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Meroitic

This book is a short guide on the Meroitic language intended for those who would like to translate from that language.

Meroitic is a dead language once spoken in the land of Kush in what is today southern Egypt and northern Sudan. It was used in writing from the 3rd century BCE to the 4th century CE. It is presumed to have gone extinct sometime after its last attestation in the late 4th century. The language was displaced in that region by Coptic and Old Nubian.

Meroitic is known from several hundred texts, the overwhelming majority being funerary tablets. The remainder consists of royal stelae, captions to works of art, ostraca, graffiti and temple inscriptions. Three substantive texts of historiographical interest exist: the "Kharamadoye inscription" from Kalabsha, the "Great Stele of Akinadd" from Hamadab and the "Taneyidamani inscription" from Jebel Barkal.

This guide will follow the outline of the language set in Francis Llewellyn Griffith's "Karanog IV: The Meroitic Inscriptions of Karanog and Shablul", published in 1911, and now in the public domain. That book serves as a an adequate introduction to the language and gives treatment to Meroitic funerary stele and ostraca from Karanog, Shablul, cataloged as REMs 201> <400. The book repeatedly makes references to Griffith's earlier treatment of Meroitic from Arika.

This guide will apply the Saharan theory but it will also prove useful to those translating according to Northern Eastern Sudanic and Afro-Asiatic grammarticalisations of the language. The first few lessons are universal as they deal with the language's inherent features.

Before beginning
To view characters encoded in Meroitic cursive and hieroglyphic as well as other missing Unicode characters you will need to install a font such as Unifont.

Lessons

 * Lesson 1: Phonology and Orthography

Appendices

 * Vocabulary

title: Meroitic


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Meroitic/Lesson1/Phonology and Orthography

Orthography
The Meroitic script has 4 vowel signs: a, e, i, o, 15 open consonant signs and 4 closed consonant signs (se, ne, te, to). The vowel "a" can only occur at the start of a word. e and i can only start words in early Meroitic. Otherwise, words always start with a consonant sign.

ɣ (voiced velar fricative), c (voiceless palatal stop) and j (voiced palatal stop) are all foreign sounds to English. The Meroitic l and w are phonetically different from their English counterparts.

There are different approaches to actually vocalizing Meroitic.

Traditional Approach
The Meroitic script can be read as a strict alphasyllabary. This means consonant signs open syllables and vowels close them. An closed consonant already has an inherent vowel and so cannot be followed by a vowel sign.

Examples
 * abr /abara/ - "man"
 * mk /maka/ - "god"
 * ant /anata/ - "priest"
 * perite /perite/ - "agent"

The traditional approach prevailed because Meroitic remained a poorly undeciphered language. It allowed for easy, speculative vocalization that was not expected to reflect how the language was actually pronounced. One problem with the traditional approach is that it does not allow words to end with a consonant sound although we know for a fact that some Meroitic place names did end in consonants. Better approaches were developed from expert study of Meroitic words and names with known Greek, Coptic and Egyptian translations.


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Perspectives of Aquatic Toxicology/Preface

“It is the supreme art of the teacher to awaken joy in creative expression and knowledge” - Albert Einstein

The Wikibook - Perspectives in Aquatic Toxicology – is primarily written by graduate students of Iowa State University. This Wikibook is the result of the Experimental Course - Aquatic Toxicology (A ECL 444/544X / TOX 444/544X) implemented, and designed by me (the editor) in spring 2019. During the many years of previous studies in my youth, I often felt constrained by the boundaries of textbooks that the teachers were imposing on me. I felt as there was no room to expand the knowledge beyond the colorful hardcovers of a textbook and it’s content. There was no reason for me to be creative, to want more, to ask questions, to seek answers, as it was already predetermined that all I, and thousands of other bright minds, need to know was already in the textbook. Even, there was no need for the teacher. All that was required was the textbook. My homework would be discarded and stowed away in some box, never again to see the sunlight, no matter how creative I tried to be. But, I wanted more...

I created the Aquatic Toxicology course to follow the open pedagogy approach “in which students are active and visible participants in the construction of knowledge” (DeRosa & Robison, 2017, p. 115). This time the students create their own textbook, selecting, and writing their own chapters while transferring the knowledge to each other in the class. There are no hardcovers or boundaries. The book is free and accessible to any student in the world. This collaborative work between the course instructor/editor and the students aimed to present perspectives in Aquatic Toxicology and to establish authors a theoretical foundation for the experience.

While Aquatic Toxicology is a post-World War II discipline, it was practiced for thousands of years. Ancient scholars were conducting research on water pollution oblivious of the fact that such philosophy branch would be dubbed the Aquatic Toxicology many years later. It was Aristotle who invented “aquatic toxicity test” – the foundation methodology behind Aquatic Toxicology designed to assess the potential for damage to an aquatic environment. Around the year 350 BC Aristotle noticed the putrid smell arising from Athens sewage effluent streams and the downstream change in color where the effluent conjoined the pristine stream. He questioned the safety of the Athens drinking water supply and transferred midge fly (chironomids) larvae from pristine stream to the effluent in order to monitor their survival. Some 2,500 years later exposure of model species to effluents is still the basis of many toxicity tests. Later, perhaps what is known to be the first man made aquatic environmental catastrophe occurred in Ancient Rome. The elaborated network of drinking water supply and sewage disposal in large Roman towns were lead pipes that over the course of centuries leached lead into the Tiber River and build-up its deposits in the Portus – an Ancient Rome harbor. It was only recently that scientists discovered that the lead deposits in the sediments beneath Portus around 250-100 AD were many times higher than what was the natural background. Such concentration likely and significantly altered the community of benthic organisms at the harbor. Concomitantly the Roman drinking water had lead concentrations roughly 100 times higher than natural spring water, all while Ancient Romans were unaware of these facts and any associated potential health and environmental issues. History was repeating itself over the course of time and the environmental and health effects of mercury, DDT, dioxins, and other chemicals once thought to be safe, become apparent years after its introduction in the aquatic environment.

Today, Aquatic Toxicology, as a discipline, is getting an increase in global attention due to the explosive growth of the human population, dwindling clean water resources, water pollution, eutrophication, global decline in biodiversity, and increase in the number of newly synthesized chemicals evaluated by the regulatory agencies. Therefore, Aquatic Toxicology is not of interest only to toxicologist but has a much wider audience comprised of ecologists, chemists, risk managers, and environmental scientists, among others.

This Wikibook was supported by The Miller Faculty Development Fund of Iowa State University, specifically The Miller Open Education Mini-Grant. The Miller Fund was made possible by the generosity of F. Wendell Miller, who left his entire estate jointly to Iowa State University and the University of Iowa. The Miller Open Education Mini-Grants aim to support faculty development through the use of new and innovative resources in the classroom. They provide faculty with opportunities to enhance their scholarship of teaching and learning by integrating Open Educational Resources (OER) into their teaching.

This book is not finished and it will be updated with new chapters and new information each time the course is offered. I wish to extend my acknowledgments to Dr. Evrim Baran, the open pedagogy advisor, and to Dana AlZoubi, technical editor of this book.

In Ames, Iowa, on June 30, 2019

Boris Jovanovic, PhD - Editor


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Perspectives of Aquatic Toxicology/Aquatic Toxicity Tests

= Chapter One: Aquatic Toxicity Tests =

INTRODUCTION
Aquatic species are vital to our planet. Phytoplankton, algal plankton, and kelp are major sources of the planet’s oxygen. They absorb and store carbon dioxide, and maintain a hospitable climate. They also play an important role in the global nitrogen cycle and support aquatic animals such as fish, mollusks, sponges, and corals. Aquatic species help maintain the earth’s ecosystem and help preserve its rich biodiversity as well as providing food, medicine, livelihoods, tourism, and recreational opportunities1.

It is therefore essential to protect the planet’s rich and diverse aquatic life, and combat the many threats facing aquatic organisms including climate change, habitat destruction, overfishing, the introduction of invasive species, and chemical pollution2. This chapter will focus on chemical pollution. The risks to aquatic life can be minimized and better managed by understanding how chemicals impact it.

There are more than 140,000 man-made chemicals in the environment3, with the United States alone producing 2000 new chemicals every year4. It is conceivable that aquatic species are exposed to many of these chemicals on an acute (short-term) and chronic (long-term) basis, although there is an absence of data to indicate how many of these chemicals are released into various water bodies. Chemical exposure can affect organisms’ growth, development, fecundity, behavior, and survival, among other biological processes. Hence, it is important to test chemical toxicity before it is released into the environment in order to determine maximum acceptable toxicant concentrations (see section II of this chapter) and to protect species from potential harm.

Toxicity testing is done to identify the degree to which chemicals can damage living organisms in a controlled environment. It has four major objectives:

a.    To obtain toxicity and exposure data for various chemicals

b.    To aid in estimating and managing risks posed by various chemicals

c.     To aid in setting chemical regulations and environmental standards

d.    To classify chemicals based on how toxic they are to various species

The dose makes the poison in toxicology. It is possible to determine safe and unsafe doses, or concentrations, for nearly every chemical. For example, the most toxic substance on earth, the bacteria-produced botulinum toxin, can kill humans with a very small dose, but it can be used safely in Botox5.

Risk is a function of toxicity and exposure. A chemical can be very toxic, but it will have zero risk to aquatic organisms if it never enters water bodies (i.e., there is no exposure). The maximum allowable concentration for a chemical in the environment is based on the risk it poses to various species. An acceptable “safe” concentration is usually one that does not harm 95% of the species.

Many questions can be answered by carrying out toxicity tests:

a.    At what concentration is a chemical non-toxic to an organism? At what concentration is it toxic?

b.    What effects can be observed from short-term and long-term chemical exposure?

c.     Which chemicals are the most and least toxic to an organism?

d.    Which organisms are the most or least sensitive to a chemical?

e.    Are some life stages of an organism more sensitive?

f.     Do certain environmental conditions make a chemical more toxic?

g.    Is the toxicity of a chemical similar in lab and in the outside environment?

h.    What is the effect of a mixture of chemicals?

