Optimization of Novel Culturing and Testing Procedures for .../67531/metadc699989/m2/1/high_res_d/thesis.pdfTisbe biminiensis is an epibenthic harpacticoid copepod that live s on bottom

APPROVED:

Duane Huggett, Major Professor Ed Dzialowski, Committee Member Mark Burleson, Committee Member Sam Atkinson, Chair of the

Department of Biological Sciences Mark Wardell, Dean of the Toulouse

Graduate School

OPTIMIZATION OF NOVEL CULTURING AND T ESTING PROCEDURES FOR ACUTE EFFECTS

ON Acartia tonsa AND Tisbe biminiensis

Erin J. Ussery

Thesis Prepared for the Degree of

MASTER OF SCIENCE

UNIVERSITY OF NORTH TEXAS

December 2014

Ussery, Erin J. Optimization of Novel Culturing and Testing Procedures for Acute Effects

on Acartia tonsa and Tisbe biminiensis. Master of Science (Biology), December 2014, 50 pp., 24

tables, 11 figures, references, 43 titles.

Copepods comprise an ecologically important role in freshwater and marine

ecosystems, which is why they are often considered an important ecotoxicological model

organism. The International Organization for Standardization’s (ISO) 14669 protocol is the only

guideline for the determination of acute toxicity in three European marine copepod species:

Acartia tonsa. The goal of this project was to assess the feasibility of establishing and

maintaining cultures of Acartia tonsa, as well as to refine current culturing and egg separation

methods. Initial culture methodology proved difficult for consistent production of eggs and

collection of nauplii. The development of an airlift system for the separation of eggs from

nauplii and adults, based on size, successfully increased the availability of eggs, nauplii and

adults. The sensitivity and relative conditions of the copepod species was assessed by running a

series of 48h acute toxicity tests with the reference toxicants 3,5-dichlorophenol, 4,4’-

methylenebis(2,6-di-tert-butylphenol. The acute 48 hour median lethal dose concentration

(LC50), the no observed effect concentration (NOEC), and the lowest observed effect

concentration (LOEC) was analyzed for the three reference compounds for of A. tonsa.

Copyright 2014

by

Erin J. Ussery

ii

ACKNOWLEDGEMENTS

I gratefully acknowledge the opportunity and financial and academic support of Dr.

Duane Huggett, without whom, this research would not have been possible. I would also like to

give a special thanks to my lab mates and Dr. David Hala for everything they have done as

friends, as well as to aid in my research. Thank you, also, to my committee members Dr. Mark

Burleson and Dr. Ed Dzialowski. Your participation and guidance is greatly appreciated!

I would also like to thank my family and friends outside of graduate school for their

support. Funding for this project was provided by Wildlife International Ltd., Easton, MD. Thank

you Hank Krueger for your support.

iii

TABLE OF CONTENTS

Page

ACKOWLEDGEMENTS ..................................................................................................................... iii

LIST OF TABLES ................................................................................................................................ ix

LIST OF FIGURES ............................................................................................................................. vii

CHAPTER 1 INTRODUCTION ............................................................................................................ 1

1.1 Background ..................................................................................................................... 1

1.1.1 Importance of the Mass Culturing of Acartia tonsa and Tisbe biminiensis ...... 1

1.2 Phenols ........................................................................................................................... 4

1.2.1 3-5-dichlorophenol ........................................................................................... 6

1.2.2 4,4’-methylenebis(2,6-di-tert-butylphenol) ..................................................... 7

1.3 Antifolates ...................................................................................................................... 7

1.3.1 5-Fluorouracil .................................................................................................... 9

1.4 Rationale for Study ...................................................................................................... 10

1.4.1 Objectives and Hypotheses............................................................................. 10

CHAPTER 2 MATERIALS AND METHODS ....................................................................................... 12

2.1 Development of Novel Culture Chamber and Egg Collecting System .......................... 12

2.1.1. Culture of Acartia tonsa and Tisbe biminiensis .............................................. 12

2.1.2 Culture Chamber ............................................................................................. 13

2.2 Materials and Methods ................................................................................................ 17

2.2.1 Chemicals ........................................................................................................ 17

2.2.2 Research Organisms ........................................................................................ 17

iv

2.2.3 Algae ............................................................................................................... 17

2.3 Test Procedures ............................................................................................................ 17

2.3.1 Collection of Eggs ............................................................................................ 17

2.3.2 Chemical Preparations .................................................................................... 18

2.3.3 Toxicity Testing ............................................................................................... 19

2.4 Analytical Chemistry ..................................................................................................... 21

2.4.1 AN2: 4,4’-methylenebis(2,6-di-tert-butylphenol) .......................................... 21

2.4.2 DCP: 3,5-dichlorophenol ................................................................................. 23

2.4.3 5-FU (5-fluorouracil) ....................................................................................... 25

2.5 Data Analysis ................................................................................................................ 26

CHAPTER 3 RESULTS ...................................................................................................................... 27

3.1 Culturing and Egg Collecting Chamber ......................................................................... 27

3.1.1 Culture Chamber ............................................................................................. 27

3.1.2 Egg Collection Method .................................................................................... 29

3.2 Toxicity Test Results ..................................................................................................... 29

3.2.1 3,5-Dichlorophenol ......................................................................................... 30

3.2.2 4,4’-methylenebis(2,6-di-tert-butylphenol) ................................................... 32

3.2.3 5-Fluorouracil .................................................................................................. 34

3.3 NOEC and LOEC Data .................................................................................................... 36

3.3.1 3,5-dichlorophenol .......................................................................................... 37

3.3.2 4,4’-methylenebis(2,6-di-tert-butylphenol) ................................................... 37

3.3.3 5-fluorouracil .................................................................................................. 38

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3.4 LC50 Data ...................................................................................................................... 39

CHAPTER 4 DISCUSSION AND CONCLUSION ................................................................................. 41

4.1 Introduction .................................................................................................................. 41

4.2 Culturing and Egg Collecting Methods ......................................................................... 42

4.3 Results .......................................................................................................................... 42

4.4 Compounds .................................................................................................................. 43

4.5 Conclusion .................................................................................................................... 44

REFERENCES .................................................................................................................................. 46

vi

LIST OF TABLES

Page

Table 1 DCP properites (Sigma Aldrich, 2014) ........................................................................... 6

Table 2 4,4’-methylenebis(2,6-di-tert-butylphenol) properties (Sigma Aldrich, 2014) ............. 7

Table 3 5-FU properties (Sigma Aldrich, 2014) .......................................................................... 9

Table 4 Culture conditions for Acartia tonsa and Tisbe biminiensis ........................................ 13

Table 5 Specifics on numbered parts of egg collecting chamber............................................. 15

Table 6 Container conditions for the 48 hour toxicity test of Acartia tonsa ........................... 21

Table 7 Liquid chromatography and mass spectrometer conditions for 4,4’-methylenebis(2,6-di-tert-butylphenol) ................................................................ 22

Table 8 Liquid chromatography and mass spectrometer conditions for 3,5-dichlorophenol ...................................................................................................... 24

Table 9 Culture conditions for 60 liter glass fish tanks for the species Acartia tonsa and Tisbe biminiensis ......................................................................................................... 27

Table 10 3,5-dichlorophenol, test 1 survivor data .................................................................... 31

Table 11 3,5-dichlorophenol, test 2 survivor data .................................................................... 31

Table 12 4,4’-methylenebis(2,6-di-tert-butylphenol), test 1 survivor data .............................. 33

Table 13 4,4’-methylenebis(2,6-di-tert-butylphenol), test 2 survivor data .............................. 33

Table 14 5-Fluorouracil test 1 survival data .............................................................................. 35

Table 15 5-Fluorouracil test 2 survival data .............................................................................. 35

Table 16 Test 1 for 3,5-dichlorophenol on Acartia tonsa ......................................................... 37

Table 17 Test 2 for 3,5-dichlorophenol on Acartia tonsa ......................................................... 37

Table 18 Test 1 for 4,4’-methylenebis(2,6-di-tert-butylphenol) on Acartia tonsa ................... 37

Table 19 Test 2 for 4,4’-methylenebis(2,6-di-tert-butylphenol) on Acartia tonsa ................... 38

vii

Table 20 Test 1 for 5-fluororacil on Acartia tonsa .................................................................... 38

Table 21 Test 2 for 5-Fluororacil on Acartia tonsa .................................................................... 39

Table 22 Median lethal dose concentration (LC50) .................................................................. 39



viii

LIST OF FIGURES

Page

Figure 1 Molecular structure of 3,5 dichlorophenol ................................................................. 6

Figure 2 Molecular structure of 4,4’-methylenebis(2,6-di-tert-butylphenol) ........................... 7

Figure 3 Molecular structure of 5-Fluorouracil (Brayfield, 2013) .............................................. 9

Figure 4 Culture tank and egg collecting apparatus ................................................................. 14

Figure 5 Filter designed for egg collecting system ................................................................... 16

Figure 6 Egg collecting cone made of 60 micron mesh ............................................................ 16

Figure 7 4,4’-methylenebis(2,6-di-tert-butylphenol) LC-MS/MS chromatogram .................... 23

Figure 8 3,5-dichlorophenol LC-MS/MS chromatogram .......................................................... 25

Figure 9 Test 1 graphical analysis ............................................................................................. 32

Figure 10 Test 1 graphical analysis ............................................................................................ 34

Figure 11 Test 1 graphical analysis ............................................................................................ 36

ix

CHAPTER 1

INTRODUCTION

1.1 Background

1.1.1 Importance of the Mass Culturing of Acartia tonsa and Tisbe biminiensis

Copepods are a valuable research organism for acute toxicity testing, as most species of

copepods are the main nutritional source for larval fish, crustaceans, sea horses and

invertebrates (Stottrup et al. 1986). Copepods are a group of small crustaceans found in the sea

and nearly every freshwater habitat, causing them to be of particular interest (Dürbaum and

