AQUACULTURE
TOXICOLOGY

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AQUACULTURE
TOXICOLOGY
Edited by
FREDERICK S.B. KIBENGE
Department of Pathology and Microbiology, Atlantic
Veterinary College, University of Prince Edward Island,
Charlottetown, PE, Canada
BERNARDO BALDISSEROTTO
Full Professor, Department of Physiology and
Pharmacology, Federal University of Santa Maria,
Santa Maria, Rio Grande do Sul, Brazil
ROGER SIE-MAEN CHONG
Registered Veterinary Specialist of Fish Health and
Production, Royal College of Veterinary Surgeons, London,
United Kingdom
Registered Specialist of Veterinary Aquatic Animal Health,
Queensland Veterinary Surgeons Board; Senior Veterinarian
(Aquatic Pathology) Biosecurity Sciences Laboratory, QLD,
Australia
Current affiliation: Australian Commonwealth Scientific
Industrial Research Organization (CSIRO), Brisbane, QLD,
Australia

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Contents
Contributors ix
About the editors xi
Preface xiii
1. Introduction to aquaculture 1
Frederick S.B. Kibenge
1.1 Introduction 1
1.2 Structure of the global aquaculture industry 2
1.3 Mollusk aquaculture 9
1.4 Crustacean aquaculture 10
1.5 Chemicals in aquaculture 10
1.6 Governance of aquaculture 11
References 12
2. General introduction to toxicology of aquatic animals 17
Bernardo Baldisserotto
2.1 Introduction to toxicology 17
2.2 Water quality criteria/guidelines 18
2.3 Intraspecies variation of toxicity 20
2.4 Models to predict toxicity of contaminants 21
References 22
3. Antifoulants and disinfectants 25
Samantha Eslava Martins and Camila de Martinez Gaspar Martins
3.1 Overview 25
3.2 Definitions and uses 26
3.3 Mode of action 28
3.4 Ecotoxicity and biological effects 33
3.5 Ecological risks and regulation 46
3.6 Further considerations 49
References 50
v

4. Metals 59
Claudia B.R. Martinez, Juliana D. Simonato Rocha, and Paulo Cesar Meletti
4.1 Introduction 59
4.2 Biochemical effects 62
4.3 Physiological effects 67
4.4 Behavioral effects 69
References 73
5. Agrochemicals: Ecotoxicology and management
in aquaculture 79
Vania Lucia Loro and Bárbara Estevão Clasen
5.1 Water and soil contamination by agrochemicals 79
5.2 Environmental contamination by agrochemicals and risk assessment
in aquaculture: Effects on aquatic organisms and food for human
consumption 82
5.3 Mitigation of agrochemicals 94
5.4 Agrochemicals banned from use in agriculture and aquaculture 98
5.5 Regulatory process for new chemicals and good agricultural
practices 98
References 101
6. Pharmaceutical pollutants 107
Helena Cristina Silva de Assis
6.1 Introduction 107
6.2 Pharmaceuticals in the aquatic environment 108
6.3 Pharmaceutical sources and pathway to the environment 109
6.4 Pharmaceutical exposure effects in nontarget species 111
6.5 Final considerations 123
References 124
7. Oil and derivatives 133
Helen Sadauskas-Henrique, Luciana Rodrigues Souza-Bastos, and
Grazyelle Sebrenski Silva
7.1 Oil and derivatives and the aquatic contamination 133
7.2 Aquaculture and the problem of oil and derivative
contamination 137
vi Contents

7.3 Effects of oil and derivatives on fish species 140
7.4 Effects of oil and derivatives on mollusks and crustaceans 160
7.5 Interaction of oil and derivatives with water characteristics 169
7.6 Future perspectives on oil and derivative contamination and
aquaculture 172
References 173
8. Ecotoxicological effects of microplastics and associated
pollutants 189
Fábio Vieira de Araújo, Rebeca Oliveira Castro, Melanie Lopes da Silva,
and Mariana Muniz Silva
8.1 Introduction 189
8.2 Impacts of microplastic on marine animals 190
8.3 Plastic additives 191
8.4 Microplastic and persistent organic pollutants (POPs) 195
8.5 Microplastics and metals 203
8.6 Microplastics and microorganisms: The plastisphere 205
8.7 Microplastics and other compounds 213
8.8 Final considerations 214
References 216
Index 229
vii
Contents

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Contributors
Bernardo Baldisserotto
Department of Physiology and Pharmacology, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil
Bárbara Estevão Clasen
Department of Environmental Sciences, State University of Rio Grande do Sul, Tr^
es Passos, RS, Brazil
Rebeca Oliveira Castro
Programa de Pós Graduação em Biologia Marinha e Ambientes Costeiros, Universidade Federal Fluminense,
Niterói, RJ, Brazil
Camila de Martinez Gaspar Martins
Universidade Federal do Rio Grande—FURG, Instituto de Ci^
encias Biológicas, Rio Grande/RS, Brazil
Fábio Vieira de Araújo
Faculdade de Formação de Professores, Universidade do Estado do Rio de Janeiro, São Gonçalo, RJ, Brazil
Grazyelle Sebrenski Silva
Departamento de Morfologia, Universidade Federal do Amazonas (UFAM), Manaus, Brazil
Luciana Rodrigues Souza-Bastos
Instituto de Tecnologia para o Desenvolvimento (LACTEC), Laboratório de Toxicologia e Avaliação
Ambiental, Ambiental, Curitiba, Brazil
Mariana Muniz Silva
Programa de Pós Graduação em Biologia Marinha e Ambientes Costeiros, Universidade Federal Fluminense,
Niterói, RJ, Brazil
Melanie Lopes da Silva
Laboratório de Ecologia e Din^
amica B^
entica Marinha, Faculdade de Formação de Professores, Universidade do
Estado do Rio de Janeiro, São Gonçalo, RJ, Brazil
Frederick S.B. Kibenge
Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island,
Charlottetown, PE, Canada
Vania Lucia Loro
Laboratory of Aquatic Toxicology, Department of Biochemistry and Molecular Biology, Federal University of
Santa Maria (UFSM), Santa Maria, RS, Brazil
Paulo Cesar Meletti
Laboratory of Animal Ecophysiology, Department of Physiological Sciences, State University of Londrina,
Londrina, Parana, Brazil
Claudia B.R. Martinez
Laboratory of Animal Ecophysiology, Department of Physiological Sciences, State University of Londrina,
Londrina, Parana, Brazil
Samantha Eslava Martins
Norwegian Institute for Water Research (NIVA), Ecotoxicology and Risk Assessment Section, Oslo, Norway;
Universidade Federal do Rio Grande—FURG, Instituto de Ci^
encias Biológicas, Rio Grande/RS, Brazil
Helena Cristina Silva de Assis
Department of Pharmacology, Federal University of Paraná, Curitiba, Paraná, Brazil
ix

Helen Sadauskas-Henrique
Universidade Santa Cecı́lia (Unisanta), Laboratório de Biologia de Organismos Marinhos e Costeiros
(LABOMAC), Santos, Brazil
Juliana D. Simonato Rocha
Laboratory of Animal Ecophysiology, Department of Physiological Sciences, State University of Londrina,
Londrina, Parana, Brazil
x Contributors

About the editors
Dr. Frederick S.B. Kibenge is Professor of Virology at the Atlantic Vet-
erinary College, University of Prince Edward Island, Charlottetown, PEI,
Canada, where he has been Chairman of the Department of Pathology
and Microbiology for several years, and teaches veterinary virology in
the second year of the DVM curriculum. He has been working with
animal viruses for more than 30years in addition to prior extensive postdoc-
toral research experience in virology in the United Kingdom and the United
States. He is a Diplomate of the American College of Veterinary Microbiol-
ogists, ACVM (subspecialty Immunology). He has published extensively on
the detection and virology of fish viruses. He is editor of the accompanying
two textbooks on the theme of aquaculture: Aquaculture Pathophysiology and
Aquaculture Pharmacology published by Elsevier Inc., Academic Press.
Dr. Bernardo Baldisserotto is a full Professor of Physiology of the
Departamento de Fisiologia e Farmacologia at the Universidade Federal
de Santa Maria, in South Brazil. He supervises graduate students from the
programs of Animal Husbandry, Pharmacology, and Animal Biodiversity
at this university and has published several articles dealing with toxicology
of aquatic animals, mainly fish. He is the coauthor on some of the book
chapters and has published a book regarding Pharmacology applied to aqua-
culture (in Portuguese). He is editor of the accompanying two textbooks on
the theme of aquaculture: Aquaculture Pathophysiology and Aquaculture Phar-
macology published by Elsevier Inc., Academic Press.
Dr. Roger Sie-Maen Chong is a veterinary specialist in Australia and
the United Kingdom, with expertise in fish and shellfish pathology as applied
to the health and biosecurity of aquacultured species. He is officially regis-
tered as a specialist by the Queensland Board of Veterinary Surgeons for
Veterinary Aquatic Animal Health (Australia) and by the Royal College
of Veterinary Surgeons for Fish Health and Production (United Kingdom).
He is also a certified fish pathologist recognized by the Fish Health Section of
the American Fisheries Society. He has worked in Hong Kong with the
Department of Agriculture, Fisheries & Conservation, in Queensland with
the Biosecurity Queensland and is presently a research fish pathologist with
the Australian Commonwealth Scientific and Industrial Research
Organization (CSIRO). He is editor of the accompanying two textbooks
on the theme of aquaculture: Aquaculture Pathophysiology and Aquaculture
Pharmacology published by Elsevier Inc., Academic Press.
xi