And more.

Aquatic organisms can be exposed to chemicals when effluents and sewage are released into water bodies. Sometimes chemicals inadvertently enter water through oil spill or runoffs from agricultural fields. Chemicals present in the air can be deposited into water bodies either directly (dry deposition) or through rainfall, snowfall, and fog (wet deposition). Some of the chemicals commonly found in water bodies include detergents, fertilizers, pesticides, pharmaceuticals, food and cosmetic preservatives, chemicals used in kitchenware and plastic, and metals6-8. Aquatic animals such as fish can take up these chemicals via their gills, absorb them through their integument, and/or ingest them. Aquatic plants that have vascular systems can absorb chemicals through their epidermal surface and/or roots. Plants that are not completely submerged in water can take up chemicals in the air through their stomata.

Chemical properties and type of aquatic species determine how chemicals are taken up, distributed, stored, metabolized, and excreted. Hydrophobic (fat-loving) chemicals are more likely to enter a fish’s body, and warm temperatures typically increase the uptake as the fat become more fluid-like. Smaller, uncharged molecules also cross membranes more easily. Hydrophilic (water-loving) chemicals are more likely to be transported by the circulatory system. On the other hand, hydrophobic chemicals are more likely to bind to molecules and accumulate in fat bodies. While chemical storage is protective in the short term (they are not free to move and act), they can be released later and cause toxicity. This usually happens when an organism breaks down fat for greater energy needs, i.e., during illness, starvation, or reproduction.

A species’ metabolic enzymes often modify a chemical in order to detoxify its effects, but this modification can sometimes make a chemical more toxic. Chemicals with many halogen atoms such as chlorine, and fluorine are often difficult to modify. Many aquatic animals eliminate chemicals through their gills or skin. Further details on chemical biotransformation can be found in the Biotransformation of Xenobiotics Chapter of this book.

Chemical exposure can kill or harm aquatic organisms directly through such means as growth reduction, delayed development, decreased fertility, and behavioral changes, or can reduce or eliminate its food supply by killing its prey, or limiting its shelter through habitat destruction. This can lead to increased competition for food and shelter, disrupting the food web, and altering the ecological balance.

Units of concentration
Concentration is used more commonly than dose in aquatic toxicology. This is because water chemical concentration is easier to measure than the amount of chemical taken up by fish through its gills, integument, and mouth. Concentration of a solution is defined as the ratio of solute to solvent, or the ratio of solute to total solution. This can be either expressed as mass of chemical per unit volume (e.g. mg/mL) or the number of moles of chemicals per liter of solution (e.g. mol/L).

Terms like ppm, ppb and ppt are also often used to describe units of concentration:

a.    Parts per million (ppm) corresponds to 1 mg of chemical/L of solution. The amount of chemical is six orders of magnitude lesser than the amount of solution. This is like emptying a large soft drink bottle of a chemical into an Olympic-sized swimming pool.

b.    Parts per billion (ppb) corresponds to 1 µg of chemical/L of solution. The amount of chemical is nine orders of magnitude lesser than the amount of solution. This is like putting half a teaspoon of a chemical into an Olympic-sized swimming pool.

c.     Parts per trillion (ppm) corresponds to 1 ng of chemical/L of solution. The amount of chemical is twelve orders of magnitude lesser than the amount of solution. This is like putting one-twentieth of a drop of chemical into an Olympic-sized swimming pool.

Concentration and response
There is a relationship between the chemical concentration to which an organism has been exposed and the resultant nature and degree of harmful effects. However, it is important to note that the chemical concentration that enters an organism is typically higher than the concentration that causes a toxic effect. This could be due to the organism producing enzymes or molecules that break down or bind to the chemical. This reduces the availability of the chemical to the body, and thus a lower concentration binds to the site of action and exerts a toxic effect.

Assumptions in a concentration-response relationship are as follows:

a.    It is a cause-and-effect relationship, i.e., the response occurs due to the organism’s exposure to a chemical.

b.    The response is due to a chemical interacting at the site of action.

c.     The concentration of a chemical at the site of action is a function of how much chemical the organism was exposed to.

d.    Above the threshold concentration (concentration at which a response can be detected), the magnitude of response is proportional to the amount of chemical interacting at the site of action.

e.    The response can be measured and reproduced under similar conditions.

The duration of an organism’s chemical exposure is also significant. If a fish is exposed to a low chemical concentration for only one day, it may be unaffected, but if it is exposed to the same concentration for months it may develop cancer, skeletal abnormalities, issues with fertility, etc.

The response to a chemical for any given species usually follows a normal distribution or bell-shaped curve (see Figure 1). This means that some organisms of the same species are very sensitive to a given chemical, some are very resistant, while most are neither very sensitive nor very resistant. Standard deviation diagram.svg

Creating a toxicity curve
A toxicity curve or a concentration-response curve is a graph which plots the results obtained from a traditional concentration-response toxicity test. The toxicity test should ideally fulfill the following criteria:

a.    There should be five different concentrations of a chemical plus a negative control.

b.    The concentrations should be equally spaced.

c.     The concentrations should cause a range of effects, from 0% effect to 100% effect.

d.    The study should be replicated at least three times.

The toxicity curve generated from such an experimental design should have the following features in most cases (see Figure 2):

a.    The x-axis should be the concentration and the y-axis should be the response.

b.    The curve should be sigmoidal in shape above the threshold dose.

c.     The average cumulative response from the three replicated studies should be plotted with the 95% confidence intervals.

The slope of a generated toxicity curve can indicate several things. A very steep slope indicates that small increases in concentration caused large increases in response; a flat slope indicates that large increases in concentrations caused small increases in response. The curve can also be used to estimate a concentration that causes a particular response (for example, a 10% response). Typically, the concentration which causes a 50% response is calculated, as it has the least variability/noise.

A non-standard toxicity curve is observed for essential chemicals such as water, oxygen, and vitamins. There is an optimal range for these chemicals: a very low concentration causes deficiency and death, and a very high concentration causes toxicity and death (see Figure 3). Another kind of non-standard toxicity curve is seen with chemicals found in such materials as plastics. These chemicals have different modes of action at different concentrations: they can disrupt hormones at low concentrations but not at high concentrations9. Some toxicity curves can be bimodal if males and females have different toxicity thresholds to a chemical.

Measures of toxicity
Toxicity is commonly measured in toxicological studies as follows:

Lethal Concentration 50 (LC50): Concentration at which 50% of the organisms in a population are killed following chemical exposure.

Effective Concentration 50 (EC50): Concentration at which 50% of the organisms in a population are affected following chemical exposure. The effects observed could be reduced growth, delayed development, etc.

Inhibitory Concentration 50 (IC50): Concentration at which 50% of organisms in a population are inhibited following chemical exposure. The chemical could have inhibited a specific biological or biochemical function.

Often LC/EC/IC 10 (harm to 10% of population) and LC/EC/IC 90 (harm to 90% of population) are also calculated.

No Observed Effect Concentration (NOEC): Highest chemical concentration that does not cause a toxic effect in the treated population.

Lowest Observed Effect Concentration (LOEC): Lowest chemical concentration that causes a toxic effect in the treated population.

Maximum Acceptable Toxicant Concentration (MATCC): Concentration between NOEC and LOEC, or is a geometric mean of the two. It is calculated for chronic studies only.

TOXIC EFFECTS OF CHEMICALS
Chemicals can exert toxic effects on organisms through various mechanisms:

a.    Binding: Chemicals can bind to molecules on the surface of cells and disrupt communication between cells. If an organism’s cells do not communicate with each other they will not function normally. Chemicals can also bind to enzymes and prevent them from carrying out essential activities such as digestion and metabolism, as well as bind to DNA and change the amount of proteins produced. A correct number of proteins are needed to build cells, produce hormones, and maintain immunity.

b.    Bioaccumulation: This occurs when an organism takes up a chemical faster than it eliminates it. In bioaccumulation the chemical is taken up by contact, respiration, and ingestion. The term bioconcentration is used when the chemical is taken up through contact and respiration only. A chemical accumulation in living tissue can poison tissue, and subsequently, organs.

c.     Interaction: Two or more chemicals in an organism can interact with one another. Usually, the combined chemical effect will equal the sum of their individual effects (1 + 2 = 3). This is called an additive effect and can be observed when aspirin and acetaminophen are taken together. They both act in a similar manner and their combined effect is comparable to taking two doses of one drug. Another kind of interaction occurs when the combined effect of two chemicals are greater than the sum of their individual effects (1 + 2 = 5), and can happen if chemical A increases the activity of chemical B. This is called the synergistic or potentiation effect and can be observed when acetaminophen and alcohol are taken together. Both are broken down by the liver and their combined presence taxes the organ, making it more vulnerable to failure. The final kind of interaction occurs when the combined effect of two chemicals is less than the sum of their individual effects (1 + 2 = 1). This occurs if chemical A hinders the activity of chemical B, and is called an antagonistic effect. A classic example of this effect is anti-venom drugs canceling the effects of a snake bite.

The intracellular effects of a chemical can also be described with the help of a toxicity pathway. It is a sequence of events, starting with a chemical entering an organism. A proportion of the chemical reaches the target tissue and interacts with it: for example, by binding to cell receptors on the tissue. This causes a perturbation, or disturbance, in normal cell function. If the cell starts to alter and the organism corrects the change in time, there will not be a problem; however, if the organism does not alter and effect change, the cell will be permanently altered/injured10 (see Figure 4).

Adverse Outcome Pathways (AOPs) encompass toxicity pathways and beyond. That is, they include chemical-molecular interactions and cellular changes, and determine how this leads to changes in organs, organisms, and populations. In Figure 5 an AOP has been drawn for a male fish exposed to a chemical that activates the estrogen receptor. At the cellular stage this could lead to the transcription and production of abnormal proteins. These proteins could cause both ovaries and testis to develop, altering the secondary sex characteristics of the fish and impairing its fertility. The sex ratio could become skewed if many males in a population become feminized and infertile11.

