the environmental fate and effects of sea lice

The Environmental Fate and Effects of Sea Lice

Chemotherapeutants used in Canadian Salmon

Aquaculture

by

Fauve Strachan

B.Sc., University of Calgary, 2012

Project Submitted in Partial Fulfillment of the

Requirements for the Degree of

Master of Environmental Toxicology

in the

Department of Biological Sciences

Faculty of Science

© Fauve Strachan 2018

SIMON FRASER UNIVERSITY

Fall 2018

Copyright in this work rests with the author. Please ensure that any reproduction or re-use is done in accordance with the relevant national copyright legislation.

ii

Approval

Name:

Degree:

Title:

Examining Committee:

Fauve Strachan

Master of Environmental Toxicology

The Environmental Fate and Persistence of Sea Lice Chemotherapeutants used in Canadian Salmon Aquaculture

Chair: Gordon Rintoul

Associate Professor

Date Defended/Approved:

Chris Kennedy Senior Supervisor Professor

Vicki Marlatt Supervisor Assistant Professor

Rostam Namdari External Examiner Senior Director Translational Drug Development Xenon Pharmaceuticals Inc.

May 23, 2018

iii

Ethics Statement

iv

Abstract

In Canada, five formulations have been used to treat sea lice infestations in salmon

aquaculture. This research investigated the environmental partitioning, persistence, and

acute toxicity to marine organisms of Slice® (AI: emamectin benzoate [EB]), Salmosan®

(AI: azamethiphos [AZ]), Alphamax® (AI: deltamethrin [DM]), Excis® (AI: cypermethrin

[CP]), and Interox® Paramove 50 (AI: hydrogen peroxide [HP]). EB, CP and DM

partitioned mainly to the sediment in sediment-water microcosms; AZ and HP remained

mainly in the water. The persistence of chemicals in water was reported: CP > DM > AZ

> HP. In sediment, CP > EB > DM was observed. Toxicity tests indicate a lack of trends

however the information is useful for identifying risks. Some reported values for

echinoderms, kelp, and topsmelt toxicity are below the recommended treatment

concentrations. This research provides insight into the environmental fate and associated

risks to non-target marine organisms in the vicinity of salmon aquaculture sites.

Keywords: toxicity; sea lice; aquaculture; pesticide; fate, persistence; partitioning;

chemotherapeutants

v

Acknowledgements

I must extend gratitude to my supervisor, Dr. Chris Kennedy, for allowing me the

freedom to take on this project as my own and for his invaluable guidance (and patience)

along the way. Without your help, the successful completion of this project would not have

been possible. This research was supported by a grant from the National Contaminants

Advisory Group of Fisheries and Oceans Canada to Dr. Chris Kennedy.

I owe much of the success of my toxicological testing to the people at Nautilus

Environmental, Burnaby BC. They provided me with a wealth of knowledge and

experience while preparing and performing my toxicological tests at their facility. I am

indebted to Emma Marus, Yvonne Lam, Karen Lee and Jeslin Wijaya who went above-

and-beyond the call of duty repeatedly, lending a hand and providing me with their

expertise and friendship. Special thanks must also be given to Krysta Pearcy for always

lending a hand, whenever possible.

The distractions, well-wishes, and advice of my lab-mates in the Kennedy lab were

always a special treat. A few amongst them were kind enough to volunteer their time when

I needed it most. The help of Tina Johnson, Vinicius Cavicchioli Azevedo, Kate Mill, and

Jessica Banning was instrumental to the success of my tests. Vini’s help extended beyond

lab-work. His positive attitude, kind spirit and friendship helped to keep me sane in the lab.

Special thanks must also go to Jill Bennett without whom the completion of the

environmental fate and persistence testing would not have been possible.

Thanks to the Gobas lab, Frank Gobas and Victoria Otton, for sharing their facilities

and expertise, lending an ear when needed, and for all their help in the early stages of the

project.

Last but not least, I am eternally grateful for the support of my friends and family

without which I most certainly would not have made it this far – I love you all. I am

especially thankful for the support of my parents, Rob and Josée, and my sister, Jessica,

for not only being my number one fans, supporting me in everything that I do, but also for

coming to visit me!

vi

Table of Contents

Approval .......................................................................................................................... ii Ethics Statement ............................................................................................................ iii Abstract .......................................................................................................................... iv Acknowledgements ......................................................................................................... v Table of Contents ........................................................................................................... vi List of Tables .................................................................................................................. ix List of Figures.................................................................................................................. x List of Acronyms ............................................................................................................. xi

Chapter 1. Introduction .............................................................................................. 1 1.1. Aquaculture .......................................................................................................... 1 1.2. Canadian aquaculture ........................................................................................... 2

1.2.1. Overview ....................................................................................................... 2 1.2.2. Aquaculture economics ................................................................................. 3 1.2.3. Regulation and management ......................................................................... 4 1.2.4. Environmental concerns ................................................................................ 5

1.3. Salmon aquaculture .............................................................................................. 7 1.3.1. History of salmon aquaculture ....................................................................... 7 1.3.2. Salmon aquaculture in Canada ...................................................................... 7 1.3.3. Environmental concerns of salmon aquaculture ............................................. 8 1.3.4. Disease and infection in salmon aquaculture ............................................... 10

1.4. Sea lice............................................................................................................... 10 1.4.1. Sea lice biology ........................................................................................... 10 1.4.2. Sea lice concerns in aquaculture ................................................................. 12 1.4.3. Sea lice treatments ...................................................................................... 13 1.4.4. Sea lice treatment in Canada ....................................................................... 16

1.5. Emamectin benzoate .......................................................................................... 17 1.5.1. Canadian usage .......................................................................................... 18 1.5.2. Chemotherapeutant efficacy ........................................................................ 18 1.5.3. Environmental fate of EB ............................................................................. 19 1.5.4. EB mechanism of action and effects on non-target species ......................... 19

1.6. Hydrogen peroxide ............................................................................................. 24 1.6.1. Canadian usage .......................................................................................... 24 1.6.2. Chemotherapeutant efficacy ........................................................................ 24 1.6.3. HP environmental fate ................................................................................. 25 1.6.4. HP mechanism of action and effects on non-target species ......................... 25

1.7. Pyrethroids ......................................................................................................... 29 1.7.1 Canadian usage .......................................................................................... 29 1.7.2 Chemotherapeutant efficacy ........................................................................ 29 1.7.3 Pyrethroid fate in the environment ............................................................... 30 1.7.4 Pyrethroid mechanism of action ................................................................... 30 1.7.5 CP effects on non-target species ................................................................. 31

vii

1.7.6 DM effects on non-target species ................................................................ 36 1.8. Azamethiphos ..................................................................................................... 40

1.8.1. Canadian usage .......................................................................................... 40 1.8.2. Chemotherapeutant efficacy ........................................................................ 40 1.8.3. AZ environmental fate and persistence ........................................................ 41 1.8.4. AZ mechanism of action and effects on non-target species ......................... 41

1.9. Risk of sea lice pesticide use .............................................................................. 46

Chapter 2. Environmental Fate and Effects of Sea Lice Pesticides used in Canadian Salmon Aquaculture ......................................................................... 47

Abstract ......................................................................................................................... 48 2.1. Introduction ......................................................................................................... 49 2.2. Materials and methods ....................................................................................... 51

2.2.1. Organisms ................................................................................................... 51 2.2.2. Chemicals .................................................................................................... 51 2.2.3. Fate and persistence ................................................................................... 52 2.2.3 Toxicity tests ................................................................................................ 53

2.2.3.1 Giant kelp germination and growth ....................................................... 53 2.2.3.2 Topsmelt survival .................................................................................. 54 2.2.3.3 Mysid survival ....................................................................................... 55 2.2.3.4 Bivalve embryo survival and development ............................................ 55 2.2.3.5 Echinoderm fertilization ........................................................................ 56

2.2.4 Chemical analysis ........................................................................................ 57 2.2.5 Calculations and statistics ........................................................................... 60

2.2.5.1 Partitioning and chemical persistence ................................................... 60 2.2.5.2 Toxicity tests ......................................................................................... 61

2.3 Results and discussion ........................................................................................ 61 2.3.1 Chemical partitioning and persistence ......................................................... 61 2.3.2 Toxicity tests ................................................................................................ 68

2.3.2.1 Giant kelp germination and growth ....................................................... 68 2.3.2.2 Topsmelt survival .................................................................................. 69 2.3.2.3 Mysid survival ....................................................................................... 69 2.3.2.4 Bivalve embryo survival and development ............................................ 70 2.3.2.5 Echinoderm fertilization ........................................................................ 71

2.4 Conclusions ......................................................................................................... 73

Chapter 3. Overall conclusions and future directions ........................................... 74 3.1. Overall Conclusions ............................................................................................ 74 3.2. Future Directions ................................................................................................ 75

References ................................................................................................................... 77

Appendix A. Summary of key properties for test substances ............................ 93

Appendix B. Raw Data: Fate and persistence testing ......................................... 94

viii

Appendix C. Raw Data: Toxicity tests .................................................................. 96

ix

List of Tables

Table 1 : Comparison of the effective versus lethal dose of sea louse treatment substances used in salmon aquaculture (Bright and Dionne, 2005; Roth, 2000). ..................................................................................................... 17

Table 2: Data from emamectin benzoate acute and chronic exposures to marine and freshwater invertebrates and fish species. ....................................... 21

Table 3: Data from hydrogen peroxide acute and chronic exposures to marine and freshwater invertebrates and fish species............................................... 27

Table 4: Data from cypermethrin acute and chronic exposures to fresh water and marine invertebrates and fish species. ................................................... 32

Table 5: Data from deltamethrin acute and chronic exposures to fresh water and marine invertebrates and fish species. ................................................... 37

Table 6: Data from azamethiphos acute and chronic exposures to freshwater and marine invertebrates and fish species. ................................................... 43

Table 7: Summary key information pertaining to the use of formulations in Canada to treat sea lice infestations: AI, recommended treatment dose, or concentration, and recommended duration of treatment. ........................ 50

Table 8: Summary of nominal EB, CP, DM, AZ, and HP test concentrations used in

toxicity tests (g L-1). Range of nominal concentration (Range), number of concentrations used in testing (Number) and the dilution factor (DF) are reported. ................................................................................................ 53

Table 9: Summary of sediment and water characteristics used in partitioning and persistence study. .................................................................................. 62

Table 10: Half-life (and p-values) for AZ, CP, DM, EB, and HP in water and sediment calculated using first-order rate equations. P-value <0.05 indicates relationships between the variables that are statistically significant. ....... 64

x

List of Figures

Figure 1: Visual representation of the major types of aquaculture practiced around the world, both marine and freshwater, including sea ranching, surface lines, subsurface lines, bottom culture, racks, cages, ponds and hatcheries (DFO, 2012). ............................................................................................ 2

Figure 2: Map of open-net pen salmon farms (indicated by yellow dots) and wild salmon migration routes (indicated by red lines) in southwest British Colombia (BC) (Morton, 2015). ................................................................ 6

Figure 3: Life cycle of sea lice, Caligus elongatus. Legend: 1 Nauplius I; 2 Nauplius II; 3 Copepodid; 4 Chalimus I; 5 Chalimus II; 6 Chalimus III; 7 Chalimus IV; 8 Pre-adult; 9 Adult (female); 10 Adult (male) (Haya et al., 2005). .......... 11

Figure 4: Chemical structures of the compounds that have been used to treat sea lice infestations worldwide: A) dichlorvos B) malathion C) trichlorfon D) azamethiphos E) pyrethrum F) cypermethrin G) deltamethrin H) ivermectin I) emamectin benzoate J) doramectin K) hydrogen peroxide L) teflubenzuron and M) diflubenzuron. ...................................................... 15

Figure 5: Chemical structure of emamectin benzoate, where when R=methanol (CH3) it is MAB1b and when R=ethanol (CH2CH3) is it is MAB1a. ......... 18

Figure 6: Chemical structure of hydrogen peroxide. .............................................. 24

Figure 7: Chemical structure of A) cypermethrin and B) deltamethrin. ................... 29

Figure 8: Chemical structure of azamethiphos. ..................................................... 40

Figure 9: Distribution of chemotherapeutants among sediment (grey bars) and water (white bars) at different sampling times presented as a percentage (%) of the total administered dose. A) AZ; B) EB; C) CP; and D) DM. ............... 64

Figure 10: Decay figures for A) AZ; B) CP; C) DM; and D) HP in water shown as concentration (µg L-1 [AZ, CP and DM] and mg L-1 [HP]) versus time. The trend lines represent the decay per standard first-order rate equations. . 67

Figure 11: Decay figure for A) CP; B) EB; and C) DM in sediment shown as

concentration (g 100 g-1 sediment) versus time. The trend lines represent the decay per standard first-order rate equations. .................................. 68

xi

List of Acronyms

Ach acetylcholine

AChE Acetyl-cholinesterase

AI active ingredient

AZ azamethiphos

BC British Colombia

BCF bioconcentration factor

BMP best management practice

CA California

CBSA Canada Border Services Agency

CCME Canadian Council of Ministers of the Environment

CDN Canadian dollars

CEAA Canadian Environmental Assessment Act

CETIS Comprehensive Environmental Toxicity Information System

CFIA Canadian Food Inspection Agency

CO Colorado

CP cypermethrin

d day

DF dilution factor

DFO Department of Fisheries and Oceans

DM deltamethrin

DNA deoxyribonucleic acid

EB emamectin benzoate

EC European Commission

EC50 effect concentration resulting in 50 % effects

ECCC Environment and Climate Change Canada

ED50 effect dose resulting in 50 % effects

EFSA European Food Safety Authority

ERM furunculosis, erenteric redmouth disease

FAO Food and Agriculture Organization of the United Nations

FDA Food and Drugs Act

FHL Norwegian Seafood Federation

g gram

xii

GABA gama-aminobutyric acid

h hour

HC Health Canada

HP hydrogen peroxide

IC50 Inhibiting concentration for a 50 % effect

IMTA integrated multi-trophic aquacutlure

IPN pancreatic necrosis

KOW n-octanol-water partition coefficient

L liter

LC50 lethal concentration resulting in 50 % mortality

LD50 lethal dose resulting in 50 % mortality

LOAEC lowest observed adverse effect concentration

LOAEL lowest observed adverse effect level

min minute

MOA mechanism of action

MSD minimum significant difference

NBDA New Brunswick Department of Agriculture

NOAEC no observed adverse effect concentration

NOAEL no observed adverse effect level

NWPA Navigable Water Protection Act

OECD Organization for Economic Co-operation and Develop-ment

OIE World Organization for Animal Health

ON Ontario

OP organophosphate

PCPA Pest Control Products Act

PMRA Pest Management Regulatory Agency

ROS reactive oxygen species

SAV salmonid alphavirus

SD standard deviation

SCCP Canadian Shellfish Sanitation program

SEPA Scottish Environmental Protection Agency

SFU Simon Fraser University

TOC total organic carbon

TOM total organic matter

xiii

USD U.S. dollars

US EPA U.S. Environmental Protection Agency

UT Utah

V volts

VDD Veterinary Drugs Directorate

WA Washington

1

Chapter 1. Introduction

1.1. Aquaculture

Fisheries and aquaculture play an essential role in ensuring food security

worldwide; additionally, these industries employ a large number of people thereby

contributing to the reduction of poverty (FAO, 2014). The aquaculture industry, which

consists of large-scale aquatic farming, is a growing industry that began in part, as a result

of unsustainable fishing practices and increasing worldwide seafood demand (DFO, 2012;

FAO, 2014; Naylor et al., 2000). However, despite contributing to an increase in the world

fish supplies, aquaculture of carnivorous fish requires the input of large quantity of wild

fish supply for feed which increases the demand on fisheries. Additionally, aquaculture

can negatively affect wild fisheries through habitat modification, food web interactions,

introduction of exotic species, and nutrient pollution (Naylor et al., 2000).

Aquaculture is a broad term that describes a variety of activities involved in farming

aquatic organisms. Aquaculture can be subdivided into two branches: marine aquaculture,

or mariculture, and freshwater aquaculture. Inland aquaculture generally involves

freshwater species whereas marine aquaculture typically occurs in the sea, intertidal, or

in land-based production facilities (FAO, 2014). There are a variety of different aquaculture

systems, each associated with their own list of species for which aquaculture has been

well established; common examples of marine aquaculture include sea ranching, surface

lines, subsurface lines, racks, cages, ponds and hatcheries (Figure 1) (DFO, 2012;

Queensland Government, 2013). Despite the tendency for monoculture, integrated multi-

trophic aquaculture (IMTA), a polyculture which combines aquaculture practices involving

species from different trophic levels, can result in greater production with lower

environmental impacts (Olin et al., 2011; Thierry et al., 2012). Specifically, polyculture

practices result in increased environmental sustainability by taking advantage of natural

processes and interactions between species of different trophic levels (e.g. through

nutrient cycling).

2

Figure 1: Visual representation of the major types of aquaculture practiced around the world, both marine and freshwater, including sea ranching, surface lines, subsurface lines, bottom culture, racks, cages, ponds and hatcheries

Source: Aquaculture in Canada 2012, A Report on Aquaculture Sustainability, Department of Fisheries and Oceans (2012). Used with permission.

Over 600 species are produced in aquaculture worldwide, including a variety of

finfish, molluscs, crustaceans, amphibians and reptiles, aquatic invertebrates, and algae

(FAO, 2014). Global aquaculture production in 2014 was estimated at $160.2 billion (USD)

(FAO, 2016), up from $144.4 billion in 2012 (FAO, 2014). Furthermore, it has been

estimated that over 25 % of fish consumed by humans comes from a farmed source

(Naylor et al., 2000). It is clear that aquaculture plays an important role in the nourishment

of humans worldwide.

1.2. Canadian aquaculture

1.2.1. Overview

Commercial aquaculture, which is present in all Canadian provinces and in the

Yukon territory, is a multi-million-dollar industry in Canada and worldwide (Bright and

Dionne, 2005; Costello, 2009; DFO, 2012; DFO, 2014; Olin et al., 2011; Torrissen et al.,

2013). Aquaculture in Canada can be divided into two major categories: finfish aquaculture

and shellfish aquaculture (DFO, 2017; Olin et al., 2011). Species of finfish cultured in

Canada includes primarily: Atlantic salmon, coho salmon, Chinook salmon, and steelhead

3

(marine); rainbow trout and brook trout (freshwater). Species of shellfish cultured in

Canada includes primarily: blue mussels, oysters, clams, scallops, and geoduck.

Additionally, there are a number of species, both marine and freshwater, for which there

is potential for industry expansion (DFO, 2012; Olin et al., 2011).

Depending on the species, finfish production can involve cage-based methods or

land-based cultures. Cage-based farms are typically large open-net pens that are

anchored in the water. In Canada, cage-based farming is mainly used for salmon farming

(marine) or rainbow trout (freshwater). Land-based cultures, which are used in freshwater

finfish production in locations where there are no other viable options, consist of raceways,

ponds or circular tanks with a continuous water supply (either fresh or recirculated).

Historically, shellfish culture, especially oyster culture, was primarily bottom culture

however more commonly off-bottom cultures are farmed using long lines or raft systems,

again, depending on the species. Bottom culture involves the seeding of intertidal or

subtidal beds whereas off-bottom cultures, such as suspended cultures, uses either long

lines or rafts. Off-bottom culture is generally preferred as it can result in higher yields,

however its viability depends on factors including local conditions and the species being

farmed.

1.2.2. Aquaculture economics

In Canada, where salmon is the primary contributor to aquaculture production, the

annual production value in 2015 was estimated at over $967 million (CDN); this figure

includes a number of other species that are farmed domestically including: trout,

steelhead, clams, oysters, mussels, and scallops (DFO, 2012; 2017). The second most

important species, in terms of production and value, is the blue mussel (Mytilus edulis),

however two species of oyster, the American oyster (Crassostrea virginica) and the Pacific

oyster (Crassistrea gigas) are also important in this regard. For in-land species, the

rainbow trout (Oncorhynchus mykiss) is the most commonly farmed. British Columbia

accounts for the greatest production value in Canada, followed by New Brunswick and

Newfoundland and Labrador (DFO, 2012).

4

1.2.3. Regulation and management

Fisheries and Oceans Canada (DFO) has the lead federal role in managing

fisheries and safeguarding its waters in Canada; it does so by supporting economic

growth, supporting innovation and contributing to sustainability (DFO, 2017b). Key pieces

of legislation guide the work of the department, including: the Oceans Act, the Fisheries

Act, the Health of Animals Act, the Species at Risk Act, the Coastal Fisheries Protection

Act, the Canada Shipping Act, the Fish Inspection Act, the Navigable Water Protection Act

(NWPA) and the Canadian Environmental Assessment Act (CEAA). The most important,

however, is the Fisheries Act which provides DFO with the legislative power to approve

aquaculture applications by focusing on habitat protection and pollution prevention.

Other agencies in the Federal government also play an important role in Canadian

aquaculture and include Health Canada (HC), the Canadian Food Inspection Agency

(CFIA), and the Canada Border Services Agency (CBSA). Within HC, the Pest

Management Regulatory Agency (PMRA) and the Veterinary Drugs Directorate (VDD)

play an important role in pest and animal health management. The CFIA enforces

aquaculture biosecurity (food safety) and works with CBSA to restrict imports when

necessary. Additionally, the CFIA also works with the DFO to implement the North

American Animal Health Program (NAAHP) which align with the World Organisation for

Animal Health (OIE) standards (DFO, 2012). The two of them also administer the

Canadian Shellfish Sanitation program (SCCP) along with Environment and Climate

Change Canada (ECCC). The goals of these programs are to protect wild and farmed

species from infectious diseases.

Regulatory responsibilities are shared among federal, provincial, and territorial

governments, as well as with private industry. These responsibilities can vary from

province to province; some provinces lead aquaculture site leasing and licences, whereas

in other provinces this is federally led (DFO, 2012). In addition to a long list of legislation

and a variety of programs that regulate aquaculture, best management practises (BMPs)

have been developed by industry and federal agencies. An example of the government’s

efforts is Canada’s Sustainable Aquaculture Program which was renewed in 2013. This

demonstrates the government’s commitment to the sustainable development of

aquaculture in Canada. The Aquaculture Development Strategy 2016-2019, a recent

5

publication by the DFO (2012), identifies means by which the development of sustainable

aquaculture in Canada can be encouraged.

1.2.4. Environmental concerns

As previously mentioned, a strong environmental regulatory framework is in place

in Canada. For example, environmental assessments, monitoring, and surveillance play

an important role in Canadian aquaculture (Olin et al., 2011). Despite these efforts, there

are still a variety of environmental concerns with respect to aquaculture, specifically

regarding open-net pen aquaculture, which is prominent. These concerns include: the

potential for disease/virus transmission between captive and wild fish populations,

conflicts with marine mammals, sea lice infestations, water pollution, escape of non-native

fish, displacement of local fishermen, and impacts to tourism (Morton, 2015). These

concerns are similar around the world and the industry is constantly trying to improve its

practices to reduce them (e.g. improved net-pet design, development of vaccines to treat

fish, improved husbandry and BMPs).

In Canada, the concerns about water quality resulting from chemical releases,

pathogen transfer and the potential for release and gene transfer from species to species

from non-native escapees is especially concerning as the density of farms along the coast

lines are very high (DFO, 2016; Morton, 2015). Furthermore, this is aggravated by the

proximity of the farms to similar wild species (i.e. sockeye salmon migratory routes)

(Morton, 2015). Figure 2 provides a visual representation of aquaculture sites on the

Pacific coast of Canada.

6

Figure 2: Map of marine finfish aquaculture facilities in British Columbia (BC).

Source: 2016 Marine finfish aquaculture facilities in BC, Aquaculture maps, Department of Fisheries and Oceans (2016). Used with permission.

