using heat pads and the ones that are not are considering it, or some other
form of heating. It is imperative that the temperature is at optimal, once it is a variable that is
relatively easy to maintain in a microcosm with this size and inside a laboratory environment.
Extreme temperatures can affect the metabolic rates and photosynthetic biomass development.

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3.3 Salinity
The groups all used the Instant Ocean™ salt to make artificial seawater for the microcosms,
by adjusting its salinity (Figure S1c. The composition for the Instant Ocean™ aquarium salt
that was used is described on Table ??. It is worth noting that this product not only secures the
salinity of the water, but also provides some other minerals that contribute the proper chemistry
of the water.
Uca pugilator crabs have a high tolerance to salinity variation within the optimal range,
so small variations due to evaporation and crystallization should not prevent the animal to
thrive inside the closed system. Even though some undergraduate groups state on their own
introductions that the optimal range for salinity in fiddler crabs is around 30 ‰, not one group
started with salinity around this value. Even though there is a risk for elevated salinity due to
evaporation, other interactions can take place because of the high concentration of salts which
can benefit the project, as will be discussed in Section 3.8.

3.4 Humidity
Concerning the health of the crab, the relative humidity can be as low as 75 % and as high as
above 100 %. The lower limit should be aimed in case that there is a concern with mold and/or
bacterial biofilm formation on surfaces inside the tank. The relative humidity higher than 75 %
is also important for the sand moisture to be maintained, because it is where the crabs can build
their burrows and forage for food. The high relative humidity can be assured with an appropriate
amount of photosynthesizing biomass, which will keep the water circulating properly through
respiration and evapotranspiration processes.
An interesting fact is that a very high relative humidity can cause water absorption by the
body, which means a momentary loss of the water that is circulating. Because the system is
closed, for the overall cycling this should not be a big problem, but this characteristic can be
used in favor of the crabs final weights.

3.5 Alkalinty and Hardness

While hardness is the sum of metal ions in solution, alkalinity is a measure of the solution’s
ability to neutralize acids (prevent pH drops) as a sum of carbonates, bicarbonates, and hydrox-
ides. These two parameters are highly correlated with the pH control, calcium availability and
absorption, and ion dynamics. Natural seawater has an average alkalinity of 125 to 200 ppm,

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and it is considered really hard, due to various dissolved salts, of around 6630 ppm. Alkalinity
was analyzed by groups 1 and 2, whereas Group 3 was the only one to measure water hardness
(Figure S1d).
The low alkalinity can play major role in pH variation in a small scale like the microcosm,
and should be addressed by group 2 before the 7-days closure experiment. As for the hardness
of the water, group 3 reported reasonably low hardness of water. This group measured hardness
as KH, which is a unit for hardness usually used by aquarium hobbyists, which was converted
to ppm for analytical purposes. The group is advised to re-check their measurements and
guidelines, and measure both alkalinity and hardness in ppm or mEq/L, which helps when
comparing against obtained values from literature. The values are available in Table S1. All
other groups are advised to measure this parameters. Extremes values for alkalinity or hardness
of water can impact ammonia levels (see Section 3.8 and complexation of metals, availability
of calcium, and of course, pH.

3.6 Dissolved Oxygen

According to the reports, only group 3 measured dissolved oxygen (Table S1). Even though
group 1 presented this parameter for the second closure, the values are probably swapped, once
they are virtually impossible to reach. According to the provided data, the dissolved oxygen
concentration in water of one-day closure test is in the ideal range. This parameter should be a
primary measurement during the closures, because it shows the system’s reliability to maintain
the oxygen levels. All the groups are advised to measure dissolved oxygen in water (mg/L) and
oxygen saturation in air (%).
Oxygen concentration in the air is the primary factor affecting the dissolved oxygen level
in the water. Apparently, the groups did not planted aquatic plants before the one-day closure,
so the only producer in this ecosystem would be small amounts of algae and/or cyanobacteria.
In principle, the air at the moment when the tank was closed was at 21 % oxygen, which
may be enough for a 24 or 48 hours experiment, but can be less than the necessary amount
for the 7-day experiment. A more concentrated amount of algae/cyanobacteria and plants are
necessary for this. A few groups acquired Marimo Balls, which are a form of Aegagropila
linnaei (Cladophorales), a green algae native of northern europe. The ideal is to evaluate
production-respiration rates experimentally in order to calculate the amount of Marimo Balls
needed to produce enough oxygen for all the E. pugilator crabs. Bigger terrestial plants should

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also be considered.
Oxygen deficiency is a major constraint not only in air, but also dissolved in water. An
anoxic environment under water could affect the aerobic bacteria to decompose the waste of
crabs. Also, phosphorus is in the soluble reactive form at low oxygen (see 3.7). This can cause
a bloom and lead to a critical water quality situation as well as a complete ecosystem ecosystem
collapse. Hence, the dissolved oxygen is a critical parameter to monitor the overall water quality
in the closed system. Prior to and during the seven-day closure experiment, more efforts should
be put into measuring the dissolved oxygen.