Table 1 shows how toxicity endpoints could manifest in various aquatic organisms. The length of chemical exposure (short-term vs. long-term) often determines which toxicity endpoints should be measured. For example, reproductive problems and tumors are only observed with long-term exposures.

Table 1: Toxicity endpoints for different aquatic species following exposure to chemicals

TYPES OF TOXICITY TESTS
Aquatic toxicity tests can be divided into categories as described below and summarized in Figure 6.

Based on duration of chemical exposure
a.    Acute: Short-term tests. For fish the tests are 24-96h long; but, for microalgae or bacteria, a 96h test could represent a chronic, life cycle, or multigenerational test. The test is commonly carried out to check chemical lethality.

b.    Subchronic: Prolonged acute tests. For fish the tests are typically anywhere between 28 days to 3 months long. The test is usually done to determine if a chemical impacts the growth (body mass) of a species.

c.     Chronic: Lasts for at least 10% of the tested species’ lifespan. For invertebrates, 21-day chronic studies are common. The test typically lasts longer than 6 months for fish. It is often conducted to see if a chemical causes reproductive and developmental effects.

d.    Life cycle: Lasts through an organism’s entire life cycle. This can be from egg to sexual maturity, or from egg to egg. This test is done to check if a chemical causes developmental or reproductive effects, as with chronic studies.

e.     Multigenerational: Carried out on two or more consecutive generations (parents and offspring). It is usually performed to examine if offspring are affected by parental exposure to chemicals.

f.      Early-life-stage: Done on embryos or larval stages. Different life stages of an organism can exhibit different sensitivities to a chemical, with early-life stages often more susceptible.

Based on exposure systems
a.    Static: Organisms are placed in still water containing a chemical (or in control water). The water is not changed during the test. This system is widely used, but for studies longer than 24h they may not accurately represent chemical effects. This is because the concentration of the chemical may change over time and toxic effects may be produced from a build-up of metabolic byproducts released by the organisms.

b.    Renewal: Similar to static, with the test conducted in still water. However, in this test the water containing the chemical (or control water) is regularly changed during the test, usually every 24h, ensuring that the chemical concentration remains stable and that organisms are exposed to clean and fresh water daily.

c.     Recirculation: Also similar to static, but the water containing the chemical (or control water) is filtered. This ensures that the water quality does not deteriorate over time. However, filters can add uncertainties to the study as the filter media may interact with the test chemical.

d.    Flow-through: Water containing the chemical (or control water) constantly flows in and out of the system, maintaining a high quality flow where the influent and effluent never mix. Pumps control the flow of water and dilutors ensure that the right concentration of chemical is delivered. Flow-through systems mimic the natural flow of water, and though expensive, are regularly used for this reason.

Based on toxicity endpoints
These tests are done to determine if a chemical could cause one or more toxicity endpoints in a test organism. The endpoints frequently analyzed include mortality, growth, development, reproduction, immobilization, respiration, endocrine effects, and chemical bioaccumulation. Most standardized studies have been developed with these endpoints in mind.

Based on experimental complexity
a.    Single species: Tests are conducted on a single species in a lab. They are simple and inexpensive to conduct, and constitute the most common type of test. They are often carried out in a flask, beaker, or some other glass container.

b.    Microcosm: Tests conducted on two or more species in an artificial and controlled system. They represent a simplified ecosystem. Microcosms should contain less than 1000 liters of water and can be done indoors (e.g. fish tank) or outdoors (e.g. small ponds).

c.     Mesocosms: Tests conducted on multiple species placed in experimental water enclosures. Mesocosms represent a complex ecosystem and mimic natural conditions. The volume of water in the system must exceed 1000 liters, and the test is usually done outdoors. More details on this testing method can be found in the Mesocosm Chapter of this book.

d.    Macrocosms: Tests conducted in lakes and on whole aquatic ecosystems. They are the most realistic, but they are very difficult and expensive to conduct. Canada has 58 experimental lakes that are designated for macrocosm studies only.

Based on test media
a.    Water: Water is spiked with a single chemical or a chemical mixture, and aquatic organisms are exposed to it. The vast majority of aquatic toxicity tests are done on water. The toxicity endpoints of these organisms are compared to organisms exposed to control (non-spiked) water.

b.    Whole effluent: Samples of effluents are tested by exposing aquatic organisms to them. It is important to assure that as wastewater effluents are discharged into water bodies they will not harm aquatic organisms--as prescribed by the Clean Water Act. Toxicity endpoints are measured and compared to that of organisms exposed to control water.

c.     Sediment: Determines if sediments contain concentrated toxic chemicals that will harm organisms. Sediments are the ultimate repository for many chemicals that enter water bodies. Benthic species such as worms, crabs, clams, and lobsters live in or on sediments. Benthic organisms are exposed to contaminated or spiked sediments in sediment toxicity tests, and their toxicity endpoints are compared to organisms that are exposed to control sediments.

It is important to understand that the five different kinds of aquatic tests mentioned above are not independent of one another; this is just one way to classify them. For example, an acute toxicity test can be done on a single species using the static exposure system. The endpoint could be mortality, and the test could be done to check for the presence of toxic contaminants in whole effluents.

Both the Organization for Economic Co-operation and Development (OECD) and the United States Environmental Protection Agency (USEPA) publish guidelines for conducting various kinds of aquatic toxicity tests. These have been summarized in Table 2, along with a link to each test guideline.

Table 2: The different aquatic toxicity test guidelines published by OECD and EPA

DESIGNING A TOXICITY EXPERIMENT
The results of a toxicity experiment depend largely on how it was designed. A good study design can ensure that the results obtained are valid, applicable, and reproducible. Below are the major criteria used to ensure good aquatic toxicity test design, but it is important to note that not all tests can satisfy all of the outlined criteria due to the specific nature of various toxicity tests.

a.    It should be widely accepted by the general scientific community.

b.    It should be standardized (i.e., carried out according to defined protocols) and the results must be replicable in different laboratories.

c.     It should be easy to perform and economical.

d.    The test species selected must be a well-known model organism (see below).

e.    The test should cover a range of concentrations, and at least some of these concentrations should be found in the environment.

f.     The duration of chemical exposure must be realistic and manageable (for example, some sharks can live for hundreds of years and it is not possible to expose them to a chemical throughout their lifespan).

g.    The test should be statistically sound and robust.

h.    The data obtained can be used to estimate risk.

i.      The test should be sensitive enough to detect and measure the toxic effects under investigation.

j.      The test should be able to predict effects to species outside the lab (i.e., in the environment) and also predict potential effects to similar species.

Major standardized aquatic tests were discussed in the above section, with protocols spelled out by the OECD and USEPA. The International Organization for Standardization (ISO) and the American Society for Testing and Materials (ASTM), which is now an international organization, have also published a few aquatic test protocols and can be found on their websites ( https://www.iso.org/home.html and https://www.astm.org/ respectively).

The easy to administer and economical nature of standardized tests enables them to be routinely performed. This precludes macrocosm and mesocosm tests which are very complex and difficult to perform. While all the standardized toxicity tests mentioned in Table 2 were for single species, the USEPA and OECD have published guidelines for indoor microcosms (USEPA 1900) and outdoor microcosms/mesocosms (OECD draft guidance).

The appropriate selection of test species is critical in toxicity testing. While more details on this can be found in the Model Species in Aquatic Toxicology Chapter, most toxicity tests are carried out on model species which have the following characteristics: a.    High sensitivity to various chemicals. b.    Easily available and abundant.

c.    Easy to rear and culture in the lab and allow for various types of toxicity testing.

d.    High survival rate in the lab under normal conditions.

e.    Extensive and available knowledge of the organism (information on their biology, physiology, genetics, and behavior).

There is no single aquatic species used in tests that can provide answers to all questions or evaluate all chemical impacts on an ecosystem. It is therefore imperative to test several species from different classes: from algae to invertebrates to fish. Also, it is important to test different life stages in a species as every stage has a different sensitivity and a unique response. The environment in which the organism lives should also be taken into consideration (freshwater/marine and warm/cold water).

Under the United States Endangered Species Act (ESA) it is necessary to ensure that chemicals released into the environment do not harm endangered or threatened species. Since the employment of endangered and threatened species in toxicity tests is discouraged, toxicity values obtained from the most sensitive test species are often used to estimate their risk to a chemical. Also, the acceptable risk for these species is often 10-fold lower than that of more abundant species12.

Toxicity tests should not be conducted without first identifying and including one or more available Estimated Environmental Concentrations (EEC) or Actual Environmental Concentration (AEC) of a chemical. EECs are the estimated concentrations of a chemical in an environment and are usually derived through computer modeling or simple predictions. These models were developed with data obtained from laboratory and environmental studies; two such models are available on the USEPA website13. The Pesticide in Water Calculator (PWC) is used to estimate pesticide concentrations in water bodies that result from pesticide applications to land. The KABAM (KOW (based) Aquatic BioAccumulation Model) is used to estimate potential bioaccumulation of hydrophobic carbon-based pesticides in freshwater aquatic food webs. AECs on the other hand, are based solely on empirical data. They can be measured by taking a water sample from a natural water body or a tissue sample from a wild aquatic organism, making it possible with the help of analytical instruments to find the concentration/dose of chemical in the samples. Various methods and instruments are required to extract and analyze different chemicals.

Estimating the risk to a species from an acute exposure to a chemical, particularly a pesticide, constitutes a Tier I study—a simple laboratory study where worse-case estimates are used to calculate the Risk Quotient (RQ). RQ is defined as the exposure concentration divided by the toxicity concentration. The toxicity concentration is the LC50 or EC50 for an acute exposure. The exposure concentration is the peak concentration of the pesticide in water bodies. If the RQ does not exceed 0.5 for aquatic animals (that is, the exposure concentration is half the toxicity concentration) and 1.0 for aquatic plants (that is, the exposure concentration does not exceed the toxicity concentration), the risk is considered acceptable12.