The environmental impact of salmon farming has reduced with time; significant

improvements have been made in the reduction of escapees, husbandry practices,

disease control and treatment (Ellis et al., 2016).

7

1.3. Salmon aquaculture

1.3.1. History of salmon aquaculture

Salmon aquaculture began in Norway and Scotland, in the 1960s, and the industry

subsequently expanded as a result of improved technologies, financial incentives, and

support from government agencies (Ellis et al., 2016; FHL, 2011; Naylor et al., 1998b).

Since its inception, the industry has expanded to include Canada, Australia, Chile and the

United Kingdom, among others (Ellis et al., 2016). Globally, Norway is the largest salmon

producing country, followed by Chile, the United Kingdom and Canada (~8 percent) (DFO,

2012; Ellis et al., 2016; Olin et al., 2011). Notably, shrimp and salmon are two of the most

important species in aquaculture, making salmon among the most economically valuable

species produced in aquaculture (Naylor et al., 1998b).

Altantic salmon (Salmo salar), the dominant species produced in aquaculture, are

anadromous, i.e. they live out their early life stages in freshwater before heading out to

sea and ultimately return to freshwater to reproduce. Smoltification is the combined

physiological, morphological and behavioral change that enables salmon to survive in

seawater (Ellis et al., 2016). As a result, the hatchery stage occurs in freshwater and once

smoltification has occurred, the fish can be transferred to the ocean (~ 12 months old to

24 months old) (Ellis et al., 2016; Olin et al., 2011). The grow-out stage occurs primarily

in large floating pens on coastlines and last for anywhere between ~ 18 and 24 months

(Burridge et al., 2008; Burridge et al., 2010; Ellis et al., 2016; Olin et al., 2011). Ultimately,

salmon are then harvested for processing and marketed for human consumption (Olin et

al., 2011).

1.3.2. Salmon aquaculture in Canada

Salmon farming in Canada began in the 1970s with coho (Oncorhynchus kisutch)

and Chinook (Oncorhynchus tshawytscha) salmon, however it quickly shifted to Atlantic

salmon (Salmo salar) since that species can withstand being cultured at greater densities

and grows more rapidly. Atlantic salmon were first farmed successfully in New Brunswick

in 1979 before farming of the species extended to the west coast (Burridge, 2003; Olin et

al., 2011). A small number of coho and Chinook salmon are still farmed today (Olin et al.,

2011).

8

As reflected by production values, the majority of salmon farms are located on the

west coast of Canada (DFO, 2017). On the west coast, salmon are commercially important

due to their significant contributions to the local economy (DFO, 2014a; DFO, 2014; DFO

2017; Manning and Hubley, 2015; Olin et al., 2011). Finfish aquaculture nationwide is

valued at over $877 million (CDN), the majority of which (> 75 %) can be attributed to

salmon aquaculture; in BC alone salmon aquaculture production was valued at over $485

million (CDN) (DFO, 2017). British Columbia, New Brunswick and Nova Scotia are the

three highest salmon producing provinces in Canada (DFO, 2017).

1.3.3. Environmental concerns of salmon aquaculture

Several environmental concerns have emerged as a result of salmon aquaculture

practices. Salmon aquaculture can have negative impacts on non-target organisms,

including native salmon populations, local benthic communities, and zooplankton and

phytoplankton communities (Buschmann et al., 2006; Ellis et al., 2016; Islam and Tanaka,

2004; Naylor et al., 1998; 2000). The negative impacts occur primarily as a result of

nutrient release, pollution resulting from the need to medicate farmed salmon, the effect

of escaped fish on native organisms, and the transfer of disease or pests to native salmon

populations (Bushmann et al., 2006; Ellis et al., 2016; Naylor et al., 1998; 1998b; 2000;

Torrissen et al., 2013). However, there is also some evidence of increased marine bird

numbers in areas where salmon aquaculture is prevalent which has been suggested to

pose additional adverse ecological effects (Buschmann et al., 2006). These concerns are

all amplified by the density of the farms along the coastlines.

Specifically, nutrient release from uneaten food or faecal matter can result in high

water levels of phosphorus and nitrogen (among other compounds) that can cause

harmful algal blooms leading to reduced oxygen concentrations in the water, to dangerous

levels, causing death or other adverse effects to non-target aquatic organisms. Escapees

are problematic because they are typically non-native and may interbreed with local

populations of wild salmon, affecting the gene pool of local populations (Bushmann et al.,

2006; Naylor et al., 1998b). Furthermore, sea lice infestations in farmed salmon generate

concerns about the potential for transfer to wild populations which can potentially lead to

wild population decline (Buschmann et al., 2006; Krkosek et al., 2005; Morton et al., 2011;

Torrissen et al., 2013).

9

Mentioned above, high-density farming often requires the use of a variety of

substances, including: antibiotics, vaccines, or chemotherapeutants which in themselves

may have negative effects on the environment and other local species (Bushmann et al.,

2006; Burridge et al., 2011).

The use of antibiotics in large-scale farming is of concern as it can contribute to

antibiotic resistance. In Canada, antibiotic use is highly regulated and only four products

are available for use in aquaculture (Burridge et al., 2011). Other chemicals used in

salmonid aquaculture include anaesthetics, pesticides, antifoulants and disinfectants

(Burridge et al., 2010; 2011; Burka et al., 1997). The use of these substances is highly

regulated too since their use can potentially result in adverse effects in the environment.

For example, the use of two metals in aquaculture have been shown to contribute to

environmental impacts: copper (Cu) is used as an anti-foulant and zinc (Zn) an additive in

food (Burridge et al., 2011).

Marine ecosystems are complex, consisting of numerous biotic and abiotic

components linked by a series of ecological interdependencies (NRC, 2010). Coast-lines

are particularly high in productivity / rich in biodiversity due to the presence of sunlight.

For example, kelp which is abundant along coast-lines, is known to provide very important

habitat which contributes to higher diversity and productivity (as discussed in Graham et

al. [2007]). Because of the complex web of interactions and ecological interdependencies

in the natural environment it is essential to conserve biodiversity and productivity wherever

reasonably possible. The high-density with which farms are found on coast-lines

contributes to the concerns being raised, as it magnifies other concerns and also results

in a loss of wild habitat (Naylor et al., 2000), often in areas along wild population migratory

routes (see Figure 2) and in areas of high biodiversity and productivity.

Because of the differences in substrates at the various sites in BC, ranging from

black, silty, mud to cobble or rocky, species composition also varies (Winsby et al., 1996).

However, numerous species that have been reported under or in the immediate vicinity of

salmon aquaculture sites in Canada. This includes benthic invertebrates such as starfish,

anemones, and lobsters as well as a variety of species of fish, mammals, zooplankton and

phytoplankton (WInsby et al., 1996). Therefore, salmon farms have the potential to pose

significant ecological concerns.

10

1.3.4. Disease and infection in salmon aquaculture

As with all largescale farming operations, infection, disease and parasite

infestation is often problematic (Burka et al., 1997; Burridge et al., 2010; Ellis et al., 2016;

Haya et al., 2001). In aquaculture, this is primarily the result of high fish densities and poor

water quality. Disease in salmon aquaculture was very problematic in the early 1980s with

Infectious Salmon Anemia (ISA) appearing for the first time in 1984 (FHL, 2011). Other

viral diseases in salmon aquaculture emerged including pancreatic necrosis (IPN), viral

haemorrhagic septicemia, salmonid alphavirus (SAV), and infectious hematopoietic

necrosis (Ellis et al., 2016; Gudding and Van Muiswinkel, 2013; Olin et al., 2011).

Problematic bacterial diseases, such as furunculosis, erenteric redmouth disease (ERM),

and vibiosis also emerged (Ellis et al., 2016). Pests have also been problematic,

particularly sea lice, as infestations have become an increasing area of concern (Ellis et

al., 2016; Olin et al., 2011; Torrissen et al., 2013). In 1994, disease and parasite problems

became a significant issue in Canadian aquaculture (Burridge, 2003).

These diseases and infections can contribute to significant losses in the industry.

As a result, a variety of antibiotics, vaccines, and chemotherapeutants are used to treat

or prevent disease (Burridge et al., 2008; Burridge et al., 2011; Ellis et al., 2016; Gudding

and Van Muiswinkel, 2013). The development and use of these antibiotics, vaccines,

chemotherapeutants, as well as the implementation of improved management techniques

(e.g. fallowing, monitoring) and introduction of new technologies (e.g. automatic feeding

systems, camera surveillance) has resulted in improved production, reduced costs and

increased efficiency (Ellis et al., 2016).

1.4. Sea lice

1.4.1. Sea lice biology

Sea lice are ectoparasite copepods which can cause significant health reduction

in their host, often resulting in death. This occurs directly by hemorrhaging or stress and

indirectly through increased susceptibility to infection and disease (Burridge et al., 2010;

Bright and Dionne, 2005; Costello, 2006; Mustafa et al., 2000; Pahl and Opitz, 1999;

Torrissen et al., 2013). These small crustaceans are natural parasites to wild salmon

populations (DFO, 2014a). Several hundred species of sea lice exist with two genera

11

having been reported to infect salmonids (Caligus and Lepeophtheirus). In Canada, three

species of sea lice have are reported as having infected salmon: Lepeophtheirus salmonis

(with a circumpolar distribution), Caligus elongatus (Atlantic ocean distribution) and

Caligus clemensi (Pacific ocean distribution) (Burridge et al., 2010; Bright and Dionne,

2005; DFO, 2014a; Grant, 2002; Hogans and Trudeau, 1989). A number of other species

have also been reported on farmed salmon including C. rercresseyi, C. teres (in Chile),

and C. orientalis (in Japan) (Costello, 2006).

The life cycle of most sea lice is composed of 10 stages including planktonic

naupliar stages, an infective copepodite stage, and attached stages (Figure 3) (Burridge

and Van Geest, 2014; Costello, 2006; Haya et al., 2005; Hogans and Trudeau, 1989; Roth

et al., 1993; Torrissen et al., 2013). Details of the various life stages are described in the

referenced literature and will not be detailed herein. In general, there are several factors

which influence the fecundity and survival of these year-round parasites. These include

temperature, salinity, abundance of planktonic predators, and host size and density

(Torrissen et al., 2013; Burridge and Van Geest, 2014; Haya et al., 2005; Hogans and

Trudeau, 1989).

Figure 3: Life cycle of sea lice, Caligus elongatus. Legend: 1 Nauplius I; 2 Nauplius II; 3 Copepodid; 4 Chalimus I; 5 Chalimus II; 6 Chalimus III; 7 Chalimus IV; 8 Pre-adult; 9 Adult (female); 10 Adult (male).

12

Source: A review and assessment of environmental risk of chemicals used for the

treatment of sea lice infestations of cultured salmon, Handbook of Environmental

Chemistry, vol. 5. Haya et al. (2005). Used with permission.

1.4.2. Sea lice concerns in aquaculture

Sea lice infestations were first reported in Norway in the early 1970s and have

since been reported essentially everywhere that salmon are commercially farmed (Roth,

1993; Torrisen et al., 2013). Although many of the concerns with sea lice relate to salmonid

species, other marine species can also be infected (Roth et al., 1993; Torrissen et al.,

2013). Concerns regarding direct losses to the aquaculture industry as a result of sea lice

infestations, the potential for sea lice transfer to native salmon populations, and the effect

of chemicals used to treat infestations, have been raised (Burridge and Van Geest, 2014;

Buschmann et al., 2006; Krkosek et al., 2005; Morton et al., 2011; Torrissen et al., 2013).

Additionally, sea lice transfers between farms are also cause for concern (Torrissen et al.,

2013). This issue becomes more serious in areas where the density of aquaculture sites

along coastlines is high. Global monetary losses due to sea lice infestation have been

estimated to be in the tens of millions of dollars (CDN) (DFO, 2014a). In Canada alone,

reported annual costs associated with sea lice infestations are over $15 million (CDN)

(Costello, 2009; Roth, 2000). In addition to the obvious cost of treatments, there are also

costs associated with reduced growth and losses resulting from a lower quality product as

a result of lice-induced skin damage (Mustafa et al., 2000).

Reviews by Costello (2006) and Torrissen et al. (2013) provide good summaries

of the effects on sea lice on both farmed and wild salmon. Sea lice grip their host with

specialized antennae and maxiliped after which they use their mouthparts to nourish

themselves through the removal and ingestion of mucus, skin and tissue. In fish, this

causes epithelium loss, bleeding, increased mucus discharge, altered biochemistry, tissue

necrosis and a loss of microbial protection. Additionally, host fish experience reduced

appetite, reduced growth and increased secondary infection rates. Finally, there is a risk

of bacterial or viral pathogen transfer via host to host transfer. Risk of bacterial or viral

pathogen transfer is magnified by the fact that sea lice can have a number of potential

host species. In the wild, host irritation may cause behaviours in fish that increase the risk

of predation by distraction, altered behaviours (e.g. leaping behaviours which can attract

predators), or energy losses (Costello, 2006).

13

The economics of commercial aquaculture, as well as concerns regarding negative

impacts to wild salmonid populations (i.e. transfer), necessitate the use of

chemotherapeutants, chemicals which are commonly employed to treat sea lice

infestations worldwide (Torrissen et al., 2013; Burridge et al., 2010; Burka et al; 1997;

Burridge and Van Geest 2014; Burridge et al., 2008; Grant, 2002; Haya et al., 2005; Roth

et al., 1993).

1.4.3. Sea lice treatments

In an attempt to minimize losses due to sea lice infestations, fish are often treated

with a wide range of available drugs, antibiotics and pesticides (Burridge et al., 2008;

Burridge et al. 2010; Burridge et al., 2011; Mustafa et al., 2000; Roth et al., 1993; Torrissen

et al., 2013). The use of these chemical controls is one of the major areas of concern with

respect to the environmental impacts of salmon aquaculture, especially in Canada

(Burridge et al., 2011; Burridge and Van Geest, 2014; Bushmann et al., 2006; Torrissen

et al., 2013). Several specific areas of concern include determining the magnitude of

effects to non-target organisms, assessing the potential bioaccumulation of substances

and investigating the cumulative effects resulting from concurrent or consecutive

treatments at adjacent farms (Burridge et al., 2011). These concerns have fueled a

number of research endeavours in Canada (many funded by DFO), many of which are

cited herein (e.g. Burridge et al. (2011), Lyons et al. (2014) and Bright and Dionne (2005)).

Historically, sea lice control has included a number of different treatments, most of

which have been shown to have negative impacts on the environment (Roth et al., 1993).

The first compound reported to be used to treat sea lice infestations on farms in Norway

was formaldehyde, however, its use was short lived due to its low margin of safety (Roth

et al., 1993). Subsequently, a number of compounds have been used, or are currently

used as a treatment (Burka et al., 1997; Burridge and Van Geest, 2014; Burridge et al.,

2008; 2010; 2011; DFO, 2003; 2013; Grant, 2002; Roth et al., 1993; Roth, 2000). Although

the clinically available treatments vary from country to country, 13 compounds have been

used to treat sea lice infestations worldwide: 4 organophosphates (dichlorvos, malathion,

trichlorfon and azamethiphos); 3 pyrethroids (pyrethrum, cypermethrin, and deltamethrin);

3 avermectins (ivermectin, emamectin benzoate and doramectin); hydrogen peroxide; and

2 benzoylphenyl ureas (teflubenzuron and diflubenzuron) (Figure 4) (Haya et al., 2005;

Roth, 2000).

14

Treatment options include topical, treatments applied to feed or treatments that

are administered directly to an organism (Burridge, 2013; Haya et al., 2005). Bath

treatments involve the direct application of the formulation to the cage at a particular

treatment concentration for a designated period of time. However, prior to application, the

depth of the cage is reduced and the cage is surrounded by a tarpaulin or skirt and upon

completion of treatment the tarp is removed and the formulation is left to disperse into the

surrounding water. Alternately, fish can be treated in well-boats, which often results in

reduced chemical release but can result in higher levels of stress in treated fish (Burridge

and Van Geest, 2014). If applied efficiently, the use of medicated feed can have less of

an environmental impact and be less stressful for the fish, however diseased fish may

consume less food thus resulting in insufficient treatment (Burridge et al., 2011; Haya et

al., 2005).

15

A

B

C

D

E

F

G

H

I

J

K

L

M

Figure 4: Chemical structures of the compounds that have been used to treat sea lice infestations worldwide: A) dichlorvos B) malathion C) trichlorfon D) azamethiphos E) pyrethrum F) cypermethrin G) deltamethrin H) ivermectin I) emamectin benzoate J) doramectin K) hydrogen peroxide L) teflubenzuron and M) diflubenzuron.

In addition to chemical use, there are a number different methods to address the

issue of sea lice infestations in salmon aquaculture. These include: monitoring, biological

control, immunostimulation, mechanical de-lousing, vaccination, selective breeding, and

regulatory approaches (MacKinnon, 1995; Torrissen et al., 2013; Webb et al., 2013).

Examples of current and potential biological controls include the use of cleaner fish, filter-

16

feeding shellfish, ciliates, Bacillus thuringiensis, or flatworms (Deady et al., 1995; Chopin

et al., 2012; MacKinnon, 1995; Skiftesvik et al., 2013; Torrinsen et al., 2013; Treasurer,

2002; Webb et al, 2013). Additionally, examples of regulatory approaches might include

ensuring that zones have synchronized production or designating minimum fallowing

periods. Despite the emergence of numerous alternatives and improved management

practices, it should be noted that there is still a heavy reliance on the use of

chemotherapeutants in salmon aquaculture (Burridge et al., 2011).

Depending on the availability of different treatment options, there are also a

number of other factors which may affect treatment choice. These include cost, weather,

resistance, and withdrawal period, among others (Grant, 2002).

1.4.4. Sea lice treatment in Canada

Chemotherapeutants used in salmon aquaculture for sea lice treatment are

classified as being a pesticide or a drug based on the mode of application (Burridge et al.,

20011; Burridge and Van Geest, 2014; Haya et al., 2005). In all cases, the formulations

are applied based on the concentration of the active ingredient (AI). In Canada, all use of

chemotherapeutants to treat sea lice is tightly regulated by Health Canada, whose

mandate is to promote and maintain the health of Canadians (Burridge, 2003; Burridge et

al., 2011; Burridge, 2013; Van Geest et al. 2014b). Drug approval occurs under the Food

and Drugs Act (FDA) by the Veterinary Drugs Directorate (VDD) whereas pesticide

approval occurs under the Pest Control Products Act (PCPA) by the Pest Management

Regulatory Agency (PMRA). In both cases, withdrawal times are applied so as to ensure

safe levels for human consumption. Additionally, an important factor to consider when

selecting any pesticide is the margin of safety of the product in question. As mentioned

previously, DFO has the lead federal role in managing fisheries and safeguarding its

waters in Canada. Unregulated use is not taken lightly; for example, in 2013, a company

pled guilty to the illegal use of a pesticide (AI cypermethrin) in southwestern New

Brunswick, and was fined $500,000 (CDN) (ECCC, 2013).

A number of formulations have been used, or continue to be used in Canada. This

includes the off-label use of ivermectin (Burridge, 2003) and the emergency use of

Calicide® (AI teflubenzuron) (Burridge et al., 2011; Haya et al., 2005). Slice® (AI

emamectin benzoate), Salmosan® (AI azamethiphos), Alphamax® (AI deltamethrin),

17

Excis® (AI cypermethrin), and Interox® Paramove 50 (AI hydrogen peroxide) are other

examples of products that have been used, or continue to be used in Canada, mostly

under emergency approvals (Burridge, 2003; Burridge et al., 2014; PMRA, 2016). Table

1 provides a summary of the margin of safety for each of these substances. Notably, some

substances (e.g. hydrogen peroxide and deltamethrin) have very low margins of safety

whereas others have very high margins of safety (e.g. cypermethrin). More specifics

regarding the use of the latter in Canada will be outlined in the following sections.

Table 1 : Comparison of the effective versus lethal dose of sea louse treatment substances used in salmon aquaculture (Bright and Dionne, 2005; Roth, 2000).

Substance Application Therapeutic Dose

(g L -1)

Toxic Dose to Atlantic Salmon (Salmo salar)

(g L -1)

Margin of Safety

Azamethiphos Topical (bath) 100 > 500 > 5x Hydrogen Peroxide Topical (bath) 1,500,000 1,500,000 –

4,000,000 0 – 3x

Cypermethrin Topical (bath) 5 > 500 > 100x Deltamethrin Topical (bath) 3 3 - > 10 0 – 3.5x

Emamectin Benzoate Oral (feed) 50 g kg-1 for 7 d 360 g kg-1 for 7 d 7x

1.5. Emamectin benzoate

Emamectin benzoate (EB) is a two component mixture consisting of a minimum of

90 % MAB1a and 10 % MAB1b (US EPA, 2009; Bright and Dionne, 2005) (Figure 5). It is

a broad-spectrum pesticide from the avermectin class used to control a wide variety of

pest infestations (Lumaret et al., 2012; Reddy, 2012). In general, EB is not very water-

soluble (Log KOW = 5; solubility = 5,500 g L-1 in seawater), unlikely to volatilize, unlikely

to bioaccumulate due to its large molecular size, and sorbs readily to organic matter (Bright

and Dionne, 2005; Lumaret et al., 2012; Reddy, 2012). Additional information on the

physical and chemical properties of EB, as MAB1a, are summarized in Appendix A.

18

Figure 5: Chemical structure of emamectin benzoate, where when R=methanol (CH3) it is MAB1b and when R=ethanol (CH2CH3) is it is MAB1a.

1.5.1. Canadian usage

Slice® (AI EB), is one of several chemotherapeutants used to treat salmon for sea

lice and the only treatment currently used on the west coast of Canada (Bright and Dionne,

2005; DFO, 2012; Ikonomou, 2011). On the east coast of Canada, and elsewhere, reports

of resistance among sea lice populations to EB has resulted in the use of other, more

effective, pesticides (Aaen et al., 2015; Burridge and Van Geest, 2014). The Slice®

formulation contains 0.2 % EB (Bright and Dionne, 2005) and has been used under an

emergency drug release in Canada since 1999 until getting full approval from the VVD in

2009 (Ikonomou, 2011). The optimal prescribed dose is 50 μg kg-1 day-1 applied to feed

for 7 consecutive days, as confirmed by laboratory studies involving L. salmonis and S.

salar L (Stone et al., 1999).

1.5.2. Chemotherapeutant efficacy

Laboratory and field experiments in Scotland have indicated that Slice® provides

effective treatment against adult, pre-adult and larval stages of L. salmonis and C.

elongatus (Stone et al., 2000b). However, Bravo et al. (2015) also found that the use of a

number of sea lice pesticides, including EB, can result in decreased egg survival among

C. rogercresseyi. Furthermore, a number of field and laboratory studies have investigated

numerous aspects of the drug’s efficacy or safety (e.g. Saksida et al. [2010], Stone et al.

[2000a; 2000b; 2002]). In general, these studies support that the drug is effective at

reducing sea lice burdens on infected populations and at reducing future infestations (for

upwards of 60 days in some cases). However, there have also been numerous reports of

reduced efficacy of the drug worldwide (e.g. Chile [Bravo et al., 2008], Scotland [Lees et

19

al., 2008], and Norway [Espedal et al., 2013]), even on the east coast of Canada (Park,

2013; Saksida et al., 2010). Saksida et al. (2010) noted some of the potential reasons why

resistance has not been observed on the west coast of Canada, including the presence of

large numbers of wild salmon and genetic differences within sea lice species, especially

when compared with the east coast.

1.5.3. Environmental fate of EB

In the vicinity of salmon farms, EB can enter the environment via uneaten food

pellets, through faecal matter and/or urine as either parent EB or its metabolites (Bright

and Dionne, 2005; DFO, 2012). Non-target organisms can be exposed to EB in water, the

sediment, or through ingestion of treated feed or faecal matter. EB has been detected in

sediments up to 1.5 years after treatment within a 150 m radius of farm sites and in the

water column at farm sites several days following treatment (Ikonomou, 2011).