Künnemann, 1997). It is important for scientists to know the toxicity of anthropogenic

contaminants on these organisms, as death to them can have a further impact on many other

aquatic organisms along the food web. While there has been a good deal of research on a

number of copepod species with various contaminants, an elaborate protocol for culturing any

species has not yet been validated, the majority of researchers order copepods by an age range

from larger stock centers. Not only is each order of copepods $50-100 (depending on the

species) for a bulk bag of approximately 1,000 organisms, but the exact age of the copepod is

unknown, as they are sent by age group. The International Organization for Standardization’s

(ISO) 14669 protocol is the only guideline for the culturing and determination of acute toxicity

in the copepod species, Acartia tonsa and Tisbe biminiensis. It states that copepods are to be

tested in the adult stage (stage 5). The ISO states that a series of sieves are to be used to collect

and separate copepods of different sizes, assuming that adults will be the largest. While this

assumption is understood, the concern is that all of the copepods may not be of the same

actual age, even though they are the same size. Differences in age for testing species can lead

1

to invalid toxicity results, as different ages of copepods may be more or less sensitive to

contaminants. Medina et al. found that copepods of the naupliar stages are up to one order of

magnitude more sensitive than adult copepods to various contaminants. With this knowledge,

it is apparent that collecting eggs and staging them to be hatched would be a more efficient

protocol. The current method for collecting eggs is the same as for adults. A small micron sieve

is used to separate the nauplii and adult copepods from the eggs. Copepods and their eggs are

extremely small and fragile organisms, and the risk of injury or death while being poured over

metal sieves is high. Gorbi et al. uses a different method to collect eggs, however it is quite

labor intensive. 20 male and 30 female copepods are placed in 200 milliliter petri dishes where

reproduction is anticipated to take place. Researchers then collect spawned eggs under a

dissecting microscope to be used in experimentation, a very tedious task.

Copepods, a part of the phylum Arthropoda, are very small invertebrates ranging in size

from microscopic to about 0.6 centimeters (Cervetto et al. 1995). The species live in virtually all

marine and freshwater habitats. Some species are planktonic, benthic, and some live in

swamps, springs, bogs, ponds or water-filled recesses (Boxhall and Defaye, 2008). Copepods eat

a diet of plant and animal plankton, and are considered to be one of the most abundant species

of animal on Earth, competing only with krill. Copepods are a key link in aquatic food webs,

representing an important, often dominant, member of the zooplankton community (Gorbi et

al. 2012). The two species of copepod used in this experiment were Acartia tonsa and Tisbe

beminiensis, both marine animals. A. tonsa are pelagic calanoid copepods distributed

worldwide; occurring in the Atlantic, Indian and Pacific oceans, and the Azov, Baltic, Black,

Caspian and Mediterranean seas (Marcus and Wilcox 2007). A. tonsa is the primary food source

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for most larval fish and crustaceans, seahorses and invertebrates such as corals (Stottrup et al.

1986). Tisbe biminiensis is an epibenthic harpacticoid copepod that lives on bottom substrates

such as rock or sand (Araujo-Castro et al. 2009). T. biminiensis is found mainly in sandy

estuarine waters off beaches in Brazil (Sauza-Santos et al. 2006). The adults are the primary

food source of bottom feeding fish such as gobies, dragonettes and blennies. Both species of

copepod meet the practical criteria suggested by Raisuddin et al.; ecological relevance,

sensitivity, wide distribution, possibility for laboratory culturing, and high egg production.

The use of copepods, as a species, is gradually becoming more important as the amount of

drilling in the North Sea increases. The North Sea, specifically the Norwegian Sea, is being

drilled for the liquid oil and natural gas, produced from the oil reservoirs beneath the sea.

While thousands of different marine species are found in this aquatic environment, copepods

are popular as a research organism because they are considered a sentinel species. Sentinel

species are animals used to detect (or test) risks to humans or other animals higher in the food

web. Sentinel animals are typically more susceptible or see a greater exposure to a particular

hazard that other organisms. Copepods are considered sentinel species, as they are more

sensitive to many compounds than other aquatic life.

Per the ISO 14669, sexually mature adults of all the same age, about 15-17 days old, are

ideal for toxicity testing. Maintaining a stocked culture of copepods, in house, would extremely

cut down on costs of ordering organisms each time the animals are needed. As well, creating a

low risk, high yield egg collecting system would allow for a more efficient way of collecting eggs.

Once collected, copepod eggs can be set to hatch, so the exact age of the animal will be known.

3

Maintaining a stock culture, combined with a well-developed way to collect eggs will create a

much more productive, accurate, and feasible approach to copepod toxicity testing.

The culturing of specific species of copepods may also be useful in aquaculture for farmed

fish. For example, A. tonsa is the primary food source for the larvae of golden and red snapper,

both popular fish in the human diet (Schipp et al. 1999). Many hatcheries collect adult A. tonsa

directly from the ocean as needed. Hatcheries run into problems doing this, as seasonal

declines of copepods are common due to monsoonal rains (Schipp et al. 1999). Also, with the

human population of the Earth increasing, there is a constant increase in the demand from

fisheries, therefor, a constant increase in the amount of copepods needed to sustain larval fish

cultures. An elaborate protocol for maintaining copepod cultures would also be extremely

valuable for hatcheries.

1.2 Phenols

Phenol is one of the most ubiquitous organic pollutants, and is considered a major pervasive

contaminant in aquatic environments because it is chemically stable, water soluble and

environmentally mobile (Barber et al., 1995). Phenol is produced naturally as a constituent of

coal tar and creosote, decomposing organic material, human and animal wastes, and as a

compound found in many non-foods and foods. As a manufactured compound, phenol is used

in the production of many consumer goods such as paints, varnishes, enamels, preservatives,

lubricants, disinfectants, herbicides, and pharmaceuticals (Dow Chemical Company, 2007).

Additional anthropogenic sources of phenol are from manufacturing industries such as resins,

plastics, fibers, adhesives, iron, steel, aluminum, leather, rubber, paper pulp mills and wood

treatment facilities (Ucisik and Trapp, 2006). Phenol is a high volume chemical with production

4

exceeding 6-billion pounds world wide and 3-billion pounds annually in the United States.

(Chemical:Phenol). Once these phenols are released from the industrial plants, they enter the

surrounding surface waters (streams, creeks, rivers, etc.) and ultimately deposit into the ocean

in estuarine areas, where they can further disperse. The Center for Disease Control (CDC) has

reported measured amounts of phenol in wastewater effluents (up to 53 ppm) and

groundwater (1.9->10 ppb), however the amount in drinking water is not quantified. Phenols

can also be released by the combustion of wood and auto exhaust and by the natural

degradation of organic wastes, including benzene (Wallace et. Al, 1999). Phenol is classified by

the Occupational Safety and Health Administration (OSHA) as a ‘hazardous chemical’ because of

its toxic effects on living organisms. These toxic effects are concerning, because the majority of

things in our daily lives are, in some way, constructed of phenols.

Phenols enter the surface and ground water from industrial effluent discharges (Sreejith et

al., 2014). Although phenol is degraded rapidly in air, it may persist in water for a somewhat

longer period; with half-lives ranging up to nine days in estuarine and marine water (CDC).

Biodegradation may be slow in cases of high concentrations of phenol (or other chemicals), or

by other factors such as lack of nutrients or microorganisms capable of its degradation. The

ecotoxicological action of phenol is related to the damage it does to biological membranes by

hydrophobic interaction with lipid bilayer structures (Sreejith et al., 2014). Phenol’s reactivity

with biomolecules is related to the ease with which it donates free electrons to oxidize

substrates and the oxidative stress is causes by the formation of free radicals and reactive

oxygen species (Michalowicz and Duda, 2007). Lipid peroxidation of cell membranes occurs

when highly reactive phenolic compounds quickly undergo such radical reactions (Sreejith et al.,

5

2014). Phenols have been found to penetrate the internal spaces of cells and damage the

membranes of the endoplasmic reticulum, mitochondria, nuclei, and other components like

nucleic acids and enzymes (Hayashi et al., 1999). The two phenols that were investigated in this

experiment are 3-5-dichlorophenol (DCP) and 4,4’-methylenebis(2,6-di-tert-butylphenol) (AN2).

1.2.1 3-5-dichlorophenol

Figure 1. Molecular structure of 3,5-dichlorophenol

Table 1. DCP properites (Sigma Aldrich, 2014)

DCP 3,5-dichlorophenol Water Solubility 5,380 mg/L @ 25°C

Molecular Weight 163.00 g/mol Formula C6H4Cl2O Storage Dark and dry

3-5-dichlorophenol (DCP) is used as an intermediate in the manufacture of more

complex chemical compounds, like fungicides and herbicides like 2,4-Dichlorophenoxyacetic

acid (2,4-D). As such, they are commonly released into the waterways by various industrial

waste streams (PubChem: 3,5-dichlorophenol).

6

1.2.2 4,4’-methylenebis(2,6-di-tert-butylphenol)

Figure 2. Molecular structure of 4,4’-methylenebis(2,6-di-tert-butylphenol).

