Prevention of Saprolegnia on Rainbow Trout Eggs

BSc Thesis
<strong>Prevention</strong> <strong>of</strong> <strong>Saprolegnia</strong>
on Rainbow Trout Eggs
Sølvi Espeland
&
Petra E. Hansen
NVDRit 2004:05

Heiti / Title
<strong>Prevention</strong> <strong>of</strong> <strong>Saprolegnia</strong> on rainbow
trout eggs
Fyribyrging av <strong>Saprolegnia</strong> á rognum hjá
ælabogasílum
Høvundar / Authors Sølvi Espeland og Petra E. Hansen
Vegleiðari / Supervisor Peter S. Østergård, Heilsufrøðiliga Starvsstovan
Ábyrgdarvegleiðari / Responsible Supervisor Eyðfinn Magnussen, Fróðskaparsetur Føroya
Ritslag / Report Type BSc ritgerð, lívfrøði
BSc Thesis, Biology
Latið inn / Submitted 18. juni 2004
NVDRit 2004:05
© Náttúruvísindadeildin og høvundarnir 2004
ISSN 1601-9741
Útgevari / Publisher Náttúruvísindadeildin, Fróðskaparsetur Føroya
Bústaður / Address Nóatún 3, FO 100 Tórshavn, Føroyar (Faroe Islands)
Postrúm / P.O. box 2109, FO 165 Argir, Føroyar (Faroe Islands)
@ +298 352550 +298 352551 nvd@setur.fo

TABLE OF CONTENTS
ABSTRACT .......................................................................................................................................................................... 2
1 INTRODUCTION............................................................................................................................................................ 3
1.1 THE GOAL OF PRESENT STUDY.......................................................................................................................... 4
1.2 TAXONOMY, DISTRIBUTION, AND PROPERTIES OF SAPROLEGNIA.......................................................... 4
1.2.1 Life cycle ............................................................................................................................................................. 6
1.2.2 <strong>Saprolegnia</strong> in Faroese hatcheries.................................................................................................................... 8
1.3 DESCRIPTION OF THE SUBSTANCES USED IN THIS EXPERIMENT ........................................................... 9
1.3.1 Pyceze .................................................................................................................................................................. 9
1.3.2 Ibiza salt ............................................................................................................................................................ 10
1.3.3 Hydrogen peroxide ........................................................................................................................................... 10
1.3.4 BioCare.............................................................................................................................................................. 11
2 MATERIALS AND METHODS .................................................................................................................................. 13
2.1 PRELIMINARY STUDY ................................................................................................................................................. 13
2.2 MAIN STUDY AT THE HATCHERY.................................................................................................................... 14
2.2.1 Test conditions .................................................................................................................................................. 14
2.2.2 Treating eggs in the hatchery .......................................................................................................................... 14
2.2.3 Gathering data .................................................................................................................................................. 17
2.2.4 Fungal spores in the intake water ................................................................................................................... 18
2.2.5 Statistical analysis ............................................................................................................................................ 19
3 RESULTS........................................................................................................................................................................ 21
3.1 PRELIMINARY STUDY ................................................................................................................................................. 21
3.1.1 Identification <strong>of</strong> the fungi ................................................................................................................................21
3.1.2 Testing the candidate substances..................................................................................................................... 21
3.2 MAIN STUDY AT THE HATCHERY ............................................................................................................................... 22
3.2.1 Test conditions .................................................................................................................................................. 22
3.2.2 Comparison <strong>of</strong> all substances .......................................................................................................................... 23
3.2.3 Ibiza salt ............................................................................................................................................................ 25
3.2.4 Hydrogen peroxide ........................................................................................................................................... 27
3.2.5 BioCare.............................................................................................................................................................. 29
3.2.6 Fungal spores in the intake water ................................................................................................................... 30
4 DISCUSSION.................................................................................................................................................................. 32
4.1 PRELIMINARY STUDY ................................................................................................................................................. 32
4.1.1 Identification <strong>of</strong> the fungi ................................................................................................................................32
4.1.2 Testing the candidate substances..................................................................................................................... 33
4.2 MAIN STUDY AT THE HATCHERY................................................................................................................................34
4.2.1 Test conditions .................................................................................................................................................. 34
4.2.2 Ibiza salt ............................................................................................................................................................ 34
4.2.3 Hydrogen peroxide ........................................................................................................................................... 36
4.2.4 BioCare.............................................................................................................................................................. 38
4.2.5 Fungal spores in the intake water ................................................................................................................... 39
4.3 IDEAS FOR FUTURE EXPERIMENTS .............................................................................................................................. 39
5 CONCLUSION............................................................................................................................................................... 41
6 REFERENCES ............................................................................................................................................................... 43
APPENDIX ......................................................................................................................................................................... 47
APPENDIX 1 .................................................................................................................................................................. 47
APPENDIX II.................................................................................................................................................................. 48