A Tier II study is conducted if the Tier I RQ is exceeded. Here, the species is subchronically or chronically exposed to a pesticide. The toxicity concentration is the NOEC and the exposure concentration is the average pesticide concentration over 21-60 days in water bodies. If the RQ does not exceed 1.0 (for both aquatic animals and plants), the risk is considered acceptable; a Tier III study is carried out if it is exceeded. These investigations are chronic, life cycle, or multigenerational in nature, and can be done in the laboratory or outside (i.e., microcosm studies). Tier IV are mesocosm or macrocosm studies involving multiple species. Both Tiers III and IV usually involve a comparison of the toxicity endpoints of the pesticide-exposed populations to that of the unexposed populations. The endpoints analyzed are biomass, diversity, species richness, etc. Figure 7 summarizes the risk assessment process14. Additional information can be found in the Risk Assessment Chapter.

The design of a study depends largely on the question(s) being asked. Consideration must also be given to the physiochemical properties of the chemical being tested, its mode of action, its pattern of use, the environmental conditions, and the characteristics of the test organism. The following is a brief overview:

a.    Study objective/question: An aquatic toxicity study could be done for various reasons. It could be done to test the quality of a sediment or effluent, to register a field-applied pesticide, to understand the long-term impacts of a chemical on a community, or for routine monitoring purposes. The objective determines the type and number of tests needed.

b.    Physiochemical properties of a chemical: Prior to toxicity testing it is important to collect information on the chemical’s structural formula, purity, stability in water and light, partition coefficient, vapor pressure, biodegradability, etc. because its property can influence how it moves, persists, and distributes. For example, chemicals which have poor solubility in water are more likely to accumulate in aquatic organisms or bind to sediment. However, in a lab they may instead bind to the plastic or glass container housing the test organism. Therefore, special steps need to be taken to ensure that the fate of a lab chemical mimics the fate of a chemical in the environment. Similarly, it is not worthwhile to conduct long-term toxicity studies in the lab for chemicals which degrade rapidly in the environment.

c.     Mode of a chemical action: Most chemicals found in water bodies have a specific mode of action, i.e., they bind to a specific target site and produce specific downstream effects. Knowledge of a chemical’s mode of action will help decide which endpoints are the most important to measure during and after a toxicity test.

d.    Pattern of chemical use (or discharge): A species can be exposed to a chemical only if there is an overlap in space and time. This means that the species must be present at a location where the chemical is found and present at a time the chemical is present in the environment. Some chemicals are only intermittently used and found in the environment. Conducting life cycle or multigenerational studies for such chemicals will not provide much usable information.

e.    Environmental conditions: The environment can affect the toxicity and availability of a chemical. High temperatures can more effectively dissolve chemicals, and this might increase the amount of chemical an organism is exposed to. They can also break down chemicals, which can decrease or increase a chemical’s toxicity. Other factors can influence toxicity such as percentage of dissolved oxygen, salinity, nutrient level, moisture level, and microbial community. This makes it important to mimic the natural conditions of a test organism in the lab.

f.     Characteristics of the test organism: If the objective of the study is to assess sediment toxicity, then organisms that dwell in or near the bottom of a water body should be tested rather than organisms that dwell near the top, unless a major disturbance of sediment is expected (e.g. dredging). It is necessary to choose species that can coexist together if the objective is to study how a community of aquatic species is harmed by a given chemical.

While specific guidelines for various aquatic toxicity tests were linked in Table 2, the following are general guidelines that apply to all tests:

a.    Laboratory toxicity tests should be replicated at least thrice under similar conditions to ensure results are reproducible. Mesocosm tests should be replicated at least twice, while there are no requirements for macrocosm tests (due to their complexity and scale).

b.    All toxicity tests should have a negative control. The control should have the same conditions and constituents as the treatment group, minus the chemical that is being tested. If the chemical is dissolved in a solvent prior to its introduction into the water, then the negative control should also contain the solvent. This is done to remove any potential effects of the solvent (though solvents should be tested before the study to ensure they are non-toxic).

c.     While most toxicity tests are not required to have a positive control, it is encouraged. A positive control is a substance that is known to produce a defined toxic effect in the test organism. It is used to determine if the health and sensitivity of the test organisms have changed over the course of the study. Also, it can help validate data across different labs and help assess reproducibility of the results.

d.    No less than three organisms must be treated for every concentration that is used (including control). However, the minimum sample size often depends on the study type and objective. If too few organisms are treated, it is possible to miss significant differences that might exist between treatments and controls. If too many organisms are treated (more than what is necessary), it would cause ethical issues and lead to wasted time and resources. Therefore, a power analysis is often carried out to find the minimum number of organisms needing treatment to study a particular effect.

e.    All organisms used in a test must be homogenous (unless specifically instructed otherwise in the test guidelines). They should be of similar age, life stage, body mass, size, etc. They should all be healthy (sick organisms might be more sensitive to the effects of a chemical) and must have followed similar growth patterns prior to chemical exposure.

f.     Randomization of controls and treatments should be done to account for non-chemical effects. For example, if all control organisms are placed on the top shelf where the light source is the brightest, they may grow differently than treatment organisms placed on the bottom shelf due to dissimilar light intensity. The difference could be mistakenly attributed to the chemical in the absence of random placement.

g.    Conditions such as temperature, light, oxygen concentration, and hardness of water should be maintained throughout the test environmental to avoid impacting a chemical’s toxicity and/or availability (i.e., exposure).

h.    The concentration of a chemical must be measured and maintained throughout the test. Ideally, the chemical concentration must be analyzed in the water, food, sediment, and in test organism tissues: however, most test guidelines only require that chemical concentration be measured in water.

CONCLUSIONS
While laboratory toxicity tests greatly help in understanding a chemical’s effects, the observed effects will not necessarily be the same in the natural environment. This is due to several laboratory test limitations: a.    In nature, there are seasonal fluctuations, temperature changes, diverse microbial communities, etc. that are not captured in lab studies and which can greatly impact results.

b.    Organisms in nature are exposed to multiple chemicals and stressors simultaneously. These chemicals and stressors can interact and produce unexpected results. The majority of lab toxicity tests are conducted with only one chemical and under conditions that are favorable to the test organism.

c.     In nature, organisms may not be continually exposed to chemicals. In the lab however, long-term studies are carried out where organisms are continuously exposed to the chemical throughout their life cycle. Also, the concentration and availability of the chemical may vary in the environment while they remain constant in the lab.

d.    Most lab studies involve spiking the chemical in water and exposing organisms to it. In nature, organisms are also exposed to chemicals through food and sediment.

e.    Different physical habitats of ecosystems as well as the genetic structure of populations can also influence toxicity. Most lab studies are carried out on single species, ignoring species-environment, and chemical-environment interactions.

f.     Lab studies are done on healthy organisms that are very similar to one another (due to frequent inbreeding). Organisms in nature are more genetically and physiologically diverse.

g.    The results from surrogate species tested in the lab are extrapolated to other species since it is not possible to test the thousands of aquatic species present in nature. This adds considerable uncertainty to the results.

The first known aquatic toxicity test was conducted by Aristotle in the 4th century BC, when he exposed midge fly larvae to Athens’ effluent streams to monitor their survival and behavior. The field has progressed considerably since then, especially in the last century. Since 1899, many environmental protection laws have been introduced around the globe, and several of these laws require the regulation of chemicals prior to its registration and introduction into the environment. This has led to the development and standardization of toxicity tests. The aquatic toxicity test guidelines that are currently being used were developed within the last 27 years. However, these guidelines are not set in stone. A greater understanding of the world and man-made chemicals has led to the revision of old guidelines and the addition of new ones. In addition, separate test guidelines have been written in recent years to accommodate new chemicals with unique properties.

To better comply with increasingly stringent regulatory requirements and chemical testing while raising ethical standards, organizations such as the European Center for the Validation of Alternative Methods (ECVAM), AltTox, National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), Center for Alternatives to Animal Testing (CAAT), etc., are focused on finding alternatives to testing chemical toxicity in animals.

A seminal report was released in 2007 by the National Academy of Sciences titled Toxicity Testing in the 21st Century: A Vision and a Strategy15. This report recommended that toxicologists move away from using vertebrates in toxicity tests and move toward the use of non-vertebrates and cell lines. Today it is possible to recreate organs on a chip and test chemicals on it16. The report also suggested using computer and mathematical models (examples include Quantitative Structure Activity Relationships, Read-Across17, Species Sensitivity Distribution18, Physiologically Based Pharmacokinetic modeling19) and knowledge-based pathways (examples include Toxicity Pathways and Adverse Outcome Pathways) to predict the toxicity of chemicals and to extrapolate results from cell lines to whole organisms and ecosystems. These technologies are yet to be implemented on a large scale, and it will probably take several decades for this to be realized.

GLOSSARY
Actual Environmental Concentration (AEC): The measured concentration of a chemical in the environment.

Acute exposure: Short-term exposure to a chemical.

Acute toxicity test: Short-term toxicity tests that are usually 24-96h long.

Additive effect: Combined effect of two or more chemicals is equal to the sum of their individual effects.

Adverse Outcome Pathway (AOP): A structural representation of a sequence of biological events that leads to adverse effects, from the cellular level to the population level.

AltTox: A website dedicated to advancing non-animal methods of toxicity testing.

American Society for Testing and Materials (ASTM) International: A nonprofit international organization that develops and publishes procedures for testing chemicals and materials.

Analytical instruments: Instruments that can measure the physical and chemical properties of compounds.

Annelids: Ringed or segmented worms. Examples include earthworms, leeches and ragworms.

Antagonistic effect: Combined effect of two or more chemicals is less than the sum of their individual effects.

Benthic species: Organisms that live in the lowest level of water body. They live in, on, or near sediments. Examples include worms, crabs, clams and lobsters.

Bimodal distribution: The observation of two peaks when the data is graphically represented, unlike a normal distribution where only one peak is observed.