Furthermore, several reports have also confirmed the presence of detectable levels of EB

(and its metabolites) in marine organisms in areas where Slice® is used (SEPA, 1999;

DFO, 2012; Ikonomou, 2011) confirming its bioavailability in marine organisms. Laboratory

tests in blue gill sunfish (Chukwudebe et al., 1996) demonstrated the ability for EB to

bioconcentrate but the authors though it unlikely that the substance would bioaccumulate

to any degree. It is possible to attribute EB’s potential inability to bioaccumulate to its high

molecular weight, the size of the molecule, and its polarity.

1.5.4. EB mechanism of action and effects on non-target species

The precise mechanism of action of EB is not fully understood. In invertebrates,

avermectins are thought to interfere with GABA- and glutamate-gated chloride channel

receptors in nerve and muscle cells to stimulate the influx of chloride ions (Burridge et al.

2010; Lumaret et al., 2012; McKellar & Benchaoui, 1996), resulting in paralysis and death

(Reddy, 2012; Lumaret et al., 2012; McKellar & Benchaoui, 1996). Endocrine disruption,

as a secondary mechanism of action, has also been speculated following molting induction

in lobsters (Waddy et al., 2002). EB is not mutagenic. The toxic effect of the substance is

observed at lower concentrations in invertebrates than in vertebrates, thus resulting in

relative selectivity (McKellar & Benchaoui, 1996). The specific glutamate-gated chloride

channels thought to be the target of avermectins have not been reported in mammals in

high numbers (McKellar & Benchaoui, 1996). A recent review by Lumaret et al. (2012) has

20

summarized the toxicological data in non-target organisms in both terrestrial and aquatic

environments. Additionally, Park (2013) provides a summary of the effects of EB to non-

target marine and freshwater invertebrates and fish species. Table 2 provides a summary

of information available in the literature. One noteworthy observation made by Park (2013)

is that a large portion of the body of literature on this topic is only located in confidential

reports and, as such, is not readily accessible.

In addition to safety testing in salmon, vertebrate toxicity has been investigated in

a number of species and species-specific differences in sensitivity have been observed.

Invertebrate sensitivity varies greatly, with crustaceans being the most sensitive of the

groups tested (Burridge et al., 2004; Lumaret et al., 2012). However, sensitivity among

crustaceans varies as well (Lumaret et al., 2012; Willis and Ling, 2003). For water

exposures, the most sensitive crustaceans appears to be M. bahia (mysid shrimp) with a

reported 96 h LC50 of 0.04 g EB L-1 (Lumaret et al., 2012), with most other values reported

for other species being orders of magnitude greater. Examples include C. crangon (bay

shrimp) with a reported 96 h LC50 of 242 g EB L-1 and C. volutator (mud shrimp) with a

reported 10 d LC50 of 6.32 g EB L-1 (Lumaret et al., 2012). Additionally, 7 d LC50 for H.

americanus (American lobster) of 644 g EB g-1 in feed has been reported. Although the

toxicity to lobsters is higher than many other values reported in the literature, a significant

amount of research has focused on this economically valuable species as EB has been

shown to induce molting (Waddy et al., 2002; 2007). C. virginica (eastern oyster), M.

galloprovincialus (Mediterranean mussel), and C. capitata (polychaete worm), are

examples of species with lower sensitivity to EB as compared to crustaceans (refer to

Table 2) (Lumaret et al., 2012; Park, 2013).

Tests involving micro-algae (Lemna gibba) exposed to 94 g EB L-1 for 14 days

and (Selenastrum capricornutum) exposed to 3.9 g EB L-1 demonstrated no effect

(Lumaret et al., 2012). Additionally, bacteria (V. fischeri) exposed to 5,000 g EB L-1

resulted in no effects on bioluminescence (Hernando et al., 2007). These results

demonstrate that some species have low sensitivity to EB.

21

Table 2: Data from emamectin benzoate acute and chronic exposures to marine and freshwater invertebrates and fish species.

Test Organism Endpoint Effect Measurement Notes Duration Concentration of EB Source

INVERTEBRATES

Homarus americanus

(American lobster) LOEL / NOEL Molting Feed (single dose) Chronic

effects 0.22 / 0.12 g a.i. g-1

lobster

Waddy et al. (2007)

Homarus americanus

(American lobster) EC44* Premature molting Feed (single dose) Molting

effects 1 g a.i. g-1 lobster Waddy et al. (2002)

Homarus americanus

(American lobster) LC50 Mortality Feed;

Juvenile 7 d >589 g g-1 Burridge et al. (2004)

LC50 Mortality Feed; Adult

7 d 644 g g-1

Nephrops

norvegicus (Dublin Bay prawn)

LC50 / NOEC Mortality Sea water 96 h 983 / 814 g L-1 McHenery & Mackie (1999), Schering-Plough Anim. Health (2002) – in Lumaret et al. (2012)

LC50 / NOEC Mortality Sea water 192 h 572 / 440 g L-1 LC50 / NOEC Mortality Feed 96 h > 0.0682 / 0.0682 g L-1 LC50 / NOEC Mortality Feed 192 h > 0.0682 / 0.0682 g L-1

Crangon crangon

(Bay shrimp) LC50 / NOEC Mortality Sea water 96 h 242 / 161 g L-1 McHenery & Mackie (1999), Schering-

Plough Anim. Health (2002) – in Lumaret et al. (2012)

LC50 / NOEC Mortality Sea water 192 h 161 / < 161 g L-1 Crangon crangon

(Bay shrimp) LC50 / NOEC Mortality Feed 96 h >0.0693 / 0.0693 g L-1 LC50 Mortality Feed 192 h >0.0693 / 0.0693 g L-1 LOEC/NOEC Egg production Adult 7 d 0.16 / 0.05 g L-1

Artemia salina

(Brine shrimp) IC50 Immobilization Sea water 6 h 1.73 g L-1 McHenery & Mackie (1999), Schering-


Mysidopsis bahia

(Mysid shrimp) LC50 / NOEC Mortality Sea water 96 h 0.04 / 0.02 g L-1

McHenery & Mackie (1999), Schering-Plough Anim. Health (2002)– in Lumaret et al. (2012)

Americamysis bahia (Mysid shrimp)

NOEC Growth Sea water 28 d 0.0087 g L-1 Blankinship et al. 2002b – in Park (2013)

Pseudocalanus

elongatus (Copepod) EC50 Immobilization Nauplii (N6) 48 h 0.12 g L-1 Willis & Ling (2003)

EC50 Immobilization Copepodites (C1) 48 / 96 h 0.14 / 0.17 g L-1


22


Temora longicornis

(Copepod) EC50 Immobilization Nauplii (N6) 48 h 0.23 g L-1 Willis & Ling (2003)

EC50 Immobilization Copepodites (C1) 48 h 0.41 gL-1 EC50 Immobilization Copepodites (C6) 48 h 2.8 g L-1

Oithona similis

(Copepod) EC50 Immobilization Nauplii (N6) 48 / 96 h >15.8 g L-1 Willis & Ling (2003)


EC50 Immobilization Copepodites (C6) 48 / 96 h 232 / 113 g L-1

Acartia clausi

(Copepod) EC50 Immobilization Nauplii (N6) 48 / 96 h 0.57 / 0.48 g L-1 Willis & Ling (2003)


EC50 Immobilization Copepodites (C6) 48 / 96 h 0.29 / 5.27 g L-1 Corophium volutator (mud shrimp)

LC50 / NOEC Mortality Sea water 10 d 6.32 / 3.2 g L-1 McHenery & Mackie (1999), Schering-Plough Anim. Health (2002) – in Lumaret et al. (2012)

LC50 / NOEC Mortality Sediment 10 d 193 / 115 g kg-1

Corophium volutator (mud shrimp)

LC50 Mortality Sediment 10 d 153 g kg-1 wet sediment

Mayor et al. (2008)

Daphnia magna

(Water flea) LC50 / NOEC Mortality Sea water 48 h 1.0 / 0.3 g L-1 McHenery & Mackie (1999), Schering-


EC50 / LOEC Reproduction Sea water 21 d 0.16 / 0.09 g L-1

LC50 Mortality Feed 21 d 0.13 g kg-1

Eohaustorius

estuarius

(Amphipod)


Kuo et al. (2010)

Monocorophium

insidiosum

(Amphipod)

LC50 Mortality Sediment 10 d 890 g kg-1 Tucca et al. (2014)

LOEC* Biochemical

response

(GST act./TBARS)

Sediment 10 d 100 / 50 g kg-1

Mytilus

galloprovincialus

(Mediterranean

mussel)

EC50 Development Sea water 48 h 314 g L-1 Aufderheide (2002) – in Park (2013)

LC50 Mortality Sea water 48 h > 713 g L-1

Crassostrea

virginica

(Eastern oyster)

EC50 / NOEC

Shell deposition

Sea water 96 h 530 / 260 g L-1 Zelinka et al. (1994a) – in Park (2013)

LC50 / NOEC Mortality Sea water 96 h 665 / 260 g L-1

23

*interpreted from data


Hediste diversicolor

(Rag worm) LC50 Mortality Sediment 10 d 1368 g kg-1 wet

sediment

Mayor et al. (2008)

Capitella capitata

(Polychaete worm) LC50 / NOEC Mortality Sea water 21 d 1,040 / 460 g L-1 McHenery & Mackie (1999), Schering-


Arenicola marina

(Lugworm) LC50 / NOEC Mortality Sediment 10 d 111 / 56.0 g kg-1 wet

sediment

McHenery & Mackie (1999), Schering-Plough Anim. Health (2002) – in Lumaret et al. (2012)

VERTEBRA TES

Oncorhynchus

mykiss (Rainbow trout) LC50 / NOEC Mortality Fresh water

96 h 174 / 48.7 g L-1 McHenery & Mackie (1999), Schering-


Lepomis

macrochirus (Bluegill sunfish)

LC50 / NOEC Mortality Fresh water

96 h 180 / 87 g L-1 Chukwudebe et al. (1996) – in Lumaret et

al. (2012)

Salmo salar

(Atlantic Salmon)

LOEC* Mortality Sea water;

Feed 7 d 0.00036 g kg-1 Bright and Dionne (2005)

Salmo salar

(Atlantic Salmon)

NOEC* Behavior, weight,

histology

Sea water;

Feed 7 d 54 g kg-1 Stone et al. (2002)

Salmo salar

(Atlantic Salmon)

LC50 / NOEC Mortality Sea water;

Feed 7 d 356 / 170 g kg-1 McHenery & Mackie (1999) - in Lumaret

et al. (2012) Pimephales promelas

(Fathead minnow) LC50 / NOEC Mortality Fresh water 96 h 194 / 156 g L-1

McHenery & Mackie (1999), Schering-Plough Anim. Health (2002) – in Lumaret et al. (2012) LC50 /

NOEC MATC (Maximum acceptable toxicant concentration)

Fresh water;

Early life stages 96 h 18 /12 g L-1

LOEC Fresh water;

Early life stages 96 h 28 g L-1

Cyprinodon

variegatus

(Sheepshead minnow)

LC50 / NOEC Mortality Fresh water 96 h 1,340 / 860 g L-1 McHenery & Mackie (1999);, Schering-Plough Anim. Health (2002) – in Lumaret et al. (2012)

Cyprinus carpio

(Common carp)

LC50 Mortality Fresh water 96 h 260 – 444 g L-1 Wallace (2001b) in Park (2013)

24

1.6. Hydrogen peroxide

Hydrogen Peroxide (HP) (chemical structure shown in Figure 6) is the simplest

peroxide compound. It is used in a wide variety of commercial and industrial applications

(e.g. disinfectant, detergents, dyes, and as a reactive intermediate), including aquaculture

where it is used as an anti-fungal (Gaikowski et al., 1999) and anti-parasitic treatment

(Burridge and Van Geest, 2014; Montgomery-Brock et al., 2001). Details on relevant

physical and chemical properties of HP can be found in Appendix A.

Figure 6: Chemical structure of hydrogen peroxide.


Interox® Paramove 50 (AI HP), is one of several chemotherapeutant formulations

used to treat salmon for sea lice worldwide (Burridge, 2013; Burridge and Van Geest,

2014; Grant, 2002; Haya et al., 2005). In May 2016, the PMRA of Canada granted full

registration (sale and use) to Interox® Paramove 50, for the treatment of sea lice

infestations on Atlantic salmon in aquaculture (PMRA, 2016). It can now be applied in

Canada as a bath treatment at 1,500,000 g L-1 for 20-30 min (PMRA, 2014), which is in-

line with the recommended dosage applied elsewhere: 1,200,000-1,800,000 g L-1 for 30

min (Burridge, 2013; Burridge and Van Geest, 2014; Grant, 2002). However, caution with

this treatment is advised as temperature-dependent toxicity has been observed (Bruno

and Raynard, 1994; Gaikowski et al., 1999; Johnson et al., 1993; Kiemer and Black, 1997).


Field and laboratory experiments with L. salmonis and C. elongatus suggest that

HP provides effective treatment for adult and potentially pre-adult stages of sea lice

(Treasurer and Grant, 1997). Notably, HP only effectively immobilizes the sea lice and as

such they could potentially attach to new hosts (Bravo et al., 2010), although this has not

25

been observed by all (Treasurer and Grant, 1997). Additionally, Bravo et al. (2015) found

that the use of a number of sea lice pesticides, including HP, can result in decreased egg

survival among C. rogercresseyi, in Chile. Some seasonal variation in effectiveness has

been reported by one Canadian study (Gautam et al., 2016). Furthermore, reduced

efficacy of HP has been observed in Scotland following heavy reliance for sea lice

control/treatment (Treasurer et al., 2000).

1.6.3. HP environmental fate

HP enters the aquatic environment through direct application to salmon

aquaculture farms where it is applied to treat sea lice or to bodies of water to treat algal

blooms. Notably, it is also naturally occurring in the marine environment (Hopwood et al.,

2017; Petasne and Zika, 1997; Yocis et al., 2000; Yuan and Shiller, 2004). In short, it has

a limited half-life in water (PMRA, 2014; Haya, 2005; Bruno and Raynard, 1994) and its

degradation products are water and oxygen (Burridge et al., 2008; Haya, 2005; PMRA,

2014). Furthermore, it has a low log Kow value (~0: highly water soluble) and as such it

does not readily bind to organic matter and is not expected to accumulate in sediment or

to bioaccumulate (PMRA, 2014; US EPA, 2007). As such, HP is not generally considered

an environmental concern.

1.6.4. HP mechanism of action and effects on non-target species

In addition to its natural occurrence in the marine environment, HP is naturally

occurring in biota where it is produced in cells via multiple pathways (Boveris et al., 1972;

Geiszt and Leto, 2004). HP serves several important chemical messenger roles in cells

(Boveris and Cadenas 2000; Rhee et al., 2003; Veal et al., 2007) (e.g. in apoptosis and

necrosis [Saito et al., 2006] and others [Gough and Cotter, 2011]). Numerous reviews

have explored both the natural role of HP in biota (Cabiscol et al., 2000; Geiszt and Leto,

2004; Gouch and Cotter 2011; Liou and Storz 2010) as well as its toxicity (Gouch and

Cotter, 2011; Veal et al., 2007; Valavanidis et al., 2006) in greater detail. The mechanism

of HP toxicity in cells is non-specific and not fully understood. As with other reactive

oxygen species (ROS), high concentrations have been attributed to cell damage (Cabiscol

et al., 2000), cell death (Saito et al., 2006) and carcinogenesis (Liou and Storz, 2010). In

sea lice control, HP is believed to involve paralysis, reduced egg string viability and

reduced ability to reattach following treatment (PMRA, 2014). HP has also been identified

26

as a weak mutagen (Kensese and Smith, 1989). In-vitro investigations provide some

support to the mechanical paralysis MOA with the formation of gas bubbles in the

haemolymph of sea lice treated with HP, immobilizing them and thus causing them to

detach and float to the surface (Burka et al., 1997; Bruno and Raynard, 1994; Grant 2002).

However, a variety of other MOAs have also been proposed, including: peroxidation of

lipid and cellular organelle membranes by hydroxyl radicals, and the inactivation of

enzymes and DNA replication (Burridge, 2013). Relatively few studies have reported

toxicity of HP to marine organisms (PMRA, 2014; Haya 2005); those that have focus

primarily on crustaceans or salmonids due to their economic importance (Burridge, 2013;

Burridge et al., 2014b; Van Geest et al., 2014a; Taylor and Glenn, 2008). Table 3 provides

a summary of toxicity information for HP available in the literature for aquatic invertebrates

and fish species.

HP has been shown to cause gill damage, decreased growth rate and mortality in

salmon (Johnson et al., 1993; Kiemer and Black, 1997) with a low margin of safety for its

use as a pesticide (Roth et al., 1993). One study demonstrated size and species

differences, even reporting LD50 values for juvenile salmonids at concentrations below the

recommended treatment levels (Taylor and Glenn, 2008). Gill damage and decreased

growth rate have been reported in rainbow trout as well, with effects lasting weeks post-

treatment (Carvajal et al., 2000). Additionally, the toxicity of HP to salmonids has been

shown to increase with temperature (Bruno and Raynard, 1994; Gaikowski et al., 1999;

Johnson et al., 1993; Kiemer and Black, 1997).

The results of a risk assessment by PMRA (2014) suggested that the use of HP

as a pesticide in salmon aquaculture poses a negligible risk to the environment, with the

exception of marine algae to which HP is highly toxic (Barroin and Feuillade, 1986).

However, effects have been reported in lobsters, shrimp, oligochaetes, and copepods at

or below treatment levels (Burridge et al., 2014b; Mischke et al., 2001; Van Geest et al.,

2014a), following short-term exposures.

27

Table 3: Data from hydrogen peroxide acute and chronic exposures to marine and freshwater invertebrates and fish species.

Test Organism Endpoint Effect Measurement Notes Duration Concentration of HP Source

INVERTEBRATES

Dero digitata

(Oligochaete Worm) LC50 Mortality Pond water 24 / 48 h 4,360 g L-1 Mischke et al. (2001)

Zooplankton spp.

(Copepods) LC50 Mortality Sea water 1 h + 5 h** 68,000 g L-1 Van Geest et al. (2014)

EC50 Feeding rate Sea water 1 h + 5 h** 2,600 – 10,000 g L-1 Homarus americanus

(American lobster) LC50 Mortality Sea water;

Stage 1 1 h + 95 h**

1,637,000 g L-1 Burridge et al. (2014b)

LC50 Mortality Sea water;

Adult 1 h + 95 h**

>3,750,000 g L-1

C. septemspinosa

(Sand shrimp) LC50 Mortality Sea water 1 h + 95

h** 3,182,000 g L-1 Burridge et al. (2014b)

Praunus flexuosus

Mysis stenolepsis

(Mysid spp.)

LC50 Mortality Sea water 1 h + 95 h**

973,000 g L-1 Burridge et al. (2014b)

VERTEBRATES

Salmo salar

(Atlantic Salmon) LOEC* Mortality Sea water;

Temp.

dependence;

100 % mortality

20 m 52,360 g L-1 HP Bruno and Raynard (1994)

LOEC* Mortality 2 h 13,530 g L-1 HP

Ctenolabrus

rupestris

(Goldskinny wrasse)

NOEC* Mortality Sea water 2 h 13,860 g L-1 HP

Oncorhynchus

kisutch

(Coho Salmon)

LD50 Mortality Fresh water;

1 h + 120 h**

test

96 h 231,000 g L-1 (small)

225,000 g L-1 (large)

Taylor and Glenn (2008)

Oncorhynchus

tshawytscha

(Chinook Salmon)


1 h + 120 h**

test

96 h 200,000 g L-1 (small)

106,000 g L-1 (large)

Oncorhynchus

mykiss

(Rainbow trout)


1 h + 120 h**

test

96 h 373,000 g L-1 (small)

196,000 g L-1 (large)

28

*interpreted from data **time in clean water (following exposure)

Test Organism Endpoint Effect Measurement Notes Duration Concentration of HP Source

Oncorhynchus

mykiss

(Rainbow trout)

LOEC* Growth rate

reduction Fresh water;

Single

concentration

20 m + 5 w**

1,250,000 g L-1 Carvajal et al. (2000)

Salmo salar

(Atlantic Salmon) LOEC* Mortality;

Gill histology Sea water;

Single

concentration;

Temp.

dependence

20 / 40 m 1,500,000 g L-1 Johnson et al. (1993)

Oncorhynchus

tshawytscha

(Chinook Salmon)

LOEC* Mortality;

Gill histology Sea water;

Single

concentration;

Temp.

dependence

20 / 40 m 1,500,000 g L-1 Johnson et al. (1993)

Salmo salar

(Atlantic Salmon) LC100* Mortality Sea water 20 m 2,580,000 g L-1 Kiemer and Black (1997)

NOEC* / LOEC*

Gill damage Sea water 20 m 1,370,000 / 2,520,000

g L-1

INVERTEBRATES

Dero digitata

(Oligochaete Worm) LC50 Mortality Pond water 24 / 48 h 4,360 g L-1 Mischke et al. (2001)

Zooplankton spp.

(Copepods) LC50 Mortality Sea water 1 h + 5 h** 68,000 g L-1 Van Geest et al. (2014)

EC50 Feeding rate Sea water 1 h + 5 h** 2,600 – 10,000 g L-1 Homarus americanus

(American lobster) LC50 Mortality Sea water;

Stage 1 1 h + 95 h**

1,637,000 g L-1 Burridge et al. (2014b)


Adult 1 h + 95 h**

>3,750,000 g L-1

C. septemspinosa

(Sand shrimp) LC50 Mortality Sea water 1 h + 95

h** 3,182,000 g L-1 Burridge et al. (2014b)

Praunus flexuosus

Mysis stenolepsis

(Mysid spp.)


973,000 g L-1 Burridge et al. (2014b)

29

1.7. Pyrethroids

Pyrethroids, or synthetic pyrethrins, are a class of broad-spectrum pesticides used in a

wide variety of applications (CCME, 1999; Costa et al., 2008; Haya, 1989, PMRA, 2015) due to

their high degradability, low mammalian toxicity and high toxicity to arthropods (Burridge et al.,

2008). The latter makes them an ideal candidate for use as pesticides to treat sea lice. Two

pyrethroids are used as the active ingredient in commercially available sea lice pesticides,

cypermethrin (CP) and deltamethrin (DM), whose chemical structures are presented in Figure 7.

A

B

Figure 7: Chemical structure of A) cypermethrin and B) deltamethrin.

1.7.1 Canadian usage

These two pyrethroids, deltamethrin (DM) and cypermethrin (CP), have been used in

Canada to treat sea lice infestations. AlphaMax® (AI DM) and Excis® (AI CP) are commercially

available sea lice pesticide formulations, both containing 1 % AI (Van Geest et al., 2014).

Recommended treatment with AlphaMax® is for 40 min at 2,000-3,000 g DM L-1 (Burridge and

Van Geest, 2014; Haya et al., 2005) and recommended treatment with Excis® is for 1 hour at

5,000 g CP L-1 (Burridge and Van Geest, 2014; Haya et al., 2005; Grant, 2002). In Canada,

Alphamax® was used under emergency registration in 2009/2010 (Burridge et al., 2014; DFO

2010; NBDA, 2011). Excis®, however, was only used under a research permit in 1995 (Burridge

et al., 2014).

1.7.2 Chemotherapeutant efficacy

Studies have found that CP (Hart et al., 1997; Jiminez et al., 2013) and DM (Bravo et al.,

2014) both provide effective treatment for adult and pre-adult stages of sea lice (family Caliigidae)

but are less effective against the chalimus stages. However, Whyte et al. (2014) did see some

30

inconsistent results in Canadian field and laboratory experiments, where the efficacy of DM

against adult and pre-adult L. salmonis was variable, especially with respect to differences

between males and females. However, Bravo et al. (2015) found that the use of a number of sea

lice pesticides, including DM, can result in decreased egg survival among C. rogercresseyi.