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Preface
The textbook Aquaculture Toxicology focuses on the practical principles of
toxicology of finfish (mainly), crustaceans, and mollusks, the three major
groups of aquatic animal species cultured for human consumption.
Recent books on this subject have a more general approach on aquatic tox-
icology, focusing more in chemical aspects of toxicology and pollutants
(Introduction to Aquatic Toxicology by Nikinmaa, M., Elsevier, 2014;
Microscale Testing in Aquatic Toxicology by Wells et al., CRC Press,
2018) or the specific aspects of the effects of pollutants (Aquatic Toxicology:
Molecular, Biochemical, and Cellular Perspectives by Malins D.C. and
Ostrander, G.K., 2018). Aquaculture is a rapidly growing agricultural sector
and requires updated research information on general aspects of several types
of toxicants to control or avoid their negative impacts on the culture of
aquatic animals.
The book is multiauthored and edited. All contributing authors for the
different chapters are highly respected international experts recognized in
their specific areas, who have published several articles dealing with the sub-
ject of their respective chapters; some have also written book chapters on the
subject. Each book chapter has been written from the perspective of the
expert, providing the reader with nuances of the topic without compromis-
ing on the essential and useful facts or being encyclopedic but reflecting
global research. Aquaculture Toxicology presents an overview of the practical
information on the effects and sources of most common pollutants: disinfec-
tants and antifoulants, metals, agrochemicals, pharmaceuticals, oils and
derivatives, and microplastics. The authors were encouraged to use the
same format in all chapters to facilitate ease of reading and studying, and
to maintain consistency throughout the book although the ultimate
approach chosen by different authors is reflective of the state of knowledge
on the respective topics. A list of subtitles is included at the beginning of each
chapter to provide the reader with a quick summary of what is covered in
each chapter.
This book provides the target audience with a reliable and valuable up-
to-date, “all-inclusive” reference and guide to the state of the field, serving as
a systematic and concise resource for aquaculture specialists/researchers
interested in toxicity of various agents to fish, crustaceans, and mollusks,
and particularly researchers, clinical veterinarians, and possibly government
xiii

personnel interested in aquaculture, the fisheries and comparative biology,
and how treatments may affect the aquatic animals directly. It is hoped that
having such a comprehensive easily readable book focused on aquaculture
will identify areas where more research is needed to generate more knowl-
edge to support a sustainable aquaculture industry.
The editors acknowledge the authors for their contributions.
Frederick S.B. Kibenge
Bernardo Baldisserotto
Roger Sie-Maen Chong
EDITORS
xiv Preface

CHAPTER ONE
Introduction to aquaculture
Frederick S.B. Kibenge
Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island,
Charlottetown, PE, Canada
1.1 Introduction
The Food and Agricultural Organization of the United Nations
(FAO) attributes aquatic organisms that are harvested by an individual or
corporate body which has owned them throughout their rearing period
to aquaculture. In contrast, aquatic organisms that are exploitable by the
public as a common property resource, with or without appropriate licenses,
are the harvest of fisheries (FAO, 2015). Two critical criteria of human activ-
ity, ownership of stock and deliberate intervention in the production cycle,
distinguish aquaculture from capture fisheries and also account for the envi-
ronmental concern centered on water quality/water pollution that consti-
tutes Aquaculture Toxicology. It has been noted that the environmental
impacts of aquaculture vary with species, system, management, production
intensity, location, and environmental carrying capacity to absorb impacts
(FAO, 2016). For example, the intensive rearing of fish has led to an increase
in water pollution per unit of farmed fish produced (Hall et al., 2011). On
the contrary, the filter feeding finfish typically raised in inland multispecies
aquaculture systems (mostly silver carp Hypophthalmichthys molitrix and big-
head carp H. nobilis, and most recently Mississippi paddlefish Polyodon
spathula) and marine bivalve mollusks (oysters, mussels, clams, and scallops)
raised in seas, lagoons, and coastal ponds require no artificial feeding (FAO,
2018) and improve the water quality in the production system by removing
waste materials, including waste from fed aquatic animal species, and low-
ering the nutrient load (FAO, 2018; Hishamunda et al., 2014; Parisi et al.,
2012). Deep-sea aquaculture is associated with a lower environmental
impact on the seafloor (Welch et al., 2019) because of deeper waters and
stronger ocean currents that disperse organic matter from farms, in contrast
to nearshore farming.
This introductory chapter describes the structure of the global aquacul-
ture industry (finfish, mollusks, crustaceans, in cold water and warm water,
Aquaculture Toxicology © 2021 Elsevier Inc.
https://doi.org/10.1016/B978-0-12-821337-7.00007-4 All rights reserved.
1

commercial and noncommercial, and small and big operations) and its
importance in supplying animal protein food to the growing human popu-
lation. A better understanding of the aquaculture methods will allow the
reader to recognize the full implications of the toxic effects associated with
different aquaculture environments and help improve toxicological risk
management for aquaculture. The chapters that follow provide an overview
of practical information covering five main aspects of Aquaculture Toxicology
in the three main aquaculture groups (fish, crustaceans, and mollusks): (1)
water quality criteria/guidelines used internationally and models used to
predict the toxicity of contaminants; (2) the primary disinfectants and
organic antifouling co-biocides currently used in aquaculture, pharmaceu-
ticals used in human and veterinary medicine and their by-products that
enter the aquatic environment; (3) water pollutants such as metals, oils
and their derivatives, and microplastics that are toxic to aquatic animals
but arising from nonaquaculture sources; (4) mechanisms of toxicity and
the responses to toxic agents including aspects of uptake, metabolism, and
excretion of toxicants in fish, crustaceans, and mollusks, and toxic effects
on nontarget aquatic organisms; and (5) brief overviews of the current
regulatory aspects of the use of these products.
1.2 Structure of the global aquaculture industry
The contribution of wild-catch fisheries as an animal protein source
for human consumption plateaued in the mid-1980s, and since then, most
natural stocks in marine waters have been harvested at, or near-maximum
rates, with the quantity of fish produced by capture fisheries, projected to
increase at only 0.2% per year for the period 2019–2028 (OECD/FAO,
2019). In contrast, the average growth in the aquaculture sector in the same
period is projected at 2.0% per year (OECD/FAO, 2019). Since 2015,
aquaculture has overtaken capture fisheries as the source of seafood for
human consumption, as global fisheries have remained flat and will continue
to be flat for the foreseeable future (OECD/FAO, 2019). This share of
aquaculture is projected to rise to 62% by 2030 as catches from wild capture
fisheries level off (FAO, 2014; World Bank, 2013). Fish, mollusks, and
crustaceans represent the most economically important global aquaculture
industry subsectors (Fig. 1.1), at 80.04 million tonnes in 2016 with an
estimated total farm gate value of USD 154.2 billion (FAO, 2018), and
expected to reach USD 202.96 billion by 2020 (Grand View Research
Inc., 2014).
2 Frederick S.B. Kibenge

Most of the global aquaculture production (89% by volume) is located in
countries in Asia (most notable being China, Indonesia, India, Vietnam,
South Korea, and the Philippines; production involves mostly freshwater
species, i.e., inland aquaculture). In terms of marine aquaculture, Norway’s
Atlantic salmon industry makes it the world’s largest marine aquaculture
producer, although this accounts for just 3% of total world farmed finfish
output (Cooke, 2016). Another top aquaculture producer is Egypt, whose
tilapia and shrimp farming have exponentially increased in the last two
decades, and by 2017 at approximately 1.3 million metric tons, surpassed
total production by Norway (1.19 million metric tons) and Chile
(1.11 million metric tons) (Tran, 2019).
1.2.1 Aquatic animal species in aquaculture
Among the three main aquaculture groups (fish, crustaceans, and mollusks),
there were 369 finfishes (including five hybrids), 109 mollusks, and 64 crus-
taceans species recorded by FAO as cultured in 2016 (FAO, 2018). Carp
dominates production in both China and the rest of Asia; for Europe and
South America, it is salmonids; African aquaculture production is almost
exclusively tilapias; for Oceania, shrimps and prawns dominate (Hall
et al., 2011). In the USA, catfish predominate (Cooke, 2016). The most
important farmed species (with a production of 1% or more of the total
in 2016) (FAO, 2018) are listed in Table 1.1. However, the analysis of aqua-
culture production and details about farmed species remain approximations
because many indigenous aquatic species are used in aquaculture without
(A) (B)
21%
10%
68%
1%
Mollusks
Crustaceans
Fish
Other species
11%
24%
62%
3%
Mollusks
Crustaceans
Fish
Other species
Fig. 1.1 World aquaculture by farmed species sector. (A) Aquaculture industry subsec-
tors by quantity 2016 and (B) aquaculture industry subsectors by value 2016. Adapted
from FAO, 2018. Leading species in global aquaculture production in 2016 (in million
metric tons). Statista. Statista Inc. https://www.statista.com/statistics/240268/top-global-
aquaculture-producing-countries-2010/ Accessed: July 14, 2019.
3
Introduction to aquaculture