3.7 Phosphate
The data for phosphates is really limited, and incertain. Only two groups provided data for
phosphates (see Table S1) and it is a way too high value (even with the correction to ppm).
Natural seawater has phosphate levels as low as 0.01 ppm, and the data provided by the group
is four orders of magnitude higher than this. For aquarium phosphates levels, up to 2 ppm is
acceptable, but as low as 1 ppm algae can overgrow, affecting the water use for the crabs.
Phosphates can be enclosed in sediment deposits, e.g. the sand that was brought from
the coast. In oxygenized water, phosphate tends to form phosphate complex by bonding with
iron, aluminum and calcium. In anoxic water, phosphorous exists in the form of soluble
reactive phosphorus (SRP). SRP is available nutrients for aquatic organisms. When the oxygen
concentration goes too low, large amounts of P releases from the sediment. If cyanobacteria
exist in the closed aquarium, they will uptake all available nutrients to grow extremely fast to
take place of all other photosynthesizing organisms. Thus, maintain plenty of oxygen in the
closed ecosystem can prevent extra available phosphorous released to water from sediment. The
phosphate data was insufficient to analyze, only group 1 provide the phosphate measurement.
Whether the phosphorous exceeds normal level can also be seen by observing the blue-green
algae growth.

3.8 Nitrogen
Inorganic nitrogen in the microcosms will be present in nitrites, nitrates, ammonia, or
ammonium. Nitrate is harmless and it is the non-toxic form of nitrogen. Nitrite is part of the
nitrogen cycle, but in high levels can be really toxic to the crabs. Group 2 had several problems
with the levels of nitrites, but reportedly fixed this by inoculating nitrifying bacteria into the

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system. Ammonia (N H3 ) is the toxic form of nitrogen, and the one that the groups should be
more concerned with. No ammonia at all is the ideal, and levels as high as 2 ppm can cause
death of aquatic heterotrophic organisms. The less toxic form, ammonium (N H4+ ).
Different forms of these nitrogenous compounds can be converted and circulated by aerobic
nitrification bacteria. The final product of nitrification is non-toxic nitrate. Generally, in natural
water bodies, these compounds remain very low concentration since they are continuously
removed by various microorganisms and aquatic plants.
The groups are advised to not use tap water as base for their microcosms, as opposed to
distilled water (provided by the lab), and include ammonia and nitrite levels on the monitoring
plans.

4 Final comments
One of the challenges of this project is to monitor the microcosm properly, and use the
derived data for improvements. The solutions are as good as the quality of data. The groups
have to make sure to maintain good practices during data collection, and to record it neatly.
The data needs to be recorded in units and formats that are widely used for that type of data,
to be comparable. If conversions are needed, the groups need to make sure that they are made
accurately.
It is imperative to remember that this document separated the parameters in different sections
for a better understanding, and that those are not, in any way, isolated from one another. One
variable can affect as many variables as are present in the system, directly and indirectly.

Bibliography
[1] Patrick Kangas. Ecological engineering: principles and practice. CRC Press, 2003.

[2] Peter J Hogarth. The biology of mangroves and seagrasses. Oxford University Press, 2015.

[3] Claude E Boyd. Water quality: an introduction. Springer, 2015.

[4] M Zachary Darnell, Haley S Nicholson, and Pablo Munguia. Thermal ecology of the
fiddler crab uca panacea: thermal constraints and organismal responses. Journal of
thermal biology, 52:157–165, 2015.

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 [5] Tung-Yun Chen, Gwo-Wen Hwang, Anderson B Mayfield, Chang-Po Chen, and Hsing-Juh
Lin. The development of habitat suitability models for fiddler crabs residing in subtropical
tidal flats. Ocean & Coastal Management, page 104931, 2019.

[6] Kathleen A Reinsel and Dan Rittschof. Environmental regulation of foraging in the sand
fiddler crab uca pugilator (bosc 1802). Journal of Experimental Marine Biology and
Ecology, 187(2):269–287, 1995.

[7] He Zhong. Common florida fiddler crabs (uca spp.). Public Health Entomology Research
& Education Center, 10:1–7, 2006.

[8] Birgit Wolfrath. Field experiments on feeding of european fiddler crab uca tangeri. Marine
Ecology Progress Series, 90:39–43, 1992.

[9] Glenn R Lopez and Jeffrey S Levinton. Ecology of deposit-feeding animals in marine
sediments. The quarterly review of biology, 62(3):235–260, 1987.