Numerous accounts of reduced efficacy, decreased sensitivity, or failed treatments have been

documented around the world (e.g. Sevatdal and Horsberg [2003]). Examples of countries where

decreased sea lice sensitivity to CP and DM has been confirmed with in vitro bioassays include

include Norway, Ireland and Scotland (Sevatdal et al., 2005), and Chile (Helgesen et al., 2014).

1.7.3 Pyrethroid fate in the environment

Pyrethroids can hydrolyze in water (Burka et al., 1997; Roth et al., 1993) and have

relatively short half-lives in aqueous solution, however, they may persist in sediments (Burridge

and Haya 1997; Haya et al., 2005; Muir et al., 1985). Their tendency to sorb to sediments (with a

log KOW value of ~ 5) (Ernst et al., 2014; Maund et al., 2002; PMRA, 2015) may alter the apparent

toxicity of the chemicals or result in chronic exposure to lower concentrations (Muir et al., 1985;

Clark et al., 1989). They also decompose readily due to their susceptibility to catabolic enzymes

and sunlight (Burridge and Van Geest, 2014; Roth et al., 1993). For example, in laboratory

experiments an average of 70 % loss of DM was observed over 48 h (Van Geest et al., 2014b).

Despite their rapid rate of metabolism (Alonso et al., 2012; Haya 1989; Muir et al., 1985; PMRA,

2015), some evidence of pyrethroid bioaccumulation has been documented (Alonso et al., 2012;

Van Geest et al., 2014d; Van Geest et al., 2014b). Additional details regarding the physical and

chemical properties of CP and DM can be found in Appendix A.

1.7.4 Pyrethroid mechanism of action

The most likely MOA of pyrethroids involves interference with nerve membrane function,

primarily by their interaction with sodium (Na+) channels (Soderlund and Bloomquist, 1989), which

results in depolarization of nerves. The repetitive firing of nerves resulting from Na+ channels

remaining open ultimately leads to paralysis and death (Burka et al., 1997; Crane et al., 2011;

Haya et al., 2005). Other, less likely, MOAs that have been proposed include blocked neuronal

conduction (PMRA, 2015; Sonderlund and Bloomquist, 1989) and GABA- and glutamate-

controlled channel effects (Burka et al., 1997; Soderlund and Bloomquist, 1989). CP and DM are

not mutagenic.

31

Pyrethroids are well known for their toxicity towards arthropods (Burridge et al, 2014; Haya

et al., 2005). Additionally, crustaceans have demonstrated high sensitivity to both CP and DM

(Clark et al., 1989; Haya et al., 2005; Fairchild et al., 2010; etc.). Thus, the majority of research in

this area has focused on their toxicity to economically valuable crustacean species (Burridge et

al., 2000b; Burridge et al., 2000a; Burridge et al., 2014b). Research indicates that DM is more

toxic than CP (Burridge and Van Geest, 2014; Fairchild et al., 2010; Haya, 1989; Tucca et al.,

2014; Van Geest et al., 2014a). Additional information regarding the toxicity of these two

compounds is discussed briefly in the following subsections.

1.7.5 CP effects on non-target species

Knowledge regarding the toxicity of CP to marine species is limited (Burridge et al., 2008).

Several studies have demonstrated toxicity to a variety of non-target marine species following

short-term exposure, including inhibitory effects in marine mussels (Ayad et al., 2011) and

delayed toxicity in copepods (Medina et al., 2004). Importantly, toxicity to marine organisms (e.g.

copepods, lobsters, and amphipods) has been reported at concentrations below the

recommended treatment levels following short-term exposure (e.g. Burridge et al. [2000a],

Medina et al., [2002], Willis and Ling [2004], and Van Geest et al. [2014c]). However, Burridge et

al. (2000b) concluded that a single sea lice treatment is not likely to result in lobster mortality.

A review by Clark et al. (1989) found that crustaceans to have greater sensitivity to CP

than fish and molluscs (Clark et al., 1989). The high sensitivity of benthic crustaceans and

amphipods has also been observed in whole sediment bioassays (Mayor et al., 2008; Tucca et

al., 2014). Additionally, the results of Ernst et al. (2001) demonstrated toxicity to non-target

organisms in laboratory testing using water collected from within a net pen during CP treatment

and in the vicinity of salmon aquaculture sites (up to 5 h post-release). Specifically, although the

results indicated significant short-duration toxicity with samples taken during treatment, longer

exposures resulted in toxicity for samples taken from the vicinity after release and delayed effects

were also observed (i.e. observations following transfer to clean sea water). Overall, the data

indicates that there is a potential for CP to cause adverse effects to non-target species over a

large area following treatment. More information on the toxicity of CP to non-target species is

provided in Table 4.

32

Table 4: Data from cypermethrin acute and chronic exposures to fresh water and marine invertebrates and fish species.

Test Organism Endpoint Effect Measurement Notes Duration Concentration of CP Source

INVERTEBRATES

Palaemonetes pugio

(Grass shrimp)

LC50 Mortality Sea water; Flow through

96 h 0.016 g L-1 Clark et al. (1987)

LC50 Mortality Sediment- static 96 h 175 g kg-1 LC50 Mortality Sediment flow

through 96 h 270 g kg-1

Mysidopsis bahia

(Mysid shrimp)

LC50 Mortality 96 h 0.056 g L-1 Unpublished data - in Clark et al. (1989)

Mysidopsis bahia

(Mysid shrimp)

LC50 Mortality 96 h 0.005 g L-1 Clark et al. (1989)

Panaeus duorarum

(Pink shrimp)

LC50 Mortality 96 h 0.036 g L-1 Clark et al. (1989)

Uca pugilator

(Atlantic sand

fiddler)

LC50 Mortality 96 h 0.2 g L-1 Hill (1985) - in Clark et al. (1989)

Cragnon

septemspinosa

(Sand shrimp)

LC50 Mortality Static toxicity

test

96 h 0.01 g L-1 Mcleese et al. (1980) - in Clark et al. (1989)

Homarus americanus

(American lobster)


test


Homarus americanus

(American lobster)

LC50 Mortality Stage 1 48 h 0.18 g L-1 Burridge et al. (2000b)

LC50 Mortality Stage 2 48 h 0.12 g L-1 LC50 Mortality Stage 3 48 h 0.06 g L-1 LC50 Mortality Stage 4 48 h 0.12 g L-1 LC50 Mortality Adult 24 / 48 h 0.14 / 0.081 g L-1

LC50 Mortality Adult 60 m 0.025 g L-1 Homarus americanus

(American lobster)

LC50 Mortality Larvae;

10 or 12 C

5 m -12 h 0.66 – 0.058 g L-1 Pahl and Opitz (1999)


10 or 12 C

5 m -12 h 1.69 – 0.365 g L-1


LC50 Mortality Sediment 10 d 5 g kg-1 wet sediment Mayor et al. (2008)

Acartia tonsa

(Copepod)

LC50 Mortality Adult 96 h 0.142 g L-1 Medina et al. (2002)

LC50 Mortality Naupliar 96 h 0.005 g L-1 Acartia tonsa

(Copepod)

LC50 / LOEC Mortality Eggs 48 h 0.1288 / 0.0893 g L-1 Barata et al. (2002)

LC50 / LOEC Mortality Copepodites 48 h 0.0646 / 0.0222 g L -1

33


Acartia clause

(Copepod)

EC50 Immobility Nauplii 48 h 1.1 g L-1 Willis and Ling (2004)

EC50 Immobility Copepodites 48 h 1.49 g L-1

EC50 Immobility Adult 48 h 2.67 g L-1 Pseudocalanus

elongatus

(Copepod)


EC50 Immobility Copepodites / Adult

48 h > 5 g L-1

Temora longicornis

(Copepod)


EC50 Immobility Copepodites 48 h 0.73 g L-1

EC50 Immobility Adult 48 h 0.74 g L-1 Oithona similis

(Copepod)


EC50 Immobility Copepodites 48 h 0.17 g L-1 EC50 Immobility Adult 48 h 0.24 g L-1

Zooplankton spp.

(Copepod)

EC50 Feeding Rate Formulation 1 h + 5 h** 0.098 – 0.36 g L-1 Van Geest et al. (2014a)

Echinogammarus

finmarchius

(Amphipod)

EC50 Immobility Formulation 1 h + 95 h**

0.189 g L-1

Van Geest et al. (2014c)

LC50 Mortality Formulation 1 h + 95 h**

0.220 g L-1


0.020 g L-1


0.077 g L-1

LC50 Mortality Formulation; Spiked sediment

10 d 80 g kg-1

Gammarus spp.

(Amphipod)

LC50 Mortality Sea water 96 h 0.36 g L-1 Ernst el al. (2001)

Voltulator

(Amphipod)

LC50 Mortality Spiked sediment 10 d 5 g kg-1 Mayor et al. (2008)

A. virginiana

(Amphipod)

EC50 Sea water 48 h 0.03 g L-1 Ernst el al. (2001)

LC50 Mortality Sea water 48 h 7.42 g L-1

A. virginiana

(Amphipod)

EC50 Sea water 48 h 0.0034 g L-1 Ernst el al. (2001)


LC50 Mortality Sea water 48 h + 48 h **

0.012 g L-1

34


Eohaustorius

estuarius

(Amphipod)

EC50 Immobility Field collected

samples of

formulation

48 h 0.007 – 0.08 g L-1 Ernst el al. (2001)

LC50 Mortality 48 h 1-3.6 g L-1

Monocorophium insidiosum (Amphipod)

LC50

Mortality

Spiked sediment

10 d

57 g L-1

Tucca et al. (2014)

Daphnia magna (Water flea)

EC50 48 h 2.1 g L-1 Hill (1985) - in Clark et al. (1987)

Artemia franciscana

(Artemia)

LC50 Mortality Larvae 24 h 4720 g L-1 Sanchez-Fortun and Barahona (2005)

Brachionus plicatilis

(Rotifer)

LC50 Mortality Larvae 24 h 300 g L-1 Sanchez-Fortun and Barahona (2005)

Mytilus

galloprovincialis

(Mediterranean

mussel)

EC50 Valve activity Sea water 0.5 h + 4 h 161 g L-1 Ayad et al. (2011)

Crassostrea

virginica

(Eastern Oyster)


test

96 h 370 g L-1 Hill (1985) - in Clark et al. (1989)

Crassostrea gigas

(Pacific Oyster)


test

96 h > 2300 g L-1 Hill (1985) - in Clark et al. (1989)

L. pictus

(sea urchin)

IC25 Fertilization Sea water 20 m 1330 g L-1 Ernst et al. (2001)

IC50 Fertilization Sea water 20 m 2560 g L-1

P. Commuta

(Polychaete)


27.8 g L-1 Ernst et al. (2001)

VERTEBRATES

Salmo salar

(Atlantic salmon)


test


Cypinodon

variegatus

(Sheepshead

minnow)

LC50 Mortality Sea water 96 h 1.0 g L-1 Hill (1985) - in Clark et al. (1989)

Salmo gairnerii

(Rainbow trout)

LC50 Mortality 96 h 0.5 – 0.9 g L-1 Hill (1985) - in Clark et al. (1987)

Salmo salar

(Atlantic salmon)

LC50 Mortality Juvenile 96 h 5.8 g L-1 Mcleese et al. (1980) – in Katsuji (1989)

35



Salmo gairnerii

(Rainbow trout)

LC50 Mortality Adult 24 h 54.9 g L-1 Mcleese et al. (1980) – in Katsuji (1989)

LC50 Mortality Adult 96 h 0.416 – 0.832 g L-1 Coats and O’Donnell-Jeffery (1979) – in Katsuji (1989)

LC50 Mortality Adult 96 h 2 g L-1 Shires (1983) – in Katsuji (1989)

Oncorhychus mykiss

(Rainbow trout)

NOEC* Immunological

response;

Weight

Flow through;

Fresh water

28 d 0.100 g L-1 Shelley et al. (2009)

G. aculeatus

(Sticklebacks)

LC50 Mortality Sea water 96 h 8.1 g L-1 Ernst et al. (2001)

36

1.7.6 DM effects on non-target species

As with CP, due to the increased sensitivity of crustaceans to DM, many studies have

focused their efforts on that subphylum. Toxicity testing results report acute toxicity at extremely

low concentrations (ng DM L-1 range), much lower than the recommended treatment

concentrations used in salmon aquaculture. For example, acute lethality to lobsters and shrimp

following short-term exposures has been reported 2000 x dilution of the treatment concentration

(Burridge et al., 2014b). Similar accounts of sensitivity in copepods, lobsters, shrimp, and

amphipods following 1-h exposure have also been reported (Ernst et al., 2014; Fairchild et al.,

2010; Van Geest et al., 2014a; Van Geest et al., 2014e). Other studies, which have investigated

the effects to other species, have demonstrated less sensitivity among polychaetes and

amphipods (Van Geest et al., 2014d; Tucca et al., 2014). However, results suggest that DM is

potentially harmful to bacteria (Vibrio fischeri) (Hernando et al., 2007). Table 5 provides a

summary of toxicity information available in the literature for aquatic invertebrates and fish

species. Interestingly, research by Fairchild et al. (2010) tested two different formulations of DM

(Decis® and Alphamax®) and obtained different results providing some support to the idea that

formulations can influence toxicity.

37

Table 5: Data from deltamethrin acute and chronic exposures to fresh water and marine invertebrates and fish species.

Test Organism Endpoint Effect Measurement Notes Duration Concentration of DM Source

INVERTEBRATES

Echinogammarus

finmarchius

(Amphipod)

EC50 Immobilization Formulation 1 h + 95 h**

0.047 g L-1 Van Geest et al. (2014c)


0.070 g L-1


0.0067 g L-1


0.0094 g L-1

LC50 Mortality Formulation; Spiked sediment

10 d 0.016 g g-1

Eohaustorius

estuarius

(Amphipod)

LC50 Mortality Formulation 96 h 0.00166 – 0.00799 g L-1

Fairchild et al. (2010)

EC50 Immobility Formulation 96 h < 0.00032 – < 0.0032

g L-1 LC50 Mortality Formulation 1 h + 95

h** 0.0131 g L-1


0.00552 g L-1


0.00032 g L-1


< 0.000032 g L-1

Monocorophium insidiosum (Amphipod)

LC50

Mortality

Sediment

10 d

7,800,000 g kg-1

Tucca et al. (2014)

Cragnon

septemspinosa

(Sand shrimp)

LC50 Mortality Formulation 96 h 0.0274 - 0.0453 g L-1



0.142 g L-1

LC50 Mortality Formulation 14 d 0.0151 - 0.0238 g L-1

38

Test Organism Endpoint Effect Measurement Notes Duration Concentration of DM Source Cragnon

septemspinosa

(Sand shrimp)

IC25 Growth

inhibition

Formulation 14 d 0.0104 - > 0.032 g L-

1


LC50 Mortality Formulation 48 h + 48 h

0.00032 g L-1

EC50 Immobilization Formulation 48 h + 48 h

< 0.000032 g L-1

Cragnon

septemspinosa

(Sand shrimp)

LC50 Mortality Sea water 24 h 0.027 g L-1 Burridge et al. (2014b)


0.142 g L-1

Eohaustorius

estuarius

(Amphipod)

EC50 Death and

immobilization

Field collected

samples of

formulation

(filtered)

48 h 0.64 % of sample taken within net pen

Ernst et al. (2014)

Eohaustorius

estuarius

(Amphipod)

EC50 Death and

immobilization

Field collected

samples of

formulation

(unfiltered)

48 h 0.032 % of sample taken within net pen

Ernst et al. (2014)

Praunus flecuosis

Mysis stenolepsis LC50 Mortality Sea water 24 h 0.0014 g L-1 Burridge et al. (2014b)


0.0139 g L-1

Homarus

americanus

(American lobster)

LC50 Mortality Formulation;

Stage 3 96 h 0.00374 – 0.00492 g

L-1



Stage 4 96 h 0.0282 g L-1


Stage 3 1 d + 16 d**

0.0365 g L-1


Stage 3 16 d 0.00445 g L-1

Homarus

americanus

(American lobster)

LC50 Mortality Stage 1 1 h + 95 h 0.0034 g L-11 Burridge et al. (2014)

LC50 Mortality Adult 1 h +95 h 0.0188 g L-11

LC50 Mortality Stage 1 24 h 0.0008 g L-11



LC50 Mortality Adult 24 h 0.015 g L-1

39


Test Organism Endpoint Effect Measurement Notes Duration Concentration of DM Source

Zooplankton spp.

(Copepod) EC50 Mortality Feeding rate 1 h + 5

h** 0.17 - 0.67 g L-1 Van Geest et al. (2014a)

Nereis virens

(Polychaete worm) LC50 Mortality Formulation 48 h 16 g L-1 Van Geest et al. (2014b)

EC50 All Formulation 48 h 2.7 g L-1

LC50 Immobility and

death (severe)

Formulation 48 h 5.4 g L-1

EC50 All Formulation;

Spiked

sediment

7 d 0.20 g kg-1

EC50 Immobility and

death (severe)

Formulation;

Spiked

sediment

7 d 0.23 g kg-1

EC50 All Formulation;

Spiked sand 7 d 0.13 g kg-1

EC50 Immobility and

death (severe)

Formulation;

Spiked sand 7 d 0.23 g kg-1

VERTEBRATES

Salmo salar

(Atlantic salmon) LOEC* Mortality Sea water;

100 %

mortality;

Lowest

concentration

1 h 1,000 g L-1 Sievers et al. (1995)

40

1.8. Azamethiphos

Organophosphates (OPs), are broad-spectrum pesticides used in a wide variety of pest

management applications (Costa et al., 2008; Kozawa et al., 2009). Azamethiphos (AZ), shown

in Figure 8, is one of many OP pesticides currently used worldwide to treat sea lice infestations

in salmon aquaculture. In addition to its use in salmon aquaculture, AZ is used as a pesticide to

control against a variety of insects. Some of the physical and chemical properties of AZ are

presented in Appendix A.

Figure 8: Chemical structure of azamethiphos.


Salmosan® (AI AZ) is 47.5 % AI (Van Geest et al. 2014) and is applied as a bath treatment

at 100 μg AZ L-1 for 30-60 min in well boats and tarps and at 150 μg AZ L-1 in skirt treatments

(Burka et al., 1997; Van Geest et al., 2014; Burridge et al. 1999; Burridge et al., 2010; Grant,

2002; Haya et al., 2001; Haya et al., 2005). Salmosan® was registered for use in Canada until

2002 and it has been used under emergency approval since 2009 (Burridge and Van Geest,

2014). Salmosan® was available in Canada from 1995 to 2002 (Burridge and Van Geest, 2014).

Subsequently, it was given emergency registration for use in New Brunswick in 2009 (Van Geest

et al., 2014).


Studies have demonstrated the effectiveness of AZ against adult and pre-adult stages of

Caligidae (Gautam et al., 2016; Roth et al., 1996; Whyte et al, 2016). However, some seasonal

variation in effectiveness was reported in one Canadian study (Gautam et al., 2016). Additionally,

Bravo et al. (2015) found that the use of a number of sea lice pesticides, including AZ, can result

in decreased egg survival among C. rogercresseyi. Numerous reports of decreased sensitivity

have been filed. One study reported decreased sensitivity to AZ in populations of L. salmonis from

Canada and Norway in in vitro assays (Fallang et al., 2004). Roth et al., (1996) found that

41

sensitivity plays a large role in the effectiveness of AZ on Caligidae infestations in Scotland.

Notably, in addition to sensitivity differences among life stages, sensitivity also differed between

populations. Additionally, field trials in Norway by Whyte et al. (2016) demonstrated that the

modality of treatment can influence effectiveness significantly (i.e. wellboat, skirt v. tarpaulin),

however, it should be noted that the treatment concentrations differed based on the modality of

treatment (200 ppb for well boat or tarpaulin and 300 ppb for skirt treatment). The mechanism

behind the resistance to AZ has even been investigated using Norwegian strains of L. salmonis

(Kaur et al., 2015).

1.8.3. AZ environmental fate and persistence

In brief, AZ is water-soluble, non-volatile and has a low octanol-water partitioning

coefficient (log KOW value ~ 1) (Burridge et al., 2005; Burridge, 2013). As such, it is likely to remain

in the aqueous phase (Burridge and Van Geest, 2014): this has been confirmed in field

experiments (Ernst et al., 2014). Furthermore, AZ hydrolyzes in water and is not likely to persist

or bioaccumulate (Burridge and Van Geest, 2014; Burridge et al., 2010).

1.8.4. AZ mechanism of action and effects on non-target species

Due to the widespread use and reported toxicity associated with OP use, there have been

many reviews published on this topic many of which explore the MOA of this group of chemicals

(e.g. Costa et al. [2008], Kozawa et al., [2009], Mileson et al. [1998]). Acetylcholine (Ach) and

acetyl-cholinesterase (AChE), neurotransmitters, play critical roles in neurotransmission in the

cholinergic nervous system (Costa et al., 2008; Mileson et al., 1998). ACh stimulates cholinergic

receptors and AChE hydrolyses ACh to stop the response (Costa et al., 2008; Mileson et al.,

1998). OPs (including AZ) act by inhibiting AChE activity (Abgrall et al., 2000; Burridge and Van

Geest, 2014; Canty et al., 2007; Kozawa et al., 2009; Mileson et al., 1998). When AChE is

inhibited it results in the repetitive firing of nerves (Baillie, 1985), ultimately resulting in a wide

variety of symptoms including tremors, twitching and even death (Costa et al., 2008). AZ has been

shown to be mutagenic in several in vitro tests, but not in vivo (EMEA, 1999).

Overall, there is a lack of information on the toxicity of AZ to non-target organisms (Roth

et al., 1993). As with the other chemicals discussed here, research investigating the effects of AZ

has focused on sensitive non-target crustaceans (e.g. Abgrall et al., [2000], Burridge et al. [1999]).

Table 6 provides a summary of toxicity information available in the literature for aquatic

42

invertebrates and fish species. Notably, Van Geest et al. (2014a) have concluded that the AZ

containing formulation Salmosan® is the least toxic of the bath-applied pesticides.

The extreme sensitivity of lobsters (Homarus gammarus and H. americanus), mysid

shrimp (Mysidopdid bahia), and amphipods (Eohausorius estuarius) to AZ demonstrate the risk

of using this chemical in areas where these species are present. This is especially concerning

because the LC50 values are lower than the recommended treatment concentration (100 g AZ L-

1) for short-term exposures. For example, Burridge et al. (2014b) reported an LC50 value for adult

lobsters (1 h exposure followed by a 95 h observation period) of 24.8 g AZ L-1. Additionally, the

results of Ernst et al. (2001; 2014) demonstrated toxicity (immobility or mortality) to non-target

crustaceans in laboratory testing using water collected from within the net pen during treatment

and in the vicinity of salmon aquaculture sites (up to 20 min post-release). Specifically, although

these results indicate significant short-duration toxicity with samples taken from within the net pen

during treatment, only longer exposures (48 h) resulted in toxicity for samples taken from the

vicinity after release. However, the data indicates that the potential for the use of AZ to treat sea

lice in salmon aquaculture to cause adverse effects to non-target species is low.

Other species, including bivalves, fish, gastropods, and echinoderms are less sensitive to

AZ than crustaceans (Canty et al., 2007; Ernst et al., 2001; Van Geest et al., 2014a).

43

Table 6: Data from azamethiphos acute and chronic exposures to freshwater and marine invertebrates and fish species.