Table 1.1 Major farmed species in world aquaculture.
Aquaculture
sector Species or species groupsa
% of total,
2016
Finfish
Grass carp (Ctenopharyngodon idellus) (FW) 11
Silver carp (Hypophthalmichthys molitrix) (FW) 10
Tilapias (Oreochromis niloticus) other cichlids (FW) 10
Common carp (Cyprinus carpio) (FW) 8
Bighead carp (Hypophthalmichthys nobilis) (FW) 7
Crucian carp (Carassius carassius) (FW) 6
Major (Indian) carp (Catla catla) (FW) 6
Freshwater fishes nei, Osteichthyes (FW) 4
Atlantic salmon (Salmo salar) (D) 4
Roho labeo (Labeo rohita) (FW) 3
Pangas catfishes (Pangasius spp.) (FW) 3
Milkfish (Chanos chanos) (D) 2
Torpedo-shaped catfishes nei (Clarias spp.) 2
Marine fishes nei (Osteichthyes) (M) 2
Wuchang bream (Megalobrama amblycephala) (FW) 2
Rainbow trout (Oncorhynchus mykiss) (D) 1
Cyprinids (Cyprinidae) (FW) 1
Black carp or black Chinese roach (Mylopharyngodon
piceus) (FW)
1
Northern snakehead (Channa argus) (FW) 1
Other finfishes 16
Finfish total 100
Crustaceans
White leg shrimp (Litopenaeus vannamei, formerly
Penaeus vannamei)
53
Red swamp crawfish (Procambarus clarkii) 12
Chinese mitten crab (Eriocheir sinensis) 10
Giant tiger prawn or Asian tiger shrimp (Penaeus
monodon)
9
Oriental river prawn (Macrobrachium nipponense) 4
Giant river prawn (Macrobrachium rosenbergii) 3
Other crustaceans 9
Crustaceans
total
100
Mollusks
Cupped oysters nei (Crassostrea spp.) 28
Japanese carpet shell or Manila clam (Ruditapes
philippinarum)
25
Scallops (Pectinidae) 11
4 Frederick S.B. Kibenge

being registered individually in national statistics. Numerous single species
registered in the official statistics of many countries consist, in reality, of
multiple species and sometimes hybrids (i.e., the number of finfish hybrids
in commercial production is more than five) (FAO, 2018). In China, more
than 200 species are farmed commercially, but the total production is
registered under fewer than 90 species and species groups.
Similarly, in India and Vietnam, the number of cultured species far
exceeds the number included in statistics (FAO, 2014). Moreover, for the
most widely farmed species, tilapia, the correct number of producer coun-
tries is higher than the FAO record of 135 countries because commercially
farmed tilapias are not yet reflected separately in national statistics in Canada
and some European countries (FAO, 2014). In terms of the projected
increase in aquaculture production, by 2028, carp and mollusks are projected
to remain the most significant aquaculture groups, accounting for 35.8% and
19.2%, respectively, of total production (OECD/FAO, 2019).
1.2.2 Aquaculture techniques, systems, and facilities
The techniques, systems, and facilities used in aquaculture are as diverse as
the species currently being raised and also vary by geographical region
(Bostock et al., 2010), but typically encompass three stages: incubation/
hatchery seed production, early-rearing, and on-growing (Roberts and
Shepherd, 1974). In salmon and trout aquaculture, on-growing has merged
with husbandry and breeding (Aarset and Borgen, 2015). For new farmed
Table 1.1 Major farmed species in world aquaculture—cont’d
Aquaculture
sector Species or species groups
% of total,
2016
Marine mollusks (Mollusca) 7
Sea mussels (Mytilidae) 6
Chinese razor clam or Agemaki clam or constricted
tagelus (Sinonovacula constricta)
5
Pacific oyster, Japanese oyster, or Miyagi oyster
(Crassostrea gigas)
3
Blood cockle or Blood clam (Anadara granosa) 3
Chilean mussel (Mytilus chilensis) 2
Other mollusks 10
Mollusks total 100
a
Major farmed species or species groups (with a production of 1% or more of the total in 2016) (FAO,
2018). FW, freshwater; D, diadromous (these fish spawn in freshwater and migrate to the sea for the main
growing period); M, marine.
5
Introduction to aquaculture

species like cod, mainly two methods are used: one is based on capturing
wild cod for on-growing, the other focuses on the production of cod from
hatchery seed to market size; currently, on-growing of wild cod (e.g., as in
sea ranching (Kitada, 2018)) is more economically efficient than using
farmed juveniles (Gunnarsson, 2007). For a few species, such as eels
“Anguilla spp.,” farming still relies entirely on the wild seed. Although
the Japanese eel “Anguilla japonica” life cycle in captivity has been completed,
the techniques for mass production of glass eels have not yet been established
because of various technical difficulties (Bird, 2013; Masuda et al., 2012).
A comprehensive review of the different aquaculture systems worldwide
can be found in Boyd and McNiven (2015). World aquaculture production
takes place under four main systems: (1) on land (using freshwater or saline
water) in static water ponds (e.g., for growing channel catfish and other
warm-water species), concrete raceway systems (e.g., for growing rainbow
trout), fresh water gravity tanks, or large recirculation tanks; (2) in floating
cages and net pens in a lake or river (e.g., for growing salmonids and other
cold-water species that require clean water with high oxygen levels) or on
the shoreline/nearshore in sheltered waters (mariculture or marine aquacul-
ture 3km from the shore) in floating net cages moored to the bottom in
the sea with long lines (Shepherd, 1993; White and Edwards, 2015); (3) in
coastal lagoons or brackish-water or marine ponds (e.g., for shellfish
aquaculture) (Parisi et al., 2012; Wever et al., 2015; White and Edwards,
2015); and (4) in onshore-based tanks using pumped sea water (e.g., for flat
fish farming of turbot or halibut that normally lie on the bottom and will not
shoal within the water column inside a floating cage (Shepherd, 1993)).
1.2.3 Finfish aquaculture
Seven of the top ten cultured fish species are carps; others are tilapias, Pangas
catfishes, and Atlantic salmon (Table 1.1). China, the largest producer of
farmed seafood, grows carp mostly in ponds and rice paddies, with little
or no attempt to actively nurture the animals and has been practicing this
type of aquaculture for thousands of years. Most carp farming is pond-based,
with several species stocked in the same pond (polyculture) (SPC, 2011).
Tilapia and sometimes catfish are stocked together with carp. Single-species
culture (monoculture) is rare, except in the flow-through system and cage
culture of common carp in streams or canals. There are different stocking
models for polyculture, depending on the availability of the primary source
of feed. If grasses (aquatic or terrestrial) are abundant, grass carp can be
6 Frederick S.B. Kibenge

stocked as the dominant species. The leftover feed and grass carp excreta
would sufficiently fertilize the pond water for the growth of filter feeders.
For example, the “80:20 pond fish culture” where 80% of harvest weight
comes from the pellet-fed target species (such as grass carp, crucian carp,
or tilapia) and the other 20% comes from the filter feeding “service species”
(silver carp) (White and Edwards, 2015). Pond-based carp culture has been
traditionally integrated with crop farming (rice, mulberry, fruit, and vege-
tables) and animal husbandry (ducks, swine, and chicken) in China
(White and Edwards, 2015). The practice has been widely introduced to
many other parts of the world, with some modifications to fit into local
conditions (SPC, 2011). Polyculture (the mixing of species in fish farms)
has taken a different trajectory since 2008 and is widely used in sea cages
for a different purpose in the form of using “cleaner fish” (lumpfish
Cyclopterus lumpus and wrasse (Labridae)) in marine farmed salmonids as a
biological control method for sea lice Lepeophtheirus salmonis in Europe
(Powell et al., 2018; Skiftesvik et al., 2014) and Canada; it involves the
use of wild-caught cleaner fish directly in the salmon farms or as broodstock
for hatchery-raised cleaner fish (EURLFD, 2016).
In industrial aquaculture, farmed fish are reared at high population
densities in floating open net cages nearshore (for marine aquaculture) or
in a lake or river (for inland aquaculture) (White and Edwards, 2015).
The cages are either of square/hexagonal steel platform construction that
are linked together or are of circular polyethylene rings 10–50m deep with
netting to hold in farmed fish. Expansion can simply be by increasing the
number of fish in a cage (i.e., cage volume) or by increasing the number
of cages. Formulated dry feeds supply all nutrition. Uneaten feed and feces
fall through the cages and settle to the bottom in the vicinity. Cages are
periodically moved to new locations to allow benthic communities affected
by sediment to recover—a process called fallowing (Boyd and McNiven,
2015). Current production systems operate on a single-year-class stocking
in a site, usually a bay (“All-in, all-out”) with a fallow period of 6 months
in between year classes.
Closed containment is a practice that entails enclosing fish in floating
containers or land-based farms to lessen their damage to the aquatic environ-
ment (Real, 2010). Recent improvements in offshore technology now
allow even larger floating fish cages out in the sea that are submersible with
automated feeding systems and remote monitoring. This has enabled the
expansion of aquaculture farther offshore (i.e., deep-sea aquaculture) into
high-energy sites and with a low environmental impact on the seafloor
7
Introduction to aquaculture

