ENV 3210 – Aquatic Sciences

ACADEMIC YEAR 2020/2021

SEMESTER Il – May 2021
__________________________________________________________________
FACULTY OF EARTH & ENVIRONMENTAL SCIENCES
COURSE NUMBER: ENV 3210
COURSE NAME: Aquatic Sciences
LECTURER –Ajay Persaud
Laboratory Exercise

GENERAL FORMAT

• Reports should be no more than 4000 words (about 15 pages).

• Double space all text. Put page numbers on every page.
• Report writing should be clear, concise, accurate and objective. Pay attention to details of
grammar and spelling: they make a real difference to the quality of your report.
• Good scientific writing is simple and direct. People tend to stumble when they try too hard to
sound “scientific”.
• Scientific writing normally uses the past tense to describe research, except for “permanent
truths” which are written in the present tense. (Newton showed that gravity attracts planets.)
Use the past tense exclusively to describe what you did in a research project.
• Use the active voice and the first person wherever appropriate. (“I collected 20 plants.”
instead of “Twenty plants were collected.”) The use of the passive voice is an old convention
which can lead to cumbersome English.
• Do not use slang, colloquialisms, or casual contractions (e.g., “laboratory” not “lab”). Avoid
using acronyms (DDT, EEG, GMO) unless they are necessary.

1
ENV 3210 – Aquatic Sciences

• Do not quote. Scientific writing almost never includes direct quotes from published sources,
such as: “Duckweeds are a group of small, floating, aquatic plants” (Taylor, 2016). Rephrase
literature information in your own words.
• Take advantage of computer technology and spell-check your report. Remember, however,
that spelling-checkers catch only typographical errors. Read through your report to find
mistakes in grammar and word choice.
• All reports should have the following sections
o Abstract
o Introduction
o Materials and Methods
o Results and Discussion (combine your results and discussion section; you can also
include a concluding paragraph)
o Literature Cited/References

2
ENV 3210 – Aquatic Sciences

SECTION CONTENT

Abstract

An abstract is not just a summary of the results of an experiment, but a compact summation of

the entire article. Its purpose is to allow a researcher to decide whether to read the whole paper.

Therefore, the abstract dedicates one or a few lines to summarizing each section of the report.

The abstract is usually one paragraph and should be less than 250 words. State explicit results in

the abstract (“The population doubled in 3.4 days”) rather than vague phrases like “The

population growth rate was calculated.”

Introduction

The introduction accomplishes three things:

(1) It discusses the background on the subject of the paper, to set the current work in perspective.

(2) it briefly reviews the literature on the subject, to define the problem and explain the rationale

for the present work.

(3) it lays out the hypothesis or hypotheses being tested/research questions being addressed.

Materials and Methods

This section should succinctly describe the organisms investigated (if not described in the

Introduction), the field or laboratory methods used, any instrumentation employed, and how the

data were analysed. It is always written in the past tense. It should clearly and simply explain

what you did and why you did it. As the lab is online and the practical cannot be done, this section
is provided for you. Ensure that you include the references in your literature cited section.

Results and Discussion

Present the qualitative and quantitative results of the experiments or observations in this section.

Take care to present the results clearly, accurately and in logical order. The text should lead the

3
ENV 3210 – Aquatic Sciences

reader through the data, with references to tables and figures in which the data are appropriately

summarized. Support the text with tables and figures (charts, diagrams) that present the numerical
data in a way that illustrates the major differences or trends that you found. Ensure that these
illustrations are in a logical order. Never include a table or figure that is not referred to in the text.

Use the Discussion to interpret your findings and compare them with those of other workers.

Draw conclusions about the hypotheses you set in the Introduction. If there are exceptions or

uncertainties in your results, simply note them and show how the findings agree or contrast with

previously published work. Limit speculation to what can be reasonably supported with direct

evidence. End the discussion with a short summary of the significance of the work and the

conclusions drawn.