Test Organism Endpoint Effect Measurement Notes Duration Concentration of AZ Source

INVERTEBRATES

Homarus americanus

(American lobster)


Stage 1

48 h 3.57 g L-1 Burridge et al. (1999)


Stage 2

48 h 1.03 g L-1


Stage 3

48 h 2.29 g L-1


Stage 4

48 h 2.12 g L-1


Adult

48 h 1.39 g L-1

Homarus americanus

(American lobster)

LC50 Mortality

(seasonal)

Sea water;

Adult

48 h 0.61 – 3.24 g L-1 Burridge et al. (2005)

Homarus americanus

(American lobster)


10 or 12 C;

5 m - 12 h 33.9 – 1.3 g L-1 Pahl and Opitz (1999)


10 or 12 C

5 m - 12 h 50.4 – 0.9 g L-1

Homarus americanus

(American lobster)

LC50 Mortality 3 x 3 exposures;

Adult

48 h 1.08 g L-1 Burridge et al. (2000a)

NOEC Mortality 3 x 3 exposures;

Stage 4

30 m 11 g L-1

NOEC Mortality 3 x 3 exposures;

Adult

30 m 1.03 g L-1

Homarus americanus

(American lobster)

LC100* Mortality Sea water;

Bi-weekly

exposure

1 h 10 g L-1 Burridge et al. (2008b)

Homarus americanus

(American lobster)


Stage 1

24 h 8.9 g L-1 Burridge et al. (2014b)


Adult

24 h 2.8 g L-1


Stage 1

1 h + 95 h**

> 86.5 g L-1


Adult

1 h + 95 h**

24.8 g L-1

Homarus americanus

(American lobster)

LOEC Predator

avoidance / shelter

use

Sea water;

Pulse exposure

10 m 100 g L-1 Abgrall et al. (2000)

44


C. septemspinosa

(Sand shrimp)


Adult

24 h 191 g L-1 Burridge et al. (2014b)


Adult

1 h + 95 h**

> 85.5 g L-1

C. septemspinosa

(Sand shrimp)

LC50 Mortality Field collected

samples of

formulation

24 h (mean)

19.2 g L-1 Ernst et al. (2014)

Flexuosus Mysis

stenolepsis

(Mysid spp.)


Burridge et al. (2014b)


> 85.5 g L-1

Mysis stenolepsis

(Mysid spp.)

LC50 Mortality Field collected

samples of

formulation

24 h (mean)

10.5 g L-1 Ernst et al. (2014)



Mayor et al. (2008)

Ehaustorius

estuarius

(Amphipod)

EC50 Immobility Field collected

samples of

formulation

48 h 1.4 – 12.1 g L-1 Ernst et al., 2001

Zooplankton spp.

(Copepod)

NOAEL Mortality/Feeding

rate

Sea water 1 h + 5 h** 620 g L-1 Van Geest et al. (2014)

Mytilus edulis

(Marine molusc)

IC50 Gill AChE activity Sea water 1 h 736 g L-1 Canty et al. (2007)

IC50 Heamolymph

AChE activity

Sea water 1 h 1300 g L-1

LOEC* AChE activity;

Cytotoxicity

Sea water 1 / 24 h 100 g L-1

LOEC* Immune function Sea water 24 h 100 g L-1 NOEC* Feeding rate Sea water 1,000,000 g L-1

Voltulator

(Amphipod)

LC50 Mortality Sediment 10 d 182 g kg-1 Mayor et al. (2008)

Gammarus spp.

(Amphipod)

LC50 Mortality Sea water 96 h < 5 g L-1 Ernst el al. (2001)

L. pictus

(sea urchin)

IC25 Fertilization Sea water 20 m 3340 g L-1 Ernst et al. (2001)

IC50 Fertilization Sea water 20 m 6840 g L-1 P. Commuta

(Polychaete)


2310 g L-1 Ernst et al. (2001)

45



VERTEBRATES

Salmo salar

(Atlantic Salmon)

LOEC* Mortality Sea water;

5 % mortality;

Lowest

concentration

1 h 1,000 g L-1 Sievers et al. (1995)

G. aculeatus

(Sticklebacks)

LC50 Mortality Sea water 96 h 190 g L-1 Ernst et al. (2001)

46

1.9. Risk of sea lice pesticide use

Several issues of concern have been identified regarding the use of chemotheraputants

in aquaculture (Burka et al., 1997; Bright and Dionne, 2005; Burridge & Van Geest, 2014). The

primary objective of the present work was to address several of the more important data gaps.

Specifically, this research addressed the lack of information on the toxicity of sea lice pesticides

used in salmon aquaculture to non-target marine organisms in the Pacific northwest region of

Canada as well as the lack of knowledge regarding the fate and persistence of these substances

once released to the environment. The results of this research will fill knowledge gaps and reduce

uncertainties for better risk assessments to be performed which will enable better decision making

for regulators and aquaculturists alike.

47

Chapter 2.

Environmental Fate and Effects of Sea Lice Pesticides used in Canadian Salmon Aquaculture

Fauve Strachan1, Frank Gobas2, Victoria Otton2, and Chris Kennedy1

1Department of Biological Sciences, Simon Fraser University, Burnaby, Canada

2Resource and Environmental Management, Simon Fraser University, Burnaby, Canada

Keywords: toxicity; sea lice; aquaculture; pesticide; fate, persistence; partitioning;

chemotherapeutants

48

Abstract

In Canada, five chemotherapeutant formulations, have been used or continue to be used to treat

sea lice infestations in salmon aquaculture. This research generated data on the environmental

partitioning and persistence, as well as acute toxicity of Slice® (AI: emamectin benzoate [EB]),

Salmosan® (AI: azamethiphos [AZ]), Alphamax® (AI: deltamethrin [DM]), Excis® (AI: cypermethrin

[CP]), and Interox® Paramove 50 (AI: hydrogen peroxide [HP]) to representative classes of marine

organisms. EB, CP and DM partitioned mainly to the sediment phase in sediment-water

microcosms; AZ and HP remained mainly in the water phase. The current study reports that the

persistence of chemicals in water was: CP > DM > AZ > HP. In sediment, the following trend was

observed: CP > EB > DM. Toxicity test results indicate a lack of susceptibility trends in any

species, or toxicity trends with any one chemical, however the information is useful for identifying

risks. Some of the values reported for echinoderms (IC25 values), kelp (IC50 values) and topsmelt

(LC50 values) are below the recommended treatment concentration. Together, this information

provides insight into the environmental fate and associated risks of chemotherapeutants to non-

target marine organisms that may be found in the vicinity of salmon aquaculture sites.

49

2.1. Introduction

Commercial aquaculture worldwide is a multi-million-dollar industry (Bright and Dionne,

2005; Costello, 2009; DFO, 2012; DFO, 2014; Olin et al., 2011; Torrissen et al., 2013) and in

Canada, salmon is the primary contributor to aquaculture production ($967 million CDN) (DFO,

2012; 2017). High-density farms use of a variety of regulated chemicals, including antibiotics,

vaccines and chemotherapeutants to maintain healthy stocks in order to reduce financial losses

(Bushmann et al., 2006; Burridge et al., 2011). Chemicals used in salmonid aquaculture include

anaesthetics, pesticides, antifoulants and disinfectants (Burridge et al., 2010; 2011; Burka et al.,

1997). Environmental concerns resulting from salmon aquaculture practices include conflicts with

marine mammals, escapes of non-native fish, displacement of local fishermen, impacts to tourism,

water pollution, and the potential for disease/virus/parasite (e.g. sea lice) transmission between

captive and wild fish populations (Morton, 2015).

Sea lice are ectoparasites that nourish themselves through the ingestion of mucus, skin

and tissue of their hosts (Burridge et al., 2010; Bright and Dionne, 2005; Costello, 2006; Mustafa

et al., 2000; Pahl and Opitz, 1999; Torrissen et al., 2013). Concerns regarding direct losses to the

salmon aquaculture industry as a result of sea lice infestations, the potential for sea lice transfer

to native salmon populations or between farms, and the effect of chemicals used to treat

infestations, have been raised (Burridge and Van Geest, 2014; Buschmann et al., 2006; Krkosek

et al., 2005; Morton et al., 2011; Torrissen et al., 2013). Global monetary losses in salmon

aquaculture due to sea lice infestation have been estimated to be in the tens of millions of dollars

(CDN) (DFO, 2014a). In Canada alone, annual costs associated with sea lice infestations are

estimated at over $15 million (CDN) (Costello, 2009; Roth, 2000). Although many of the concerns

regarding sea lice relate to salmonid species, other marine species can also be infected (Roth et

al., 1993; Torrissen et al., 2013). Sea lice infestations are commonly treated by a variety of

chemotherapeutants (Torrissen et al., 2013; Burridge et al., 2010; Burka et al; 1997; Burridge and

Van Geest 2014; Burridge et al., 2008; Grant, 2002; Haya et al., 2005; Roth et al., 1993) which

results in the entry of these substances into the surrounding marine environment.

A number of compounds have been used, or are currently used to treat sea lice

infestations in salmon aquaculture (Burka et al., 1997; Burridge and Van Geest, 2014; Burridge

et al., 2008; 2010; 2011; DFO, 2003; 2013; Grant, 2002; Roth et al., 1993; Roth, 2000). Clinically

available treatments vary from country to country, but essentially 13 compounds have been used

worldwide and include: dichlorvos, malathion, trichlorfon, azamethiphos [AZ], pyrethrum,

50

cypermethrin [CP], deltamethrin [DM], ivermectin, emamectin benzoate [EB], doramectin,

hydrogen peroxide [HP], teflubenzuron and diflubenzuron (Haya et al., 2005; Roth, 2000). In

Canada, five chemotherapeutant formulations have been used or continue to be used: Slice® (AI:

EB), Salmosan® (AI: AZ), Alphamax® (AI: DM), Excis® (AI: CP), and Interox® Paramove 50 (AI:

HP) (Burridge, 2003; Burridge et al., 2014; PMRA, 2016). With the exception of Slice® (which is

applied to feed), all of these formulations are applied as water bath treatments. In all cases,

formulations are applied based on the concentration of the product AI. A summary of the key

information pertaining to the use of these 5 formulations can be found in Table 7. The AIs used

in these formulations all have broad-spectrum mechanisms of toxic actions, the details of which

are explored in detail elsewhere (e.g. Grant [2002]; Burka et al. [1997]), and therefore pose a

potential risk to non-target organisms in the environment.

Table 7: Summary key information pertaining to the use of formulations in Canada to treat sea lice infestations: AI, recommended treatment dose, or concentration, and recommended duration of treatment.

Formulation Active Ingredient (AI) Dose Duration Source

Slice® Emamectin Benzoate (EB) 50 g kg-1 day-1 7 d Bright and Dionne (2005)

Salmosan® Azamethiphos (AZ) 100 g L-1 30 - 60 mins

Burridge and Van Geest (2014)

Alphamax® Deltamethrin (DM) 2 - 3 g L-1 40 mins Burridge and Van Geest (2014)

Excis® Cypermethrin (CP) 5 g L-1 60 mins Burridge and Van Geest (2014)

Interox®

Paramove 50 Hydrogen Peroxide (HP) 1,200,000 -

1,800,000 g L-1

20 - 30 mins

Burridge and Van Geest (2014); Grant (2002); PMRA (2014)

The toxicity of sea lice chemotherapeutants to non-target organisms has been the subject

of several studies, however, there exists a notable lack of information specific to non-target

species found in the Pacific north west region of Canada, where salmon aquaculture is common

(DFO, 2017), has been identified (Burridge and Van Geest, 2014). The majority of these studies

have focused on crustaceans (e.g. Burridge et al. [2014b], Ernst et al. [2014]). Significantly more

literature exists on the effects of Slice® (EB) on marine organisms compared to the other

chemicals, particularly Interox® Paramove 50 and HP. As well, there is limited information

regarding the fate and persistence of these substances in water-sediment systems as well

(Burridge and Van Geest, 2014; Benskin et al., 2016; Bruno and Raynard, 1994; Ernst et al.,

2001; Lyons et al., 2014; Meyer et al., 2013; Muir et al., 1985) which is essential for estimates of

exposure and risk assessments. Among these available studies, only Muir et al. (1985) and Meyer

et al. (2013) used a multi-phase system; studies that used water-only or sediment-only systems,

provide an incomplete understanding of the fate these substances in the environment. Lacking

51

this data, information on partitioning and environmental persistence is often inferred from

chemical properties or the results of limited field sampling.

The objectives of this study were 1) to generate data on the environmental partitioning

and persistence of these chemotherapeutants in a water-sediment microcosm, and 2) to

determine their acute toxicity to a broad representation of relevant classes of marine organisms

that may be exposed to them near aquaculture operations. The information gained here provides

valuable input to risk determinations for non-target organisms inhabiting in areas which may be

contaminated by these chemicals from salmon aquaculture operation. Additionally, comparisons

of the toxicity, environmental fate and persistence of the chemicals can easily be made between

chemicals and species.

2.2. Materials and methods

2.2.1. Organisms

Macrocystis pyrifera were obtained from A.K. Siewers (Santa Cruz, CA). Atherinops affinis

and Mysidopsis bahia were obtained from Aquatic Biosystems (Fort Collins, CO). Mytilus

galloprovincialis were obtained from Kamilche Seafarms (Shelton, WA). Strongylocentrotus

purpuratus were obtained from Nautilus Environmental (San Diego, CA). All work with animals

was conducted in accordance with Canadian Council of Animal Care (CCAC) guidelines under

permit of the Simon Fraser University Animal Care Committee.

2.2.2. Chemicals

The following chemicals were obtained from Sigma-Aldrich (Oakville, ON): AZ, (> 99 %

pure), CAS: 35575-96-3; CP, (> 98 % pure), CAS: 52315-07-8; DM, (> 99 % pure), CAS: 52918-

63-5; EB, (> 99 % pure), CAS: 155569-91-8; HP, (30 %), CAS: 7722-84-1; acetone, CAS: 67-64-

1; methanol, CAS: 67-56-1; dichloromethane, CAS: 75-09-2; chloroform, CAS: 67-66-3; and

sodium chloride (NaCl), CAS 7647-14-5. Copper chloride dehydrate (CAS: 10125-13-0);

potassium chloride (CAS: 7447-40-7), and prepared buffered formalin solutions (3% and 50%

[CAS: 50-00-0]) were sourced from Fisher Scientific (Ottawa, ON). Formalin was diluted with

deionized water and adjusted with Borax to a pH of 7.5.

52

2.2.3. Fate and persistence

Aerobic sediment and associated water was collected from Maplewood Flats

Conservation Area (North Vancouver, BC) as per OECD 308 (OECD, 2002) for use in determining

the partitioning behavior and persistence of each chemical in simple water-sediment microcosms.

Sediment collection was performed at low tide and included the entire 5 to 10 cm upper layer of

the sediment. Water was collected on the same day from the same site. Sediment and water were

stored in a cold room at 4 C in 20 L plastic buckets with free access to air for no more than 4

weeks before use. Prior to use, sediment was wet-sieved with a 2 mm sieve using excess water

collected from the site. During sampling, temperature, salinity, pH and O2 concentration of the

water were measured. Sediments collected from the site were analyzed post-handling for particle

size distribution, pH, microbial biomass, total organic matter (TOM) and total organic carbon

(TOC). At each sampling time, temperature, pH, O2 concentration, salinity and redox potential

were analyzed. At test initiation and test termination, TOC, microbial biomass (aerobic plate count

and mould plate count methods) and redox potential were also analyzed. Particle size distribution,

microbial biomass, TOM and TOC (water and sediment) in all cases were analyzed by Maxxam

Analytics (Burnaby, BC).

The procedure used to determine the partitioning and persistence of each

chemotherapeutant in the microcosms was a modified OECD 308 (OECD, 2002) methodology. A

single sediment type was used in the current study, consistent with recommendations by Ericson

(2007). Water and sediment were placed into 500 mL wide-mouth amber jars at a ratio of 3:1

(water:sediment) with a sediment depth of 2.5 cm. Prior to study initiation, sediment and water

were acclimated for 1 week in test vessels, at test conditions (10 C, in the dark, free access to

air). The initial nominal concentration of each substance in the water phase were as follows: AZ:

100 g L-1; CP: 200 g L-1; DM: 400 g L-1; EB: 100 g L-1; and HP: 100,00 g L-1. Sampling

occurred at initiation, 0.5, 1, 2, 4, 7, 14, 28, and 98 d for all substances, except HP, for which

sampling occurred at initiation, 1, 3, 6, 12, 24 and 48 h.

At the initiation of incubations, water-sediment systems were spiked with chemicals by

adding 32 L stock solution (for EB, CP and DM) or solvents (for methanol and acetone controls)

or 48.4 L 30 % HP to the water, followed by gentle swirling to avoid disturbing the sediment.

Stock solutions for all experiments were prepared using methanol (EB) and acetone (CP, DM,

and AZ) solvents. All tests included sea water controls, methanol solvent controls and acetone

53

solvent controls (0.01 %; for both). Incubations were performed such that whole units, in duplicate,

were sacrificed at each sampling time for analysis.

At each sampling period, water and sediment were carefully separated, and both were

stored at -20 C until analysis. Due to its high water solubility, HP recovery in sediment was not

considered.

2.2.3 Toxicity tests

Test chemical stock solutions for toxicity tests were prepared as described above. UV-

sterilized seawater was used for all tests. Test concentrations were prepared by serial dilutions

of the stock solution and a summary of the test concentrations used in each of the tests are

presented in Table 8. Concentrations were selected based on the results of range finding

experiments.

Table 8: Summary of nominal EB, CP, DM, AZ, and HP test concentrations used in

toxicity tests (g L-1). Range of nominal concentration (Range), number of concentrations used in testing (Number) and the dilution factor (DF) are reported.

Emamectin Benzoate

Hydrogen Peroxide

Cypermethrin Deltamethrin Azamethiphos

Kelp Range 300 – 5,000 24 – 15,000 10 – 50 0. 008 – 20 20 – 12,500

Number 5 5 5 5 5

DF 0.4 5 0.008 0.00125 0.5

Topsmelt Range 310 – 5,000 9,600 – 377,500 3.1 - 50 1.25 - 20 625 -10,000

Number 5 5 5 5 5

DF 2 2.5 2 2 2

Mysid Range 8 – 5,000 1100 – 90,000 6 –100 3 –50 180 – 3,000

Number 5 5 5 5 5

DF 5 3 2 2 2

Bivalve Range 300 – 5,000 60 – 7,500 3 –50 6 – 100 800 – 12,500

Number 5 7* 5 5 5

DF 2 2 2 2 2

Echinoderm Range 20 – 5,000 150 - 9,600 2 - 50 0. 08 - 20 10 – 12,500

Number 7 7 7 7 7

DF 2.5 2 2.5 2.5 3

*No effects up to 500 g L-1 in preliminary testing.

2.2.3.1 Giant kelp germination and growth

The giant kelp germination and growth test estimates the developmental toxicity to

zoospores and gametophytes of the giant kelp (Macrocystis pyrifera). Tests were performed

54

immediately as per US EPA (1995a) protocol, with some modifications. Sporophylls were rinsed

thoroughly with filtered seawater, blades were desiccated for 1 h and then rinsed with filtered

seawater to stimulate zoospore release and stored between paper towels until needed. Upon re-

submersion in seawater zoospores are released. The 48 h static non-renewal test was run at 15

C with 5 replicates per test concentration in petri dishes with 10 mL of test solution. CuCl2 was

used as a reference toxicant.

At test initiation, zoospores were added to test containers to achieve a concentration of ~

7,500 zoospores mL-1 in each of the randomly arranged test containers. Water quality

(temperature, pH, salinity, O2 concentration) was assessed prior to test initiation and once daily

for the duration of the test. After 48 h, tests were terminated by the addition of 3 % buffered

formalin and spores were counted directly in the vessels using an inverted microscope at 100 x

magnification.

The percent germination success was assessed for the first 100 spores encountered. The

length of the germination tube was determined, using a calibrated micrometer, for the first 10

germinated spores encountered per replicate following the initial 100 spore count (US EPA,

1995a). Germination was deemed successful if the spore was in development as determined by

the presence of a germination tube. Per the protocol (with previously mentioned modifications),

test acceptability was determined based on a mean control germination success of > 70 %, a

mean germination-tube length in the controls of > 9 m (slightly lower than the values

recommended by the protocol [10 m]), a germination-tube growth NOEC > 35 g L-1 for the

reference toxicant (CuCl2), and minimum significant difference (% MSD) < 20 % for the reference

toxicant relative to the control for both endpoints.

2.2.3.2 Topsmelt survival

This test was performed to estimate the acutely lethal toxicity of a chemical to juvenile

topsmelt (Atherinops affinis) in a 96 h static renewal test according to a modified US EPA (1995a)

protocol and recommendations found in US EPA (2002b). The US EPA (1995a) protocol was

shortened to a 96-h test to reflect an anticipated acute exposure to aquaculture chemicals in

practice. The growth endpoint was not assessed because growth changes were not anticipated

following such a short exposure. CuCl2 was used as a reference toxicant. Tests were performed

the day of organism receipt.

55

Topsmelt (9-d old) were randomly distributed into test vessels containing test solutions (5

per test vessel). The test was run at 20 C with 5 replicates in 1 L vessels containing 200 mL of

test solution. At 48 h, an 80 % solution change was performed and excess food and waste was

removed. Two h prior to test initiation and water change, fish were fed Artemia cysts cultured from

premium grade brine shrimp eggs (Brine Shrimp Direct Inc. [Ogden, UT]). Brine shrimp were

cultured using ~5 g of brine shrimp eggs in 500 mL of sea water left to hatch at 25 C overnight

with constant aeration and light in an inverted cone. Prior to use, shrimp were concentrated and

re-suspended in seawater for use in testing. Water quality (temperature, pH, salinity, O2

concentration) and mortality were assessed on each day of the test.

Tests were deemed acceptable if there was > 90 % control survival (US EPA, 1995a).

Mortality was confirmed by checking for movement following a gentle nudge with a glass rod.

2.2.3.3 Mysid survival

This acute lethal toxicity of the chemicals to mysid (Mysidopsis bahia) was examined in a

48-h static renewal test and was performed according to a modified US EPA (2002) protocol and

recommendations found in WS DOE (2008). The US EPA (2002) protocol was shortened to a 48-

h bioassay to reflect that anticipated acute exposure in practice. CuCl2 was used as a reference

toxicant. Tests were run the day of organism receipt.

Mysids (5-d old) were randomly distributed into test vessels containing test solutions (10

per vessel). This test was run at 20 C with 4 replicates for each test concentration (and 8 controls)

in 500 mL vessels containing 200 mL of a test solution. At 24 h, an 80 % solution change was

performed and excess food and waste was removed. Two h prior to test initiation and water

change, mysids were fed Artemia cysts. Water quality (temperature, pH, salinity, O2

concentration) and mortality was assessed on each day of the test.

Tests were deemed acceptable if there was > 90 % control survival (US EPA, 2002).

Mortality was confirmed by checking for movement following a gentle nudge with a glass rod.

2.2.3.4 Bivalve embryo survival and development

To estimate the acute developmental and lethal toxicity of bivalve larvae (Mytilus sp.) a

48-h static renewal test was performed according to US EPA (1995b) protocol with some

modifications. The test was conducted in the absence of sediment, in 30 mL scintillation vials

containing 10 mL of solution per replicate and 0.1 mL of larvae solution (containing 150-300

56

larvae). Because bivalve larvae are typically found in the water column the simplification to the

protocol to use a water only was deemed acceptable. CuCl2 was used as a reference toxicant.

Tests were performed on the day of organism receipt.