Table 2. 4,4’-methylenebis(2,6-di-tert-butylphenol) properties (Sigma Aldrich, 2014)

AN2 4,4’-methylenebis(2,6-di-tert-butylphenol) Water Solubility 3.99 mg/l at 25°C

Molecular Weight 424.66 g/mol Formula CH2[C6H2[C(CH3)3]2OH]2 Storage Dark and dry

4,4’-methylenebis(2,6-di-tert-butylphenol) (AN2) is used as a UV stabilizer and an

antioxidant for hydrocarbon based products ranging from petrochemicals to plastics (PubChem:

2,6-di-tert-butylphenol). In water, AN2 will absorb to sediment and particulate matter, volatilize

from the surface (P-Tert-Butylphenol. 2008), bioaccumulate in aquatic organisms, and then

oxidize in the water column (PubChem: 2,6-di-tert-butylphenol).

1.3 Antifolates

Other common chemicals found in waterways are antifolates. Antifolates are a very

common form of treatment in cancer chemotherapy, rheumatoid arthritis, lupus, asthma,

psoriasis, scleroderma, IBS, spinal fluid leukemia and sarcoma (Takimoto, 1996). Antifolates

were the first class of antimetabolites to enter the medical field over 65 years ago (Visentin et

7

al. 2012). Antimetabolites are chemicals that inhibit the use of a metabolite, a biological

chemical part of normal metabolism (Smith 1997). Antifolates specifically impair the function of

folic acids, and in many cases are dihydrofolate reductase (DHFR) inhibitors (McGuire, 2003).

DHFR inhibitors bind and inhibit the enzyme dihydrofolate reductase, preventing the formation

of tetrahydrofolate, which is essential for purine and pyrimidine synthesis (McGuire, 2003). The

deficiency of tetrahydrofolate leads to an inhibited production of DNA, RNA and proteins

(Morgan and Baggott 1995). Antifolates enter the waters via human excretion. When used as a

therapeutic, a portion of the drug is not used in the body, therefore, a small amount of the

active drug and its metabolites are excreted from the body in the form of urine and feces. This

excretion is brought to the waste water treatment plant, where the drug is not effectively

extracted from the water, and thus found in the effluent of the water treatment center. In the

United States, the actual concentrations of 5-FU in the environment are still limited, however a

recent paper by J.O. Straub summarized evaluated 5-FU concentrations in wastewater and

surface water in Europe. To be expected, the highest measured 5-FU concentrations were

found in wastewater effluent produced by oncological wards and general hospital wastewater

at 122 and 2.03 µg/L, respectively (J.O. Straub, 2009). The concentration of 5-FU recorded in

the effluent of municipal wastewater (a few ng/l – 673 ng/l) and surface water (1 ng/l) is much

lower than what is found in the effluent from water treatment centers connected to

oncological wards (J.O. Straub, 2009). The effluent of most treatment centers enters small

creeks and rivers, potentially leading to larder bodies of water, such as lakes. The antifolate

being investigated in this experiment is 5-fluoruracil (5-FU), commonly known as Capcitabine or

Xeloda.

8

1.3.1 5-Fluorouracil

Figure 3. Molecular structure of 5-Fluorouracil (Brayfield, 2013).

Table 3. 5-FU properties (Sigma Aldrich, 2014).

5-FU 5-Fluorouracil Water Solubility 12.2 g/L @ 20°C

Molecular Weight 130.08 g/mol Formula C4H3FN2O2 Storage Dark and dry

5-FU is the most common antifolate used in clinical practices to treat colorectal, anal,

breast, esophageal, stomach, pancreatic and skin cancers (Takimoto, 1996). Capcitabine, it’s

pro-drug, is an orally administered chemotherapeutic agent used in the treatment of numerous

cancers, mainly colorectal (Rossi 2013). Capcitabine is metabolized, in the body, to 5-FU

(Koukourakis et al. 2008). 5-FU is a thymidylate synthase (TS) inhibitor, which interrupts the

action of the enzyme thymidylate synthase, blocking the synthesis of the pyrimidine thymine, a

nucleoside required for DNA replication (McGuire, 2003).

9

1.4 Rationale for Study

A robust culturing method for species of copepods will cut the down the cost of ordering

and shipping animals for different practices. This study proposes a standardization method for

the culturing of two species of copepods, including a method for mass egg collection. The

experiment also follows (and adds to) a method per the ISO 14669, for an acute toxicity test (24

and 48 hour). Toxicity tests are essential tools in the biomonitoring and risk evaluation of

contaminants released in the environment. The results of acute toxicity tests are often used to

set environmental ecological standards.

1.4.1 Objectives and Hypotheses

Objective 1: To assess the feasibility of establishing and maintaining cultures of two species of

copepod: Acartia tonsa and Tisbe biminiensis, as well as to refine current culturing and egg

separation methods.

Hypothesis 1: HO: Building a culture chamber with an accompanied airlift system will not

facilitate a more efficient way to separate and collect copepod eggs, nor decrease the

effort and cost of using copepods for toxicity testing.

Objective 2: Discover the acute toxicity (LC50), no observed effect concentration (NOEC) and

lowest observed effect concentration (LOEC) of 3,5-dichlorophenol on the copepod species,

Acartia tonsa and Tisbe biminiensis. Recorded after 24 and 48 hours.

Hypothesis 2: HO: I hypothesize that 3,5-dichlorophenol will not cause a toxic effect to

the copepod species, Acartia tonsa and Tisbe biminiensis.

10


lowest observed effect concentration (LOEC) of 4,4’-methylenebis(2,6-di-tert-butylphenol) on

the copepod species, Acartia tonsa and Tisbe biminiensis. Recorded after 24 and 48 hours.

Hypothesis 3: HO: I hypothesize that 4,4’-methylenebis(2,6-di-tert-butylphenol) will not

cause a toxic effect to the copepod species, Acartia tonsa and Tisbe biminiensis.


lowest observed effect concentration (LOEC) of 5-fluorouracil on the copepod species, Acartia

tonsa and Tisbe biminiensis. Recorded after 24 and 48 hours.

Hypothesis 4: HO: I hypothesize that 5-fluorouracil will not cause a toxic effect to the

copepod species, Acartia tonsa and Tisbe biminiensis.

11

CHAPTER 2

MATERIALS AND METHODS

2.1 Development of Novel Culture Chamber and Egg Collecting System

2.1.1. Culture of Acartia tonsa and Tisbe biminiensis

Both A.tonsa and T. biminiensis reproduce sexually, however eggs of A. tonsa are

spawned freely into the water column where they typically sink (Gorbi et al. 2012), while eggs

of T. biminiensis are deposited in an egg sac attached to female genital segment and then

released before hatching (Araujo-Castro et al. 2009). A. tonsa are reported to lay about 10-20

eggs per brood (Gissi et al. 2013), while T. biminiensis are reported to lay about 10-70 eggs per

brood (Algagen LLC). The following experiments were conducted on copepods that were bred in

the laboratory, and each species was cultured completely separate from one another.

Copepods were cultured in 60 liter glass aquaria containing artificial sea water, using Instant

Ocean (www.InstantOcean.com) prepared and maintained at a salinity of 30-35 parts per

trillion. The aquaria are maintained at 20°C ± 2 via a climate controlled room, and a 16 hour

light and 8 hour dark cycle is applied. The pH is maintained at 8.0 ± 0.3. Each aquaria received

light aeration by an air stone lowered to the middle of the tank. About a handful of the salt

water algae, Chaetomorpha, was added to each aquaria, as the algae proved to be crucial for

the mass culturing of both species in the laboratory. The medium in the aquaria was renewed

once a week by siphoning approximately 80% of the original volume through 55 micron mesh,

and replacing with fresh salt water. Copepods in all aquaria are fed one and a half tablespoons

of Phycopure Copepod Blend (www.liveaquaria.com) once a day. The blend contains two

12

http://www.instantocean.com/http://www.liveaquaria.com/

species of microalgae: T- Isochrysus galbana, and Chaetoceros gracilis, the main diet for both A.

tonsa and T. biminiensis. A tabular form of the culture parameters can be found in Table 4.

Table 4. Culture conditions for Acartia tonsa and Tisbe biminiensis.

Culture incubation condition Temperature 20°C d 2 pH 8.0 ± 3 Salinity 30-35 ppt Dissolved oxygen 8-9 mg/L Photoperiod 16 h/8 h light/dark Nitrates/nitrites 0 ppm Aeration Light- placed in middle of tank Water renewal Once/week Extra Chaetomorpha added Feeding Once a day (Phycopure Copepod blend)

2.1.2 Culture Chamber

The construction of the egg collecting chamber was created on the basis of an airlift

system, and was built to fit the needs of this experiment. A 60 liter glass fish tank with a 1 inch

diameter hole drilled in one of the long sides was used as the larger housing chamber for the

airlift system. A one inch bulk head was fit into the drilled hole, where elbow PVC piping was

attached to the bulkhead on the outer side of the tank. Additional PVC was attached to follow

the tank up the side and over the top. An air stone was inserted through a hole drilled in the

elbow PVC coming from the bulkhead. The bubbles from the air stone create suction; pulling

water from the tank, through the PVC and up the side of the tank, where the water is deposited

back into the chamber. This essentially creates a system similar to what a deep well would use:

air is forced into the lower part of a pipe which transports the liquid upwards. By buoyancy, the

air, which has a lower density than the liquid, rises quickly. By fluid pressure, the liquid is taken

in the ascendant air flow and moves in the same direction as the air. Figure 4 illustrates the

13

http://en.wikipedia.org/wiki/Buoyancyhttp://en.wikipedia.org/wiki/Densityhttp://en.wikipedia.org/wiki/Fluid_pressure

setup of the large egg collecting chamber. Parts are numbered and listed in Table 5 with their

measurements and uses.

Figure 4. Culture tank and egg collecting appartatus. Explanation of numbered parts can be found in Table 5.