Literature Cited

List all publications cited in the text in this section, in alphabetical order by authors’ names. Follow
the APA format for intext referencing and for the literate cited section. You may also use the
referencing format adopted by popular peer-reviewed scientific journals, such as Nature, however,
ensure you are consistent throughout your report.

Captions for tables and figures:

Caption for a table should be placed immediately above a table.

[Example] Table 1. Water chemistry of a hot spring and control stream in Pictou Landing, Nova
Scotia. Units are mg/L unless indicated otherwise. Values for conductivity and pH in the hot spring
are ranges of four measurements in 2003; all other data are from samples taken 25 May 2003.

Caption for a figure should be placed immediately below a figure.

[Example] Figure 1. Growth of duckweed, Lemna minor, populations over two weeks under

430-W grow lights in nutrient media with (Control) and without (Low N) available nitrogen.

Each point is the mean of five replicates. Error bars are standard deviations (n = 5).

4
ENV 3210 – Aquatic Sciences

Refer to tables and figures in the text by their sequential numbers, as Figure 1, Table 3, etc. The

words “Table” and “Figure” begin with a capital letter.

Formatting for tables

• Do not include any other horizontal lines in the table. Do not include vertical lines anywhere.
• Include footnotes if necessary, to specify exceptions, present statistical comparisons or
explain
• contractions.
• Footnote texts appear at the bottom of the table
• Do not include shading, colours or other fancy refinements in your tables
• Example:

Formatting for figures

• Expand the figure until it almost fills the field of view on your monitor. If you put the graph
in a separate sheet, on the other hand, it will be uncomfortably large. In your report, graphs
should normally occupy about half a page.
• Make sure the background is white. Make the axes, the frame around the whole figure, and
the frame around the key, if there is one, several shades heavier than the default. Lines must
be more prominent in print than on a computer screen to appear equally to the eye.

5
ENV 3210 – Aquatic Sciences

• Be sure that the symbols denoting data are large enough to be distinguishable on the printed
page. If there are lines connecting points, or trend lines, make these heavier, as well as error
bars.
• Enlarge both the numbers and the labels to at least 12 point. Make sure the same font is used
for all. If the graph has a key, increase the font size there as well, and darken the box around
it.
• Make sure the axes span the appropriate range for your data. The high and low ends, as well
as the number of divisions along the axis, should all be specified.

6
ENV 3210 – Aquatic Sciences

OVERVIEW OF LAB – RESURRECTION ECOLOGY

Many living creatures adopt dormancy strategies during periods of adverse environmental
disturbances to ensure the survival of their populations (Dahms, 1995; Radzikowski et al., 2018). One
such strategy is the formation of resting stages, which is a state of arrested development (Danks,
1987; Radzikowski, 2013). For Cladocera, including Daphnia, the production of subitaneous eggs
(eggs with no dormancy stage) shift to the production of diapausing eggs (eggs that have a
dormancy/resting stage) under adverse environmental conditions (Radzikowski et al., 2018). The
resting eggs are found within ephippia, which is a chitinous shell (a modification of the carapace) that
encapsulates the eggs (Schultz, 1977). During periods of intense contamination, resting stages play
a critical role in ecosystem recovery, as they provide a temporal refuge for biota (Araújo, Lopes,
Santangelo, Petry, & Bozelli, 2013; Piscia, Tabozzi, Bettinetti, Nevalainen, & Manca, 2016; Santangelo,
Esteves, Manca, & Bozelli, 2011).

Daphnia resting eggs can remain viable (able to hatch) for several decades (Radzikowski,
2013) and even centuries (Frisch et al., 2014), and can end up trapped within the sediment matrix of
a lake basin (Burge, Edlund, & Frisch, 2018). Assuming the sediment profile is not perturbed, the
sediment layers in which the resting eggs are trapped can be dated to determine when the eggs were
produced. Because of their long-lived nature, the ephippial eggs can be extracted from the sediment
matrix and stimulated to hatch clonal lines of past populations. To stimulate the hatching process, a
combination of two stimuli are used, light/photoperiod and temperature regimes (Cuenca
Cambronero & Orsini, 2018; Radzikowski et al., 2018).