Bioaccumulation: Uptake and retention of chemicals through contact (integument), respiration (gills), and ingestion (mouth).

Bioconcentration: Uptake and retention of chemicals through contact (integument) and respiration (gills).

Biodegradability: Breakdown of chemicals by microorganisms, such as bacteria and fungi.

Biomass: The total mass of organisms in a given area or volume.

Cell lines: Cells that are isolated from organisms and grown in the lab under controlled conditions.

Cell perturbation: Alteration in the functioning of the cell.

Center for Alternatives to Animal Testing (CAAT): An academic center at Johns Hopkins University that is working to find new methods to replace, reduce, and refine animal testing.

Chemical purity: The degree to which a substance is undiluted or unmixed with extraneous material. Typically expressed as a percentage (%).

Chemical stability: Tendency of a chemical to resist change or decomposition due to physical, chemical, or biological factors.

Chronic exposure: Long-term exposure to a chemical.

Chronic toxicity test: Long-term toxicity tests that last for at least 10% of the life span of the tested species.

Clean Water Act (CWA): The United States Act that prohibits discharge of toxic pollutants in toxic amounts in water bodies.

Concentration of solution: The ratio of solute to solvent, or the ratio of solute to total solution.

Concentration-Response Curve: Graphical representation of a population’s response to a range of chemical concentrations.

Confidence interval 95%: A range of values within which the true value lies, with 95% certainty.

Crustaceans: Invertebrate animals with external skeleton and a segmented body. Examples include crabs, lobsters, shrimps, and prawns.

Dose: The amount of chemical that enters an organism.

Dry deposition: The direct deposition or settling of chemicals present in the air.

Early-life-stage toxicity test: Toxicity tests that are done on embryos or on larval (juvenile) stages of a species.

Ecological balance: The equilibrium between, and harmonious coexistence of, organisms and their environment.

Ecosystem: A biological community of interacting organisms and their physical environment.

Effective Concentration 50 (EC50): Concentration at which 50% of the organisms in a population are affected following chemical exposure.

Effluent: Liquid waste or sewage discharged into a water body.

Emergence: The appearance of adult insect from its pupal case (i.e., its chrysalis or cocoon).

Empirical data: Evidence gathered from observation or experimentation.

Endangered Species Act (ESA): The United States Act that requires federal agencies to ensure that their actions do not jeopardize the existence of any threatened or endangered species.

Epidermal surface: Tissue that covers the outer surface of plants.

Estimated Environmental Concentration (EEC): The estimated (non-measured) concentration of a chemical in the environment.

European Centre for the Validation of Alternative Methods (ECVAM): An European Union Reference Laboratory that is studying alternatives to animal testing.

Exposure (or exposure concentration): The concentration of chemical an organism encounters in the environment.

Extrapolate: A method to estimate new (unknown) values from old (known) values.

Filter media: Anything placed in a filter that changes the quality of water flowing through it.

Flow-through system: A setup in which organisms are held in continuously flowing water for the duration of the study.

Food web: A system of interdependent food chains. A food chain is a hierarchical series that shows how various organisms obtain food.

Geometric mean: The geometric mean of two numbers is the square root of the product (multiplication) of the two numbers.

Habitat: The natural home or environment of an animal, plant, or other organism.

Halogens: A group of reactive nonmetallic elements that include fluorine, chlorine, bromine, iodine, and astatine.

Histopathology: Microscopic examination of tissues in order to study damage.

Hydrophilic chemicals: Chemicals that easily dissolve in water and typically contain many hydrogen and oxygen atoms.

Hydrophobic chemicals: Chemicals that do not dissolve in water and typically contain many carbon atoms.

Inhibitory Concentration 50 (IC50): Concentration at which 50% of the organisms in a population are inhibited following chemical exposure.

Influent: Something that flows into a system.

International Organization for Standardization (ISO): An independent international organization that sets standards for many materials, products, processes, and services.

Kelp: A type of large brown seaweed.

KOW (Based) Aquatic Bioaccumulation Model (KABAM): A model that can estimate potential bioaccumulation of hydrophobic carbon-based pesticides in freshwater aquatic food webs.

Lethal Concentration 50 (LC50): Concentration at which 50% of the organisms in a population are killed following chemical exposure.

Life cycle toxicity test: Toxicity tests that last throughout the life cycle of an organism.

Lowest Observed Effect Concentration (LOEC): The lowest concentration of chemical that causes a toxic effect in the treated population.

Macrocosm: Toxicity tests that are conducted in lakes and on whole aquatic ecosystems.

Maximum Acceptable Toxicant Concentration (MATC): The concentration between NOEC and LOEC, or a geometric mean of the two.

Mesocosm: Toxicity tests that are conducted on multiple species placed in experimental water enclosures that mimic natural conditions. The system contains more than 1000 liters of water.

Metabolic byproducts (or metabolic waste): The left-over products of metabolism.

Metabolic enzymes: Enzymes that carry out a variety of critical cellular functions in organisms.

Metabolism: The chemical processes that occur within a living organism in order to maintain life.

Metamorphosis: The process of transformation of an organism from an immature form to an adult form.

Microcosm: Toxicity tests that are conducted on two or more species in an artificial and controlled system within a laboratory or outdoors. The system contains less than 1000 liters of water.

Model organism: A species that has been widely studied, is easy to maintain and breed in a laboratory, and has several other characteristics that make it ideal for experimental use.

Mode of action: The mechanism by which a substance produces an effect in living organisms or biochemical systems.

Moles: The quantity of a substance that has the same number of particles found in 12 grams of carbon-12 (6.02x1023 particles).

Mollusks: Invertebrate animals that have a soft unsegmented body. Examples include snails, slugs, mussels, and octopuses.

Multigenerational toxicity tests: Toxicity tests that are carried out on two or more consecutive generations (parents and offspring).

National Centre for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs): A UK-based scientific organization dedicated to finding alternatives to animals in research and testing.

Negative control: A group that has the same conditions and constituents as the treatment group, minus the chemical that is being tested.

No Observed Effect Concentration (NOEC): The highest chemical concentration that does not cause a toxic effect in the treated population.

Normal distribution: The distribution of many random values as a symmetrical bell-shaped graph.

Organization for Economic Co-operation and Development: An intergovernmental economic organization that considers the environmental implications of economic and social development.

Partition coefficient: The ratio of concentrations of a chemical in a mixture of two immiscible (unmixable) liquid phases at equilibrium.

Parts per billion: Corresponds to 1 µg of chemical/L of solution. The amount of chemical is nine orders of magnitude lesser than the amount of solution.

Parts per million: Corresponds to 1 mg of chemical/L of solution. The amount of chemical is six orders of magnitude lesser than the amount of solution.

Parts per trillion: Corresponds to 1 ng of chemical/L of solution. The amount of chemical is twelve orders of magnitude lesser than the amount of solution.

Peak concentration in water: The maximum concentration of chemical estimated or measured in water.

Pesticide: A substance used to destroy pests. The pests can be insects, weeds, fungi, rodents, etc.

Pesticide in Water Calculator (PWC): A model that estimates pesticide concentrations in water bodies following pesticide applications to land.

Physiochemical properties: The properties of a chemical observed from its interaction with the physical environment.

Physiologically based pharmacokinetic (PBPK) modeling: A mathematical modelling technique that predicts the absorption, distribution, metabolism, and excretion of substances in living organisms.

Plankton: A diverse collection of small organisms that live in large bodies of water and are unable to swim against a current. Examples include bacteria, algae, protozoa, and archaea.

Positive control: A group that receives a substance that is known to produce a defined toxic effect.

Potentiation effect: Combined effect of two or more chemicals is greater than the sum of their individual effects. Usually one of the chemicals produces no effect on its own.

Power analysis: A statistical analysis that helps determine the sample size required to detect an effect of a given size with a given degree of confidence.

Quantitative structure–activity relationship (QSAR) model: A model that predicts the biological activities of untested chemicals using their structures.

Randomization: The process of making something random to minimize bias.

Read-Across: A model that predicts the toxicity of untested chemicals using the toxicity information of similar but tested chemicals.

Recirculation system: A setup in which organisms are held in water that is continuously run through a filter for the duration of the study.

Renewal system: A setup in which organisms are held in still water that is regularly changed for the duration of the study.

Resistance: The natural ability of some organisms to withstand the effects of a compound.

Risk: It is the likelihood of a hazard (e.g. a toxic chemical) causing harm.

Risk Quotient: The ratio of chemical exposure to chemical effects.

Sample size: The number of organisms in an experimental sample or group.

Sediment toxicity testing: Toxicity tests where aquatic organisms are exposed to sediments to determine if the chemicals in the sediments will harm them.

Sensitivity: An organism’s susceptibility to the effects of a compound.

Sigmoidal curve: A “S” shaped curve.

Site of action: The location where a compound binds to exert its effects. This is usually a cell receptor, an ion channel or an enzyme.

Slope of curve: The steepness of a curve.

Solvent: A liquid in which a compound is dissolved to form a solution.

Species diversity: The number and distribution of species in the environment.

Species richness: The number of species in the environment.

Species Sensitivity Distribution: A mathematical model that describes the variation in chemical sensitivity between species.

Spiking: The addition of a compound to a solution.

Standard deviation: A quantity that indicates how measurements for a group are spread out from the average or expected value.

Standardization: The process of making something conform to a standard i.e. done according to specific guidelines.

Static system: A setup in which organisms are held in static water that in unchanged for the duration of the study.

Stomata: Minute pores in the epidermis of the leaf or stem of a plant which allows for exchange of gases.

Stressors: Factors that stress organisms. Examples include food shortage, parasites, predators, and changing environmental conditions.

Subchronic toxicity test: Prolonged acute toxicity tests that are typically between 28 days to 3 months long in fish.

Surrogate species: A species that is used to represent multiple species or aspects of the environment.

Swimming equilibrium: The ability that allows fish to maintain an upright position within the water column.