1

14

Table 5. Specifics on numbered parts of egg collecting chamber.

Part number Part specifics 1. 60 liter glass aquarium. One inch diameter whole drilled into one of the

long sides, approximately 1.5 inches from bottom. 2. 1 inch PVC, 18 inches long. 3. 1 inch elbow PVC with one male and one female end. Hole drilled in one

side to fit tubing for air stone. 4. 1 inch elbow PVC with both female ends. 5. 1 inch bulkhead. 6. Tubing connected to air stone inside part number 3. Other end of tubing

was attached to air pump. 7. 1 inch T shaped PVC to hang collection cone from.

In order to create the egg collecting portion of the chamber, a filter (Figure 5) was made

out of 3 inch PVC, approximately 5 and ¾ inches long. Four rectangles, evenly spaced,

measuring 1 inch by 4 inches are cut into the 3 inch PVC, and covered with 100 micron mesh.

100 micron mesh was used because only eggs are able to fit through. A flat piece of plastic is

glued to one end of the PVC, while a 1 and ½ by 3 inch converting pipe is attached to the other

end. A threaded one inch PVC is attached so the filter can be screwed into the bulkhead on the

inside of the tank. This filter is attached to the bulk head on the inside of the tank. A cone

(Figure 6) made of 60 micron mesh was created to catch the eggs at the top of the airlift, but

still allow water to flow through and recirculate. When the air stone is turned on, the bubbles

from the stone create suction, pulling water through the filter inside the tank. Eggs accompany

the water through the filter, as adults and nauplii cannot fit through the 100 micron mesh. As

the water pours out of the top of the airlift system, the 60 micron mesh cone catches all of the

copepod eggs, while the tank water filters through.

15

Figure 5. Filter desinged for egg collecting system.

Figure 6. Egg collecting cone made of 60 micron mesh, with wire loop to hang collection cone from T-shaped PVC. This cone is used to catch the eggs, while allowing water to filter through. A small hole is made at the bottom of the cone where a clip is applied to hold the eggs in until it is time to transfer.

16

2.2 Materials and Methods

2.2.1 Chemicals

AN2 (4,4’-methylenebis(2,6-di-tert-butylphenol), Cat# 277924-100G, CAS# 118-82-1),

DCP (3,5-dichlorophenol, Cat# D70600-10G, CAS# 591-35-5), 5-FU (5-fluorouracil, Cat# F6627-

1G, CAS# 51-21-8), and EB4 (2,2’-ethylidene-bis(4,6-di-tert-butylphenol, Cat#372137-250G,

CAS# 35958-30-6), were all obtained from Sigma-Aldrich Corporation (St. Louis, MO). Milli-Q

water was obtained from in house Milli-Q water system (Millipore, Billerica, MA). HPLC grade

methanol, ammonium formate and dichloromethane were obtained from Fisher Scientific

(Houston, TX).

2.2.2 Research Organisms

To start the mass culture of both Acartia tonsa and Tisbe biminiensis, two “bulk” bags of

animals were ordered from Algagen LLC (www.liveaquaria.com). Each bulk bag contained

approximately 1,000 animals of mixed sex. The animals were housed in a climate controlled

room in the aquatic toxicology laboratory at the University of North Texas.

2.2.3 Algae

The salt water algae, Chaetomorpha, was added to the culture aquarium, as the

copepods and the algae seem to have a symbiotic relationship. Chaetomorpha was purchased

from Reef Remedies in Denton, Texas (www.reefremedies.com).

2.3 Test Procedures

2.3.1 Collection of Eggs

Each species of copepod was left to fully acclimate to their culture chambers for 3

weeks before egg collecting was performed. The egg collection apparatus was run for

17

http://www.liveaquaria.com/

approximately two hours (for both species) to ensure efficient turnover of the tank water. After

the two hours, the air pump was turned off and the egg collecting cone was removed from the

system; the contents rinsed with fresh salt water into a 50 milliliter conical tube. To perform

this task, a squirt bottle filled with salt water was used in order to apply a gentle stream of fluid.

While carefully holding the collection cone over the conical tube, the clip is removed. Then a

gentle stream of salt water is applied by squirting the inside of the cone. This should rinse all of

the collected eggs into the conical tube for storage. Placing the eggs in a 4°C refrigerator keeps

the eggs dormant until they are ready to be used.

Hatching tanks were set up to create a suitable hatching environment for copepod eggs.

All parameters used for culture tanks were maintained in the hatching tanks. Eggs were poured

into the 30 milliliter beakers, which were used as “housing” for the fragile eggs. Then,

Chaetomorpha was placed on top the eggs to form a buffer between the eggs and the

environment of the hatching chamber. The beakers were then submerged, on their side, into a

4-liter glass fish tank filled with clean artificial salt water, prepared to 30-35 parts per trillion.

Light aeration was applied to the hatching tank to not disturb the eggs or nauplii. Copepods are

left in hatch tanks until they are 15 days old and ready for toxicity testing.

2.3.2 Chemical Preparations

A master stock of 100 mg/l was made of all three chemicals. This was achieved by

dissolving 25 mg of each compound in 250 milliliters of artificial salt water. The master stock of

3,5-dichlorophenol was placed in a sonicator for 60 minutes in order to fully dissolve

compound. The master stock of 4,4’-methylenebis(2,6-di-tert-butylphenol) was also placed in

the sonicator for 60 minutes, then on a stir plate for 30 minutes, followed by another 60

18

minutes of sonication. The master stock of 5-fluorouracil was placed on a hot plate, held at

70°C, for 30 minutes to fully dissolve the compound.

Once all compounds were fully dissolved in the salt water, dilutions were made to equal

the dosing concentrations. The dosing concentrations (in mg/l) for all compounds were kept the

same through the experiment, and are as follows: 0, 0.010, 0.032, 0.10, 0.32, 1.0, 3.2, and 10.

Dilutions were made following the equations below.

(1) Control – pure artificial salt water.

(2) 0.010 mg/l – 20 µl of 100 mg/l master stock + 199.98 ml of artificial salt water.




(6) 1.0 mg/l – 2,000 µl of 100 mg/l master stock + 198 ml of artificial salt water.

(7) 3.2 mg/l – 6,400 µl of 100 mg/l master stock + 193.6 ml of artificial salt water.

(8) 10 mg/l – 20 ml of 100 mg/l master stock + 180 ml of artificial salt water.

2.3.3 Toxicity Testing

Toxicity tests were set up following the guidelines listed in the ISO 14669, which

describes a method for the determination of acute lethal toxicity to marine copepods. A

preliminary test to determine the range of concentrations for each chemical to be tested in the

definitive test was performed, giving an approximate value for the definitive 48 hour LC50. The

preliminary results gave LC50’s of 0.13 mg/l and 0.12 mg/l for A. tonsa and T. biminiensis,

respectively for DCP. Because the LC50’s for the two species were so close, a decision to just

use the species, A. tonsa, for the definitive toxicity test was made. The toxicity test took place in

19

a temperature and humidity controlled room held at 20°C ± 2 under a 16 hour light and 8 hour

dark photoperiod. A total of three replicates was used for each concentration, where 10

copepods per replicate was tested. Each test series was performed twice. Equal volumes of the

test solutions were placed into the containers with a volume that facilitates 1 copepod per 0.5

milliliter of solution. The dissolved oxygen concentration, pH, temperature and salinity was

measured twice per day during the toxicity test. Addition of food to test animals is avoided, as it

can introduce an additional route of exposure to the organisms (both water and diet-borne)

and can change the bioavailability of contaminants by altering water chemistry (Pinho et al.,

2007). Water samples for every concentration was taken and analyzed using liquid

chromatography mass spectrometry (LC-MS/MS) once during the toxicity test to ensure validity

of chemical concentrations. After both the 24 and 48 hour time period, the amount of living

and dead copepods was viewed under a low powered dissecting microscope. Those showing no

swimming or appendage movements were considered dead. All abnormal appearance or

behavior was also recorded and compared with control animals. Tabular form of toxicity testing

parameters can be found in Table 6.

20

Table 6. Container conditions for the 48 hour toxicity test of Acartia tonsa.

Toxicity test chamber condition Test type Static, non-renewal Test duration 48 hours Temperature 20 °C ± 2 Test chamber size 30 mL glass beakers Test solution volume 30 mL Age of test organism 15-17 days No. of organism per test chamber 10 No. of replicate test chambers per concentration 3 No. of organisms per concentration 30 pH 8.0 ± 3 Salinity 30-35 ppt Dissolved oxygen 8-9 mg/L Photoperiod 16 h/8 h light/dark Nitrates/nitrites 0 ppm Aeration none Feeding regimen none

2.4 Analytical Chemistry

2.4.1 AN2: 4,4’-methylenebis(2,6-di-tert-butylphenol)

A one milliliter aliquot of each concentration was taken and analyzed using LC/ESI-/MS

to validate dosing concentrations. The samples underwent liquid/liquid extraction using one

milliliter of dichloromethane (DCM). The layer of DCM was removed and gently dried under

nitrogen and the residue was reconstituted to a final volume of one milliliter methanol to fix

the final concentration, however, both the 10 mg/ml (ppm) and 3.2 mg/ml (ppm) dosing

samples were reconstituted in a volume of methanol so the final concentration was 1 ppm and

0.32 ppm, respectively. The dilutions of these two samples allowed for them to fall accurately

on the standard curve, as the curve spanned a range from 0.0078 ppm to 1 ppm. The AN2

acquisition method was created using selective ion monitoring for the parent ion (m-H)- of m/z

21

423.3 using a 7 minute isocratic mobile phase (5%:95% Milli-Q water:methanol). 2,2’-

ethylidene-bis(4,6-di-tert-butylphenol) (EB4) was used as the analytical internal standard (at 10

ppb final concentration). Analytes were analyzed using SIR with m/z 423.3 (m-H)- parent ion for

AN2 and m/z 438.7 (m-H)- for EB4. Parameters for this method are listed in Table 7.