(Lovatelli et al., 2013; Welch et al., 2019). China currently has one of the
world’s biggest fully submerged net cage farming Atlantic salmon in the Yel-
low Sea (Owen, 2018); and SalMar’s Ocean Farm 1 is 40km off the coast of
Frohavet, Norway (Bennet, 2019). Moving fish farms out to sea helps to
solve several problems. The open ocean offers more space, deeper waters,
and stronger currents. Ocean currents are particularly effective for reducing
the spread of pathogens and allowing better exchange and dilution of wastes
than nearshore farming; greater distance offshore also minimizes interactions
with wild fish and reduces the risk of unwanted exposure of nontarget
organisms to aquacultural chemicals (Park et al., 2012).
On land, increased development of land-based recirculating aquaculture
system (RAS) (Espinal and Matuli
c, 2019) is leading to an ever-increasing
move to saltwater land-based aquaculture, particularly in Europe and most
recently in North America, despite the significant capital costs compared to
those for floating cage farms. RAS offers environmentally sustainable
methods for farming marine (Tal et al., 2009) and freshwater fish by having
minimal effluents compared to other systems (White and Edwards, 2015).
For example, in BC-Canada, a recent analysis for the Fraser Basin Council
found that a capital investment of $1.1 billion would be required to establish
a land-based industry capable of producing 50,000 tons of Atlantic salmon a
year on Vancouver Island (Shore, 2019). There is already extensive knowl-
edge derived from land-based hatcheries. The capital costs are compensated
by lower operating costs, for example, land-based aquaculture farms have
low pharmaceutical costs (e.g., no sea lice therapeutics) and no costs of
maintaining boats that go out to feed the fish (Kramer, 2015).
Moreover, with land-based farming, there would be no interactions
between farmed fish and wild fish (Shore, 2019). However, while land-
based aquaculture requires low antibiotic usage, there appears to be a con-
nection in antimicrobial resistance issues found in land-based aquaculture
with those established by terrestrial animal agriculture (Done et al., 2015).
Another crucial disadvantage has been the development of “off-flavor” of
the harvested fish “caused by geosmin and 2-methylisoborneol metabolites
released by microbes that grow within the land-based systems” (Sapin
et al., 2020).
Another aquaculture development that has gained traction in recent
years is the biofloc technology (White and Edwards, 2015). Biofloc technol-
ogy is a technique of enhancing water quality in aquaculture through the
manipulation of microbiota to convert the harmful waste from aquaculture
production to consumable body biomass, with many other benefits (Dauda,
8 Frederick S.B. Kibenge

2019). The technology has recently gained attention as a sustainable method
to maintaining optimum water quality parameters under a zero-water
exchange system, thus preventing eutrophication and effluent discharge into
the surrounding environment, thereby increasing biosecurity, as there is no
water exchange except sludge removal (Ahmad et al., 2017). The technol-
ogy has the added value of producing proteinaceous feed in situ, which
results in higher productivity with less impact on the environment
(Bossier and Ekasari, 2017). It has been considered the new “blue revolu-
tion” in aquaculture (Emerenciano et al., 2017). It may act as a complete
source of nutrition for aquatic organisms, along with some bioactive
compounds that will enhance growth, survival, and defense mechanisms
and acts as a novel approach for health management in aquaculture by
stimulating the innate immune system of animals. Beneficial microbial bac-
terial floc and its derivative compounds such as organic acids, polyhydroxy
acetate, and polyhydroxy butyrate could resist the growth of other
pathogens and thus serve as a natural probiotic and immunostimulant. This
technology is economically viable, environmentally sustainable, and socially
acceptable (Ahmad et al., 2017; Daniel and Nageswari, 2017).
1.3 Mollusk aquaculture
In the case of mollusk (oysters, clams, scallops, abalone, mussels,
cockles, and related species) aquaculture, there are many varieties of culture
and grow-out methods that are particular to species and locations (Boyd
et al., 2005; Dı́az et al., 2011; Dumbauld et al., 2009; Elston, 1999;
Elston and Ford, 2011; Parisi et al., 2012). For example, Parisi et al.
(2012), in discussing the diversification of Italian shellfish culture, reviewed
the most promising mollusk species and their culture in general (refer to
Table 1.1 for a list of the most notable farmed mollusk species worldwide).
Most mollusk culture requires no feed inputs (algae are used to feed the
larvae and juveniles in hatcheries to provide spat for mollusk culturing) but
may require control of fouling and predators. Seed bivalves are produced
either in land-based hatcheries (Robert, 2009) or from natural populations
(wild spat) (Parisi et al., 2012). They can be transported over considerable
distances to grow-out sites in estuaries and even to different countries
(FAO, 2013). Farming mollusks is limited in several countries because of
limited capacity producing seed from mollusk hatcheries and nurseries
(FAO, 2014), relying on the wild seed. The use of wild seed is impossible
if the natural populations of mollusks are not suited to culture.
9
Introduction to aquaculture

Intensification will require a reliable, plentiful, and inexpensive supply of
hatchery seed, requiring the selection and management of broodstocks
and the controlled production of high-quality seed stocks (Elston and
Ford, 2011). This would reduce the environmental biosecurity concern
of the translocation of non-native species (Parisi et al., 2012).
1.4 Crustacean aquaculture
The most recent dramatic change in aquaculture has been the explo-
sive growth in shrimp farming in Southeast Asia (Cressey, 2009). The
production has increased almost exponentially since the mid-1970s
(FAO, 2010). This rapid increase largely reflects the dramatic increase in
white leg shrimp Litopenaeus vannamei (formerly Penaeus vannamei) culture
in Asia (China, Thailand and Indonesia) and Latin America (Ecuador and
Mexico) (Bondad-Reantaso et al., 2012), where the penaeid shrimps have
tended to dominate due to high-value, short-production cycles and
accessible technologies (Bostock et al., 2010). Crustacean farming, similarly
to marine fish aquaculture, requires a high-quality diet, usually containing
fish meal and often fish oil (Bostock et al., 2010).
1.5 Chemicals in aquaculture
There is a long list of chemicals that are used in aquaculture, including
agricultural limestone, lime, fertilizers, oxidants, coagulants,
osmoregulators, algicides, herbicides, fish toxicants, antifoulants, therapeu-
tics, disinfectants, anesthetics, agricultural pesticides, and hormones (Arthur
et al., 2000; Boyd and Massaut, 1999; Boyd and Tucker, 1998; Noga, 2010;
Schnick, 2001). To mitigate the environmental impact associated with the
use of aquacultural chemicals, various regulations have been advocated
(FAO, 1997). In many countries, governmental regulations on chemical
use in aquaculture have been established (Cabello, 2006; Schnick, 1988,
2001) (refer also to Martins and Martins (2020) in Chapter 3 (Section 3.4),
Loro (2020) in Chapter 5 (Section 5.4), and de Assis (2020) in Chapter 6 in
this book. Most importing countries also have food safety regulations that
involve the inspection of incoming shipments of seafood products for
specific chemical residues. A companion textbook, Aquaculture Pharmacology
(Kibenge et al., 2020), has a comprehensive review of most aquaculture
pharmaceuticals. The pharmaceuticals used in human and veterinary
medicine (including aquaculture) and their by-products enter the aquatic
10 Frederick S.B. Kibenge