ABSTRACT
2
ABSTRACT
In spring 2004 pre-eyed eggs <strong>of</strong> rainbow trout (Oncorhynchus mykiss) were treated against
saprolegniasis with Ibiza salt, hydrogen peroxide, and BioCare at different concentrations and
exposure durations twice a week. The fungistatic effects, hatching rates, and cripple rates were
compared to those <strong>of</strong> negative control, which received no treatment, and to those <strong>of</strong> positive control,
which was treated with Pyceze, currently considered the best treatment option against
saprolegniasis. No treatments were better than Pyceze. However, hydrogen peroxide treatments
were statistically as good as Pyceze with the exception <strong>of</strong> fungal infection which was higher for the
hydrogen peroxide treatments. Ibiza salt and BioCare can not be recommended as Ibiza salt
treatments were no better than no treatments at all and BioCare seemed to be toxic to the eggs.
Fungus infecting salmon eggs was identified to be <strong>Saprolegnia</strong> sp. Although not identified it
was assumed that it was this same species that also infected the rainbow trout eggs. The number <strong>of</strong>
fungal spores in the intake water decreased during the treatment period but there was no connection
between number <strong>of</strong> spores and infection <strong>of</strong> the eggs.
SAMANDRÁTTUR
Á vári 2004 vórðu eygarogn hjá ælabogasílum viðgjørd fyri at fyribyrgja soppi (<strong>Saprolegnia</strong>).
Til hetta vórðu evnini Ibiza salt, brintyvirilta og BioCare í ymiskum styrkjum og tíðum nýtt.
Viðgjørt varð tvær ferðir um vikuna. Fyri hvørja viðgerð varð funnð útav, hvussu væl hon forðaði
soppavøkstri, hvussu klekiprosentið var og hvussu nógv prosent vórðu kryplar. Hesi úrslit vórðu
samanborin við úrslit fyri negativan kontroll, sum onga viðgerð fekk, og við positivan kontroll, sum
fekk viðgerð við Pyceze, sum í løtuni verður mett at vera besta viðgerðarevnið móti soppi á
rognum. Eingi av evnunum vóru betri enn Pyceze, men brintyvirilta hevði hagfrøðiliga sæð eins góð
úrslit og Pyceze, undantikið fyri soppismittu, sum var hægri fyri viðgerðirnar við brintyviriltu. Ibiza
salt og BioCare kunnu ikki sigast at vera vælegnað at viðgera við, tí viðgerðirnar við Ibiza salti
høvdu á leið somu úrslit sum eingin viðgerð og BioCare sá út til at vera eitrandi fyri rognini.
Soppur, sum hevði smittað laksarogn, varð staðfestur at vera av slagnum <strong>Saprolegnia</strong> sp.
Hóast tað ikki varð staðfest, varð gingið út frá, at soppurin, sum smittaði sílarognini, eisini var av
hesum sama slagnum. Tal av soppasporum í inngangandi vatninum øktist meðan viðgerðirnar
vardu, men einki samband var millum sporatalið og smittu av rognunum.