Resurrection ecology is predicated on there being viable resting eggs within the sediment
matrix. However, viable resting eggs can be extremely rare. For example, in examining the resting
egg bank of six lakes in the High Tatra Mountains, Marková et al. (2006) only encountered 94
potentially viable eggs from a total of 4,948 ephippia.

While there is much more to be understood about cladoceran resting egg banks, their
usefulness in interpreting the responses of Cladocera to ecosystem changes, including industrial
contamination cannot be understated.

For this lab, you will be examining the impacts of lead (Pb) pollution on Daphnia resting egg
production in in a hypothetical Guyanese lake, FEES Lake, where a history of gold mining has directly
resulted in the legacy contamination by lead and metal(loid)s such as arsenic and antimony. A multi-
proxy paleolimnological study of FEES Lake documented striking ecological impacts at multiple
trophic levels coincident with the onset of lead pollution in the lake. Notably, the subfossil record of

7
ENV 3210 – Aquatic Sciences

FEES Lake revealed the appearance of Daphnia at the time of lead (Pb) enrichment [during mining],
a species previously absent from the lake [pre-mining], suggesting a high tolerance of Daphnia to
lead. As lead levels peaked, Daphnia was inferred to have been functionally extirpated [post mining].

PURPOSE AND OBJECTIVES

The purpose of this lab is to examine trends in Daphnia ephippia production during the pre-mining
period, the mining period, and the post-mining period, to highlight the mechanisms underlying
Daphnia responses to lead pollution. Specifically, the objectives are to 1) investigate if sediment lead
toxicity compromised the integrity of the Daphnia resting egg bank in FEES Lake; and 2) assess the
viability of resting eggs in FEES Lake sediments for use in future resurrection ecology studies.

METHODS

FIELD METHODS

A sediment core was collected from FEES Lake using a Uwitec gravity corer (Mondsee,
Austria). The 25 cm long core was sectioned into 0.5 cm intervals for the first 15 cm intervals and
then at 1 cm intervals for the remainder of the core, using a modified Glew (1988) vertical extruder.
Sediments were stored cold (4°C) or frozen until analysis at the University of Guyana.

LABORATORY METHODS

Daphnia resting eggs were isolated from the sediment matrix using a modified Onbé-Marcus
sugar flotation method (Vandekerkhove et al. 2004; Marcus, 1990; Onbé, 1978). Every other 0.5 cm
interval was analyzed fully from 0 to 14.5 cm, an average sediment volume of 22 mL per interval;
however, sediments were extracted from five intervals at the 8.0-11.5 cm section of the core (median
core depths of 8.25, 8.75, 9.75, 10.25 and 11.25 cm) to intensify counting efforts at sediment depths
estimated to represent the period in which Daphnia dominated the zooplankton assemblage of FEES
Lake. For core depths below 14.5 cm, which were divided into 1 cm intervals, sediments were
extracted from every other interval, and had an average volume of ~46.4 mL per interval.

Sediment intervals were placed into separate centrifuge tubes. Sugar and water were mixed
in a 1:1 ratio and added to the tubes with sediments. Sediments mixed with the sugar solution were
centrifuged at 3000 rpm for 3 minutes to homogenize the mixture. The supernatant from the

8
ENV 3210 – Aquatic Sciences

centrifugation was then then rinsed with Milli-Q water through a 45 µm sieve to remove the sugar.
The filtrate was checked regularly to ensure that no ephippium was lost at this filter size. Following
filtration, small aliquots of the wet sediment were transferred to petri dishes and resuspended with
the addition of Milli-Q water. Sediments were then analyzed under a Motic SMZ-161 R2LED Stereo
Zoom Microscope at X10 magnification to identify ephippia. Ephippia were separated into separate
petri dishes and were decapsulated based on methods in Cuenca Cambronero & Orsini (2018) to
determine the physical condition of the ephippia and viability of the resting eggs based on the
categories established in Marková et al. (2006). Ephippia were also identified to the species complex
level using a taxonomic guide. There was no predetermined maximum count for ephippia; all
ephippia from each selected interval were analyzed and corrected for volume of sediment used. As
no viable resting eggs were identified, resurrection ecology methods could not be used.