environment as pollutants in a variety of ways, causing adverse effects on
nontarget aquatic organisms. The aquatic environment may also be contam-
inated by chemicals such as agrochemicals, metals, oils and their derivatives,
and microplastics as a result of erosion of surface deposits of metal minerals,
as well as from human activities, such as agriculture, mining, smelting, fossil
fuel combustion, and industrial and commercial uses of metals (Nordberg
et al., 2007; Pandey et al., 2019). Martinez et al. discuss metal pollutants that
are toxic to aquatic animals in Chapter 4, Loro discusses agrochemical
pollutants in Chapter 5, de Assis discusses pharmaceutical pollutants in
Chapter 6, Sadauskas-Henrique et al. discuss oil and derivatives pollutants
in Chapter 7, and de Araújo et al. discuss microplastics and associated
pollutants in Chapter 8 in this book. Additional information on chemicals
in aquaculture and the contribution of aquaculture to chemical pollution
and other adverse impacts of chemical use by humans can be found in
Boyd and McNiven (2015).
1.6 Governance of aquaculture
The FAO (2017) technical guidelines on “aquaculture governance
and sector development” define aquaculture governance as “the set of
processes by which a jurisdiction manages its resources concerning aquacul-
ture, how its stakeholders participate in making and implementing decisions
affecting the sector, how government personnel are accountable to the
aquaculture community and other stakeholders, and how the respectful rule
of law is applied and enforced.” Most countries have some form of legisla-
tion governing aquaculture operations (with regard to animal welfare,
animal diseases, the environment, water use, land use, and waste disposal),
with the competent authorities residing in various government ministries
and agencies. Given the diversity of countries and aquaculture systems, it
is beyond the scope of this chapter to consider legislation in each country.
Instead, the reader is referred to a series of comparative national overviews of
aquaculture laws and regulations, “National Aquaculture Legislation
Overview (NALO),” from aquaculture producing countries that are
published by the FAO as NALO Fact sheets (FAO, 2019). These fact sheets
are updated every 2 to 3 years (FAO, 2019). At the national level, aquacul-
ture operations require various licenses and permits to operate, ensuring
environmental protection relating to the waste generated, prevention,
and control of aquatic animal diseases, and the well-being of aquaculture
animals (FAO, 2019). There are also Policy Frameworks mandated
11
Introduction to aquaculture

nationally, regionally, or internationally on how aquaculture activities are
developed and managed. These guidelines and codes of conduct provide
good practices for a variety of aquaculture operations and how the activities
are regulated in both freshwater and marine environments as well as for
land-based aquaculture. The ultimate purpose of governance of the aqua-
culture sector should be to support sustainable aquaculture by emphasizing
environmental sustainability and social responsibility. Enhancement of
aquaculture’s growth needs the right policy decisions regarding the use of
natural resources (water, land, seed, and feed), as well as sound environmen-
tal management. Good governance is fundamental to the successful
formulation and implementation of aquaculture development policies,
strategies, and plans (FAO, 2016; Hishamunda et al., 2014). Three main
environmental actions arising from good governance are: (1) management
of effluent and nutrient loading, (2) improvement of aquaculture legislation,
and (3) mandatory environmental impact assessment (EIA) (FAO, 2016;
Hishamunda et al., 2014).
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tropical Caribbean. J. World Aquacult. Soc. 50, 299–316. https://onlinelibrary.wiley.
com/doi/full/10.1111/jwas.12593.
Wever, L., Krause, G., Buck, B.H., 2015. Lessons from stakeholder dialogues on marine
aquaculture in offshore windfarms: perceived potentials, constraints and research gaps.
Mar. Policy 51, 251–259.
White, P., Edwards, P., 2015. Types of culture systems and aqua-ecosystems. Available at
http://aquaculture.management/2015/types-of-culture-systems-and-aqua-
ecosystems/ (Accessed March 26, 2020).
World Bank, 2013. Fish to 2030: Prospects for Fisheries and Aquaculture (English). Agricul-
ture and Environmental Services Discussion Paper; no. 3. World Bank Group, Washing-
ton, DC. http://documents.worldbank.org/curated/en/458631468152376668/Fish-
to-2030-prospects-for-fisheries-and-aquaculture. (Accessed 3 October 2019).
16 Frederick S.B. Kibenge

CHAPTER TWO
General introduction to toxicology
of aquatic animals
Bernardo Baldisserotto
Department of Physiology and Pharmacology, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil
2.1 Introduction to toxicology
Laboratory studies dealing with exposure to pollutants can last for
hours to a few days (acute experiments) and are usually used to calculate
the concentration that affects (effective concentration—EC50) or kills (lethal
concentration—LC50) 50% of test organisms. Chronic studies are used to
determine threshold concentrations (no observed effect concentration, or
NOEC; lowest observed effect concentration, or LOEC). These values
can be used to compare the effect of a given pollutant in different species
or different pollutants between themselves.
These values are used worldwide to derive water quality guidelines for
the protection of aquatic life, i.e., concentrations that should result in a neg-
ligible effect on aquatic biota. Two basic guideline derivation methodolo-
gies are in use throughout the world to calculate these concentrations
(Nugegoda and Kibria, 2013): one is the assessment factor method, which
involves multiplying the lowest value of a set of toxicity data by a factor
to arrive at a criterion value. For example, the LC50 value is multiplied
by 0.01 and the NOEC value by 0.1. The assessment factor can vary
according to the country and the amount of toxicological studies available.
The other method is the species sensitivity distribution, a statistical extrap-
olation involving the use of individual toxicity data for a range of species to
determine the criterion value. This method estimates the concentration of a
substance that is predicted to protect 95% of all aquatic species extrapolating
from an equation or curve that considers the relationship between probabil-
ity x log LC50 and NOEC. Some countries use either one of the methods,
and others use a combination of both methods.
Aquaculture Toxicology © 2021 Elsevier Inc.
https://doi.org/10.1016/B978-0-12-821337-7.00004-9 All rights reserved.
17

2.2 Water quality criteria/guidelines
The United States Environmental Protection Agency (USEPA) has
water quality criteria/guidelines for water quality, and its website contains
tables with the highest concentration of the most common metals, pesticides
(mainly insecticides), and other pollutants or contaminants that are not
expected to pose a significant risk to the majority of species in a given envi-
ronment (USEPA, 2019). The same agency maintains the ECOTOX
Knowledgebase Explore function (https://cfpub.epa.gov/ecotox/explore.
cfm), which is an interactive way to examine search paths by chemical, spe-
cies, and effects.
The guidelines of the Canadian Council of Ministers of the Environment
(CCME, 2003) explain that some water quality parameters can affect the
toxicity of contaminants. Some are described later.
– pH: At alkaline pH, many metals form insoluble hydroxides or carbon-
ates that precipitate, while at acidic pH, a higher release of metals from
the sediments can occur (CCME, 2003). Water pH may also influence
toxicity in pharmaceuticals because the toxicity of sertraline and fluox-
etine was higher and diclofenac was lower at pH8.2 than at pH5.8, but
the toxicity of ethinyl estradiol was not affected by pH. Probably these
differences are due to the higher uptake of sertraline at pH8.2 and no
change in ethinyl estradiol uptake by pH (Alsop and Wilson, 2019).
The piscicide 3-trifluoromethyl-4-nitrophenol (TFM), which is applied
in the Great Lakes to control streams infested with larval sea lamprey
(Petromyzon marinus), has 5.5-fold higher uptake rates at low pH
(6.86) compared to alkaline pH (8.78). This higher uptake is most likely
because the unionized, lipophilic form of TFM exists in greater amounts
at a lower pH. On the contrary, the elimination rates are 1.7–1.8-fold
higher at pH8.78 than at pH6.86, indicating that TFM is more effective
at low pH (Hlina et al., 2017).
– Hardness and alkalinity: The increase of water hardness usually reduces
the toxicity of many metals, largely due to the formation of metal-
carbonate complexes and Ca2+
and/or Mg2+
antagonism (Blewett
and Leonard, 2017). CCME (2003) presents equations in which the
values of water hardness are used to predict the concentrations of zinc
and copper to protect aquatic life. The toxicity of the insecticides endosul-
fan (Thiodan; 1,4,5,6,7-hexachloro-8,9,10-trinorborn-5-en-2,3-yelen)
18 Bernardo Baldisserotto

(dimethyl) sulfite and methiocarb [Mesurol; 3,5-dimethyl-4-(methylthio)
phenyl methylcarbamate] to rainbow trout, Oncorhynchus mykiss, is lower
at higher alkalinity (40–121mg CaCO3/L) than at lower alkalinity
(19–20mg CaCO3/L), but hardness ranging from 50 to 147mg
CaCO3/L had no effect (Altinok et al., 2006; Capkin et al., 2006).
Hardness (6–309mg CaCO3/L) also did not affect the toxicity of another
insecticide, fenvalerate, to bluegill (Lepomis macrochirus) (Dyer et al., 1989).
The increase of hardness increases surfactant toxicity to aquatic species in
some cases, but the outcome is compound and species-specific
(Lewis, 1992).
– Salinity: The toxicity of metals in general decreases with the increase of
salinity due to complexation with seawater ions (Blewett and Leonard,
2017; Tan et al., 2019). For example, nickel may complex with SO4
2
and Cl and free nickel reduces (Blewett and Leonard, 2017). The same
complexation with Cl can be observed with cadmium (Tan et al.,
2019). The uptake of cadmium and zinc by the teleost Acanthopagrus
schlegelii reduced with the increase of salinity, probably due to the higher
waterborne Ca2+
levels at higher salinities, because both metals use the
Ca2+
uptake pathway (Zhang and Wang, 2007). Salinity does not affect
LC50 of fenvalerate to bluegill, but the accumulation of this insecticide
decreased with the increase of salinity (Dyer et al., 1989).
– Total dissolved solids: It can change the complexing and precipitation
processes of metals (CCME, 2003). The presence of suspended solids
and naturally occurring dissolved substances decreases the bioavailability
of cationic surfactants but not that of anionic and nonionic surfactants
(Lewis, 1992).
– Temperature: Besides the direct effect of temperature on the metabo-
lism of ectothermic animals, it can also affect the toxicity of several pol-
lutants. The effect depends on the pollutant (CCME, 2003).
– Dissolved oxygen: The toxicity of pollutants can be magnified by low
dissolved oxygen levels (CCME, 2003).
– Dissolved organic matter (DOM): This can also affect the toxicity of
metals. The major chemical components of DOM are humic substances
(humic and fulvic acids). Its protective effect varies according to its ori-
gin and composition. Overall, darker organic matter is expected to be
more protective against metal toxicity (Al-Reasi et al., 2013). DOC also
decreased uptake and toxicity of sertraline, but not of ethinyl estradiol
(Alsop and Wilson, 2019).
19
General introduction to toxicology of aquatic animals