1 INTRODUCTION
INTRODUCTION
One <strong>of</strong> the main types <strong>of</strong> fungal diseases in farmed salmonid fish is saprolegniasis, caused by
species in the genus <strong>Saprolegnia</strong>. It causes considerable economic problems in the fish farming
industry (Bly et al. 1992; Pottinger & Day 1999), infecting both fish and fisheggs (Bruno & Wood
1999). In the past this problem was solved with the extremely effective fungicide (compound which
kills fungi) malachite green (Pottinger & Day 1999). The use <strong>of</strong> malachite green began in 1933 and
it was one <strong>of</strong> the cornerstones used in treatment <strong>of</strong> fish against a range <strong>of</strong> parasites (Foster &
Woodbury 1936 quoted from Meyer & Jorgenson 1983). It has been used extensively by the
aquaculture industry in Europe and throughout the world for many years in the absence <strong>of</strong> an
authorized veterinary medicinal alternative. It has proved effective as a fungicide on farmed fish
(Fitzpatrick et al. 1995; Kitancharoen et al. 1997). Malachite green acts as a respiratory enzyme
poison (Werth 1967, Werth & Boiteaux 1967a quoted from Alderman 1985), damaging the cell’s
ability to produce energy to drive vital metabolic processes (Werth & Boiteaux 1967b quoted from
Alderman 1985). Malachite green is considered carcinogenic (Culp & Beland 1996 quoted from
Pottinger & Day 1999), mutagenic (Committee on toxicity <strong>of</strong> chemicals in food, consumer products
and the environment 1999), and teratogenic (Meyer & Jorgenson 1983). Carcinogenic substances
are agents capable <strong>of</strong> causing cancer. Mutagenic substances can cause changes in amount or
chemical structure <strong>of</strong> DNA resulting in changes in the characteristics <strong>of</strong> an organism or an
individual cell. Teratogenicity means the cabability <strong>of</strong> a substance to cause malformations during
embryonic development (Lawrence 2000). Meyer & Jorgenson (1983) treated eggs <strong>of</strong> rainbow trout
(Oncorhynchus mykiss) with malachite green and observed deformities in larvae at rates three to
five times those observed in larvae developed from untreated eggs. Meinertz et al. (1995) concluded
that undetectable residues <strong>of</strong> malachite green would still remain in fish grown from eggs which had
been exposed to the chemical until they reached market size. In UK the Committee on toxicity <strong>of</strong>
chemicals in food, consumer products and the environment (1999) found residues <strong>of</strong> malachite
green and <strong>of</strong> its conversion product leucomalachite green in market size fish, and because <strong>of</strong>
concerns regarding the consumers and the health implications <strong>of</strong> the operators at the fish farms the
use <strong>of</strong> it was restricted. Malachite green is not listed as a veterinary medicine in EU´s lists <strong>of</strong>
approved substances which can be used on food-producing animals (Council Regulation
2377/90/EC 1990, Annexes I, II, and III), and is therefore not permitted for use in fish-farming in
EU. The FDA (United States Food and Drug Administration) has also banned the use <strong>of</strong> malachite

4
INTRODUCTION
green in fish-farming (Schreier et al. 1996) because <strong>of</strong> its teratogenic effects (Meyer & Jorgenson
1983). The loss <strong>of</strong> this extremely effective substance in the fish-farming industry has driven
scientists to look for a less hazardous substance which is as effective as malachite green.
1.1 THE GOAL OF PRESENT STUDY
The goal <strong>of</strong> this project was to investigate three different ecologically friendly substances as a
treatment option against saprolegniasis on salmonid eggs. The substances were Ibiza salt, hydrogen
peroxide, and BioCare. The project involved two main parts:
I. A preliminary study in a laboratory involving:
1) identification <strong>of</strong> the fungus,
2) testing the fungistatic effect <strong>of</strong> the three candidate substances.
II. The main studies at a hatchery (P/F Fiskaaling) which included:
treatment <strong>of</strong> rainbow trout eggs with the candidate substances and gathering data.
The treatments in part two were compared based on:
• how their fungistatic properties were (how well they inhibited growth <strong>of</strong> the fungi),
• how they affected the hatching rate,
• whether they led to fry abnormality.
The treatments were compared to negative control which received no treatment, and to
treatment with Pyceze which was used as positive control. Pyceze is considered the best and safest
alternative to malachite green that has yet been found (Pottinger & Day 1999).
The second part <strong>of</strong> the project was somewhat limited by the lack <strong>of</strong> cylinders for treating the
eggs in so that the range <strong>of</strong> concentrations was not so wide as originally planned.
1.2 TAXONOMY, DISTRIBUTION, AND PROPERTIES OF SAPROLEGNIA
The genus belongs to the class Oomycotea which are also called water moulds (Bruno &
Wood 1999). Moulds are mycelium-forming micr<strong>of</strong>ungi which spread by spores, conidia or hyphal
fragments (Nordic Committee on Food Analysis 1995).