Bivalves were rinsed in seawater scrubbed clean of fouling organisms with a sponge and

spatula. Gamete release was induced by heat shock (28 C) and spawning organisms were

placed in individual beakers containing seawater at 15 C for approximately 30 mins. Gametes

were checked for viability and top quality gametes were pooled for testing. The test was run at 15

C with 4 replicates per test concentration (8 for the controls). Eggs were fertilized and once 90

percent cell division was achieved they were added to test vessels (0.1 mL; 1500 - 3000 eggs

mL-1) signifying test initiation. Water quality (temperature, pH, salinity, O2 concentration) was

assessed on each day of the test. Tests were terminated by the addition of 50 % buffered formalin

to each vial for preservation and embryos were counted directly in the vessels using an inverted

microscope under 100 x magnification. Additionally, day 0 counts were obtained by seeding 6

vessels and immediately preserving them.

Embryos were counted and scored as being normal or abnormal and the difference

between starting counts and the sum of the normal and abnormal embryos were assumed dead.

Development was determined based on the shape of the embryo; abnormal embryos were

circular whereas normal embryos were “D-shaped”. Tests were deemed acceptable if 70 % of

all control embryos survived and developed normally, the minimum significant difference for the

reference toxicant relative to the control is < 25 %, and the coefficient of variance for the initial

count vessels was 15 %. (US EPA, 1995b).

2.2.3.5 Echinoderm fertilization

The acute toxicity of chemicals to echinoderm gametes (Strongylocentrotus purpuratus.)

in a 20 min static non-renewal test according to Environment Canada (EC) (2011) protocol was

performed. CuCl2 was used as a reference toxicant. Tests were performed on the day of organism

receipt.

Gamete release was induced by the injection of 0.5 - 1 mL of 0.5 M potassium chloride

(KCl) through the peristomal membrane. All viable male gametes and all viable female gametes

were pooled for testing. The test was run at 15 C with 5 replicates for each test concentration

(and 3 for the controls) in 30 mL scintillation vials containing 10 mL of solution per vessel. Water

quality (temperature, pH, salinity, O2 concentration) was assessed prior to test initiation. Sperm

57

solution (100 L) was introduced to each of the test vessels, 10 min later, egg solution (1 mL,

2000 eggs mL-1) was added and incubated for an additional 10 min. Tests were terminated by the

addition of 50 % buffered formalin to each vial for preservation and fertilization was assessed

using an inverted microscope under 100 x magnification.

The first 100 eggs viewed under a microscope were scored as either fertilized or

unfertilized to and the percentage of fertilized eggs was reported. Successfully fertilized eggs can

be distinguished from unfertilized eggs by their raised fertilization membrane (EC, 2011). Tests

were deemed acceptable if the average success of fertilization in the control organisms is

between 60 - 98 % (EC, 2011).

2.2.4 Chemical analysis

Water samples from toxicity tests (100 mL) were collected in amber jars (100 mL). DM

and CP samples were collected and preserved with dichloromethane (~ 5 % v/v), shaken and

then stored at 4 C until analysis as described in Van Geest et al. (2014a). AZ samples were

collected and preserved with 2 g NaCl and 5 mL chloroform, shaken and then stored at 4 C until

analysis as described in Burridge et al. (1999). EB samples were collected and stored at -20 C

until analysis as described in Park (2013).

Hydrogen Peroxide. HP samples were analyzed, as soon as possible, on the day of

sampling using a Flurometric Hydrogen Peroxide Assay Kit, read at ex = 540/ em = 590 nm

(reporting limit: 0.1 uM, Sigma-Aldrich).

Cypermethrin and Deltamethrin. To assess water extraction efficiencies, samples

(deionized water spiked with 1 to 5 µg L-1 DM or CP), sediments, or water were added to round-

bottom flasks with 10 µL of PCB-155 (0.135 µg mL-1) added as an internal standard. Water was

extracted twice with 60 mL dichloromethane (DCM) using vigorous shaking for 5 min followed by

sonication and shaking at intervals of 5 s for another 5 min. Extracts were combined and allowed

to settle for 2 h and evaporated in a rotory evaporator until the volume was approximately 3 mL.

The remaining extract was transferred to a glass test tube and dried down to completeness under

nitrogen. Sediments were air-dried, and then transferred to a glass centrifuge tube for extraction

with DCM (60 mL) with shaking for 2 h followed by 5 min of alternating sonication and shaking.

The extract was centrifuged at 1,600 x g for 15 min. The supernatant was removed and the

extraction process repeated 2 times further. Supernatants were combined (Maund et al., 2002)

58

and allowed to settle for 2 h and evaporated in a rotory evaporator until the volume was

approximately 3 mL. Extracts were transferred to a glass test tube and dried down to

completeness under nitrogen gas. Water and sediment extracts were reconstituted to 5 mL in

methanol and co-extracted matrix interferences were removed by loading samples onto stacked

graphitized carbon and alumina SPE cartridges. DM or CP were eluted from the SPE cartridge

with DCM (Hladik et al., 2012), dried down to completeness under nitrogen gas. Dried extracted

water or sediment samples were reconstituted with 0.5 mL acetonitrile and placed into

autosampler vial. A Hewlett-Packard 5890 GC equipped with a 63Ni electron-capture detector, a

splitless injection port and a 30 m x 0.25 mm x 2.65 DB-5 column (Agilent Technologies,

Mississauga, ON) was used with the following conditions: an oven temperature program of 80 ºC

for 0.5 min, 20 ºC min-1 to 270 ºC, which was held for 5 min, then 15 ºC min-1 to 290 ºC, which

was held for 5 min. The injection port temperature and detector temperatures were 240 ºC and

310 ºC, respectively, and the carrier gas was helium (1 mL min-1). Argon/methane was used as

the make-up gas. Injections (2 µL) were done manually, using a gas-tight syringe with a Teflon-

tipped plunger. Peaks were integrated using Chemstation (Hewlett Packard, Mississauga, ON)

software. The GC was calibrated with each new sample batch. A number of calibration standards

were run using a minimum of five and up to seven. DM and CP recoveries were determined by

comparing 5 spiked samples with one another. DM or CP were spiked into several water and

sediment matrices at 10 and 100 ng per 1 L of water or 5 g (dry weight) of sediment. The detection

limit for DM and CP in water was 0.05 µg L-1. The detection limit for DM and CP in sediment was

0.10 µg kg-1. Recoveries from water and sediment were 92% and 86%, with between-day

variability of 6.1%.

Azamethiphos. To assess water extraction efficiencies, samples (deionized water spiked

with 10 to 50 µg AZ L-1), sediment, or water to assess extraction efficiencies were extracted with

DCM (Van Geest et al. 2014). Water samples were extracted as follows: 2 g of NaCl was added

to each 100 mL of water (Burridge et al. 1999), followed by the addition of 50 mL DCM and

vigorous shaking for 5 min followed by sonication and shaking at intervals of 5 s for another 5

min. DCM and water phases were separated using centrifugation at 1500 x g. The supernatant

was removed and the extraction process repeated 2 further times with 60 mL DCM. Supernatants

were combined and allowed to settle for 2 h and evaporated in a rotory evaporator until the volume

was approximately 3 mL. Extracts were transferred to a glass test tube and dried down to

completeness under nitrogen gas. Water and sediment extracts were reconstituted to 5 mL in

methanol and co-extracted matrix interferences were removed by loading samples onto stacked

59

graphitized carbon and alumina SPE cartridges. AZ was eluted from the SPE cartridge with DCM,

dried down to completeness under nitrogen gas. The residue taken up in 1 mL of high pressure

liquid chromotography (HPLC) grade acetonitrile into a 2-mL amber sample vial.

This sample was analyzed by HPLC on a Hewlett Packard Model 1050 with an HP Model

1046A programmable fluorescence detector (FLD) and HP 3396 Series II Integrator (Hewlett-

Packard). The analyte is determined by LC using a C18 column, ACN-H2O (32 + 68) mobile

phase, (Pfenning et al. 1999) under the following conditions: Column, Thermo Scientific™

Syncronis™ C18 reversed phase (100 Å, 5 µm, 250 × 3.2 mm id); mobile phase, water:acetonitrile

68:32 at 1.0 ml/min (isocratic); Fluorescence settings at 230 and 345 nm for excitation and

emission, respectively, with a 280 nm cutoff filter; detector temperature: 50 °C; column pressure,

220 bar; injection volume, 100 µL. Calibration was carried out by the analysis of a series of

standard AZ solutions, prepared in seawater or sediment and extracted by the above method.

Calibration standards were prepared weekly from technical AZ (Ciba Geigy Analytical Master

Standard). AZ recovery was determined by comparing 5 spiked samples with one another. AZ

was spiked into several water and sediment matrices at 10 and 100 ng per 1 L of water or 5 g

(dry weight) of sediment. The detection limit for AZ using 1-L water samples is 1.5 µg L-1. The

detection limit for AZ in sediment was 5 µg kg-1. Recoveries from water and sediment were 96%

and 94%, with between-day variability of 4.5%.

Emamectin Benzoate. To assess water extraction efficiencies, samples (deionized water

spiked with 10 to 50 ng L-1 EB), sediment, or water to assess extraction efficiencies were extracted

with DCM. Water samples were extracted as follows: samples were adjusted to pH 4 with

orthophosphoric acid (1% v/v) and extracted with 50 mL DCM by vigorous shaking for 5 min

followed by sonication and shaking at intervals of 5 s for another 5 min. DCM and water phases

were separated using centrifugation 1500 x g and the DCM removed. The supernatant was

removed and the extraction process repeated 2 further times with 60 mL DCM. Supernatants

were combined and allowed to settle for 2 h and evaporated in a rotory evaporator until the volume

was approximately 3 mL. Extracts were transferred to a glass test tube and dried down to

completeness under nitrogen gas. Sediments were air-dried, and then transferred to a glass

centrifuge tube for extraction with DCM (60 ml) with shaking for 2 h followed by 5 min of alternating

sonication and shaking. The extract was centrifuged at 1,600 x g for 15 min. The supernatant

was removed and the extraction process repeated 2 further times. Supernatants were combined

and allowed to settle for 2 h and evaporated in a rotory evaporator until the volume was

approximately 3 mL and transferred to a glass tube. Extracts were eluted through anhydrous

60

sodium sulphate, dried by rotary film evaporation (35 °C). The evaporated residues were reacted

with derivatisation reagents according to the following processes: 200 mL of N-NMIM/acetonitrile

(1:1, v/v) were added to the tube and vortexed briefly, followed by the addition of 300 mL

TFAA/acetonitrile (1: 2, v/v). The reaction mixtures were stored in the dark for at least 0.5 h. The

derivatisation reagents were made fresh every day. To avoid the decrease of fluorescent signal,

the analysis of derivates was completed within 80 h. The derivatisation solutions were dried down

under N2 and and the residue taken up in 1 mL of high pressure liquid chromotography (HPLC)

grade acetonitrile (Fisher Scientific) (1 mL) with ultrasonication (Xie et al. 2011).

This sample was analyzed by HPLC on a Hewlett Packard Model 1050 with an HP Model

1046A programmable fluorescence detector (FLD) and HP 3396 Series II Integrator (Hewlett-

Packard). The analyte was determined by LC according to Xie et al. (2011) under the following

conditions: Column, Waters Xbridge C18 column (250 x 4.6mm i.d., 5 µm) with a guard column

(20 x 4.6 mm i.d., 5 µm); mobile phase, gradient program of mobile phase with acetonitrile (solvent

A), methanol (solvent B) and water (solvent C) carried out at a flow rate of 1.0 ml min-1; gradient

elution program started with acetonitrile/methanol/water (10:80:10, v/v/v) followed by a linear

adjustment for acetonitrile/methanol (20/80, v/v) in 10 min; the last condition was kept constant

for 5 min; Fluorescence settings at 254 and 400 nm for excitation and emission, respectively;

detector temperature: 40 °C; column pressure, 220 bar; injection volume, 20 µL. Calibration was

carried out by the analysis of a series of standard EB solutions, prepared in seawater or sediment

and extracted by the above method. EB recovery was determined by comparing 5 spiked samples

with one another. EB was spiked into several water and sediment matrices at 1 and 50 ng per 1

L of water or 5 g (dry weight) of sediment. The detection limit for EB using 1-L water samples is

4.8 ng L-1. The detection limit for EB in sediment was 7.9 ng kg-1. Recoveries from water and

sediment were 89 and 86%, respectively with between-day variability of 6.7%.

2.2.5 Calculations and statistics

2.2.5.1 Partitioning and chemical persistence

Chemical analysis results from sediment and water in the microcosm experiment at each

sampling time are reported as the quantity recovered, concentration, the percent of the quantity

recovered, and the percent remaining of the applied concentration. The half-lives (t1/2) of the

chemicals were calculated using a standard first-order rate equation (1) (Ericson, 2007; Kwon

61

and Armbrust, 2006; Tariq et al., 2014), applied to the data following the observance of a peak

concentration to ensure more conservative results.

[𝐴] = [𝐴]0𝑒−𝑘𝑡 (1)

Where: [A] : concentration of the substance at time t; [A]0 : concentration of the substance

at time 0; and k : rate of decay.

And, the half-life is estimated as follows:

𝑡12

=ln 2

𝑘 (2)

Half-life values are reported with p-values to assess the fit of the model used in the

analysis. All statistical analyses were done using RStudio (version 1.0.44).

2.2.5.2 Toxicity tests

Calculations and statistical analyses were performed using the Comprehensive

Environmental Toxicity Information System CETIS (version 1.8.7.16, Tidepool Scientific LLC.).

Point estimate techniques were used to calculate endpoints (IC50, LC50, and EC50) and

LOEC/NOEC values were determined using appropriate hypothesis testing techniques per the

test protocols.

2.3 Results and discussion

2.3.1 Chemical partitioning and persistence

The fate and persistence testing was done based on the OECD 308 protocol (OECD,

2002). Modifications to the protocol were made in order to simplify testing, similar to

methodologies reported or recommended in the literature (Benskin et al., 2016; Ericson, 2007;

Lyons et al., 2014), as noted in the methodology section. A summary of the sediment and water

characteristics used in the current study is provided in Table 9. Sediment characteristics vary from

site to site, as confirmed by the results of field sampling in British Columbia (Winsby et al., 1996;

Wright et al., 2004). Notably, a reference site, as used in the current study might differ from

sediment under an aquaculture site in that sediment collected from beneath farms is more likely

to be anoxic, have high levels of sulphides, ammonia or methane or be as high in organic carbon

(Winsby et al., 1996; Wright et al., 2007).

62

Table 9: Summary of sediment and water characteristics used in partitioning and persistence study.

Characteristic (units) Average value

Sediment Moisture (%) 29

pH 7.5

Temperature (ºC) 10

Redox (mV) -20.1

Total organic carbon (TOC) (mg kg-1) 9350

Total organic matter (TOM) (%) 4.1

Microbial Biomass - Aerobic Plate Count (CFU g-1) 10300

Microbial Biomass - Mould Plate Count (CFU g-1) 590

Sand by hydrometer (%) 63

Silt by hydrometer (%) 32

Clay content (%) 5

Water pH 7.6

Redox (mV) -29.3

02 (mg L-1) 6.7

Salinity 23.5

Total organic carbon (TOC) (mg L-1) 5.2

The partitioning of the chemicals with time in sediment and water media are presented in

Figure 9. In short, substances with lower partition coefficients (log KOW) and higher solubility in

water (i.e AZ with a log KOW value of 1.05 and water solubility of 1,100,000 g L-1 [Burridge et al.,

2005; Burridge and Van Geest, 2014] and HP with a log KOW value of <0 and high affinity for water

[Lyons et al., 2014]) showed greater tendency to be recovered from the water phase, whereas,

more hydrophobic substances with higher partitioning coefficients showed greater tendency to be

recovered from the sediment phase (i.e. CP with a KOW value of 5.9 and water solubility of 10 -

200 g L-1 [Clark et al., 1987], DM with a log KOW value of 4.6 and water solubility of < 5 g L-1

[EC, 2002] , and EB with a log KOW value of 5 and water solubility of 5,500 g L-1 in seawater

[Lumaret et al., 2012]). Additionally, the results obtained in the current study are consistent with

the results of field sampling where sampling from aquaculture sites using EB-based treatments

(Ikonomou, 2011) and pyrethroids-based treatments (Benskin et al., 2016; Lao et al., 2012; Muir

et al., 1985; Weston et al., 2004; 2011) have detected these compounds in sediments; the

presence of AZ has been detected in water samples collected from the vicinity of aquaculture

sites following treatment with this chemical as well (Ernst et al., 2001).

AZ, which is generally believed to remain mostly in the aqueous phase (Burridge, 2013),

was almost undetectable in the sediment throughout the experiment (0.1 - 0.4 % of the applied

dose) and was almost undetectable in water by the end of the experiment (0.1 % of the applied

63

dose remained at 98 d). The partitioning of pyrethroids to sediment has been reported by others

(Muir et al., 1985; Palmquist et al., 2012). In the current study, 49 % of the applied DM dose had

partitioned into the sediment after 24 h and by test termination almost all that remained of the

substance had partitioned to the sediment (92 %). Similarly, at termination only 3 % of the applied

dose of CP remained in water, with 49 % of the applied dose remaining in sediment. Surprisingly

however, partitioning of CP into the sediment phase was slower than anticipated (slower than the

partitioning of DM). Given their similar chemical properties, more similar results would have been

expected. Finally, for EB, which has a tendency to sorb into particulate matter (Bright and Dionne,

2005; Lumaret et al., 2012; Reddy, 2012), 89 % of the applied dose had partitioned into the

sediment within the first 24 h and by the 48 h sampling time, it was undetectable in water.

Partitioning can be influenced by a number of factors, the primary influences being the

characteristics of the sediment, water and the substance itself (Zhou et al., 1995); it is therefore

difficult to compare the results of the present research to other studies without consideration of

these factors. However, given that the current study was done using the same conditions for all

of the chemicals tested the results provide an opportunity to compare the results.

64

Figure 9: Distribution of chemotherapeutants among sediment (grey bars) and water (white bars) at different sampling times presented as a percentage (%) of the total administered dose. A) AZ; B) EB; C) CP; and D) DM.

The half-life estimates for the chemicals were obtained assuming first-order kinetics and

are summarized in Table 10. The decay for each substance in water and sediment, is presented

in Figure 10 and Figure 11, respectively.

Table 10: Half-life (and p-values) for AZ, CP, DM, EB, and HP in water and sediment calculated using first-order rate equations. P-value <0.05 indicates relationships between the variables that are statistically significant.

AZ CP DM EB HP

Half-life (water)

12.7 d (p=0.00002)

19.8 d (p=0.001)

17.9 d (p=0.003)

N/A 8.9 h (p=0.004)

Half-life (sediment)

N/A 557.2 d (p=0.03)

45.2 d (p=0.004)

230.0 d (p=0.001)

N/A

65

In the current study HP was the least persistent of all the chemicals tested. An estimated

half-life of 8.9 h was obtained which is similar to half-life values reported elsewhere (1.3 - 5.3 h in

aerobic biotic conditions [US EPA, 2007]). However, depending on the test conditions, reported

half-lives vary and include values on the order of days in seawater (between 1 and 28 d) (Bruno

and Raynard, 1994; Lyons et al., 2014; Pentasne and Zika, 1997; US EPA, 2007). HP

decomposition increases with increasing pH (US EPA, 2007), is susceptible to photolysis (US

EPA, 2007), and shows temperature dependent decay (Bruno and Raynard, 1994; Lyons et al.,

2017) and therefore variability among reported results is not unexpected. Persistence in sediment

was not estimated as a result of low partitioning to that media. Rapid decomposition via biological

and chemical pathways provide explanation for the short half-life of HP in water reported in the

current study (PMRA, 2014). Photolysis is another major decomposition pathway for HP,

however, the current study as performed in the absence of light thus making the results more

conservative.

AZ was the next least persistent substance with an estimated half-life of 12.7 d in water,

which is similar to the value of 8.9 d in seawater reported by Burridge (2013). Persistence in

sediment was not estimated as a result of low partitioning to that media. AZ’s physical/chemical

properties (i.e. octanol-water partitioning coefficient) and susceptibility to hydrolysis in water

provide explanation for the results reported in the current study (Burridge and Van Geest, 2014;

Burridge et al., 2010).

DM was found to have a half-life of 17.9 d in water and 45.2 d in sediment, values that are

similar to reported values (e.g. 11.7 - 44.6 d in sediment for aerobic water-sediment systems

[Meyer et al., 2013]). Others have reported half-lives as short as 2 - 4 h in small artificial outdoor

ponds (Muir et al., 1985).

The calculated half-life of CP in water (19.8 d) is much shorter than the half-life in sediment

(557.3 d). This is consistent with reports of CP having a shorter half-life in water than in sediment

where it is more persistent (Burridge and Haya 1997; Haya et al., 2005; Muir et al., 1985).

However, the result obtained in the current study is much longer than the values reported by

Meyer et al. (2013) (half-life in sediment of 3 - 14.1 d in aerobic water-sediment systems). Again,

a number of factors, including temperature, moisture content, soil type, dissolved organic matter

(DOM), and presence of microorganisms and metals, among others have been shown to affect

the half-life of CP in sediment and soil (Ismail et al., 2012; Palquist et al., 2012; Rafique and Tariq,

2015; Remucal, 2014).

66

Pyrethroids have a tendency to sorb to sediments (Ernst et al., 2014; Maund et al., 2002;

PMRA, 2015) where they are more persistent (Burridge and Haya 1997; Haya et al., 2005; Muir

et al., 1985) than in water where they tend to hydrolyze (Burka et al., 1997; Roth et al., 1993).

They also decompose readily due to their susceptibility to catabolic enzymes and sunlight

(Burridge and Van Geest, 2014; Roth et al., 1993), and therefore, the results presented in the

current study are conservative.

The slow disappearance of EB in sediment resulted in an estimated half-life of 230 d which

is similar to values reported by others (193.4 – 250 d) in aerobic soil [Burridge et al., 2010; SEPA

1999; SEPA 2004; US EPA, 2009]), however others have reported values as short as 63 - 71 d

in aerobic soils in the dark (EFSA, 2012). Photodegradation is a known decomposition pathway

for EB (Tariq et al., 2014) and therefore studies performed in the presence of light may have half-

lives shorter than similar experiments performed in the dark. As previously noted, there are also

a number of other factors which can contribute to the stability of the AI and result in differences

between studies. A half-life was not estimated in the water phase due to low partitioning in that

media. These results can be explained by EB’s low water solubility (Bright and Dionne, 2005;

Lumaret et al., 2012; Reddy, 2012).

Additives in formulations are thought to influence the properties of an AI (e.g. solubility,

toxicity, fate, persistence, etc.). The following half-lives of the AIs in formulation have been

reported in the literature: Salmosan® (AI: AZ), 9 - 50 d (Mayor et al., 2008); Excis® (AI:CP), 35 -

80 d (Mayor et al., 2008); Alphamax® (AI:DM), 285 d (Benskin et al., 2016); Paramove® 50 (AI:

HP), 8 - 19 d (Lyons et al., 2014); and Slice® (AI: EB), 164 - 175 d (Mayor et al., 2008) and > 400

d (Benskin et al., 2016). Consistent with general belief that AIs in formulation are more stable than

the chemicals alone (Burridge, 2013; Lyons et al., 2014; Mayor et al., 2008; Meyer et al., 2013;

US EPA, 2007), the majority of the half-lives reported herein are shorter than most of those

reported in the literature for the AIs in formulation. Notably however, one study reported no

statistical differences between the half-life of EB in Slice® and EB alone or DM in Alphamax® and

DM alone (Benskin et al., 2016).

Overall, the persistence of chemicals from greatest to least in water was: CP > DM > AZ

> HP. In sediment, the following trend was observed: CP > EB > DM. As with partitioning, a

number of factors influence the breakdown of a substance, primarily the physical/chemical

properties of the substance itself and the environment. Examples include temperature (Lyons et

al., 2014), the presence of biotic communities (Lyons et al., 2014), organic content (OECD, 2002)

67

and UV exposure (Tariq et al., 2014), among others. Because the current study was done using

the same conditions for all of the chemicals tested the results provide a unique opportunity to

compare the results more directly.