Table 7. Liquid chromatography and mass spectrometer conditions for 4,4’-methylenebis(2,6-di-tert-butylphenol).

Liquid chromatograph conditions Column Sunfire C18-column (Waters #186002533), 50 mm

× 2.1 mm inner diameter (3.5µm stationary phase) Column temperature 30°C Eluent Solution A: Milli-Q Water

Solution B: Methanol Gradient conditions Time (min) A(%) B(%) 0.00 5 95 7.00 5 95

Flow rate 0.2 ml/min Amount injected 20 µl Mass spectrometer conditions Ionization method Electrospray negative ion mode (ESI-) Detection method Selective ion recording (SIR) Precursor ion m/z 423.3 (m-H)- parent ion Source temperature 145°C Desolvation temperature 175°C Capillary voltage -4.0 kV Cone voltage -60 V Low Mass/High Mass 1 2.5 Entrance/Exit 5 Low Mass/High Mass 2 2.5 Ion Energy 1/2 0.5

22

Figure 7. 4,4’-methylenebis(2,6-di-tert-butylphenol) LC-MS/MS chromatogram.

2.4.2 DCP: 3,5-dichlorophenol

A one milliliter aliquot of each concentration was taken and analyzed using LC/ESI-/MS

to validate dosing concentrations. The samples underwent the same liquid/liquid extraction

process as AN2 using one milliliter of dichloromethane (DCM). The layer of DCM was removed

and gently dried under nitrogen and the residue was reconstituted to a final volume of one

milliliter methanol to fix the final concentration. Just like in the AN2 method, both the 10

mg/ml and 3.2 mg/ml dosing samples were reconstituted in a volume of methanol so the final

concentration equaled 1 ppm and 0.32 ppm, respectively. The dilutions of these two samples

allowed for them to fall accurately on the standard curve. The DCP acquisition method was

created using multiple reaction monitoring for the parent ion (m-H)- of m/z 163.0 and daughter

ion (m-H)- of m/z 125.0 using a 35 minute method. 2,2’-ethylidene-bis(4,6-di-tert-butylphenol)

(EB4) was used as the analytical internal standard (at 10 ppb final concentration) with the

parent ion (m-H)- of m/z 438.7 and daughter ion (m-H)- of m/z 205. Parameters for this method

are listed in Table 8.

23

Table 8. Liquid chromatography and mass spectrometer conditions for 3,5-dichlorophenol.

Liquid chromatograph conditions Column Sunfire C18-column (Waters #186002533), 50 mm

× 2.1 mm inner diameter (3.5µm stationary phase) Column temperature 30°C Eluent Solution A: 0.1% Formate in Milli-Q Water

Solution B: 0.1% Formate in Methanol Gradient conditions Time (min) A(%) B(%) 0.00 70 30 2.00 70 30

20.00 0 100 25.00 0 100 30.00 70 30 35.00 70 30 Flow rate 0.2 ml/min Amount injected 30 µl Mass spectrometer conditions Ionization method Electrospray negative ion mode (ESI-) Detection method Multiple Reaction Monitoring (MRM) Precursor ion m/z 163.00 (m-H)- parent ion Source temperature 145°C Desolvation temperature 175°C Capillary voltage -4.0 kV Cone voltage -60 V Low Mass/High Mass 1 2.5 Entrance/Exit 5 Low Mass/High Mass 2 2.5 Ion Energy 1/2 0.5

24

Figure 8. 3,5-dichlorophenol LC-MS/MS chromatogram.

2.4.3 5-FU (5-fluorouracil)

Because 5-fluorouracil has a high polarity, the compound elutes too close to the solvent

front, even when the mobile phases contain little or no organic solvent, such as methanol or

acetonitrile. An analytical method using hydrophilic interaction liquid chromatography (HILIC)

(Pisano et al., 2005) would need to be used, which is unavailable at the study’s present facility.

The nominal concentrations for 5-FU was used to quantify the median lethal dose, no observed

effect concentration, and the lowest observed effect concentration because of the inability to

create an in house method.

25

2.5 Data Analysis

The median lethal dose (LC50) data for each compound is analyzed using the software

C.E. Stephan which calculates the LC50 value and 95% confidence interval by probit analysis.

The no observed effect concentration (NOEC) and the lowest observed effect concentration

(LOEC) are generated by post-analysis of variance (ANOVA) multiple comparison tests, using the

SAS created, JMP Software (www.jmp.com). Because each compound was tested twice, an

ANOVA was also run between the concentrations of each test to ensure that there was no

significant difference between the multiple tests for each compound.

26

CHAPTER 3

RESULTS

3.1 Culturing and Egg Collecting Chamber

3.1.1 Culture Chamber

Following multiple trials to discover the best lab culture optimization method for Acartia

tonsa and Tisbe biminiensis, a well-defined method for both species was feasibly found.

Through research and trial and error, a suitable environment was created, not only for adult

copepods, but also to sustain the successful reproduction of both species. The set parameters

for all culture media is found in Table 9.

Table 9. Culture conditions for 60 liter glass fish tanks for the species Acartia tonsa and Tisbe biminiensis.

Culture incubation condition Temperature 20°C ± 2 pH 8.0 ± 3 Salinity 30-35 ppt Dissolved oxygen 8-9 mg/L Photoperiod 16 h/8 h light/dark Nitrates/nitrites 0 ppm Aeration Light- placed in middle of tank Water renewal Once/week Extra Chaetomorpha added (handful) Feeding 1 ½ table spoon, once a day (Phycopure Copepod blend)

The feasibility to maintain and operate an in house culture chamber for the two species

of copepod, is respectable. During prior toxicity research in this laboratory, bulk bags of

copepods were ordered at the start of each toxicity test. To be specific, two bulk bags of

animals, ranging from $50 for A. tonsa to $100 for T. biminiensis each, would be shipped to the

lab from Algagen LLC. This totals $100-200, depending on the species being shipped and the

27

availability of the species of interest. Chaetomorpha was also purchased each time a new batch

of animals was shipped, as the algae could not be recycled from mass stocks in order to prevent

any type of contamination. In my personal experience, this was performed 8 times over the

course of a single set of toxicity tests, totaling approximately $880 for A.tonsa work and $1680

for T. biminiensis for animals and algae. This cost does not include the price of salt to make

artificial salt water, water quality instruments, or any lab equipment used during

experimentation. Through the developmental process, it was found that the creation and

perfection of a culture chamber for the species A. tonsa and T. biminiensis decreased this

overall cost by a factor of 8. The total to originally start a mass culture per species ranged from

$110 for A. tonsa to $210 for T. biminiensis. Again, this cost does not include the price for salt

for artificial saltwater, water quality instruments, or lab equipment, as the cost for these

essentials do not vary between each condition.

All of the animals used in the toxicity tests came from the mass stock culture,

eliminating the need to order animals a second time throughout the duration of this

experiment. Constructing and using an in house culture cuts down on many of the inevitable

obstacles that come along with ordering and shipping animals. For one, there is always a risk of

mortality when animals (of any kind) are being shipped because of the added stress of not only

the action of transport, but also being uprooted and transferred from the organism’s

environment they are accustomed to. In house stock cultures eliminate this and the need to

acclimate the animals to the new environment, as acclimation itself is very time consuming and

can also cause a large degree of mortality.

28

3.1.2 Egg Collection Method

Construction of the egg collecting apparatus is extremely important for the precision of

copepod toxicity testing. Not only is the apparatus easy to run and maintain, but it significantly

cuts down the amount of manual labor needed to efficiently collect eggs and decreases the risk

of injury and death to adults and eggs, alike. The apparatus increases the validity of toxicity test

results, as all of the copepods used during experimentation essential go through a controlled

hatch. Each nauplii hatch within 24 hours of each other, allowing copepods of known age to be

used during test protocols. Roughly 1,000 copepod eggs are collected in two hours each time

the apparatus is run (every 2 days). This is an estimate based off counting how many eggs were

found after suspending the collected volume (which was diluted in 50 milliliters of salt water),

sampling off one milliliter and counting the amount of eggs found in the sample. This was then

multiplied by 50 to represent the final collection volume of 50 milliliters. The discovery that

eggs have the ability to stay dormant for some time in a 4°C environment allows for the option

of back collection of eggs to use for larger experiments. Eggs can remain dormant for up to one

month. The animals used in the entirety of this experiment were eggs collected using the

constructed apparatus.

3.2 Toxicity Test Results

Two tests were performed for each compound containing three replicates per

concentration, per test in order to illustrate the success of the culturing and collection

apparatus and to gather accurate results for the 48-hour toxicity test. The median lethal dose

(LC50) data for each compound was analyzed using the software C.E. Stephan which calculates

the LC50 value and 95% confidence interval by probit analysis. The no observed effect

29

concentration (NOEC) and the lowest observed effect concentration (LOEC) were generated by

post-analysis of variance (ANOVA) multiple comparison tests.

The amount of survivors for each replicate in each concentration of 3,5-dichlorophenol,

4,4’-methylenebis(2,6-di-tert-butylphenol) and 5-fluorourcil was recorded at 24 and 48 hours.