5
INTRODUCTION
There is much confusion about how to name the pathogenic <strong>Saprolegnia</strong> species. In 1923
Coker was the first to describe S. parasitica growing on fish at a hatchery. His work was not good
taxonomic practice as he did not observe any sexual structures on which species delimitations were,
and are, traditionally based. Still scientists adopted the name S. parasitica to any fungus isolated
from a living fish and which did not produce sexual structures under laboratory conditions. This is
bad practice as many other <strong>Saprolegnia</strong> species living on fish do not produce sexual structures if
they are in abundance on the host. Moreover, Coker did not leave any type material for the future.
In 1932 Kanouse was able to describe the sexual structures <strong>of</strong> S. parasitica but there is doubt as to
whether it really was the right species, Coker´s S. parasitica, which she described. In 1976 Neish
observed that all S. parasitica isolates which produced sexual structures could be assigned to S.
diclina Humphrey, a species described by Humphrey in 1893. Thus was born the <strong>Saprolegnia</strong>
diclina-<strong>Saprolegnia</strong> parasitica complex which included 5 species, S. kauffmania, S. shikotsuensis,
S. australis, S. diclina and S. parasitica. In 1978 Willoughby divided the complex into three
subgroups on the basis <strong>of</strong> the lenght/width ratios <strong>of</strong> the oogonia. One was called S. Diclina Type I.
and included fungal parasites <strong>of</strong> salmonid fishes. This complex seemed a good solution to the
species problem but since then some went back to the term S. parasitica Coker and currently the
situation is very confusing, some using one name and some another name for the same species. In
present study it was decided to use the term <strong>Saprolegnia</strong> sp. for all non-sexual isolates <strong>of</strong>
<strong>Saprolegnia</strong> as scientists recently have concluded that it is best to name all non-sexual isolates <strong>of</strong>
<strong>Saprolegnia</strong> from fish as <strong>Saprolegnia</strong> sp. (Hughes 1994).
<strong>Saprolegnia</strong> is found naturally in all freshwater (Poppe 1999). Some species are pathogenic,
and some are not (Langvad 1999). All pathogenic species have got bent hairs on their secondary
cysts (figure 1.1), possibly for attachment (Håstein et al. 1999; Langvad 1999). <strong>Saprolegnia</strong> species
can infect dead fish eggs (Pottinger & Day 1999). From these eggs the fungus can spread to live
eggs via positive chemotaxi (Bruno & Wood 1999) meaning that some chemical signal from the
live eggs causes the fungus to move towards them (Lawrence 2000). When first established the
fungus produces further zoospores which infect more eggs. Therefore it is important continuously to
remove dead eggs (Kitancharoen et al. 1997). The fisheggs are very fragile the first 175 daily
temperature units (in the pre-eyed stage) and can not be handled. Hence, one can not remove dead
eggs in this period. In this period it is therefore important to treat the eggs with some fungistatic in
order to inhibit the fungus from infecting dead eggs and spreading (Refstie 1993).

6
INTRODUCTION
Studies have shown that pathogenic <strong>Saprolegnia</strong> species have adapted, so that their thermal
tolerance is similar to that <strong>of</strong> their host fish. For example <strong>Saprolegnia</strong> isolates from cold water
salmonids grow better at lower than at higher temperatures (Pickering & Willoughby 1982). There
are also other conditions that render eggs in hatcheries susceptible to infection by the fungus. For
example fungal spores are highly resistant to heat, drying, and disinfectants (Klinger & Francis-
Floyd 1996), so it is difficult to exclude them from the intake water in fish-farms. Moreover,
handling (Bruno & Wood 1999), poor water quality, such as water with low circulation, low
dissolved oxygen, and high ammonia content (Klinger & Francis-Floyd 1996), crowding, stress,
and decreasing temperatures (Poppe 1999) all help the fungus to establish. Generally there are more
fungal outbreaks after a sharp decrease in water temperature, to levels near the physiological
minimum for the particular fish species (Bruno & Wood 1999). Poppe (1999) states that
<strong>Saprolegnia</strong> infections seem to come in turns which vary in time. And <strong>of</strong>ten there seems to be no
good explanation for this. Langvad (1999) observed that in recent years <strong>Saprolegnia</strong> had shown
unusually agressive attacks. In Norway it has been observed that <strong>Saprolegnia</strong> has got a summer and
an autumn bloom, when the water temperature lies around 11-12 ºC. In these periods the danger <strong>of</strong>
infection is at its peak (Håstein et al. 1999). According to Langvad (1999), there normally were 50-
200 spores/L, but in spring and autumn this number increased by a factor 20.
1.2.1 Life cycle
The life cycle <strong>of</strong> <strong>Saprolegnia</strong> includes both sexual and asexual reproduction (figure 1).
<strong>Saprolegnia</strong> is homothallic, meaning that one single individual contains both male and female sex
organs. The mycelium is comprised <strong>of</strong> several hyphae, and each hypha is like one big cell with
many nuclei, because <strong>of</strong> its lack <strong>of</strong> cell walls. On the hyphae sit the male and the female sex organs,
the antheridium and the oogonium, respectively. In these meiosis occurs to produce male nuclei and
female eggs. The antheridia grow toward the oogonia and produce fertilization tubes that penetrate
the oogonia. Fertilization occurs when the male nuclei travel down these tubes to the female eggs
and fuse with the female nuclei. This produces several thick-walled zygotes, called oospores. Each
oospore germinates into a new hypha which will produce a zoosporangium. From the
zoosporangium the asexual reproduction occurs which is the main type <strong>of</strong> reproduction.