Water quality guidelines in China are in the “Surface Water Environmental
Quality Standards” GB3838-2002 (http://www.codeofchina.com/
standard/GB3838-2002.html). This guide considers that class II water is
for “precious fish reserves” and up to class III water is adequate for “winter-
ing grounds of fish and shrimp, migration channels, aquaculture areas of
fishing waters.” The fourth amendment of this guideline used the data from
native species and the assessment factor method and/or species sensitivity
distribution to calculate concentrations that should result in a negligible
effect on aquatic biota (Zhao et al., 2018). However, the eutrophication
control management is incomplete compared to developed countries
because it is based only on total nitrogen and total phosphorus values
(Su et al., 2017).
South Africa has a very recent and complete guideline of water quality
for coastal marine waters for use to mariculture with maximum values for
several pollutants, based on the values from USEPA, CCME, and EU
(2013), as well as from other African countries (RSA-DEA, 2018). From
Latin America, Brazil follows the resolution from the Brazilian National
Environment Council (CONAMA, 2005), which has specific tables with
maximum values for contaminants for fisheries and aquaculture for fresh-,
brackish-, and saline waters. Mexico, on the contrary, uses only four water
quality parameters (biochemical and chemical demand of oxygen, fecal coli-
forms, total suspended solids) to indicate water quality (Comisión Nacional
del Agua, 2018). This country has also a law that states maximum values of
fats, oils, and a few metals (NOM, 1998).
2.3 Intraspecies variation of toxicity
The effects of metals and pharmaceuticals in fish can be affected by sex,
size, “personality” (defined as bold or shy, according to their risk-taking
behaviors), and social status. For example, different responses were observed
in males and females of zebrafish, Danio rerio, to cocaine, and subordinate
rainbow trout are more vulnerable than dominant ones to copper and silver
exposure (Demin et al., 2019). Survival of rainbow trout exposed to endo-
sulfan was significantly increased with increasing fish size (Capkin et al.,
2006), but the opposite was observed in those exposed to methiocarb
(Altinok et al., 2006).
20 Bernardo Baldisserotto

2.4 Models to predict toxicity of contaminants
2.4.1 Biotic ligand model (BLM)
The USEPA uses the BLM to predict dissolved metal concentrations that
correspond to lethal accumulations of a metal on biotic ligands for unique
water compositions, i.e., depends on the concentrations of some cations
(e.g., K+
, Na+
, Ca2+
, Mg2+
, and H+
). BLMs are computational models that
determine metal speciation and predict metal toxicity to biota in aqueous
systems and combine an equilibrium geochemical speciation model, a
metal–organic binding model, and a toxicological model (Smith et al.,
2015). This methodology was applied mainly for copper (USEPA, 2007),
but there are data from the USA and Canadian species for other metals
and there are also proposals to analyze the mixture of metals (Balistrieri
and Mebane, 2014). The binary metal interactions tested (silver, cadmium,
copper, nickel, lead, and zinc) demonstrated interactions, mostly inhibitory,
nonreciprocal, and caused by silver or copper (Cr
emazy et al., 2019). Nev-
ertheless, BLMs must be constructed for different water qualities and species.
For example, LC50 for copper in cardinal tetra Paracheirodon axelrodi from the
ion-poor blackwater Rio Negro, Amazon (high level of DOC) was not
predicted properly using BLM models developed using temperate DOC
and temperate species (Cr
emazy et al., 2016).
2.4.2 Toxicokinetic-toxicodynamic (TK-TD) models
These models consider the processes that lead to toxicity at the organisms
over time instead of at a certain endpoint (96h, for example), as the
BLM. Nevertheless, these models use BLM-estimated binding constants cal-
culated with toxicity data. The TK-TD models simulate the temporal
aspects of toxicity and provide a conceptual framework to better understand
the causes for variability in different species’ sensitivity to the same com-
pound as well as the causes for different toxicity of different compounds
to the same species (Giesy et al., 2010). Recent models can predict metal
toxicity as a function of the waterborne concentrations of some cations
(Feng et al., 2018a) and the mixture of metals (Gao et al., 2016). The inter-
actions between metals may be time-dependent (Feng et al., 2018b). These
models are also useful to extrapolate toxicity of pesticides from relatively
21
General introduction to toxicology of aquatic animals

constant to relatively variable exposure profiles, because in the environment,
they generally occur in fluctuating and highly variable patterns (Ashauer
et al., 2013).
2.4.3 Principal component analysis (PCA)
The PCA is a multivariate statistical procedure that uses an orthogonal trans-
formation to convert a set of correlated variables into a set of values of lin-
early uncorrelated variables called principal components, where the first
principal component has the largest possible variance and each succeeding
component in turn has the highest variance possible. Plotting the principal
components can elucidate potential correlations between the set of variables
(Marques et al., 2019).
2.4.4 Generalized additive modeling (GAM)
The GAM approach is an extension of the generalized linear model (GLM).
It is a generalized linear model with a linear predictor involving a sum of
smooth functions of covariates that allows a flexible description of complex
responses to environmental changes. Therefore, it can allow statistical infer-
ence to nonlinear correlations between the contaminant levels and bio-
marker responses. Consequently, it provides a model to predict the
bioaccumulation of chemical contaminants based on biomarker responses
and vice-versa, besides other influencing factors, such as seasonality and gra-
dients in water chemistry parameters (Marques et al., 2019).
References
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solved organic matter (DOM): mechanistic explanations for protective effects against
metal toxicity and direct effects on organisms. Environ. Int. 59, 201–207.
Alsop, D., Wilson, J.Y., 2019. Waterborne pharmaceutical uptake and toxicity is modified by
pH and dissolved organic carbon in zebrafish. Aquat. Toxicol. 210, 11–18.
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22 Bernardo Baldisserotto

CCME (Canadian Council of Ministers of the Environment), 2003. Canadian Water Quality
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Demin, K.A., Lakstygal, A.M., Alekseeva, P.A., Sysoev, M., Abreu, M.S., Alpyshov, E.T.,
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Dyer, S.D., Coats, J.R., Bradbury, S.P., Atchison, G.J., Clark, J.M., 1989. Effects of water
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24 Bernardo Baldisserotto

CHAPTER THREE
Antifoulants and disinfectants
Samantha Eslava Martinsa,b
and Camila de Martinez Gaspar Martinsb
a
Norwegian Institute for Water Research (NIVA), Ecotoxicology and Risk Assessment Section, Oslo, Norway
b
Universidade Federal do Rio Grande—FURG, Instituto de Ci^
encias Biológicas, Rio Grande/RS, Brazil
3.1 Overview
In the past 50years, the primary aquatic food supply for humans chan-
ged from wild-caught to production in farms. Aquaculture increased not
only in the levels of production, but in the types of species cultivated, driven
by high demand in a globalized environment. In 2014, the contribution of
aquaculture to the production of fish for human consumption outweighed
the acquisition of fish through wild fishing. Currently, the aquaculture sec-
tor plays an important role in food security and livelihood, being the source
of income and social development. Fish and fishery goods represent one of
the most-traded segments of the world food sector, with a high international
trade competition. For several countries and for numerous coastal and riv-
erine regions, production of fish and fishery products is essential to their
economies, accounting for more than 40% of the total value of traded com-
modities in some countries, mainly islands. This represents a value 9% of
global agricultural exports and 1% of world commodity trade (FAO,
2016). This impressive growth of the aquaculture sector was possible
through the development of technologies that confer improved productiv-
ity, allowing the production of aquatic animals in line with the increasing
demand for seafood as a source of animal protein. However, major con-
straints impinging aquaculture include the biofouling on aquaculture equip-
ment and infrastructure, and the losses from diseases.
Biofouling is of major concern in both shipping and aquaculture indus-
tries. In the shipping industry, biofouling brings economic constraints
because fouled organisms result in the deformation of the shape of hulls
and hence the hydrodynamics of the boat, leading to an increase in the fric-
tional drag and thereby fuel consumption (Abbott et al., 2000). Biofouling is
a problem especially for sea cage-based finfish aquaculture where impacts are
well documented. In particular, a major concern with the fouling of cages is
the occlusion of netting mesh, which restricts water flow resulting in
Aquaculture Toxicology © 2021 Elsevier Inc.
https://doi.org/10.1016/B978-0-12-821337-7.00005-0 All rights reserved.
25