Synergistic effect: Combined effect of two or more chemicals is greater than the sum of their individual effects.

Threshold concentration: The lowest concentration of a chemical that elicits a response.

Tiered Testing: A toxicity testing method where lower tiered studies (representing simple worse-case scenarios) are followed by higher tiered studies (representing complex realistic scenarios).

Toxic effects: The adverse effects produced by exposure to a substance.

Toxicity: The degree to which a substance can harm living organisms.

Toxicity curve: A graph in which chemical concentration (or dose) is plotted against an organism response.

Toxicity endpoint: The measurements and observations taken during or after a toxicity study.

Toxicity Pathway: A sequence of intracellular events that, if disturbed, can cause an adverse outcome at the cellular level.

Toxicity test: A test conducted to find the degree to which a substance can harm living organisms.

Toxicity thresholds: The lowest concentration of a chemical that causes toxicity in organisms.

Uncertainty: Undetermined factors that can produce unreliable results.

United States Environmental Protection Agency (USEPA): A federal agency in the United States that is responsible for registering and regulating chemicals, including pesticides.

Validate: Check or prove the accuracy of (something).

Vapor pressure: A quantity which informs the tendency of a chemical to evaporate.

Vascular system: The presence of xylem and phloem tissues in plants that are responsible for transporting water, nutrients, and food.

Wet deposition: The deposition or settling of chemicals in the air through rainfall, snowfall, and fog.

Whole effluent toxicity testing: Toxicity tests where aquatic organisms are exposed to wastewater effluents to determine if chemicals in the effluents will harm them.


text: User talk:23.28.183.102

title: User talk:23.28.183.102


text:
Programming in 1L a/Flattened 1L a

A way to create 1L_a programs is to write the corresponding Brainf*** program first, and then convert it into 1L_a. So it's time for a new language, Flattened 1L_a, which took place between 1L_a and Brainf***.

Brainf*** and its minimalizations
As you might know, Brainf*** is a well-known and one-dimensional programming language. Like 1L_a, there's DP and IP too, yet the DP only moves in a line, and the DP moves on a character array instead of a bit array. It is Turing complete and has 8 instructions. Here are them:

Yet, as a Turing-Tarpit(languages that are designed to be both have only a few instructions and is Turing-Complete), 8 instructions are a bit too much. So we may create Brainf*** equivalents that have less instructions.

We call a language Brainf*** equivalent if you can implement every instructions in the language in Brainf*** and implement every instructions in Brainf*** in the language.

Boolf***
It's pretty much obvious that we can reduce Brainf*** to 7 instructions by operating on an array of bits(like 1L_a!) instead of an array of characters, so that the instruction + and - become one: the toggle operation, notated as @. It is possible to use our powerful flow control to simulate a character array, but it would be bulky and I'm too lazy to do that. Luckily, someone have already done it for us:

Brainf*** The new language - >        >>>>>>>>> <         <<<<<<<<< +         >>>>>>>>[<]@>[@>]<<<<<<<<<[@] -         @>>>>>>>>@[<@]>[>]<<<<<<<<<[@] [         @[>>>>>>>>@[<@]>[>]<<<<<<<<<[@]>>>>>>>>[<]@>[@>]<<<<<<<<<@[@ ]         >>>>>>>>>@<<<<<<<<<]>>>>>>>>>[<<<<<<<<<@>>>>>>>>>@]<<<<<<<<<] .         >.<,         >,<

We call this language Boolf***. Since Brainf*** is Turing-Complete, and you can translate any Brainf*** program into Boolf*** program, Boolf*** is Turing-Complete too.

title: Programming in 1L a/Flattened 1L a


text: Perspectives of Aquatic Toxicology/Contributors' Biographies


text:
Baroque Macedonia and the Macedonian Revolts/Chapter 3 : The gathering of troops

Chapter 3
The gathering of troops

The Macedonians had also attempted a revolt back in the mid 16th century and this was one of the very first revolts ever to occur. The local uprising took place in 1564, it began in the Mariovo region and spread to the Prilep plains and from there to the town of Prilep. Known as the Mariovo and Prilep revolt, it is unknown why this revolt began, but it is clear that three peasants and two priests from the Mariovo district started it. No sooner had the trouble started when the Sultan, through a decree dated October 3, 1564, ordered that the leaders of the revolt be put to death while the followers were to be sent to serve as oarsmen on Turkish galleys. Before the decree could be enforced, however the perpetrators fled causing the Sultan to order another decree for their capture. The Mariovo and Prilep rebellion was one that was unsuccessful and the rebellion was suppressed by the Ottoman troops. Prilep soon became a place known for demonstrations, when the Ottoman court ruled in favour of a Pasha in a dispute with the peasants. According to a document dated December 1565 a revolt broke out inside the town of Prilep. The situation began when the Prilep court, in settling a dispute between the peasants and Mustafa Pasha, ruled in favour of the Pasha. Later the news hit the streets and more than a thousand rebels from the surrounding villages, armed with garden shovels, and long barrelled muskets assembled and stormed the court. It is unknown how this situation or incident ended. As explained previously the peasants eventually had no chance in further causing trouble as the large Ottoman forces greatly enforced order upon the peasant rebels.

According to historical sources General Piccolomini was currently in the town of Nis, Ottoman Serbia, he arrived there on the 24th of September, 1689 in the early hours of the morning. With him was also the Austrian commander, Louis William, Margrave of Baden-Baden. Accompanying the two commanders was Pavle Nestorović who was a Serbian commander that assisted Piccolomini, during the capture of Nis. Nis was a quite town located in the southern part of Ottoman Serbia, it had a garrison of approximately 40,000 soldiers or most likely over 40,000. Louis William had a total of about 16,000 troops, most of them were Austrian however there were also Serbian troops. When Louis William learned that there were no Ottoman defence positions on Vinik he ordered Pavle to attack. Eventually Pavle was successful and the Battle of Nis ended with an Austrian victory. Pavle was promoted and later Louis William had decided to leave for Vidin while Piccolomini was left to keep an eye on Nis. The next month Piccolomini left for Ottoman Macedonia, and when this happened the Ottoman troops once again gained possession of the Sanjak of Nis.

On the right we see a photo of a Macedonian rebel holding a heavy musket. These types of guns were very popular during the first half of the 17th century. A musket is a muzzle-loaded long gun that appeared as a smoothbore weapon in the early 16th century, at first as a heavier variant of the arquebus capable of penetrating heavy armor if fired correctly. Distinguished features of a heavy musket was a very long barrel, the reason for this extremely long barrel was to gain a good or reasonable range. Evidence of the musket as a type of firearm does not appear until 1521, the long-barreled, musket-caliber weapons had been in use as wall-defense weapons in Europe and in other continents. By 1650 the heavy musket eventually become obsolete due to its size and weight, as a result of this the musket was improved. By 1690 the wearing of knights armor dramatically decreased, this was probably due to the increased power of certain types of guns.

There are two sources regarding the arrival of Silvio Piccolomini to the city of Uskup (Skopje). General Piccolomini arrived in Uskup in late October, 1689. One historical source explains that when General Piccolomini entered Uskup he was greeted by the people of Uskup, and there was also a fanfare playing at the time, while the other historical source explains that when he arrived to the city, the city was most likely abandoned and there was only a few people. When General Piccolomini set out from the Habsburg Empire he had a total of 4,000 soldiers most of them were cavalry, after the battle of Nis, his armies were reduced as some of them were killed in action or were injured and had to remain behind. Overall his armies now were a small number, and this greatly affected his abilities to guard or even defend a large city such as Uskup.

With General Piccolomini in Uskup, what happened to the Ottoman garrison that was stationed in Uskup ? Did they withdraw from the city of Uskup after witnessing the Austrian armies entering the city, or was the city unguarded and no Ottoman troops were positioned within the streets of Uskup ? These are just some questions that have puzzled the Macedonian historians. The answer to this question is that there were no Ottoman soldiers stationed within Uskup (Skopje) at the time, because if there was then a fierce battle would break out. There was no armed conflict between the Ottoman troops and the Austrian cavalry units within Uskup.

While staying in Skopje, Piccolomini started feeling very ill. He was unaware that he had contracted some form of disease. According to historical accounts, Piccolomini had cholera and his health was beginning to deteriorate. Piccolomini had the following symtoms : diarrhea, vomiting, dehydration, and discoloration of the skin. It is unclear how Piccolomini contracted cholera, it was most likely through the drinking of contaminated water or the eating of food that was contaminated with the bacterium. Back in the late 17th Century (1601-1700) the medical knowledge relating to cholera was limited, doctors were uncertain as to what caused the disease, and their current medicines provided no relief or prevention from the bacterium. Piccolomini was beginning to feel very weak due to the cholera, on the 26th of October, 1689 he ordered his armies to burn the city of Skopje. His troops placed gunpowder all around some of the buildings within the centre of Skopje and a fire was started. The Skopje fire of 1689 lasted two whole days, burning much of the wooden structures and buildings. The fire later spread towards the Jewish quarter of the city, the Jewish synagogue and a Jewish church was completely destroyed in the fire. However many of the stone buildings remained, but were blackened by the raging fire. Due to General Piccolomini's health he had decided to disband from his armies, heading to Ottoman Albania.

General Piccolomini's reason for going to Ottoman Albania was so that he can board a ship and head back to Austria. In the cartographic map of Europe which is shown on the right, the Habsburg Empire also known as the Imperial Austrian Empire had access to the adriatic sea. It was a small region just north of Ottoman Bosnia where Austria had a number of ports, here the galleons would arrive and unload. The prospects for Austrian arms in Macedonia and Albania looked promising, but a few days later Piccolomini died of the plague and his successor duke Georg Christian Von Holstein was unable to capitalize on the so called " Karposh Uprising " of the Christian peasantry which broke out in Macedonia at about this time. Eventually assistance from the Austrian armies ended and the continuation of the Karposh uprising went until December, 1689.