Each beaker housed 10 live animals at the start of the toxicity test. The survivor count can be

found in Tables 10-15. An analysis of variance (ANOVA) showed that there was no significant

difference in mortality of Acartia tonsa per concentration between the two tests for each

compound. ANOVA’s are statistical models used to analyze the differences between group

means. Figures 9-11 show graphical analysis of the toxicity data, generated by Graphpad

Software, Inc. (San Diego, CA). The graphs illustrate the mortality of Acartia tonsa based on the

concentrations of the three compounds. Both tests for each chemical are depicted on the

graphs.

3.2.1 3,5-Dichlorophenol

Recorded survivors for 48 hour toxicity test using 3,5-dichlorophenol are displayed in

Tables 10 and 11. The table depicts the measured amount of DCP via LC-MS/MS for each

concentration. The control salt water also went through the same extraction method as all of

the other concentrations and was run on the LC-MS/MS showing that it was below the lower

limit of detection for the machine.

30

Table 10. 3,5-dichlorophenol, Test 1 survivor data.

24

Hours 48

Hours Concentration R1 R2 R3 R1 R2 R3

Control 10 10 10 10 10 9 0.012 mg/l 10 9 10 10 9 8 0.025 mg/l 9 9 8 7 6 6 0.109 mg/l 6 7 5 4 3 2 0.310 mg/l 4 4 3 2 1 1 0.766 mg/l 2 2 1 1 0 0 2.990 mg/l 0 1 0 0 0 0 10.09 mg/l 0 0 0 0 0 0

Per the tables, there is no difference in the amount of mortality seen in each

concentration for the two tests. Odd swimming behavior was seen in many of the copepods at

the 48 hour mark in the concentrations 0.025 mg/l, as well as 0.109 mg/l.

Table 11. 3,5-dichlorophenol, Test 2 survivor data.

24

Hours 48



31

The mortality of Acartia tonsa increased as the concentration of 3,5-dichlorophenol

increased, as can be seen in Figure 9A and B.

A). B).

Figure 9. A). Test 1 Graphical analysis of A. tonsa 48 hour mortality based on dose concentration of 3,5-dichlorophenol. B) Test 2 Graphical analysis of A. tonsa 48 hour mortality based on dose concentration of 3,5-dichlorophenol. Each concentration contained an n of 30 for each test.

3.2.2 4,4’-methylenebis(2,6-di-tert-butylphenol)

Recorded survivors for 48 hour toxicity test using 4,4’-methylenebis(2,6-di-tert-

butylphenol) are depicted in Tables 12 and 13. The table depicts the measured amount of AN2

via LC-MS/MS for each concentration. The control salt water also went through the same

extraction method as all of the other concentrations and was run on the LC-MS/MS showing

that it was below the lower limit of detection for the quantification machine.

32

Table 12. 4,4’-methylenebis(2,6-di-tert-butylphenol), Test 1 survivor data.

24

Hours 48


Control 10 10 10 9 10 9 0.006 mg/l 8 9 9 7 9 8 0.040 mg/l 9 7 8 7 6 6 0.087 mg/l 7 6 6 5 4 3 0.250 mg/l 6 5 6 2 3 2 0.960 mg/l 5 2 6 1 0 1 2.790 mg/l 4 5 3 1 0 0

13.111 mg/l 3 3 2 0 0 0


concentration for the two tests. It should be noted that many copepods in in the 0.040 mg/l

and 0.087 mg/l concentrations seemed to be dead at the 48 hour mark, however, when nudged

with a glass stir rod, they began to swim.

Table 13. 4,4’-methylenebis(2,6-di-tert-butylphenol), Test 2 survivor data.

24

Hours 48



33

The mortality of Acartia tonsa increased as the concentration of 4,4’-methylenebis(2,6-

di-tert-butylphenol) increased, as can be seen in Figure 10A and B.

A). B).

Figure 10. A). Test 1 Graphical analysis of A. tonsa 48 hour mortality based on dose concentration of 4,4’-methylenebis(2,6-di-tert-butylphenol). B) Test 2 Graphical analysis of A. tonsa 48 hour mortality based on dose concentration of 4,4’-methylenebis(2,6-di-tertbutylphenol). Each concentration contained an n of 30 for each test.

3.2.3 5-Fluorouracil

Recorded survivors for 48 hour toxicity test using 5-Fluorouracil are depicted in Tables

14 and 15. Nominal concentrations for 5-Fluorouracil were used instead of measured values

because of the inability to create a valid method using LC-MS/MS. 5-FU has too high of a

polarity causing the compound to elute too close to the solvent front, calculating an average

recovery of about 45% of the analyte. The table depicts the nominal amount of 5-FU for each

concentration. Although measured values are ideal, this research is a valuable source of

preliminary data for the toxicity of 5-FU on aquatic life.

34

Table 14. 5-Fluorouracil, Test 1 survival data.

24

Hours 48


Control 10 10 10 10 9 10 0.010 mg/l 10 9 9 8 7 6 0.032 mg/l 10 8 9 5 5 4 0.100 mg/l 9 8 8 2 3 3 0.320 mg/l 7 8 8 1 1 0

1.0 mg/l 6 7 8 1 0 0 3.2 mg/l 6 6 4 0 0 0 10 mg/l 4 2 3 0 0 0


concentration for the two tests. All of the living copepods at the end of the 24 hour testing

period seemed to be healthy, however, odd swimming behavior could be seen in copepods at

the 3.2 mg/l and 10 mg/l concentrations. At the 48 hour mark in the 0.032 mg/l concentration,

the living copepods appeared to be dead until they were nudged with a glass stirring rod, where

they then showed healthy swimming habits.

Table 15. 5-Fluorouracil, Test 2 survival data.

24

Hours 48


Control 10 9 10 10 9 9 0.010 mg/l 10 9 8 7 7 8 0.032 mg/l 8 9 9 4 5 6 0.100 mg/l 9 8 7 3 2 2 0.320 mg/l 8 7 6 2 0 0

1.0 mg/l 7 6 6 1 0 0 3.2 mg/l 6 4 3 0 0 0 10 mg/l 3 1 2 0 0 0

35

The mortality of Acartia tonsa increased as the concentration of 5-fluorouracil

increased, as can be seen in Figure 11A and B.

A). B).

Figure 11. A). Test 1 Graphical analysis of A. tonsa 48 hour mortality based on dose concentration of 5-Fluorouracil. B) Test 2 Graphical analysis of A. tonsa 48 hour mortality based on dose concentration of 5-Fluorouracil. Each concentration contained an n of 30.

3.3 NOEC and LOEC Data

The no observed effect concentration (NOEC) and the lowest observed effect

concentration (LOEC) are generated by post-analysis of variance (ANOVA) multiple comparison

tests. The graphs below show both the 24 hour and 48 hour results for NOEC and LOEC for each

compound. In aquatic toxicology, the NOEC is specifically the highest tested concentration of a

compound at which no adverse effect is found on the exposed organism. Concentrations higher

than the NOEC result in noticeable adverse effects. The LOEC is simply the lowest

concentration, or amount of a substance, found by experimentation that warrants an adverse

alteration of an organism’s health. These effects can be morphology, function, capacity, growth,

development or lifespan. In this experiment, specifically, mortality is used as the endpoint.

Most federal agencies use the NOEC and LOEC concentrations to set environmental

toxicological standards.

36

3.3.1 3,5-dichlorophenol

Below is the 48 hour toxicity test results for DCP on Acartia tonsa. At 0.006 mg/l, no

observed effect (NOEC) was seen. 0.026 mg/l was the lowest concentration where an observed

effect (LOEC) was seen. Test one is depicted in Table 16.

Table 16. Test 1 for 3,5-dichlorophenol on Acartia tonsa.

NOEC LOEC α p-value 24-H 0.027 mg/l 0.111 mg/l 0.05

The AN2 results in Test two gave a no observed concentration value of the control water

treatment, and is below the level of detection for the machine. The lowest observed effect

concentration of 0.007 mg/l. Results for Test two can be found in Table 19.

Table 19. Test 2 for 4,4’-methylenebis(2,6-di-tert-butylphenol) on Acartia tonsa.

NOEC LOEC α p-value 24-H 0.007 mg/l 0.029 mg/l 0.05 0.0255 48-H

Table 21. Test 2 for 5-Fluororacil on Acartia tonsa.

NOEC LOEC α p-value 24-H 0.1 mg/l 0.32 mg/l 0.05 0.0291 48-H 0 mg/l (Control) 0.01 mg/l 0.05 0.0126

3.4 LC50 Data

The median lethal dose concentration (LC50) represents the standard toxicity measure

of a compound in a medium that will kill half of the sample or research population of a species

in a specified period of time through a selected type of exposure. In the case of this

experiment, absorption would be the route of exposure in a 48 hour testing time period. Like

the NOEC and LOEC, the median lethal dose concentration is used to set environmental

toxicological standards.

Table 22. Median lethal dose concentration (LC50) results after 48 hour acute toxicity test for Test one and two for A. tonsa exposed to 3,5-dichlorophenol.

Test # Endpoint Concentration (mg/l) Lower 95% Upper 95%

1 24-h LC50 0.40 0.30 0.54 48-h LC50 0.13 0.09 0.18

2 24-h LC50 0.69 0.43 1.10 48-h LC50 0.13 0.09 0.18

The 48 hour LC50 for 3,5-dichlorophenol is 0.13 mg/l for test one, equal to the LC50

value of 0.13 mg/l, for test two. The 95% confidence intervals for both test one and test two are

0.09-0.18 mg/l.

39

Table 23. Median lethal dose concentration (LC50) results after 48 hour acute toxicity test for Test one and two for A. tonsa exposed to 4,4’-methylenebis(2,6-di-tert-butylphenol).