Course instructor’s note: While the methods are provided you are required to view the article and
video provided at this link: https://www.jove.com/t/56637/resurrection-of-dormant-daphnia-
magna-protocol-and-applications .Additionally, please view the document provided in the
methodology folder. These will help you in understanding the methodology and developing your
discussion and introduction.

RESULTS AND DISCUSSION

Based on the methods outlined the following data was acquired:

Mass of Sediment Volume of Sediment Empty Empty egg

Depth (cm)
(g) (mL) ephippia membrane

0-0.5 14.50 13 0 0

1.0-1.5 22.86 21 0 0

1.5-2.0 23.94 22 0 0

2.5-3.0 22.55 21 6 0

3.5-4.0 20.45 19 3 0

4.5-5.0 23.15 21 13 1

6.0-6.5 22.11 20 9 2

7.0-7.5 23.64 22 8 6

9
ENV 3210 – Aquatic Sciences

Mass of Sediment Volume of Sediment Empty Empty egg

Depth (cm)
(g) (mL) ephippia membrane

8.0-8.5 26.23 24 5 3

8.5-9.0 27.19 25 5 0

9.5-10.0 25.41 24 26 0

10.0-10.5 26.58 25 10 0

11.0-11.5 20.16 19 9 0

12.0-12.5 24.61 23 23 0

13.0-13.5 28.50 26 25 1

14.0-14.5 23.10 21 2 0

15-16 49.12 45 7 0

17-18 47.95 44 3 0

19-20 51.82 48 3 0

21-22 48.29 45 10 0

23-24 53.92 50 10 0

The previous paleolimnological study of FEES lake based on subfossil analysis found the following:

Table 2.

Depth (cm) Lead (ug/g) Daphnia

0.25 730 0 Pre mining

1.25 1100 0

1.75

2.25 1700 0

2.75

3.25 1800 0

10
ENV 3210 – Aquatic Sciences

Depth (cm) Lead (ug/g) Daphnia

3.75

4.25 1800 0

4.75

5.25 1400 0

6.25 1700 0

7.25 6900 0

8.25 20000 40 During mining

8.75

9.25 33000 58

9.75

10.25 29000 11.1

11.25 9700 0

12.25 3300 4.5

13.25 1900 0 Peak of contamination

and post mining
14.25 2200 0
period
15.25 1800 0

15.5

16.25 1400 0

17.25 1100

17.5

18.25 1100

19.25 1000

19.5

20.25 790

21.5

11
ENV 3210 – Aquatic Sciences

Depth (cm) Lead (ug/g) Daphnia

23.5

24.25 640

****Note: you did not acquire this hypothetical data from your hypothetical study. Source the
information in figure(s) using “Data from Doe, J. (unpublished data)”, or “Adapted from Doe,
J. (unpublished data)”***

INSTRUCTIONS:

• Using the data provided you are required to describe and analyze the trends in the
number of empty ephippia and egg membranes (corrected for volume of sediment
used based on Table 1). In addition, you must compare these results to the trends in
lead (Pb) concentrations and % Daphnia recreated from data presented by Doe, J. in
table 2.
o Findings should be visually presented using an appropriate figure(s). Example:

Source: (Persaud, 2020)

12
ENV 3210 – Aquatic Sciences

• Discuss the following (some relevant materials provided in Discussion folder):

o Temporal trends in Daphnia ephippia production
o Trends in ephippia production versus results presented by Doe, J., accounting
for differences, if any.
o Viability of resting eggs in FEES Lake for future resurrection ecology studies
o Impacts of contamination on the Daphnia resting egg bank of FEES Lake