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Perspectives of Aquatic Toxicology/Chapter Two: Bio-transformations of Xenobiotics

= Chapter 2: Biotransformations of Xenobiotics =

Introduction
Fish and other aquatic organisms are exposed life-long to the combined effluents of human sources, erosion runoff, and natural excretions from plants and animals. Exposure of aquatic organisms to the chemical mixture is very different from that of terrestrial species like humans. For example, while both humans and aquatic organisms might be exposed to the water-soluble herbicide atrazine, in humans the exposure would most likely be through ingestion of contaminated food or drinking water. Fish would be exposed through their skin and gills. Ingested atrazine first travels to the liver whereas atrazine taken up through gills go directly to the bloodstream. In this way, the environment of terrestrial and aquatic organisms plays a significant role in exposure to various environmental chemicals.

All organisms have defenses to help them deal with and survive potentially harmful chemicals originating from outside the body known as xenobiotics. A large group of these defenses take the form of enzymes that transform a xenobiotic into a different molecule, ideally one that will no longer pose a threat to the host organism. This process is called biotransformation (Figure 1). Biotransformation is the method for metabolic detoxification of xenobiotics. Aquatic organisms have evolved an array of methods to perform biotransformation when they encounter a potent mix of chemicals dissolved in their aquatic environment.

ADME: Absorption, Distribution, Metabolism and Excretion
Absorption, Distribution, Metabolism, and Excretion (ADME) are the four steps used to describe the ways a toxicant will interact with an organism to allow or disallow it from inducing a toxic outcome. ADME describes where, when, and how much of a potential toxicant is present (the toxicokinetics), but it does not describe how the toxicant causes harm (the toxicodynamics). Many of the principles of ADME are very similar between related terrestrial and aquatic organisms; however, absorption can be very different due to varying potential exposures when a toxicant crosses an aqueous medium. This differential absorption--based on the physical properties of the xenobiotic compounds--determines which chemicals will be engaged in the third part of the acronym: metabolism.

One physical property that largely influences how a potential toxicant will interact with a body is its propensity to associate with water or lipids. A measure that describes this physical property is the partition coefficient (P) which is often reported as log P. Log P is sometimes written as log Kow where P = Kow = the octanol/water partition coefficient as the measurement is taken while performing a liquid/liquid separation with octanol and water. Higher values of log P mean that a given molecule will spend more time associated with non-polar conditions such as octanol over a highly polar aqueous environment. This has major implications for fish and other aquatic organisms that have fat rich tissues separated from the water-based environment by complex membranes having both non-polar and polar characteristics.

Physical properties of gills: The gills of fish are highly specialized oxygen and ion exchange tissues that include high surface area lamella and extensive vasculature. Even with these specializations, some of the properties of fish gills (e.g. thin membrane size and partial permeability to water, gases, and solutes) allow general comparison to membranes of other taxa such as insect gills that do not have pulmonary vasculature. The gill cells in direct contact with the environment are epithelial cells of various kinds, including lamellar cells that facilitate oxygen exchange and chloride cells that are essential for ion balance (Evans, 1987). Fluids near a solid surface create a slow-moving layer called a boundary layer. When a xenobiotic interacts with the boundary layer of water around a gill, it is called an aqueous diffusion layer. The xenobiotic must cross this layer via diffusion rather than being carried by the flow turbulence due to the relative stillness of this boundary layer (Figure 2). Erickson, et al. (1990) explored how potential xenobiotics cross the aqueous diffusion layer and the cell membrane of the gills in rainbow trout. They found that chemicals of high lipophilicity (log KOW > 3) had low uptake due to an inability to readily diffuse across the aqueous diffusion layer. Chemicals of particularly low lipophilicity (log KOW less than 1) also had lower uptake because it is more difficult to cross the cell membrane which contains lipids. Uptake of chemicals peaked in a Goldilocks zone of moderate lipophilicity (log KOW between 1 and 3), as they could cross both barriers at higher rates. It is likely that other taxa of organisms with gills or permeable skin would have similar chemical uptake profiles in relation to the compound’s log KOW.

The excretion of xenobiotics in aquatic life and land dwelling counterparts is very different. Terrestrial animals typically have some mechanism to withdraw excess water from fecal material to reduce water lost during defecation. Nitrogen waste in the urine depends on the common availability of water to a species. Bursell (1967) describes several different strategies for nitrogen waste removal in insects. Terrestrial insects that reside in dry climates such as the migratory locust (Locusta migratoria), pack their nitrogen waste in the form of insoluble crystals of uric acid and excrete a mostly dry waste. Insects that spend part of their life history in water such as Aeshna cyanea larva, a species of the hawker dragonfly, are more similar to fish in that they produce the potentially toxic nitrogen product, ammonia, but allow it to diffuse into the surrounding water before it builds to harmful levels. Humans take a middle route. We have no sink that allows ammonia to simply diffuse away when it occurs, and we require a relatively high water intake to partially eliminate the need to spend energy packing nitrogen into uric acid. Instead, humans and many other species make urea: a compound with lower toxicity and solubility in-between ammonia and uric acid (OpenStax, 2013). The general excretion strategy used by aquatic organisms is to make use of the surrounding environment by creating a sufficiently water-soluble molecule that will diffuse away from the body.

Biotransformations, Phase I
The major metabolic pathways responsible for biotransformation of potential toxicants are called phase I and phase II, although they do not necessarily occur in that order or in sequence at all. Phase I bio-transformations typically employ water or oxygen as an enzymatic factor in reactions such as oxidation, hydrolysis, and reductions. Phase II bio-transformations often form covalent bonds to existing biomolecules in the body. Bio-transformations typically lead to detoxification of a xenobiotic, most commonly by making it more readily excretable. Once a chemical is excreted from the body it is much less likely to cause harm. Changes to the chemical structure can also make a xenobiotic less toxic, even if it is retained in the body.

Oxidation: Many phase I bio-transformations are performed by mixed-function oxidases (MFOs) which are common in the liver as well as in other tissues. A major family of MFOs is called cytochrome P450 (Klaassen, 2013). Cytochrome P450 enzymes perform oxidations (the number in the name refers to the wavelength of light absorbed by the enzyme and helps in their characterization). Many different cytochrome P450’s have been discovered and have a wide range of specificity and substrate compatibility (Figure 3). Stegeman et al. (1991) explored how several carcinogenic toxicants are metabolized by different Cytochrome P-450’s in fish. Distribution of these enzymes were spread out over the locations, liver, kidney, and gills in Scup fish. Despite subtle differences in the specific enzyme used, cytochrome P-450’s are highly conserved. Moktali et al. (2012) trace the evolution of these detoxification enzymes and show that it is more ancient than any terrestrial lineage.

Reductions: Xenobiotics of an elevated oxidation state (certain metals and functional groups such as carbonyls, disulfides, quinones, azo, and nitro groups) can be reduced directly or enzymatically using reducing agents often referred to in consumer foods as “anti-oxidants.” These reducing agents or cofactors include glutathione, FAD (Figure 4), FMN, and NAD(P) (Klaassen, 2013). Many of these cofactors are modifications of basic essential molecules: glutathione is a modified polypeptide and NAD(P) are modifications of dinucleotides. Some enzymes considered oxidases can perform reductions: cytochromes P450 can sometimes use a xenobiotic as the oxidizing cofactor which will effectively perform a reduction on the xenobiotic to oxidize something else. Reductases can vary in specificity. Nitrate reductases are important for the nitrogen cycle and allow plants to utilize nitrate fertilizers. Timmermans, et al. (1994) investigated the effects of available iron on nitrate reductases in phytoplankton and found that the ability to produce a functional enzyme requires the presence of dissolved iron in the environment.

Hydrolysis: Hydrolysis is the process of adding water across a chemical bond. The most generic example is breaking a C-O bond, and making a new C-O bond and an O-H bond (Figure 5). Other bonds can be hydrolyzed as well. The defining characteristic is the consumption of water to split a bond, as shown in blue in Figure D. The process leads to a net zero change in oxidation state: it is neither an oxidation nor reduction reaction. Some compounds undergo hydrolysis spontaneously in water or in plasma at appreciable rates, but living organisms have evolved enzymes that can speed up this reaction. Some of these enzymes include esterases which hydrolyze esters, peptidases which hydrolyze protein peptide bonds, and phosphatases which hydrolyze phosphoester bonds. Chlorine metabolism: While humans are the main generators of organic chlorines in terrestrial environments, producing chemicals and legacy pollutants such as polychlorinated biphenyls (PCB’s) and insecticides (Figure 6), some living organisms produce chlorinated organic compounds as well, particularly those in aquatic environments. Gribble (1996) discussed “the diversity of natural organochlorines in living organisms” and offered examples, including a chemical produced by the freshwater fungus Kirschsteiniothelia sp., 3,3’-oxybis(2,4-dichloro-5-methylphenol) (Figure 7).

Organochlorines or chlorinated organic molecules are chemicals containing a C-Cl bond. The presence of chlorine on the molecule tends to make it more lipophilic and more difficult to degrade via phase I biotransformations or energy metabolism by microorganisms. These two characteristics together, high lipophilicity and persistence, promote retention of these chemicals within biological tissues and can lead to toxic buildup. Removing chlorines from organic molecules, dehalogenation, is energetically less favorable than removing common heteroatoms such as sulfur or nitrogen (Dugat-Bony, 2016). The enzymes that detoxify organochlorines are rare in life; they are usually relegated to microorganisms that use compounds for energy metabolism. Dugat-Bony, et al. (2016) described various methods used to dehalogenate the organocholrines which overlap with other detoxification processes described above. Monooxygenases such as cytochromes P-450 can both oxidatively or reductively eliminate chlorine. Glutatione S-transferase (see next section, phase II biotransformations) can reductively eliminate chlorine. Detoxification of organochlorines are undoubtedly useful for the host microbe, but they have also been studied as potential solutions to human environmental contamination involving persistent organochlorine pollutants (Jugder, 2016).