reduced water quality (De Nys and Guenther, 2009). Another major chal-
lenge is the development of effective disease treatment and prevention.
Studies have shown that caged salmon may come in contact with nemato-
cysts of fouling cnidarians when nets are cleaned and hydroids are released
into the water (Bloecher et al., 2018; Floerl et al., 2016). Hydroid’s nema-
tocysts lead to irritation and pathological damage to salmon gills and may
facilitate the spread of the amoebic gill disease (AGD), an increasing threat
to Atlantic salmon farming (Bloecher et al., 2018). In this sense, in order to
maintain optimum culture conditions and ensure aquaculture as a profitable
industry, proper husbandry techniques are mandatory, requiring that anti-
fouling biocides are used to minimize biofouling.
About 90% of the aquaculture occurs in developing countries, and the
production losses due to disease reach up to 6 billion dollars per annum
(Assefa and Abunna, 2018; Leung and Bates, 2013). The proliferation of
opportunistic pathogens (bacteria, virus, fungi, or protozoa) is driven by
the high density of fish stocks and the feed wastes; thus, the use of quick
and effective antipathogens is necessary (Castillo-Rodal et al., 2012;
Twiddy et al., 1995). Therefore, disinfection is commonly used in aquacul-
ture facilities.
3.2 Definitions and uses
3.2.1 Antifoulants
When an object is placed into water, microorganisms followed by algae and
animals start to adhere to it. This process is termed biofouling, defined as the
settlement and attachment of organisms on the external surfaces of sub-
merged or semisubmerged objects (Lewis, 1998). Antifouling technologies
have developed to protect structures against biofouling.
Antifouling coatings have been used for many centuries. The Phoeni-
cians and Carthaginians were the first to use pitch and likely copper sheeting
to prevent the settlement of organisms on the bottom of wooden ships
(Hellio and Yebra, 2009). From the late 18th century, the use of metals
in coatings increased successfully and these compounds, particularly copper,
are still incorporated into modern coatings (Dafforn et al., 2011). The effi-
cacy of metals as antifoulants can be enhanced by co-biocides (or booster
biocides) in the painting formulations. Such co-biocides found their market
from the early 1960s when organotin-based paints were introduced as
marine antifoulants and believed to be the solution to preventing biofouling
26 Samantha Eslava Martins and Camila de Martinez Gaspar Martins

(Yebra et al., 2004). However, severe impacts on the marine environment
occurred following the introduction of tributyltin (TBT), including the
well-known phenomenon of imposex, which decimated the marine gastro-
pod Nucella lapillus in coastal areas of Southwest England (Gibbs and Bryan,
1996). The severity of adverse ecological effects of organotin-based anti-
foulants culminated in the gradual restriction until the global ban of TBT
as an active ingredient in antifouling paints, in 2008 (AkzoNobel, 2000;
Dafforn et al., 2011). Subsequently, organotin-free technologies flooded
the paint market, turning paint formulations into a mixture of inorganic bio-
cides (typically cuprous oxide) and one or more organic/organometallic co-
biocides (Hellio and Yebra, 2009), such as Irgarol 1051, Diuron, DCOIT
(Seanine), chlorothalonil, dichlofluanid, TCMTB, thiram, zinc pyrithione
(ZnPT), and copper pyrithione (CuPT) (Castro et al., 2011; Hellio and
Yebra, 2009). Such co-biocides are not expected to leach into the water
bodies to trigger toxic concentrations to nontarget species (Hellio and
Yebra, 2009). However, it is already known that several organisms may
be very sensitive to such antifoulants, particularly during the early life stages,
including farmed aquatic animals (Martins et al., 2018).
3.2.2 Disinfectants
Disinfectants are used in intensive cultures, particularly in finfish and shrimp
hatcheries and grow-out ponds in routine practices to disinfect culture facil-
ities and equipment, to maintain hygiene throughout the production cycle,
and often to treat bacterial disease outbreaks. A disinfectant is an agent that
destroys infection-producing organisms. In this sense, disinfectants can act
on microorganisms in two distinct ways: (1) through growth inhibition
(bacteriostasis and fungistasis) or (2) through lethal action (bactericidal, fun-
gicidal, or virucidal effects). The particular reason for disinfection will deter-
mine the disinfection strategy used and how it should be applied.
General practices of the disinfection of aquaculture systems involve the
application of chemical compounds in sufficient concentrations, and for suf-
ficient periods, to kill or reduce the pathogenic organisms, mainly virus and
fungus, avoiding the loss of production. Essential disinfection protocols
include: (1) removal of all aquatic animals (both dead and alive) from the
facility; (2) a deep cleaning and washing to eliminate remaining organic mat-
ter adhered to the surfaces; (3) the application of disinfectants on equipment
and installations; (4) a final wash; and (5) the neutralization using chemical
products, if needed. The disposal of diseased animals should not be done in
27
Antifoulants and disinfectants

receiving waters due to the risk of contamination of wild populations or
neighboring farms that use the same water supply. Therefore, the animals
must be euthanized and collected by a company designed to treat or store
hazardous waste. The disinfectants must be stored in such a way that they
will not pose direct or indirect dangers to animal or human health and
the environment (OIE - World Organisation for Animal Health, 2009).
Among the most used disinfectants are formaldehyde, hydrogen perox-
ide, chlorine, potassium permanganate, isopropyl alcohol, iodophors,
peracetic acid and quaternary ammonium compounds (Bowker et al.,
2014; Scarfe et al., 2006). In general, they are characterized by high solubility
and low persistence in the aquatic environment. The efficacy and character-
istics of the commonly used disinfectants are summarized in Table 3.1 (Kohn
et al., 2017; Rico et al., 2012).
3.3 Mode of action
3.3.1 Antifoulants
The mode of action of antifouling biocides depends on the target organisms
for which the formulation was designed for. Antifouling herbicides such as
Irgarol 1051 and Diuron act by inhibiting the transport of electrons during
photosystem II (Hall et al., 1999), affecting photosynthetic organisms. Anti-
fouling fungicides may act through different mechanisms. Chlorothalonil
acts through the inhibition of glycolysis or depleting glutathione (Caux
et al., 1996); despite being designed to act as a fungicide, the presence of
multiple reactive electrophilic centers makes chlorothalonil extremely toxic
Table 3.1 Efficacy and characteristics of commonly used disinfectants.
Disinfectant Efficacy
Target organism
Virus Bacteria
Bacterial
spore Fungi
Spore-forming
protozoan
Formaldehyde High + + + + +
H2O2 High + + VA + +
PAA High + + + + +
Chlorine High + + VA + LA
Iodophors Intermediate + + + LA
QACs Low VA VA +
Ozone High + + VA + LA
QACs, quaternary ammonium compounds; PAA, peracetic acid; +, effective; , nonrecommended;
VA, variable activity; LA, limited activity. Information on specific efficacies followed Scarfe et al.
(2006) and AQUAVETPLAN (2008).
28 Samantha Eslava Martins and Camila de Martinez Gaspar Martins

to nontarget aquatic organisms (Castro et al., 2011), causing effects in ani-
mals and plants. Dichlofluanid is a potent inhibitor of fungal spore germina-
tion (PPDB, 2007–2017), but its degradation products may play a major role
in toxicity since dichlofluanid quickly undergoes hydrolysis in water
(Schouten et al., 2005). Thiram inhibits spore germination and mycelial
growth (PPDB, 2007–2017) as well as nontarget species for being a multisite
inhibitor (KEMI, 2015).
The organometallic antifouling biocides such as zinc pyrithione (ZnPT)
and copper pyrithione (CuPT) show microbiocidal activity. There is a lack
of information on the mode of action of pyrithione salts, but it has been
reported that ZnPT and CuPT catalyze the electroneutral exchange of
H+
and other ions with K+
across cell membranes, disrupting the proton
motive force in target organisms. As a consequence, the transport of nutri-
ents across membranes is impaired, leading organisms to starvation and even-
tual death (KEMI, 2014).
Some antifoulants act as broad-spectrum biocides with efficacy against
either weeds or fungi (Fernández-Alba et al., 2002). For instance, the anti-
fouling co-biocide DCOIT (4,5-dichloro-2-octyl-1,2-thiazol-3-one)
reacts with the proteins of fouling specimens when they find the painting
surface, breaking metabolic processes, and hence preventing the attachment
of the organism to solid surfaces. Another important broad-spectrum co-
biocide is TCMTB (1,3-benzothiazol-2-ylsulfanylmethyl thiocyanate) that
inhibits the mitochondrial electron transport chain (Fernández-Alba et al.,
2002) in a wide range of nontarget organisms.
In addition to herbicides, fungicides, microbiocides, and broad-spectrum
biocides, some antifoulants are categorized as “emerging compounds”
because they have a relatively new use as an antifouling biocide encompassing
mainly regional markets. Among them, tralopyril, medetomidine, and TPBP
(pyridine-triphenylborane) should be highlighted as synthetic biocides.
Tralopyril is used in coatings to enhance the antifouling efficacy of
copper-free antifouling paintings by uncoupling mitochondrial oxidative
phosphorylation (EU, 2014; International, 2014). Medetomidine acts
through the activation of analogous octopamine leading to an anti-settling
effect (EU, 2015) and is designed to protect against shell-building marine
organisms. TPBP has been largely used in Japan (Mochida et al., 2012),
but its mode of action is still unknown (Wendt et al., 2016). Capsaicin is a
natural co-biocide extracted from chili peppers, which acts on the nervous
system and also disrupts metabolism and damages membranes (Gervais
et al., 2008). The capsaicin derivative nonivamide acts the same way.
29
Antifoulants and disinfectants