Figure 10: Decay figures for A) AZ; B) CP; C) DM; and D) HP in water shown as concentration (µg L-1 [AZ, CP and DM] and mg L-1 [HP]) versus time. The trend lines represent the decay per standard first-order rate equations.

68

Figure 11: Decay figure for A) CP; B) EB; and C) DM in sediment shown as

concentration (g 100 g-1 sediment) versus time. The trend lines represent the decay per standard first-order rate equations.

2.3.2 Toxicity tests

2.3.2.1 Giant kelp germination and growth

The results of the giant kelp germination and growth test are presented in Table 10. NOEL,

LOEL and IC50 values (with 95 % CI) are presented for the growth endpoint; and NOEL, LOEL

and EC50 values (with 95 % CI) are presented for the germination endpoint. In the current study

only HP demonstrated any measureable toxicity to kelp. HP has been considered for use as a

treatment to control harmful algal blooms (Barroin and Feuillade, 1986; Burson et al., 2014) and

is known for its toxicity to algae (PMRA, 2014). Toxicity in the current study was observed at

concentrations that are much lower than the recommended treatment concentration of 1,200,000-

1,800,000 g L-1 (EC50 4,500 g L-1 and IC50 3,700 g L-1) demonstrating potential risk for kelp in

69

the vicinity of salmon aquaculture sites using HP to treat sea lice infestations, a fact consistent

with suggestions by others in the past (Barroin and Feuillade, 1986). For the other 4 chemicals at

the concentrations tested no negative impacts on kelp germination and growth were seen. Several

researchers have suggested that, in general, pesticides may either have negative effects on spore

germination in algae (e.g. organophosphates [Agrawal, 2009]) however some have reported

growth stimulation in phytoplankton (e.g. ivermectin [Garric et al., 2007] and CP [Wang et al.,

2010]).

The results of the current study, and of others, suggests that when used to treat sea lice

infestations, HP may result in toxicity to algal species in the vicinity. Toxicity to macroalgal species

should be avoided as they often provide valuable coastal habitats for a multitude of organisms

(XXX). The use of HP may however help control harmful algal blooms that may occur under farms.

2.3.2.2 Topsmelt survival

The results of the topsmelt survival test are presented in Table 10. NOEL, LOEL and LC50

values (with 95 % CI) are presented for the survival endpoint. For the control treatment group,

survival was 90 %. The results of the current study suggest that topsmelt are sensitive to the

chemicals tested in the current study with the greatest sensitivity being to CP followed by DM >

EB > AZ > HP. LC50 values obtained for HP and DM are lower than the recommended treatment

concentrations (Table 6). Extreme sensitivity was observed for CP exposure where complete

mortality was observed after 24 h; it was not possible to determine an LC50 value. The lowest

concentration tested for CP (3 g CP L-1) is almost half the recommended application

concentration (5 g CP L-1). This was unexpected based on range finder experiments where 100%

survival was observed at 5 g CP L-1 after 48 h. No studies in the literature have reported the

effect of any chemotherapeutant or chemical used in salmon aquaculture on topsmelt.

The results of the current study suggest that the use of HP, DM and CP may introduce

unwanted risks to topsmelt in the vicinity. This is combination with the low margin of safety for the

use of DM (0 – 3.5x) and HP (0 – 3x) in the treatment of Atlantic salmon suggests that the entire

Actinoptrerygii class of fish might be at risk. Their use in areas where wild salmon populations (or

other ray-finned fish) are present should be minimized.

2.3.2.3 Mysid survival

The results of the mysid test are presented in Table 10. NOEL, LOEL and LC50 values

(with 95 % CI) are presented for the survival endpoint. For the control treatment group, survival

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was 92 %. A 96-h LC50 value of 610 g EB L-1 was obtained in the current study; a similar study

reported a 96-h LC50 value of 0.04 g EB L-1 in formulation for the same species (Lumaret et al.,

2012). The differences observed may be attributable to the use of the formulations rather than

the chemical alone. Subtle differences in the test conditions (e.g. age of the organisms) could

also contribute to these differences. A 96-h LC50 value of 40 g CP L-1 was obtained in the current

study; a similar study reported 0.05 g CP L-1 for the same species (Clark et al., 1989). 96-h LC50

values of 16 g DM L-1, 7,700 g HP L-1, and 1,218 g AZ L-1 were also obtained in the current

study; for HP, DM, and AZ, no comparable values have been published in the literature. Overall,

these results show that mysids have lower sensitivity to HP and AZ than to the other chemicals

tested. The LC50 values reported in the current study for HP, DM and CP are below the

recommended treatment concentrations for their respective formulations but not for AZ (Table 6).

The results of the current study are unsurprising given that sea lice and mysids are both

crustaceans. Toxic effects to crustaceans following the use of sea lice pesticides have already

been documented by others (e.g. Burridge et al. [1999]). In general, because substances that

sorb to sediment have a tendency to be more persistent, benthic organisms are at greater risk of

toxicity .

2.3.2.4 Bivalve embryo survival and development

The results of the bivalve test are presented in Table 10. NOEL, LOEL and EC50 values

(with 95 % CI) are presented for the normality endpoint and combined endpoint of normal survival;

and NOEL, LOEL and LC50 values (with 95 % CI) are presented for the survival endpoint. The

results of the current study indicate low sensitivity to CP, DM and AZ. CP and DM did not have

an effect on bivalve survival and development at the concentrations tested (up to 500 g CP L-1

and 100 . g DM L-1). Other studies using bivalves have also demonstrated low sensitivity to CP

(Clark et al., 1989) and AZ (Canty et al., 2007; Roth et al., 1993; Van Geest et al., 2014a), however

the results presented by Canty et al. (2007) suggest that more subtle effects may be occurring

(e.g. neurotoxicity and immunotoxicity). For AZ, a 48-h LC50 value of > 12,500 g AZ L-1 and a 48-

h LC50 value of 2,9000 g HP L-1 was obtained in the present study. A 48-h LC50 of 1,605 g EB

L-1 was determined which demonstrates a relatively low sensitivity of bivalves to EB, a fact

reported elsewhere (a 48-h LC50 of > 713 g EB L-1 has been reported for M. galloprovincialus

[Park, 2013]). For the chemotherapeutants that are applied as a water bath, the reported LC50

values are all greater than the doses recommended in aquaculture practices (Table 6). Both the

71

information in the literature and the results of the current study suggest that bivalves are not very

sensitive to the toxic effects of chemicals used to treat sea lice in salmon aquaculture.

2.3.2.5 Echinoderm fertilization

The results of the echinoderm fertilization test are presented in Table 10. NOEL, LOEL

and IC25 values (with 95 % CI) are presented for the fertilization endpoint. For the control treatment

group, the average success of fertilization was 86 %. Fertilization was affected by all of the

chemicals tested, however, fertilization effects were insufficient to calculate an IC25 value for the

AZ treatments. The only information available in the literature in this regard was a report of low

sensitivity of echinoderms to AZ (Burridge and Van Geest, 2014; Ernst et al., 2001). A 20-min

fertilization IC25 value of 3340 g AZ L-1 in formulation was reported for effects on fertilization in

L. pictus (Ernst et al., 2001). This suggests that L. pictus may be more sensitive than S. purpuratus

(in this study: IC25 of > 12,500 g AZ L-1). However, formulations were used in the study by Ernst

et al. (2001). For all other chemicals that are applied in a water bath, effects on fertilization

occurred at concentrations below those used in salmon aquaculture (Table 6). Echinoderms are

benthic organisms that release their gametes into the water column. Ultimately the gametes settle

to the substrate. As such, substances dissolved in the water column and sorbed to sediment may

pose a risk, if bioavailable. Given the short duration of this test, the results of the current study

demonstrate real potential risk for echinoderms in the vicinity of aquaculture sites.

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Table 10: Summary of results (in g L-1) for the 20-min echinoderm (S. purpuratus) fertilization assay, the 48-h kelp (M. pyrifera) germination and growth assay, the 96-h topsmelt (A. affinis) survival assay, performed using 9 d-old topsmelt, the 96-h mysid (M. bahia) survival assay, performed using 5 d-old mysids, and for the 48-h bivalve (M. galloprovincialis) embryo survival and development assay. Results are presented for all five chemicals tested: emamectin benzoate (EB), hydrogen peroxide (HP), cypermethrin (CP), deltamethrin (DM) and azamethiphos (AZ). This includes LC50, IC25, or EC50 values (presented with 95 % CI), NOELs and LOELs for each of the endpoints that were assessed.

Species Endpoint Result EB HP CP DM AZ

Echinoderm (Strongylocentrous purpuratus)

Fertilization IC25 2,100 (1,400-2,500) 2,800 (2,700-3,000) 5 (3-6) 4.2 (3-5) >12,500

NOEL 800 600 <0.2 2 17

LOEL 1,260 1,200 0.2* 5.1 50

Kelp (Macrocystis pyrifera)

Germination EC50 >5,000 4,500 (4,100-4,800) > 50 > 20 >12,500

NOEL 5,000 600 50 20 < 20

LOEL >5,000 3,000 > 50 > 20 20

Growth IC50 > 5,000 3,700 (3,200-4,200) > 50 > 20 >12,500

NOEL <33 < 24 < 1.3 <0.08 < 20

LOEL 33* 24* 1.3* 0.08* 20*

Topsmelt (Atherinops affinis)

Survival LC50 350 (290-420) 172,000 (140,000-211,000)

< 3 1.6 (1-2) 980 (800-1,200)

NOEL 300* 51,180 < 3 1* 600*

LOEL 600 144,970 3* 2 1,250

Mysid (Mysidopsis bahia)

Survival LC50 617 (480-780) 7,700 (5,800-10,100)

41 (36-45) 16 (14-19) 1,218 (1,080-1,370)

NOEL 200 1,110 25 6 750

LOEL 1,000 3,330 50 13 1,500

Bivalve (Mytilus galloprovincialis)

Combined Proportion Normal

EC50 1,060 (1,040-1,080) 2,530 (2,500-2,550) > 500 > 100 5,350 (5,210-4,490)

NOEL 600 1,900 500* 100* 3,100

LOEL 1,250 3,750 > 500 > 100 6,200

Proportion Normal

EC50 1,030 (1,010-1,050) 2,020 (1,970-2,060) > 500 > 100 6,010 (5,800-6,220)

NOEL 6,00 100 500* 100* 3,100

LOEL 1,250 200 > 500 > 100 6,200

Survival LC50 1,605 (1,580-1,630) 2,900 (2,800-2,920) > 500 > 100 >12,500

NOEL 1,250 1,900 500* 100* 3,100

LOEL 2,500 3,750 > 500 > 100 6,200

*lowest concentration tested

73

2.4 Conclusions

Understanding the fate and persistence of the 5 chemicals used prominently as

AIs to treat sea lice infestations in water-sediment systems is the first step to identifying

the organisms most at risk in the marine environment following their application, and the

potential exposure durations that they may face in real world scenarios. The results

confirm that EB, CP and DM partition mainly to marine sediments (benthic organism at

risk), and that AZ and HP will remain associated with water phase (pelagic organisms at

risk). The chemicals that partitioned mainly into sediment are relatively persistent

indicating that exposure durations for benthic species are expected to be much longer

than for pelagic species exposed to more water soluble chemicals. Future research may

consider focusing on the bioavailability of these substances to various organisms in water

and when sorbed to the sediment.

The results of bioassays are useful in calculating the risks to non-target organisms

using toxicity tests with representative classes of marine organisms that may be exposed

near aquaculture operations. A lack of susceptibility trends for any species, or toxicity

trends with any one chemical were found, but, generally, toxicity occurred at water

concentrations that were above the target treatment concentrations. However, some of

the values obtained for echinoderms (IC25), kelp (IC50), and topsmelt (LC50) are below the

recommended treatment concentrations indicating a high potential risk to those species.

These risks are amplified for more persistent chemicals that have a tendency to partition

into the sediment. The information generated here indicates that consideration of the

bioavailability and toxicity of sediment-bound chemicals to benthic species should be

made to adequately determine the consequences of chemotherapeutant use in

aquaculture in this regard.

74

Chapter 3. Overall conclusions and future directions

3.1. Overall Conclusions

The current study provides much needed information regarding the fate and

persistence in water-sediment systems of 5 active ingredients in chemotherapeutants that

are or have been used in salmon aquaculture while also providing information on their

toxicity to various non-target organisms. Information on the environmental fate and

persistence of these chemicals is critical to understanding exposure risks associated with

their use in salmon aquaculture and toxicity tests help to identify toxic thresholds to to non-

target organisms that live near aquaculture operations.

The results of the environmental fate and persistence testing confirm what is

reported in the literature such that it highlights the tendency for EB, CP and DM to partition

into the sediment; AZ and HP remained in the water phase and do not associate with

sediments. In sediment, CP was found to have the greatest half-life (557.2 d), followed by

EB (230.0 d), with DM being the least persistent in that media (45.2 d). In water, CP had

the greatest half-life (19.8 d), followed by DM (17.9 d), AZ (12.7 d), with HP (8.9 h) being

the least persistent. This this partitioning behaviour explains why EB (Ikonomou, 2011)

and pyrethroids (Benskin et al., 2016; Lao et al., 2012; Muir et al., 1985; Weston et al.,

2004; 2011) are found in sediments and AZ (Ernst et al., 2001) in water during field

sampling activities. The combined environmental fate and persistence data in the current

study show that the chemicals that have a tendency to partition into sediment are more

persistent than the more hydrophilic chemicals indicating higher potential risk to benthic

dwelling species.

Marine ecosystems are complex, consisting of numerous biotic and abiotic

components linked by a series of ecological interdependencies. Although some species

play more important roles than others (e.g. keystone species vs. redundant species), the

complex web of interactions in the natural environment makes it very important to protect

as many non-target organisms from toxic effects as reasonably possible. This reinforces

the need to better understand risks associated with the use of sea lice pesticides. In the

current study, differences in sensitivity to sea lice pesticides from species to species were

75

observed for all chemicals tested. As previously mentioned, the current study shows the

lack of susceptibility trends in any species, or toxicity trends with any one chemical. This

information is useful for identifying high risks to specific groups of organisms including

echinoderms, kelp and some developing fish (topsmelt) for which reported endpoints (IC25,

IC50 and LC50, respectively) are below the recommended treatment concentrations for

some of the pesticide formulations. The high sensitivity to topsmelt along with the low

margins of therapeutic safety for salmon (Bright and Dionne, 2005), raises concerns about

the potential for sea lice pesticides to elicit adverse effects to non-target fish species in

general.

Typically studies focus on one, or several pesticides, chemicals or organisms,

whereas, the current study included multiple chemicals and organisms. Because these

individual tests were done using similar procedures, it provides a means to do side-by-

side comparisons of the different chemicals while reducing uncertainty.

3.2. Future Directions

The current study investigated chemicals used as AIs in several

chemotherapeutants, however, other studies have used the formulations themselves (e.g.

Lyons et al. [2014], Mayor et al. [2008], and Burridge et al. [2014b]). As previously

mentioned, the formulations often behave differently than the active ingredient alone

(Burridge, 2013; Lyons et al., 2014; Mayor et al., 2008; Meyer et al., 2013; US EPA, 2007).

Future work should include simultaneous studies with formulations and the AI’s alone.

This would aid in understanding if synergistic or antagonistic effects of the ingredients

contained within the formulations exist. In nature, non-target organisms are likely exposed

to a multitude of toxicants both from the aquaculture sites (e.g. antibiotics, pesticides,

antifungals, etc.) as well as from other anthropogenic sources (e.g. sewage, spills, etc.)

and so mixture exposures may be necessary. For example, one study reported complex

toxic interactions in testing using sediment samples that were contaminated with a variety

of pyrethroid chemotherapeutants; it was noted that these interactions make it very difficult

to predict toxicity (Lao et al., 2012). The toxic loading that non-target organisms are

exposed to is difficult to predict, however a better understanding of the complexity of the

interactions between toxicants could help enable us to make better decisions and provide

greater protection to the non-target organisms.

76

The current study found that chemicals that have a tendency to partition into

sediment are more persistent than the more hydrophilic chemicals. Therefore, it is

recommended that future studies focus on the bioavailability of these chemicals in

sediment (in water-sediment systems). Similarly, more work to understand the

bioavailability of EB in fish feed would help understand the link between the water route

of exposure commonly used in toxicity tests and the exposure via faeces and food pellets

in nature. This information is critical to interpreting the environmental risks.

The results of the current study indicate that future studies may also examine

subtle sublethal endpoints, including behavioral endpoints (as these can have important

adverse impacts in nature). This recommendation was also listed in a review published by

the DFO (Burridge and Van Geest, 2014). For example, one study examined shelter use

among juvenile lobsters and found that repeated short-term pulse exposure to AZ resulted

in increased shelter use, a behavior which decreases vulnerability towards predators while

putting a halt to foraging behavior (Abgrall et al., 2000).

To assess the environmental consequences of aquaculture chemical use, it is

important to generate data on their environmental persistence, and acute and sublethal

toxicity to marine organisms. This research targeted specific research experiments to

yield information that would be useful to ensure the proper and safe use of aquaculture

chemicals, and to appropriately regulate this important industry.

77

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93

Appendix A. Summary of key properties for test substances

Table A1: Summary of Key Properties for Test Substances.

Azamethiphos Cypermethrin Deltamethrin Emamectin Benzoate

Hydrogen Peroxide

CAS 35575-96-3 52315-07-8 52918-63-5 155569-91-8 7722-84-1

Molecular Formula

C9H10ClN2O5PS C22H19Cl2NO3 C2H19Br2NO3 C56H81NO15 H2O2

Structural Formula

Molecular Weight (g mol-1)

324.68 416.3 505.2 994 - 1008.24 34.01

Purity 99.5% >98% 99.6% 99.4% 30%

Source Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich

Log Kow 1.05 f, g 5.9 e 4.6a 5 c < 0 d

Solubility (mg L-1)

1100 f, g < 0.009 b (20°C) 0.01-0.2 e

0.0002 a < 0.005 a

(25 & 20°C)

24-320 c 5.5 (in seawater)

high affinity for water d

Completely miscible h

Vapour Pressure (Pa)

low 2.3 x 10-7 b

(at 20°C) 1.24 x 10-8 a

(at 25°C) 4 x 10-6 c

200 h

(30 °C)

pKa - does not dissociate b

does not dissociate a

- 11.62 h

Photolysis in Water (days)

- 5.1 – 221 b (pH 7-8)

stable (pH 7) 31 (pH 8) a

0.7 – 35.4 c -

Half-Life (days)

8.9 g 48-88 b (20-22°C)

26 a (at 25°C)

174 c 8 hrs - > 6 days d

Formulation Dose (mg L-1)

0.1 f, g

0.005 g 0.002 - 0.003 g 0.05 mg/kg/day i (for 7 days)

1200 - 1800 d

a EC (2002) b EC (2005) c Lumaret et al. (2012) d Lyons et al. (2014) e Clark et al. (1987) f Burridge et al. (2005) g Burridge and Van Geest (2014) h PMRA (2014) i Bright and Dionne (2005)

94

Appendix B. Raw Data: Fate and persistence testing

Table B1: Summary of results from the fate and persistence test (HP).

Time (h) Concentration (mg L-1)

0.5 75.2

1 62.2

5 50.1

10.5 13.8

27.5 3.5

46.25 2.5

95

Table B1: Summary of results from the fate and persistence tests (AZ, CP, DM, and EB).

96

Appendix C.

Raw Data: Toxicity tests

Table C1: Raw data for kelp toxicity test – hydrogen peroxide (nominal).

Concentration Rep

No. Germi-nated

No. Not Germi-nated

Tube Length (m)

1 2 3 4 5 6 7 8 9 10

SW Control A 76 24 11 9 11 12 10 8 11 10 8 10 B 80 20 9 7 8 10 15 9 11 7 11 10 C 79 21 10 8 12 9 9 9 10 12 11 10 D 67 33 8 10 10 10 14 9 10 8 9 10

E 66 34 8 9 10 11 8 10 9 10 10 11

HP C5 A 82 18 6 7 8 7 11 12 10 8 10 8 (0.024 mg L-1) B 61 39 10 12 10 6 8 10 8 8 9 11

C 67 33 10 11 8 12 8 6 9 11 8 10 D 78 22 8 12 10 8 8 12 10 12 7 9 E 75 25 9 9 8 10 12 9 10 8 8 10

HP C4 A 63 37 6 6 8 8 8 8 10 10 8 9 (0.12 mg L-1) B 74 26 8 7 9 8 6 10 12 8 8 10

C 68 32 12 6 10 7 8 7 7 9 7 9 D 67 33 8 6 9 8 10 8 9 9 8 8 E 60 40 8 8 13 11 8 11 10 8 8 9

HP C3 A 71 29 6 10 10 8 10 7 10 8 7 8 (0.6 mg L-1) B 72 28 6 8 10 8 8 10 7 5 8 9

C 79 21 8 10 8 5 6 9 6 10 7 11 D 70 30 9 7 10 6 8 8 6 8 11 9 E 50 50 11 6 6 8 10 6 8 9 10 7

HP C2 A 64 36 6 7 4 7 4 7 5 6 8 7 (3 mg L-1) B 72 28 5 6 5 4 6 5 6 7 4 4

C 66 34 4 5 4 7 4 4 6 5 7 6 D 53 47 8 4 5 6 6 8 5 6 6 5 E 64 36 7 5 6 4 6 4 6 5 6 6

HP C1 A 0 100 0 0 0 0 0 0 0 0 0 0 (15 mg L-1) B 0 100 0 0 0 0 0 0 0 0 0 0

C 0 100 0 0 0 0 0 0 0 0 0 0 D 0 100 0 0 0 0 0 0 0 0 0 0 E 0 100 0 0 0 0 0 0 0 0 0 0

97

Table C2: Raw data for kelp toxicity test – emamectin benzoate (nominal).

Concentration Rep

No. Germi-nated

No. Not Germi-nated

Tube Length (m)

1 2 3 4 5 6 7 8 9 10

SW Control A 74 26 9.5 8 10 10 10 10 10 8 10 12 B 81 19 8 11 10 12 12 10 7 8 11 10

C 76 24 15 9 10 8 13 10 11 10 11 8 D 67 33 9 8 11 11 9 11 8 9 11 10

E 61 39 10 10 12 12 6 8 10 7 12 8

MeOH Control A 71 29 8 7 13 8 10 10 10 9 10 8 B 78 22 9 10 12 8 9 11 10 8 13 8 C 73 27 10 9 11 10 8 10 9 10 8 11 D 78 22 10 11 7 9 10 10 12 7 8 9 E 62 38 10 12 12 9 10 7 10 10 9 8

EB C5 A 70 30 12 8 8 8 11 8 10 8 8 10 (0.03 mg L-1) B 72 28 12 10 9 8 7 10 10 8 8 12

C 70 30 8 7 10 9 8 11 6 9 5 12 D 74 26 11 9 10 8 7 8 9 10 8 9 E 70 30 10 9 10 11 7 8 8 7 8 10

EB C4 A 77 23 9 11 8 12 11 8 9 8 7 8 (0.1 mg L-1) B 65 35 8 9 8 10 11 9 8 8 7 8

C 81 19 11 8 9 10 10 7 12 10 9 8 D 83 17 8 10 8 8 7 10 8 9 10 10 E 80 20 9 13 14 6 9 6 8 8 9 6

EB C3 A 78 22 10 8 12 9 11 10 7 10 6 10 (0.4 mg L-1) B 78 22 8 8 9 8 8 8 9 10 11 10

C 80 20 8 9 8 10 9 10 7 10 10 11 D 86 14 11 7 8 8 9 11 10 12 8 12 E 88 12 5 8 8 8 10 7 10 8 8 7

EB C2 A 88 12 10 7 10 7 10 8 6 12 11 12 (1.4 mg L-1) B 86 14 12 11 10 8 12 11 8 6 11 8

C 84 16 8 13 9 8 10 10 7 8 8 10 D 85 15 6 8 8 8 7 1 48 9 6 7 E 84 16 10 10 10 10 12 10 9 5 8 12

EB C1 A 76 24 7 10 8 9 9 7 12 6 8 8 (5 mg L-1) B 67 33 8 10 8 11 8 10 6 7 10 9

C 76 24 8 14 11 9 7 10 8 8 6 7 D 73 27 8 10 8 10 10 12 10 8 10 9 E 69 31 7 7 11 11 8 10 8 5 8 7

98

Table C3: Raw data for kelp toxicity test – cypermethrin (nominal).