[10] Pablo D Ribeiro and Oscar O Iribarne. Coupling between microphytobenthic biomass and
fiddler crab feeding. Journal of Experimental Marine Biology and Ecology, 407(2):147–
154, 2011.

[11] William Mitsch. Ecological engineering a cooperative role with the planetary life-support
system. Environmental Science & Technology, 27(3):438–445, 1993.

[12] HT Odum. Man in the ecosystem. in proceedings lockwood conference on the suburban
forest and ecology. Bull. Conn. Agr. Station, 652:57–75, 1962.

[13] Howard T Odum and Nils Peterson. Simulation and evaluation with energy systems blocks.
Ecological Modelling, 93(1-3):155–173, 1996.

[14] William J Mitsch. Ecological engineering—the 7-year itch. Ecological engineering,

10(2):119–130, 1998.

[15] James W Green, Mary Harsch, Lloyd Barr, and C Ladd Prosser. The regulation of water
and salt by the fiddler crabs, uca pugnax and uca pugilator. The Biological Bulletin,
116(1):76–87, 1959.

[16] Jocelyn Crane. Fiddler crabs of the world: Ocypodidae: genus Uca, volume 1276.
Princeton University Press, 2015.

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5 Supplementary Material

(a) Water pH data. (b) Water temperature data.

(c) Water salinity data. (d) Hardness and Alkalinity data.

Figure S1: Data from closure experiments of 1 and 2 days of 4 different undergraduate
groups in designed microcosms for Uca pugilator fiddler crab. Ideal range for fiddler
crabs explicit. 1c: 1 day closure experiment; 2c: 2 days closure experiment; Asterisks (*)
indicate lack of data, hardnes is represented in deci ppm to facilitate understanding. See
text for details.

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Figure S2: Relationship between the ammonia to ammonium ratio and pH.

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 Table S1: Compilation of all data provided by the undergraduate groups for the experiments of 1 and 2 days of closure.
Experiment Parameter (unit) Group 1 Group 2 Group 3 Group 4 Ideal range
intial 7.1 7.0 7.2 7.3
pH 8.1 - 8.3
final 7.3 7.0 7.8 7.2
intial 21.6 23.8 22.9 24.4
Temperature (°C) 21.0 - 27.0
final 21.6 23.3 22.9 23.3
intial 9.8 (*) 3.5 7.8
Salinity (ppm) 30 - 33
final 180 (*) 3.6 8.3
intial (*) (*) 98 (*)
Relative humidity (%) 80 - 100
final (*) (*) 90 (*)
intial 160 80 (*) (*)
Alkalinity (ppm) 125 - 200

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final 180 80 (*) (*)
1-day closure
intial 160 (*) 425 (*)
Hardness (ppm) 350 - 390
final 180 (*) 425 (*)
intial (*) (*) 6 (*)
Dissolved Oxygen (mg.L−1 ) 6.0 - 9.0
final (*) (*) 9 (*)
intial 0.25 (*) (*) (*)
Phosphate (ppm) 0 - 0.1
final 0.3 (*) (*) (*)
intial 0.25 0.5 (*) 1.5
Nitrite (ppm) 0 - 0.5
final 0.7 0.5 (*) 1.5
intial (*) 20 (*) (*)
Nitrate (ppm) 0 - 25
final (*) 20 (*) 20
 Table S1 continued from previous page
Experiment Parameter (unit) Group 1 Group 2 Group 3 Group 4 Ideal range
intial 7.1 7.0 (*) 7.4
pH 8.1 - 8.3
final 7.3 7.0 (*) 7.1
intial 24.1 25.0 (*) 22.8
Temperature (°C) 21.0 - 27.0
final (*) 25.7 (*) 23.1
intial 9.0 14.0 (*) 7.4
Salinity (ppm) 30 - 33
final 8.8 15.0 (*) 7.4
intial (*) (*) (*) (*)
Relative humidity (%) 80 - 100
final (*) (*) (*) (*)
initial 180 80 (*) (*)
Alkalinity (ppm) 125 - 200

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final 160 80 (*) (*)
2-day closure
initial (*) (*) (*) (*)
Hardness (ppm) 350 - 390
final (*) (*) (*) (*)
intial (*) (*) (*) (*)
Dissolved Oxygen (mg.L−1 ) 6.0 - 9.0
final (*) (*) (*) (*)
intial 0.4 0.3 (*) (*)
Phosphate (ppm) 0 - 0.1
final 0.4 0.3 (*) (*)
intial (*) 5 (*) 1.6
Nitrite (ppm) 0 - 0.5
final (*) 5 (*) 2.0
intial 1.5 40 (*) (*)
Nitrate (ppm) 0 - 25
final 1.5 80 (*) (*)