1 24-h LC50 5.09 3.32 8.34 48-h LC50 0.21 0.11 0.35

2 24-h LC50 2.55 1.62 4.00 48-h LC50 0.13 0.08 0.20

The 48 hour LC50 for 4,4’-methylenebis(2,6-di-tert-butylphenol) is 0.061 mg/l for test

one, compared to 0.046 mg/l for test two. The 95% confidence intervals are 0.036-0.095 mg/l

and 0.028-0.071 mg/l respectively.

Table 24. Median lethal dose concentration (LC50) results after 48 hour acute toxicity test for Test one and two for A. tonsa exposed to 5-fluorouracil.


1 24-h LC50 5.97 4.59 8.22 48-h LC50 0.08 0.04 0.13

2 24-h LC50 5.33 4.00 7.58 48-h LC50 0.08 0.04 0.13

The 48 hour LC50 for 5-Fluorouracil is 0.08 mg/l for test one, equivalent to the LC50 of

0.08 mg/l, for test two. The 95% confidence intervals for both test one and test two are 0.04-

0.13 mg/l.

40

CHAPTER 4

DISCUSSION AND CONCLUSION

4.1 Introduction

This study optimized and successfully applied a method to culture and efficiently collect

eggs for two marine copepod species, Acartia tonsa and Tisbe biminiensis. This research also

optimized and applied methods for an acute toxicity test. Alterations to previously found

culture conditions provided a reliable and healthy environment for the culturing of test

organisms.

Acute toxicity testing, where animals are given doses of a test compound to find the

lethal (or toxic) dose, are important because these studies give most of the basic information

about the fate compounds have on living organisms. Copepods are a valuable research

organism for this testing, as most species of copepods are the main nutritional source for laval

fish, crustaceans, sea horses and invertebrates (Stottrup et al., 1986). Because copepods can be

found in virtually every water supply, they also play a valuable role in assessing the health of a

particular body of water. It is important for scientists to know the toxicity of anthropogenic

contaminants on these tiny organisms, as death to them can have a further impact on many

other aquatic organisms along the food web. Daphnia magna, commonly known as the water

flea, has a much higher median lethal dose concentration for the compounds used in this study

than Acartia tonsa. D. magna has a LC50 value of 36 mg/l to 5-fluorouracil (Zounkova et al.,

2007) and a LC50 value of 30.59 mg/l to phenols (Kim et al., 2002). Although the phenols used

in the article by Kim et al. are not the specific phenols used in this study, they are still indicative

of the sensitivity of D. magna to phenolic compounds. Comparing the LC50s between A. tonsa

41

and D. magna, it is obvious why copepods are considered important sentinel species, as they

are much more sensitive to compounds than other organisms.

4.2 Culturing and Egg Collecting Methods

This study optimized and successfully applied an acute marine copepod test. Through

the developmental process, it was found that the creation and perfection of a culture chamber

for the species Acartia tonsa and Tisbe biminiensis decreased the original cost by a factor of 8

from approximately $880-1680 to about $110-210. Through experimentation, the cost

remained unchanged, as all of the animals used in this research came from the mass stock

culture, eliminating the need to order more animals. In house stock cultures eliminate the

many obstacles that are met when animals are ordered and travel to new destinations.

Construction of the egg collecting apparatus is also extremely important for the

precision and efficiency of copepod toxicity testing. The apparatus significantly cuts down the

amount of manual labor needed to properly collect eggs, and eliminates the risk of injury and

death to adults and eggs, alike. The apparatus increases the validity of toxicity test results, as all

of the copepods used during experimentation essentially go through a controlled hatch; all eggs

hatching within 24 hours of each other. This collection method could also be valuable for early

life stage testing, as mass quantities of eggs can be collected and stored for experimentation.

4.3 Results

The no observed effect concentration (NOEC), lowest observed effect concentration

(LOEC), and the concentration causing 50% lethality (LC50) for 3,5-dichlorophenol for the first

48 hour toxicity test were 0.006 mg/l, 0.026 mg/l, and 0.125 mg/l respectively. An ANOVA

42

showed that there was no difference between the 3,5-dichlorophenol test one and test 2. Test

two results are as follows, NOEC was found to be 0.009 mg/l, LOEC was found to be 0.026 mg/l

and 0.124 mg/l was the 48 hour LC50. Test one and test two for 4,4’-methylenebis(2,6-di-tert-

butylphenol) were also compared using an ANOVA, and no significant difference between the

test results was found. The NOEC, LOEC and LC50 for test one were found to be 0.006 mg/l,

0.040 mg/l, and 0.21 mg/l respectively. Test two results are as follows, NOEC was found to be 0

mg/l mg/l, LOEC was found to be 0.007 mg/l and 0.13 mg/l was the 48 hour LC50. An ANOVA

was performed to compare the differences between test one and test two for 5-Fluorouracil,

and no significant difference was found. The NOEC, LOEC and LC50 for test one were found to

be 0 mg/l, 0.01 mg/l, and 0.027 mg/l respectively. Test two results are as follows, NOEC was

found to be 0 mg/l mg/l, LOEC was found to be 0.01 mg/l and 0.030 mg/l was the 48 hour LC50.

Looking at the 50% lethality concentration to compare the three chemicals, 5-

fluorouracil appears to be the most toxic compound with LC50s of 0.027 mg/l and 0.030 mg/l

for test one and two, respectively; followed by 3,5-dichlorophenol with LC50 values of 0.125

mg/l and 0.124 mg/l for test one and two, respectively. Lastly, 4,4’-methylenebis(2,6-di-tert-

butylphenol) appears to be the least toxic between the three compounds with calculated LC50s

of 0.21 mg/l and 0.13 mg/l for test one and two, respectively.

4.4 Compounds

Many thousands of anthropogenic chemicals are released into the environment every day,

which include endocrine-disrupting (Vandenberg et al., 2009) or developmentally disrupting

compounds. Endocrine-disrupting compounds are defined as exogenous chemicals or chemical

mixtures that impact endocrine system structure or function and cause adverse effects (US

43

Environmental Protection Agency, 2007). Endocrine systems regulate a multitude of

developmental, metabolic and reproductive processes. These processes include embryonic

development, gonadal formation, sex differentiation, growth and digestion. Plasticizers are

chemicals implicated in endocrine disruption (Markey et al., 2001). The phenols 3,5-

dichlorophenol and 4,4’-methylenebis(2,6-di-tert-butylphenol) were chosen as toxicity research

compounds for this study because of their potential endocrine disrupting qualities and the

prevalence of phenols in the environment.

Other anthropogenic compounds released into the environment have medical

implications, most of which have positive therapeutic uses but can have unwanted on the

environment. Antifolates are one of these compounds. 5-fluorouracil certainly has a positive

and important therapeutic use as a very common form of treatment in cancer chemotherapy,

rheumatoid arthritis, lupus, asthma, psoriasis, scleroderma, IBS, spinal fluid leukemia and

sarcoma (Takimoto, 1996). At therapeutic doses, 5-FU is meant to essentially prevent the

production of a diseased cell’s DNA, RNA and proteins in an attempt to treat the patient.

However, the small doses released into the environment by human excretion and disposal has

negative impacts on aquatic organisms, as shown by the results in this experiment. 5-FU was

chosen as the third research compound because of its value and common usage as a

pharmaceutical.

4.5 Conclusion

Acute toxicity testing is essential to monitor the health of our environment and to

humans; so it should be equally important to continually question and update current protocols

that are set in place for culturing and testing. The work done in this experiment shows that

44

certain steps should be taken to help update these practices so more species and more

compounds may be analyzed.

45

REFERENCES

Araujo-Castro C., Sauza-Santos L.P., Torreiro A., Garcia K.S. 2009 Sensitivity of the marine

Benthic copepod Tisbe biminiensis (copepod, harpacticoida) to potassium dichromate

and sediment particle size. Brazilian Journal of Oceanography 57(1):33-41.

Barber, J.T., Sharma, H.A., Ensley, H.E., Polito, M.A., Thomas, D.A., 1995, Detoxification of

phenol by the aquatic angiosperm Lemna gibba. Chemosphere 31: 3567-3574.

Boxhall, G.A. and Defaye, D. (2008). Global diversity of copepods (Crustacea: Copepoda) in

freshwater. Hydrobiologia 595(1): 195-207.

Brayfield, A. (2013). Fluorouracil. Martindale: The Complete Drug Reference. Pharmaceutical

Press.

Cervetto G., Pagano M., Gaudy R. 1995. Feeding behavior and migrations in a natural

Population of the copepod Acartia tonsa. Hydrobiologia 300/301:273-248.

Crane, M., Newman, M. 2000. What level of effect is a no observed effect? Environmental

Toxicology and Chemistry. 19: 516-519.

Dow Chemical Company: Product Safety Assesment: Phenol.

http://msdssearch.dow.com/PublishedLiteratureDOWCOM/dh_007e/0901b8038007e693.

pdf?filepath=productsafety/pdfs/noreg/233-00308.pdf&fromPage=GetDoc

Dürbaum, J. & Künnemann, T. (1997). Biology of Copepods: An Introduction. Carl von Ossietzky

University of Oldenburg.