3.3.2 Disinfectants
Formaldehyde is one of the most applied disinfectants in intensive aquacul-
ture, being frequently used to control parasitic infections on fish and crus-
tacean surfaces, and it is also used for treatment against water mold (fungus)
on fish eggs (Boyd and Massaut, 1999). Although its use is encouraged in
hatcheries to control the presence of “fungus,” it is not recommended for
ponds because it kills algae present in pond water, reducing the production
of oxygen by phytoplankton and augmenting the organic matter and
decomposition (Francis-Floyd and Pouder, 1996). Formaldehyde when
diluted in water form is known as formalin. Formalin may be applied as a
prophylactic measure or for therapeutic purposes and is extremely effective
against most protozoan parasites (Ichthyophthirius spp., Costia spp., Epistylis
spp., Chilodonella spp., Scyphidia sp., and Trichodina spp.) and monogenetic
trematodes (Cleidodiscus spp., Gyrodactylus spp., and Dactylogyrus spp.)
(FDA, 1995; Francis-Floyd and Pouder, 1996; Shao, 2001). Due to its elec-
trophilic character, formalin can react with functional groups of several bio-
logical macromolecules, such as proteins, DNA and RNA, polysaccharides,
and glycoproteins (Leal et al., 2018).
Hydrogen peroxide (H2O2) and chlorine are potent oxidizing agents not
only used as broad-spectrum disinfectants in finfish and shellfish produc-
tions, but can also be used to treat fungal infections and as pesticides during
the pond preparation between production cycles (Rico et al., 2012). Chlo-
rine gas and powdered forms such as calcium hypochlorite and sodium
hypochlorite are used to disinfect water supplies in fish and shrimp hatch-
eries. They can be used in ponds after physical sediment removal, also
between production cycles (AQUAVETPLAN, 2008). The chlorine and
H2O2 oxidize electrons from susceptible chemical groups and become
themselves reduced in the process. Oxidizing agents are usually low-molec-
ular-weight compounds ready to easily pass through cell walls/membranes
and then react with internal cellular components disrupting them, and lead-
ing to apoptotic and necrotic cell death (Finnegan et al., 2010). At cellular
level, low levels of oxidation can be reversible and prokaryotic organisms
have developed many defenses against these effects. Recently, Pedersen
et al. (2019) showed a relation between H2O2 decomposition and
particle-associated bacterial activity, suggesting that the quantification of
H2O2 in water samples can be used as a rapid and feasible indicator of micro-
bial activity in fresh and saltwater aquaculture systems, ranging from pond
farming to intensive recirculating systems.
30 Samantha Eslava Martins and Camila de Martinez Gaspar Martins

Iodophors are polyvinylpyrrolidone-iodide-iodine complexes in aque-
ous solution, used as disinfectants for nonhardened fish eggs to prevent
spreading pathogens from the broodstock fish to the offspring
(Lahnsteiner and Kletzl, 2016). Iodine acts by decreasing the oxygen
requirements of aerobic microorganisms. It acts on microorganisms’ respi-
ratory chain by blocking the transport of electrons through electrophilic
reactions with the enzymes of the respiratory chain (Maris, 1995). The opti-
mum concentration is 200mgL1
free iodine with a contact time of 2min
and 100mgL1
free iodine for cleaned and dried equipment. In a nonfood-
contact application, the concentration may rise to 500–800mgL1
. Iodo-
phors should be used with caution because they are highly toxic to fish
(Boyd and Massaut, 1999).
Ammonia treatment in sanitation is a relatively new approach. In contrast
to oxidants, ammonia is not consumed during the treatment. The ammonia
effect on sanitation is sustained for prolonged periods, while also reducing
the risk for regrowth (Kohn et al., 2017). Quaternary ammonium com-
pounds (QACs) impair membrane permeability by irreversibly binding to
phospholipids and proteins of the membrane. One of the commonly used
products is benzalkonium chloride, applied to inhibit bacterial growth
and the development of mucus in the gills of salmon (Burka et al., 1997),
thereby allowing an adequate absorption of oxygen. The capability of the
bacterial cell to absorb such molecules influences sensitivity. These com-
pounds are used in finfish and shellfish production to treat bacterial, proto-
zoan, and monogenean infections and as fungicides in shrimp hatcheries
(Rico et al., 2012).
Ozone is a powerful oxidizing agent widely used in aquaculture for dis-
infection of pathogens and for water quality enhancements since it oxidizes
organic wastes and nitrite. The oxidation of pathogens and other material by
ozone occurs extremely rapidly, through reactions of molecular ozone with
oxidizable compounds, and also due to the reaction of the oxidizable com-
pounds with reactive oxygen species (ROS) formed by ozone decomposi-
tion in water (AQUAVETPLAN, 2008; Gonçalvez and Gagnon, 2011;
Scarfe et al., 2006; Spiliotopoulou et al., 2018; Summerfelt and
Hochheimer, 1997; Tyrell et al., 1995). As an oxidative disinfectant, it is
consumed by organic matter; thus, initial ozone concentration decays faster
in waters with higher concentrations of organic matter as wastewater. For
this reason, it is important to apply an ozone dose that remains in the system
for enough time for the oxidation demands, whether inactivation of path-
ogens or degradation of the organic matter present, without affecting farmed
31
Antifoulants and disinfectants

species. Nevertheless, monitoring ozone performance is still a challenge
(Spiliotopoulou et al., 2018). Ozonation is usually used in recirculating cul-
ture systems that consume less water per kg of fish produced and allow solid
removal and effluent treatment (Gonçalvez and Gagnon, 2011). The liter-
ature reports a wide range of ozone dosages in recirculating systems,
according to the number of animals and feed ratio (Bullock et al., 1997;
Summerfelt et al., 2009; Summerfelt and Hochheimer, 1997). A drawback
of ozone use is that its application is energy-consuming and requires a trained
operator. Even though ozone is more reactive than chlorine in demand-free
solutions toward all microorganisms, it has shown to be more efficient
against viruses, but less efficient against bacteria than chlorine (Tyrell
et al., 1995), although both can inactivate bacterial spores, depending on
the conditions of the application and the origin of the pathogen
(Broadwater et al., 1973; Rose et al., 2005; Tyrell et al., 1995).
Nanomaterials are gaining space as disinfectants for aquaculture pur-
poses. For example, they have been used to improve water quality in shrimp
aquaculture, reducing the rate of water exchange, increasing shrimp survival
rate and yield (Wen et al., 2003). Silver (Ag) nanoparticles (NPs) (nAg) are
the most investigated antibacterial compounds. The nAg was used for the
treatment of fungal infections in rainbow trout eggs showing an inhibitory
effect on fungi growth ( Johari et al., 2015). Dananjaya et al. (2016) inves-
tigated the antibacterial function of chitosan-Ag nanocomposites (CagNCs)
against the fish pathogen Aliivibrio salmonicida. The CagNCs inhibited A. sal-
monicida growth with a minimum inhibitory concentration (MIC) and min-
imum bactericidal concentration (MBC) at 50 and 100mgL1
, respectively.
Similarly, nZnO exhibited antibacterial activity disrupting bacterial cell
membrane integrity, reducing cell surface hydrophobicity and down-
regulating the transcription of oxidative stress-resistance genes (Pati et al.,
2014). Furthermore, M€
uhling et al. (2009) showed that nTiO2 and nAg
reduced the buildup of bacteria in estuarine water ().
Another disinfectant that has been recognized to be suitable for aquacul-
ture is peracetic acid (PAA) which is considered an alternative sanitizer to
formaldehyde. PAA is a highly reactive peroxygen with widespread antimi-
crobial effects (Liu et al., 2015, 2016; Pedersen et al., 2009, 2013). It
degrades entirely within several hours after application, resulting in trace
concentration residuals that are not readily measured (Pedersen et al.,
2009). The test kits are not sensitive enough to analyze PAA at low ranges
(below 0.2ppm). Only a few studies have described analytical measurement
of the commercial PAA compound (Pedersen et al., 2013). In this sense,
32 Samantha Eslava Martins and Camila de Martinez Gaspar Martins