Biotransformations, Phase II
Phase II biotranasformations are characterized by additions, called conjugations, of small bio-molecules to the xenobiotic such as sugars and amino acids. These additions, larger in size than hydroxyl additions from phase I oxidations, both potentially increase the hydrophilicity of the toxicant and potentially block the molecule from its site of toxic action by changing how it moves through tissues and cell membranes. While Phase II does not always follow phase I, the functional groups created in phase I transformations such as a hydroxyl –OH group, can be used as a target for conjugation reactions of phase II biotransforamtions. Conjugations are usually more effective than phase I biotransformations in regards to increasing hydrophilicity and decreasing toxic effects. Many examples of conjugations can be found including methylations and acetylation (Figure 8); however, this chapter focuses on the most well studied: glucuronidation, sulfonation, and glutathione conjugation. Glutathione: The glutathione S-transferases (GST) is a highly conserved conjugation enzyme family. Glutathione, a tripeptide capable of acting as a reducing agent for phase I biotransformations on its own, can be used with a GST to bind with a xenobiotic via the sulfur group on the cysteine moiety of a reduced glutathione molecule. This greatly increases the polarity and molecular weight of most target substrates, allowing them to be excreted with water soluble waste. Stenersen et al. (1986) investigated nine different animal phyla, examining both terrestrial and aquatic organisms for the presence and activity of GST. They studied aquatic and non-aquatic vertebrates, insects, crustaceans, and mollusks and found that nearly all the animals had GST activity, though activity was significantly higher for terrestrial organisms than for aquatic life. As described by Edwards, et al. (2005), plants have a small subset of water-soluble GST enzymes. Glutathione is very important for the glutathione-ascorbate cycle and reduction of hydrogen peroxide in plants, and it can also play a part in plant defense against pathogens.

Glucuronidation: Sugars are a common biomolecule and are highly water soluble, making them ideal conjugates to increase the solubility of a xenobiotic when carbohydrates are in abundance. Glucuronic acid, a derivative of glucose, is a major cofactor of phase II biotransformations and are used by the enzyme family: uridine 5’-diphospho-glucuronosyltransferase (UDP-glucuronosyltransferase, UGT). Glucuronic acid itself is initially transformed into uridine diphosphate glucuronic acid which is the sugar derivative covalently bonded to another biomolecule through a diphosphate linkage. The diphosphate raises the energy of the molecule (similar to why Adenosine triphosphate is used as energy by the cell) which allows the UDP-glucuronosyltransferase enzyme to proceed with the conjugation by releasing the energy stored in the phosphate linkage of the cofactor. In this way the cofactor acts as both conjugate and energy source (Klaassen, 2013). Enzymes without active sources of energy drastically slowdown in response to temperature decline. UDP-glucuronosyltransferase is also affected by temperature, with an optimal range dependent on the organism and its environment, but not to the same extent due to energetics. As such, UDP-glucuronosyltransferase is helpful to aquatic organisms that experience temperature changes in the water. HÄNNINEN, et al. (1984) describe how rainbow trout lose CYP-450 enzyme functionality at colder stream temperatures, but maintain the use of UDP-glucuronosyltransferase to allow continued detoxification processes in colder climates.

Sulfonation: Sulfates can be conjugated to open hydroxyl groups on xenobiotics. These are performed by sulfotransferases which take a sulfonate group (SO3-) from the cofactor 3’-phosphoadenosine-5’-phosphosulfate, which is a modified nucleotide similar to ATP (adenosine triphosphate) but with a sulfate instead of a phosphate (Figure 9). Sulfonation is an important way to biotransform the active estrogen, 17β-estradiol. Exposure to exogenous estrogens can lead to deformations of the gonads in aquatic organisms. Wang et al. (2007) explored what can happen when the sulfonation of these estrogens is blocked by other environmental contaminants. The authors found that polychlorinated biphenyls (PCBs) with hydroxyl groups (OH-PCBs) inhibited the activity of sulfotransferases to sulfonate 17β-estradiol. In this case, products of phase I biotransformations (OH-PCBs) were detrimental to the biotransformation of other xenobiotics (estrogen), essentially making the PCB more toxic or toxic in a different way. See the following section for more examples of biotransformations increasing the toxicity of a xenobiotic.

Biotransformations that Increase Toxicity
Biotransformations typically result in changing a xenobiotic so that it will not harm the organism. The range of xenobiotics that can affect an organism are diverse however, and the same biotransformation that causes one xenobiotic to become harmless could transform a different xenobiotic into something much more harmful. A human example is alcohol dehydrogenase. Ethanol, which can have harmful effects on the body or behavior on its own, undergoes an oxidative biotransformation catalyzed by alcohol dehydrogenase or other enzymes (Figure 10). The ethanol becomes ethyl aldehyde (or acetaldehyde) which is toxic to the liver and other organs (NIH NIAAA, 2007). The aldehyde is further oxidized to acetic acid (the tangy part of vinegar) which is much less toxic. A xenobiotic that becomes more toxic after a biotransformation is called a pro-toxin. The above example illustrates that alcohol is both a toxin and pro-toxin for humans. An endangered fish species found in Mexico’s freshwater environments, Chirostoma riojai, is an example. Vega-Lo´pez et al. (2011) did a study on how naturally occurring halomethanes could be affecting this species. Halomethanes are single carbon molecules with some number of halogens, and occur naturally in water. The halomethanes underwent biotransformation with a subset of cytochrome P450s and were oxidized (see section on chlorine metabolism). The oxidized halomethanes caused oxidative stress to the fish, which can further lead to oxidative damage. The oxidation caused the xenobiotic to become more reactive and increased the exposure risk. This is an occasional downside to the broad specificity of some enzymes such as mix function oxidases.

Enzyme Induction
Types of pollutants and xenobiotics can vary according to location and time. Keeping a suit of enzymes to answer every potentially harmful xenobiotic would be energetically expensive and potentially counterproductive when there is only a finite subset of xenobiotics to address at any one time. The key to surviving and thriving is to have the correct enzymes in place when needed. Enzymes are proteins which are produced constantly by reading DNA. Cells self-regulate which DNA genes are being read and how much protein to create, depending on signals that tell the cell what is currently needed. Genes corresponding to detoxification enzymes can be turned on or “upregulated” by a process called enzyme induction. When an organism encounters a particular xenobiotic it signals a response to induce the production of detoxification enzymes to address that specific xenobiotic or closely related group. This process is not instantaneous and means that when encountering a new xenobiotic, it will likely be harmed. If an organism survives the initial encounter it will induce the production of enzymes to protect itself from subsequent encounters, similar to the immune system’s reaction to a virus. This could result from switching food sources where a new plant food is high in secondary metabolites--in this case the enzymes induced may be of general detoxifying function. A review of biotransformation systems in fish by Chambers et al. (1976) compared the prevalence of enzyme induction of mixed function oxidases between mammals and fish. Fish were found to upregulate these enzymes following a change in diet or pesticide exposure, but at lower levels than that found in mammals.

Many of the enzyme groups discussed in phase I and II biotransformations have some members that act broadly on a wide range of molecules. Mixed function oxidases are named for their broad specificity and have more than one function. More specific enzymes can be induced once a xenobiotic is encountered, and they may possess more potent activity due to their specialization. Hence, organisms can balance the tradeoff between specificity and activity while maintaining the highest chance of evolutionary success.

Glossary
Aquatic organism – An organism that spends a part of its life-cycle (e.g. larval stage) or a whole life-cycle in the water. For this chapter it includes both freshwater and saltwater environments. Some literature uses the term “marine organism” for saltwater environments.

Xenobiotic – A molecule that originates from outside an organism that is not naturally produced (synthetic) or expected to be present in the organism. They are not always toxic (many are beneficial) but this chapter will use the term to refer to a potentially toxic substance.

Biotransformation – The process of a living organism changing a chemical. A biotransformation is often carried out by an enzyme.

Enzyme – A protein that catalyzes a chemical reaction. This means it facilitates and speeds up chemical reactions. Enzymes are unchanged after the reaction, allowing them to repeat the process; but, may be limited by the availability of cofactors.

Cofactor – A biomolecule that is required by an enzyme to function. In biotransformations the cofactor is used up by the enzyme to change the xenobiotic.

Oxidation/Reduction – Terms for chemical reactions in which electrons are transferred from one molecule to another. These reactions go in pairs because if one molecule is gaining electrons another must lose them. The names in Phase I biotransformations refers to the effect on the xenobiotic. I.e. “oxidation reactions” are where the xenobiotic is oxidized (looses electrons); the cofactor is reduced and the enzyme stays the same.

Enzyme Induction – The upregulation of transcription for a certain protein due to internal signaling in an organism. In this chapter it refers to the upregulation of detoxifying enzymes after a poisoning to counteract further poisonings.

Conjugation – The chemical process of joining two molecules together.

Toxicokinetics – Movement of a toxic chemical through a living organism. This includes absorption, distribution, metabolism and excretion. Toxicokinetics is sometimes referred to as, “How the body acts on the chemical.”

Toxicodynamics – The way a toxic chemical exerts its toxic effect. How the toxin interacts with the target site of action. Toxicodynamics is sometimes referred to as, “How the chemical acts on the body.”

Pollutant – A substance in the environment at least in part as a result of human origin or activity which has deleterious effect on living organisms.

Toxin – A chemical that causes harm to an organism. Any chemical can be a toxin depending on the dose/concentration and exposure.

Metabolism – The breaking down or conjoining of molecules by an organism.

Energy metabolism – Metabolism involved in generating energy for an organism to live; it is also called “catabolism.”

Co-metabolism – Metabolic processes used to break down molecules, but do not yield energy for the organism.

Conserved (evolution) – A characteristic of a gene or morphological feature. Conserved genes/morphologies stay the same or relatively unchanged across different branches of the evolutionary tree. For example, if there is an enzyme that is the same in rainbow trout and humans, that enzyme and the corresponding gene is “conserved between rainbow trout and humans.” If the same enzyme is present in many other taxa as well, it would be called “generally conserved” or “highly conserved.”