Figure 1.1: Life cyclus <strong>of</strong> <strong>Saprolegnia</strong> sp. (Raven et al. 1999).
7
INTRODUCTION
The zoosporangium releases zoospores with two flagella which swim for a while before they
encyst. Each <strong>of</strong> these zoospores eventually becomes a secondary zoospore, which also encysts,
before it germinates into a new mycelium, on which sexual reproduction occurs, thus starting the
reproduction cycle anew (Raven et al. 1999). The secondary cysts can also release new secondarylike
zoospores which are able to encyst again and so on. These repeated cycles <strong>of</strong> encystment and
release <strong>of</strong> respectively secondary zoospores and cysts are called polyplanetism. Polyplanetism
contributes to the fungus´ pathogenicity by helping it to make several attempts locating a suitable
culture medium to live on before settling down for good (Beakes 1983). This, and the fact that the

8
INTRODUCTION
secondary zoospores are more motile than the primary zoospores and also motile for a longer
period, contributes to the presumption that the secondary zoospores are the main dispersion phase in
<strong>Saprolegnia</strong>´s life cycle (Pickering & Willoughby 1982). After they have encysted the secondary
zoospores release hairs for attachment (Beakes 1983). Possibly these hairs also decrease the
sedimentation rate and serve as fungal-host recognition response. These long hairs on the secondary
cysts are sometimes called boathooks (Beakes 1983).
Pathogenic and non-pathogenic species <strong>of</strong> <strong>Saprolegnia</strong> germinate under different conditions
and nutrient levels. Pickering and Willoughby (1982) observed that pathogenic species grew faster
compared to non-pathogenic, especially in an enriched water supply (the effluent from a trout
hatchery). Moreover, they observed that the pathogenic species frequently germinated in an indirect
way. This indirect germination is characterized by an attached zoospore and very fine initial germ
tube with several cross walls and both contain no cytoplasmic contents, while in direct germination,
the germ tube is cytoplasm-filled. In very concentrated and very dilute nutrient solutions direct
germination occurred whereas indirect germination occurred at intermediate concentration. L. G.
Willoughby (unpublished observations) has observed firm attachment <strong>of</strong> empty cyst cases and
empty portions <strong>of</strong> the initial germ tube to petri dishes while no such attachment <strong>of</strong> the cytoplasmic
regions <strong>of</strong> the hypha was observed so perhaps indirect germination is important as a means af firmer
attachment to the host (Pickering & Willoughby 1982).
1.2.2 <strong>Saprolegnia</strong> in Faroese hatcheries
As in other countries, eggs in Faroese hatcheries also suffer from saprolegniasis. The question
whether the cause is <strong>Saprolegnia</strong> or some other fungi is not fully answered, although some
identification has been done. In 1994 S. parasitica was isolated from the kidneys <strong>of</strong> some diseased
smolts at a hatchery and identified by Finn Langvad, Norway (pers. comm. Jürgens 2004).
In the Faroe Islands there are two main hatcheries (P/F Fiskaaling and P/F Fútaklettur) and
both used malachite green before but because <strong>of</strong> its danger <strong>of</strong> causing cancer it is no longer in use
(pers. comm. Hansen 2004; pers. comm. Gregersen 2004). Although P/F Fiskaaling has done some
tests on different substances, up to date no antifungal agent has been found that is as effective as
malachite green (pers. comm. Hansen 2004). The one that works best, and the only one in use now,
is Pyceze. The current situation is that P/F Fútaklettur uses Pyceze to control fungus on the eggs
(pers. comm. Gregersen 2004) and P/F Fiskaaling does not use any antifungal agent at all, partly
because <strong>of</strong> uncertainty regarding how the substances affect the fish later in life. Even though no