Concentration Rep

No. Germi-nated

No. Not Germi-nated

Tube Length (m)

1 2 3 4 5 6 7 8 9 10

Acetone A 76 24 11 9 11 12 10 8 11 10 8 10 Control 1 B 80 20 9 7 8 10 15 9 11 7 11 10 C 79 21 10 8 12 9 9 9 10 12 11 10 D 67 33 8 10 10 10 14 9 10 8 9 10

E 66 34 8 9 10 11 8 10 9 10 10 11

Acetone A 81 19 8 12 9 9 8 10 10 12 10 11 Control 2 B 72 28 14 10 7 8 11 8 11 9 10 9 C 77 23 6 10 10 11 8 11 9 9 12 10 D 67 33 8 12 12 9 7 10 8 10 11 10 E 73 27 10 8 11 10 11 11 7 11 8 8

CP C5 A 70 30 7 8 12 8 9 8 10 10 8 10 (0.001 mg L-1) B 70 30 8 10 7 8 7 7 9 8 7 10 C 76 24 8 11 6 9 9 12 10 8 12 8 D 78 22 8 10 9 10 8 10 7 8 10 7 E 74 26 8 7 13 8 7 11 10 10 10 7

CP C4 A 76 24 7 10 11 6 7 10 10 10 7 10 (0.003 mg L-1) B 70 30 10 8 8 10 6 8 7 10 6 9

C 80 20 8 12 10 9 6 10 7 9 10 12 D 90 10 10 6 14 10 6 10 10 11 9 8 E 71 29 10 7 12 8 9 8 7 8 8 10

CP C3 A 71 29 11 8 9 8 8 10 9 8 7 10 (0.008 mg L-1) B 80 20 10 6 11 7 8 12 8 6 8 10 C 74 26 8 7 6 12 8 8 12 10 10 8 D 64 36 10 9 6 7 8 10 7 11 10 8 E 51 49 8 8 6 8 12 7 10 10 7 10

CP C2 A 71 29 12 12 10 7 8 9 8 8 8 10 (0.02 mg L-1) B 70 30 10 7 8 9 9 8 10 7 10 11

C 82 18 9 7 6 10 12 7 14 8 9 10 D 86 14 10 10 9 10 10 9 7 12 7 10 E 51 49 6 8 7 12 9 6 8 8 6 7

CP C1 A 81 19 11 9 10 9 8 11 10 10 6 10 (0.05 mg L-1) B 65 35 8 10 8 10 9 6 8 12 8 8 C 67 33 8 6 8 8 8 9 11 9 6 8 D 75 25 8 6 10 12 6 8 11 9 8 7 E 90 10 9 9 10 7 10 8 12 7 9 10

99

Table C4 - Raw data for kelp toxicity test – deltamethrin (nominal).

Concentration Rep

No. Germi-nated

No. Not Germi-nated

Tube Length (m)

1 2 3 4 5 6 7 8 9 10


E 66 34 8 9 10 11 8 10 9 10 10 11


DM C5 A 66 34 9 10 11 8 9 8 9 9 6 6

(0.00008 mg L-1)

B 70 30 8 11 9 8 7 8 8 9 7 8

C 74 26 13 10 8 8 8 6 8 8 6 9 D 78 22 10 10 10 8 7 8 8 8 8 8 E 70 30 9 10 9 11 7 8 10 12 8 8

DM C4 A 70 30 8 8 12 12 10 6 8 12 8 7

(0.0003 mg L-1)

B 60 40 7 8 10 14 8 10 6 8 8 8

C 88 12 12 7 8 10 8 10 7 8 6 11 D 69 31 8 9 6 10 12 6 7 8 8 7 E 80 20 8 7 10 7 7 7 11 8 10 6

DM C3 A 66 34 8 6 8 9 7 8 6 8 6 8 (0.001 mg L-1) B 78 22 11 7 9 8 8 12 8 8 8 9 C 76 24 8 10 8 9 8 8 6 8 11 8 D 80 20 11 6 8 8 8 9 7 10 6 7 E 75 25 9 8 7 8 8 12 7 8 9 10

DM C2 A 70 30 8 9 9 7 10 11 10 6 10 7 (0.005 mg L-1) B 79 21 8 8 7 9 7 13 11 9 6 6 C 65 35 8 10 6 8 7 8 6 12 8 11 D 77 23 7 9 8 12 8 12 9 7 10 6 E 73 27 10 6 8 10 9 6 7 8 10 9

DM C1 A 78 22 10 7 9 7 8 8 8 10 9 6 (0.02 mg L-1) B 66 34 11 6 10 10 6 7 8 7 6 8 C 62 38 6 8 7 11 9 8 7 10 10 8 D 75 25 8 9 10 6 10 8 10 10 6 10 E 72 28 8 8 8 7 10 6 12 11 8 8

100

Table C5 - Raw data for kelp toxicity test – azamethiphos (nominal).

Concentration Rep

No. Germi-nated

No. Not Germi-nated

Tube Length (m)

1 2 3 4 5 6 7 8 9 10


E 66 34 8 9 10 11 8 10 9 10 10 11


AZ C5 A 69 31 7 6 8 10 8 11 7 10 10 10

(0.02 mg L-1)

B 69 31 11 7 12 10 8 8 10 8 7 10

C 85 15 9 11 11 8 10 5 8 10 8 8 D 62 38 11 8 10 5 6 8 10 7 11 8 E 18 82 8 8 9 8 8 12 13 10 10 8

AZ C4 A 74 26 9 8 8 10 9 8 8 7 10 9 (0.1 mg L-1) B 68 32 6 6 8 8 11 10 6 6 9 6

C 83 17 9 9 8 7 9 8 6 6 7 12 D 48 52 8 8 6 9 9 9 6 8 12 6 E 68 32 6 12 10 9 7 9 8 8 7 9

AZ C3 A 70 30 6 8 6 9 12 9 9 6 7 9 (0.5 mg L-1) B 67 33 9 7 8 11 9 8 8 12 8 9 C 80 20 8 7 10 8 8 8 8 8 12 8 D 81 19 9 10 8 6 12 8 8 12 6 10 E 72 28 10 6 13 9 7 8 9 6 14 10

AZ C2 A 65 35 10 8 10 6 6 6 12 8 10 14 (2.5 mg L-1) B 78 22 5 6 7 10 6 12 10 10 6 11 C 69 31 7 8 10 9 8 8 8 7 8 10 D 71 29 10 8 9 6 8 10 8 6 10 9 E 79 21 10 6 11 8 6 10 7 8 8 6

AZ C1 A 66 34 8 8 10 7 10 9 10 7 9 8 (12.5 mg L-1) B 62 38 8 8 6 5 8 8 7 10 7 9 C 62 38 8 8 10 7 6 10 8 6 8 6 D 66 34 6 8 6 6 8 9 8 8 8 7 E 65 35 8 8 6 6 8 8 8 7 6 6

101

Table C6: Raw data for topsmelt toxicity test – azamethiphos (nominal).

102

Table C7: Raw data for topsmelt toxicity test – deltamethrin (nominal).

103

Table C8: Raw data for topsmelt toxicity test – cypermethrin (nominal).

104

Table C9: Raw data for topsmelt toxicity test – emamectin benzoate (nominal).

105

Table C10: Raw data for topsmelt toxicity test – hydrogen peroxide (nominal).

106

Table C11: Raw data for mysid toxicity test – deltamethrin (nominal).

Concentration Rep Survival Temperature (°C) Dissolved oxygen (mg L-1)

pH Salinity (ppt)

Time (h) 24 48 0 24 48 0 24 48 0 24 48 0 24

DMC5 A 10 10 20 19 20 7.4 7.1 56.1 7.6 7.6 7.6 28 28

(0.003 ug L-1) B 10 10

C 10 10

D 10 9

DMC4 A 10 9 20 19 19 7.4 7.1 6 7.6 7.6 7.6 28 28

(0.006 ug L-1) B 10 9

C 10 10

D 10 8

DMC3 A 10 7 19 19 20 7.4 7.1 6 7.6 7.6 7.6 28 28

(0.013 ug L-1) B 9 6

C 10 5

D 9 8

DMC2 A 9 1 19 19 19 7.4 7.1 6 7.6 7.6 7.7 28 28

(0.025 ug L-1) B 9 3

C 10 3

D 8 2

DMC1 A 9 1 20 19 20 7.4 7.2 6.1 7.6 7.6 7.7 28 28

(0.05 ug L-1) B 10 1

C 7 0

D 9 0

107

Table C12: Raw data for mysid toxicity test – azamethiphos (nominal).


pH Salinity (ppt)

Time (h) 24 48 0 24 48 0 24 48 0 24 48 0 24

AZC5 A 10 9 20 19 20 7.4 7.1 6.1 7.6 7.6 7.6 28 28

(0.18 ug L-1) B 9 10

C 10 10

D 10 10

AZC4 A 10 10 20 19 19 7.4 7.1 6 7.6 7.6 7.6 28 28

(0.375 ug L-1) B 10 10

C 10 10

D 10 10

AZC3 A 10 8 19 19 20 7.4 7.1 6 7.6 7.6 7.6 28 28

(0.75 ug L-1) B 10 9

C 10 10

D 10 9

AZC2 A 9 0 19 19 19 7.4 7.1 6 7.6 7.6 7.6 28 28

(1.5 ug L-1) B 10 5

C 9 3

D 9 3

AZC1 A 2 0 19 19 20 7.4 7.1 6.1 7.6 7.6 7.6 28 28

(3 ug L-1) B 1 0

C 3 0

D 3 0

Cont SW 1 A 10 8 20 19 20 7.4 7 6.3 7.6 7.6 7.6 28 28

B 10 9

C 10 10

D 10 10

Cont SW 2 E 10 10

F 10 10

G 10 10

H 10 10

108

Table C13: Raw data for mysid toxicity test – cypermethrin (nominal).


pH Salinity (ppt)

Time (h) 24 48 0 24 48 0 24 48 0 24 48 0 24

CP C5 A 10 10 19 19 20 7.4 7.1 5.7 7.6 7.6 7.6 28 28

(0.006 ug L-1) B 9 9

C 10 10

D 10 10

CPC4 A 9 9 19 19 20 7.4 7.1 5.6 7.6 7.6 7.6 28 28

(0.01 ug L-1) B 9 9

C 10 10

D 10 10

CPC3 A 10 9 20 19 20 7.4 7.1 5.9 7.6 7.6 7.6 28 28

(0.025 ug L-1) B 10 8

C 10 10

D 10 10

CPC2 A 9 2 20 19 19 7.4 7.1 6.2 7.6 7.6 7.7 28 28

(0.05 ug L-1) B 9 2

C 9 2

D 10 3

CPC1 A 8 1 20 19 19 7.4 7.2 6.2 7.6 7.6 7.7 28 28

(0.1 ug L-1) B 8 0

C 8 0

D 8 0

Control A 10 10 20 19 20 7.4 7.1 6.4 7.6 7.6 7.6 28 28

Acetone 1 B 10 9

C 10 10

D 10 10

Control E 10 10

Acetone 2 F 10 10

G 10 10

H 10 10

109

Table C14: Raw data for mysid toxicity test – emamectin benzoate (nominal).


pH Salinity (ppt)

Time (h) 24 48 0 24 48 0 24 48 0 24 48 0 24

Control SW A 10 10 20 19 19 7 6.9 6.4 7.7 7.7 7.6 28 29

B 10 9

C 10 9

D 10 9

Control MeOH

A 10 10 20 19 19 7 6.9 6 7.7 7.7 7.6 28 29

B 10 9

C 10 10

D 10 10

EB C5 A 10 10 20 19 19 7 6.5 5.3 7.7 7.6 7.6 28 29

(0.008 mg L-1)

B 10 10

C 9 9

D 10 10

EB C4 A 9 9 20 19 19 7 6.6 5.7 7.7 7.6 7.6 28 29

(0.04 mg L-1) B 10 10

C 10 10

D 10 10

EB C3 A 10 10 20 19 19 7.8 6.6 5.4 7.7 7.7 7.6 28 29

(0.2 mg L-1) B 10 10

C 10 10

D 6 7

EB C2 A 10 3 20 19 19 7.8 6.7 5.1 7.7 7.7 7.6 28 29

(1 mg L-1) B 9 3

C 10 2

D 8 1

EB C1 A 0 0 20 19 19 7 6.8 6.2 7.7 7.6 7.6 28 29

(5 mg L-1) B 1 0

C 0 0

D 0 0

110

Table C15: Raw data for mysid toxicity test – hydrogen peroxide (nominal).


pH Salinity (ppt)

Time (h) 24 48 0 24 48 0 24 48 0 24 48 0 24

Control SW A 10 10 20 19 19 7 6.9 6.1 7.7 7.7 7.6 28 29

B 10 10

C 10 10

D 10 10

HP C5 (1.11 mg L-1)

A 10 10 20 19 19 7 6.9 6.1 7.7 7.7 7.6 28 29

B 10 8

C 10 10

D 9 9

HP C4 A 7 7 20 19 19 7 7.2 6.1 7.7 7.7 7.6 28 29

(3.33 mg L-1) B 10 8

C 7 7

D 8 7

HP C3 A 8 0 20 19 19 7 8.5 6.1 7.7 7.7 7.6 28 29

(10 mg L-1) B 10 7

C 8 2

D 9 5

HP C2 A 4 0 20 19 19 7 8.4 9.4 7.7 7.7 7.6 28 29

(30 mg L-1) B 6 0

C 6 0

D 7 0

HP C1 A 0 0 20 19 19 7 8.6 8.7 7.7 7.7 7.6 28 29

(90 mg L-1) B 0 0

C 0 0

D 0 0

111

Table C16: Raw data for bivalve toxicity test – azamethiphos (nominal).

Concentration Rep No. Normal No. Abnormal

AZ C5 A 165 67

(0.8 mg L-1) B 126 53

C 161 65

D 170 48

E 139 59

AZ C4 A 130 46

(1.6 mg L-1) B 136 66

C 176 55

D 145 50

E 158 56

AZ C3 A 131 51

(3.1 mg L-1) B 104 67

C 148 60

D 163 65

E 130 67

AZ C2 A 64 110

(6.2 mg L-1) B 48 130

C 49 121

D 48 119

E 54 117

AZ C1 A 10 101

(12.5 mg L-1) B 14 97

C 9 98

D 9 102

E 9 95

Control A 154 28

SW AZ B 152 48

C 147 39

D 131 45

E 143 48

Control A 167 56

Acetone AZ B 169 75

C 147 54

D 158 43

E 136 51

112

Table C17: Raw data for bivalve toxicity test – cypermethrin (nominal).


CP C5 A 134 25

(0.03 mg L-1) B 143 43

C 151 68

D 129 54

E 138 43

CP C4 A 160 67

(0.06 mg L-1) B 155 44

C 136 62

D 146 69

E 178 54

CP C3 A 144 65

(0.1 mg L-1) B 145 77

C 154 66

D 161 65

E 145 71

CP C2 A 152 59

(0.2 mg L-1) B 151 64

C 153 55

D 127 47

E 171 66

CP C1 A 154 71

(0.5 mg L-1) B 125 62

C 158 55

D 156 51

E 166 59

Control A 133 41

SW CP B 177 60

C 131 78

D 141 54

E 166 43

Control A 155 42

Acetone CP B 139 56

C 151 60

D 169 52

E 128 39

113

Table C18: Raw data for bivalve toxicity test – deltamethrin (nominal).


DM C5 A 165 65

(0.006 mg L-1) B 145 59

C 153 49

D 152 47

E 136 40

DM C4 A 135 46

(0.01 mg L-1) B 122 50

C 155 73

D 147 61

E 148 52

DM C3 A 161 38

(0.02 mg L-1) B 136 67

C 140 61

D 133 66

E 161 52

DM C2 A 142 45

(0.05 mg L-1) B 167 55

C 167 58

D 141 52

E 158 60

DM C1 A 131 72

(0.1 mg L-1) B 149 58

C 145 65

D 179 96

E 128 75

Control A 171 71

SW DM B 176 73

C 153 73

D 166 82

E 156 64

Control A 151 50

Acetone DM B 124 53

C 138 77

D 183 72

E 181 82

114

Table C19: Raw data for bivalve toxicity test – emamectin benzoate (nominal).


EB C5 A 144 79

(0.3 mg L-1) B 159 66

C 164 66

D 143 63

E 152 70

EB C4 A 161 60

(0.6 mg L-1) B 170 83

C 188 75

D 153 65

E 178 69

EB C3 A 32 152

(1.25 mg L-1) B 38 127

C 41 146

D 65 152

E 34 162

EB C2 A 0 0

(2.5 mg L-1) B 0 4

C 0 1

D 0 2

E 0 2

EB C1 A 0 0

(5 mg L-1) B 0 0

C 0 0

D 0 0

E 0 0

Control A 143 39

SW EB B 156 55

C 153 69

D 190 76

E 156 84

Control A 126 77

Methanol EB B 146 67

C 149 73

D 150 79

E 166 69

115

Table C20: Raw data for bivalve toxicity test – hydrogen peroxide (nominal).

Concentration Rep

No. Normal

No. Abnormal

Concentration Rep

No. Normal

No. Abnormal

HP Control A 214 46 HP C5 A 181 75

SW B 200 46 (0.94 mg L-1) B 214 69

C 188 46 C 179 42

D 182 40 D 191 59

E 179 41 E 200 48

F 172 40 HP C4 A 208 62

HP C9 A 194 36 (1.9 mg L-1) B 183 54

(0.06 mg L-1) B 211 45 C 159 72

C 199 12 D 174 59

D 184 36 E 205 62

E 205 37 HP C3 A 0 38

HP C8 A 179 44 (3.75 mg L-1) B 0 48

(0.1 mg L-1) B 204 43 C 0 30

C 168 50 D 0 37

D 181 36 E 0 24

E 228 40 HP C2 A 0 24

HP C7 A 205 53 (7.5 mg L-1) B 0 33

(0.23 mg L-1) B 186 58 C 0 31

C 177 69 D 0 18

D 182 54 E 0 22

E 170 59 HP C1 A 0 20

HP C6 A 225 62 (15 mg L-1) B 0 26

(0.5 mg L-1) B 211 68 C 0 22

C 186 60 D 0 23

D 182 61 E 0 12

E 202 78

116

Table C21: Raw data for echinoderm toxicity test – azamethiphos (nominal).

Concentration Rep No. Fertilized Eggs No. Unfertilized Eggs

AZ C1 A 77 23

(12.5 mg L-1) B 74 26

C 85 15

D 84 16

E 77 23

AZ C2 A 85 15

(4.1 mg L-1) B 85 15

C 87 13

D 84 16

E 83 17

AZ C3 A 84 16

(1.4 mg L-1) B 80 20

C 78 22

D 81 19

E 84 16

AZ C4 A 86 14

(0.5 mg L-1) B 85 15

C 80 20

D 78 22

E 77 23

AZ C5 A 82 18

(0.15 mg L-1) B 76 24

C 83 17

D 80 20

E 76 24

AZ C6 A 79 21

(0.05 mg L-1) B 84 16

C 77 23

D 75 25

E 78 22

AZ C7 A 77 23

(0.02 mg L-1) B 81 19

C 80 20

D 88 12

E 81 19

117

Table C22: Raw data for echinoderm toxicity test – cypermethrin (nominal).


CP C1 A 21 79

(0.05 mg L-1) B 23 77

C 24 76

D 25 75

E 23 77

CP C2 A 47 53

(0.02 mg L-1) B 41 59

C 42 58

D 32 68

E 35 65

CP C3 A 55 45

(0.008 mg L-1) B 51 49

C 55 45

D 59 41

E 52 48

CP C4 A 71 29

(0.003 mg L-1) B 79 21

C 70 30

D 67 33

E 65 35

CP C5 A 80 20

(0.001 mg L-1) B 83 17

C 84 16

D 78 22

E 85 15

CP C6 A 78 22

(0.0005 mg L-1) B 80 20

C 82 18

D 77 23

E 86 14

CP C7 A 81 19

(0.00002 mg L-1) B 80 20

C 80 20

D 76 24

E 76 24

118

Table C23: Raw data for echinorderm toxicity test – deltamethrin (nominal).


DM C1 A 51 49

(0.02 mg L-1) B 42 58

C 52 48

D 56 44

E 34 66

DM C2 A 26 74

(0.008 mg L-1) B 18 82

C 16 84

D 14 86

E 20 80

DM C3 A 79 21

(0.003 mg L-1) B 72 28

C 76 24

D 84 16

E 75 25

DM C4 A 75 25

(0.001 mg L-1) B 79 21

C 85 15

D 83 17

E 75 25

DM C5 A 75 25

(0.0005 mg L-1) B 77 23

C 78 22

D 79 21

E 85 15

DM C6 A 90 10

(0.0002 mg L-1) B 79 21

C 81 19

D 82 18

E 83 17

DM C7 A 81 19

(0.00008 mg L-1) B 81 19

C 85 15

D 89 11

E 79 21

119

Table C24: Raw data for echinoderm toxicity test – emamectin benzoate (nominal).


EB C1 A 8 92

(5 mg L-1) B 8 92

C 16 84

D 6 94

E 4 96

EB C2 A 68 32

(2 mg L-1) B 51 49

C 77 23

D 75 25

E 65 35

EB C3 A 80 20

(0.8 mg L-1) B 88 12

C 87 13

D 86 14

E 83 17

EB C4 A 85 15

(0.3 mg L-1) B 84 16

C 82 18

D 77 23

E 83 17

EB C5 A 86 14

(0.1 mg L-1) B 93 7

C 87 13

D 85 15

E 76 24

EB C6 A 90 10

(0.05 mg L-1) B 79 21

C 84 16

D 83 17

E 79 21

EB C7 A 91 9

(0.02 mg L-1) B 84 16

C 85 15

D 84 16

E 83 17

120

Table C25: Raw data for echinoderm toxicity test – hydrogen peroxide (nominal).


HP C1 A 4 96

(9.6 mg L-1) B 9 91

C 8 92

D 11 89

E 3 97

HP C2 A 11 89

(4.8 mg L-1) B 16 84

C 13 87

D 14 86

E 6 94

HP C3 A 50 50

(2.4 mg L-1) B 63 37

C 52 48

D 57 43

E 53 47

HP C4 A 74 26

(1.2 mg L-1) B 79 21

C 84 16

D 76 24

E 82 18

HP C5 A 91 9

(0.6 mg L-1) B 86 14

C 81 19

D 88 12

E 81 19

HP C6 A 83 17

(0.3 mg L-1) B 83 17

C 91 9

D 86 14

E 86 14

HP C7 A 85 15

(0.15 mg L-1) B 87 13

C 80 20

D 88 12

E 84 16

121

Table C26: Raw data for echinoderm toxicity test – controls.


Control A 88 12

SW B 84 16

C 85 15

D 88 12

E 83 17

Control A 82 18

MeOH B 83 17

C 83 17

D 85 15

E 84 16

Control A 85 15

Acetone B 88 12

C 84 16

D 85 15

E 88 12

the environmental fate and effects of sea lice

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