Gissi, F., Binet, M. T., & Adams, M. S. 2013. Acute toxicity testing with the tropical marine

copepod Acartia sinjiensis: optimisation and application. Ecotoxicology and Environmental

Safety, 97, 86–93. doi:10.1016/j.ecoenv.2013.07.008

46

http://msdssearch.dow.com/PublishedLiteratureDOWCOM/dh_007e/0901b8038007e693.pdf?filepath=productsafety/pdfs/noreg/233-00308.pdf&fromPage=GetDochttp://msdssearch.dow.com/PublishedLiteratureDOWCOM/dh_007e/0901b8038007e693.pdf?filepath=productsafety/pdfs/noreg/233-00308.pdf&fromPage=GetDochttp://www.uni-oldenburg.de/zoomorphology/Biologyintro.htmlhttp://en.wikipedia.org/wiki/Carl_von_Ossietzky_University_of_Oldenburghttp://en.wikipedia.org/wiki/Carl_von_Ossietzky_University_of_Oldenburg

Gorbi G., Invidia M., Savorelli F., Faraponova O., Giacco E., Cigar M., Buttino I., Leoni T., Prato E.,

Lacchetti I., Ser S. 2012. Standardized methods for acute and semichronic toxicity tests

with the copepod Acartia tonsa. Env Toxicol and Chem 31: 2023-2028.

Hayashi, M., Nakamura, Y., Higashi, K., Kato, H., Kishida, F., Kaneko, H., 1999. A quantitative

structure-activity relationship study of the skin irritation potential of phenols. Toxicol. In

Vitro 13: 915-922.

Kim, B.C., Park, K.S., Kim, S.D., Gu, M.B., 2002. Evaluation of a high throughput toxicity

biosensor and comparison with a Daphnia magna bioassay. Biosensors and Bioelectronics.

18: 821-826.

Klaassen, C., & Watkins III, J. (2010). Essential of Toxicology (2nd ed.). McGraw-Hill.

Kosjeck T., Perko S., Zigon D., Heath E. 2013. Fluorouracil in the environment: Analysis,

occurrence, degradation and transformation. Journal of Chromatography A. 1290: 62-72.

Koukouarakis, G.V., Kouloulias, V., Koukourakis M.J., Zacharias, G.A., Zabatis, H., Kouvaris, J.

(2008). Efficacy of the Oral Fluorouracil Pro-drug Capecitabine in Cancer Treatment: a

Review. Molecules. 13(8), 1897-1922.

Marcus, N., Wilcox, J. 2007. A guide to the meso-scale production of the copepod Acartia tonsa.

Markey, C.M., Michaelson, C.L., Sonnenschein, C., Soto, A.M., 2001. Alkylphenols and

bisphenol-A as environmental estrogens. In: Metzler, M. (Ed.), The Handbook of

Environmental Chemistry. Part L. Endocrine Disrupetors, Part I. M, vol. 3.

Springer-Verlag, Berlin.

McGuire, J., 2003. Anticancer antifolates: Current status and future directions. Current

Pharmaceutical Design. 9, 2593-2613.

47

Michalowicz, J., Duda, W., 2007. Phenols-sources and toxicity. Polish J. Environ. Stud. 16, 347-

362.

Medina M., Barata C., Telfer T., Baird DJ. 2002. Age- and sex-related variation in sensitivity

to the pyrenoid cypermetrin in the marine copepod Acartia tonsa Dana. Arch Environ

Contam Toxicol 42:17-22.

Morgan S.L., Baggott J.E., 1995. Folate antagonists in nonneoplastic disease: proposed

mechanisms of efficacy and toxicity. Folate in Health and Disease (New York: Marcel

Dekker). pp. 405–433.

Moriarty, F. (1999). Ecotoxicology: The Study of Pollutant in Ecosystems (3rd ed.). London:

Academic Press.

Newman MC. 1995. Quantitative Methods in Aquatic Ecotoxiclogy. Lewis, Boca Raton, FL, USA.

Pinho, G.L.L., Pedroso, M.S., Rodrigues, S.C., Souza, S.S.D., Bianchini, A., 2007. Physical effects

of copper in the euryhaline copepod Acartia tonsa: waterborne versus waterborne plus

dietborne exposure. Aquatic Toxicology. 84: 62-70.

Pisano, R., Breda, M., Grassi, S., James, C.A., 2005. Hydrophilic interaction liquid

chromatography-APCI-mass spectrometry determination of 5-fluorouracil in plasma and

tissues. Pharmaceutical and Biomedical Analysis. 38: 738-745.

PubChem: 2,6-di-tert-butylphenol. NCBI.

http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=31405

PubChem: 3,5-dichlorophenol. NCBI.


Raisuddin S., Kwok KWH, Leung KMY, Schlenk D., Lee J. 2007. The copepod Tigriopus: A

48


promising marine model organism for ecotoxicology and environmental genomics.

Aquatic Toxicology 83:161-173.

Remaud G., Boisdron-Celle M., Morel A., Gamelin A. 2005. Sensitive MS/MS-liquid

chromatography assay for simultaneous determination of tegafur, 5-fluorouracil and 5-

fluorodihydrouracil in plasma. Journal of Chromatography B. 824: 153-160.

Rossi, S. (2013). Australian Medicines Handbook. Adelaide: The Australian Medicines Handbook

Unit Trust. ISBN 978-0-9805790-9-3.

Sauza-Santos, L.P., Pastor, J., Ferreira, N.G., Costa, W.M., Araujo-Castro, C., Santos, P., 2006.

Developing the harpacticoid copepod Tisbe biminiensis culture: testing for salinity

tolerance, ration levels, presence of sediment and density dependent analysis. Aquatic

Research. 37: 1516-1523.

Scorecard: The Pollution Information Site, “Chemical: Phenol,”

http://www.scorecard.org/chemical-profiles/summary.tcl?edf_substance_id=108-95-2.

Schipp, G.R., Bosmans, J., Marshall, A.J. (1999). A method for hatchery culture of tropical

calanoid copepods, Acartia species. Aquaculture 174: 81-88.

Smith, A.L. 1997. Oxford dictionary of biochemistry and molecular biology. Oxford

[Oxfordshire]: Oxford University Press. pp. 43. ISBN 0-19-854768-4.

Sreejith, K., Youn-Jung, K., Hoon, C., Mi-Sung, K., Aream, P., Jihae, P., Woongghi, S,. Taejun, H.,

2014. A rapid phenol toxicity test based on photosynthesis and movement of the

freshwater flagellate, Euglena agilis Carter. Aquatic Toxicol. 155: 9-14.

Stottrup, J.G., Richardson, K., Kirkegaard, E., Phil, N.J., 1986. The Cultivation of Acartia tonsa

Dana for use as a live food source for marine fish larvae. Aquaculture, 52: 87-96.

49

http://www.scorecard.org/chemical-profiles/summary.tcl?edf_substance_id=108-95-2

Takimoto, C. (1996). New Antifolates: Pharmacology and Clinical Applications. The Oncologist, 1(1 & 2),

68–81. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/10387971

Ucisik, A.S., Trapp, S., 2006. Uptake, removal, accumulation, and phytotoxicity of phenol in

Willow trees (Salix viminalis). Environ. Toxicol. Chem. 25: 2455-2460.

US Environmental Protection Agency, 2007. Memorandum to EDSTAC Members RE: Definition

of “Endocrine Disruptor”. Washington, D.C., USA.

Vandenberg, L.N., Maffini, M.V., Sonnenschein, C., Rubin, B.S., Soto, A.M., 2009. Bisphenol-A

and the great divide: a review of controversies in the field of endocrine disruption.

Endocrin. Rev. 30: 75-95.

Visentin, M, R Zhao, and D Goldman, 2012. The Antifolates. National Institute of Health.

(PMCID: PMC3777421): http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3777421/

Wallace G, Fry S. 1999. Action of diverse peroxidases and laccases on six cell wall-related

phenolic compounds. Phytochemistry. 52: 769-773.

Zounkova, R., Odreska, P., Dolezalova, L., Hilscherova, K., Marsalek, B., Blaha, L. 2007.

Ecotoxicity and genotoxicity assessment of cytostatic pharmaceuticals. Environmental

Toxicology and Chemistry, 26: 2208-2214.

50

ACKNOWLEDGEMENTSLIST OF TABLESLIST OF FIGURESCHAPTER 1 INTRODUCTION1.1 Background1.1.1 Importance of the Mass Culturing of Acartia tonsa and Tisbe biminiensis

1.2 Phenols1.3 Antifolates1.4 Rationale for Study1.4.1 Objectives and Hypotheses

CHAPTER 2 MATERIALS AND METHODS2.1 Development of Novel Culture Chamber and Egg Collecting System2.1.1. Culture of Acartia tonsa and Tisbe biminiensis2.1.2 Culture Chamber

2.2 Materials and Methods2.2.1 Chemicals2.2.2 Research Organisms2.2.3 Algae

2.3 Test Procedures2.3.1 Collection of Eggs2.3.2 Chemical Preparations2.3.3 Toxicity Testing

2.4 Analytical Chemistry2.4.1 AN2: 4,4’-methylenebis(2,6-di-tert-butylphenol)2.4.2 DCP: 3,5-dichlorophenol2.4.3 5-FU (5-fluorouracil)

2.5 Data Analysis

CHAPTER 3 RESULTS3.1 Culturing and Egg Collecting Chamber3.1.1 Culture Chamber3.1.2 Egg Collection Method

3.2 Toxicity Test Results3.2.1 3,5-Dichlorophenol3.2.2 4,4’-methylenebis(2,6-di-tert-butylphenol)3.2.3 5-Fluorouracil

3.3 NOEC and LOEC Data3.3.1 3,5-dichlorophenol3.3.2 4,4’-methylenebis(2,6-di-tert-butylphenol)3.3.3 5-fluorouracil

3.4 LC50 Data

CHAPTER 4 DISCUSSION AND CONCLUSION4.1 Introduction4.2 Culturing and Egg Collecting Methods4.3 Results4.4 Compounds4.5 Conclusion

REFERENCES

Documents

Optimization of Novel Culturing and Testing Procedures for .../67531/metadc699989/m2/1/high_res_d/thesis.pdfTisbe biminiensis is an epibenthic harpacticoid copepod that live s on bottom