REFERENCES

Araújo, L. R., Lopes, P. M., Santangelo, J. M., Petry, A. C., & Bozelli, R. L. (2013). Zooplankton resting
egg banks in permanent and temporary tropical aquatic systems. Acta Limnologica
Brasiliensia, 25(3), 235–245. https://doi.org/10.1590/s2179-975x2013000300004

Burge, D. R. L., Edlund, M. B., & Frisch, D. (2018). Paleolimnology and resurrection ecology: The
future of reconstructing the past. Evolutionary Applications, 11(1), 42–59.
https://doi.org/10.1111/eva.12556

Cuenca Cambronero, M., & Orsini, L. (2018). Resurrection of Dormant Daphnia magna: Protocol and
Applications. Journal of Visualized Experiments : JoVE, (131). https://doi.org/10.3791/56637

Dahms, H. U. (1995). Dormancy in the Copepoda - an overview. Hydrobiologia, 306(3), 199–211.

https://doi.org/10.1007/BF00017691

Danks, H. V. (1987). Insect dormancy: an ecological perspective. Insect Dormancy: An Ecological

Perspective. Retrieved from https://www.cabdirect.org/cabdirect/abstract/19870543089

Frisch, D., Morton, P. K., Chowdhury, P. R., Culver, B. W., Colbourne, J. K., Weider, L. J., & Jeyasingh, P.
D. (2014). A millennial-scale chronicle of evolutionary responses to cultural eutrophication in
Daphnia. Ecology Letters, 17(3), 360–368. https://doi.org/10.1111/ele.12237

Marková, S., Černý, M., Rees, D., & Stuchlík, E. (2006). Are they still viable? Physical conditions and
abundance of Daphnia pulicaria resting eggs in sediment cores from lakes in the Tatra
Mountains. Biologia, 61(18), 135–146. https://doi.org/10.2478/s11756-006-0126-5

Persaud, A. A. (2020). Paleo-Ecotoxicology of Yellowknife (Northwest Territories, Canada) Lakes

Impacted by Historic Gold Mining Activities. York University, Toronto, Canada. Retrieved from
https://yorkspace.library.yorku.ca/xmlui/handle/10315/37974

Piscia, R., Tabozzi, S., Bettinetti, R., Nevalainen, L., & Manca, M. M. (2016). Unexpected increases in
rotifer resting egg abundances during the period of contamination of Lake Orta. Journal of

13
ENV 3210 – Aquatic Sciences

Limnology, 75(2S), 76–85. https://doi.org/10.4081/jlimnol.2016.1300

Radzikowski, J. (2013). Resistance of dormant stages of planktonic invertebrates to adverse

environmental conditions. Journal of Plankton Research, 35(4), 707–723.
https://doi.org/10.1093/plankt/fbt032

Radzikowski, J., Krupińska, K., & Ślusarczyk, M. (2018). Different thermal stimuli initiate hatching of
Daphnia diapausing eggs originating from lakes and temporary waters. Limnology, 19(1), 81–
88. https://doi.org/10.1007/s10201-017-0520-4

Santangelo, J. M., Esteves, F. D. A., Manca, M., & Bozelli, R. L. (2011). Abundance, composition and
spatial variation in the egg bank of a tropical zooplankton community. Studies on Neotropical
Fauna and Environment, 46(3), 225–232. https://doi.org/10.1080/01650521.2011.632672

Schultz, T. W. (1977). Fine Structure of the Ephippium of Daphnia pulex (Crustacea: Cladocera).
Transactions of the American Microscopical Society, 96(3), 313.
https://doi.org/10.2307/3225860

Vandekerkhove, J., Niessen, B., Declerck, S., Jeppesen, E., Conde Porcuna, J. M., Brendonck, L., & De
Meester, L. (2004). Hatching rate and hatching success with and without isolation of
zooplankton resting stages. Hydrobiologia, 526(1 SPEC. ISS.), 235–241.
https://doi.org/10.1023/B:HYDR.0000041598.68424.fc

14