9
INTRODUCTION
antifungal agent is used at P/F Fiskaaling fungus is not always a very big problem. But it can cause
serious problems. It depends on the quality <strong>of</strong> eggs. Poor quality eggs (overripe eggs) make it easier
for the fungus to establish, as there are many dead eggs among them, so these eggs can have a
mortality rate twice as high as that <strong>of</strong> good quality eggs (pers. comm. Hansen 2004).
To conclude, further investigation on different antifungal substances is needed, both on how
their antifungal properties are and on possible secondary effects later in the fish’ lives. But it is not
enough only to treat the eggs against fungus. Other measures need also to be taken, such as
minimizing the number <strong>of</strong> pathogens in intake water and removing dead eggs as soon as they reach
the eyed stage, thus preventing the fungus from spreading further. Both hatcheries do this. UV rays
are the means used to control the amount <strong>of</strong> pathogens in intake water (pers. comm. Hansen 2004;
pers. comm. Gregersen 2004). However, in order to minimize the amount <strong>of</strong> fungal spores as much
as possible it would be better to treat the intake water with ozon provided that the water was well
oxidized afterwards (pers. comm. Østergård 2004).
1.3 DESCRIPTION OF THE SUBSTANCES USED IN THIS EXPERIMENT
The criteria for the substances chosen were that:
• they had to be legal for use on food- producing animals,
• they had to have a fungistatic effect,
• they had to be relatively cheap so that the hatcheries actually could afford to use them if they
proved to be effective,
• there had to be a likelihood for approval <strong>of</strong> the substances in organic aquaculture.
1.3.1 Pyceze
Pyceze was used as positive control because it is the best authorised veterinary fungistatic
substance available for preventing saprolegniasis in fish eggs today. It is produced by Novartis
Animal Vaccines and approved by the EU in Annex II <strong>of</strong> Council Regulation (EEC) 2377/90
(Standing Committee on the Food Chain and Animal Health 2003). For substances in this list it is
not necessary to establish a maximum residue limit for the protection <strong>of</strong> public health. The active
ingredient <strong>of</strong> Pyceze is bronopol (2-bromo-2-nitropropane-1,3-diol). Bronopol is an antimicrobial
preservative which is used in food-contact materials, medical and pharmaceutical products,
cosmetics, and shampoos (Pottinger & Day 1999; EMEA (European Agency for the Evaluation <strong>of</strong>

10
INTRODUCTION
Medicinal Products) Committee for veterinary medicinal products 2001). It is believed to have a
dual toxic action in bacteria, with growth suppression being ascribed to the catalytic oxidation <strong>of</strong>
accessible thiols while cell death is thought to be caused by the generation <strong>of</strong> free radicals
(Pottinger & Day 1999 quoted Shepherd et al. 1988).
Bronopol is a broad-spectrum biocide. It presents no serious toxicological hazard to humans
(Bryce et al. 1978, Croshaw & Holland 1984 quoted from Pottinger & Day 1999) or fish (Pottinger
& Day 1999). A detailed Environmental Risk Assessment has assessed the risk <strong>of</strong> bronopol and the
formulated product Pyceze and the conclusion was that any potential for toxic impact should be
transient and localized. Amongst other following observations were made: bronopol is unlikely to
adsorb to sediment or suspended solids, it is unlikely to persist or bioaccumulate in the
environment, degradation products are less toxic than the parent compound and final breakdown
products are naturally occurring endogenous compounds (Novartis 2002).
1.3.2 Ibiza salt
Salt (sodium chloride) is granted low regulatory approval by the FDA (2002) and is therefore
not considered a harmful chemical. It is concidered a safe, common substance which has
antimicrobial properties (Kitancharoen et al. 1997). Salt is a mineral that exists everywhere in the
environment. The salt content in most oceans is between 34 and 36 ppt (parts per thousand)
(Hansen 2000). By evaporation <strong>of</strong> sea or lake water rock salt is formed (Murck & Skinner 1999).
Salt also has an important role in cells where the ion movement <strong>of</strong> Na + and Cl - across cell
membranes plays an essential part in many cell processes (Alberts et al. 1998).
Salt is more expensive than hydrogen peroxide. In this project Ibiza salt which is seasalt was
preferred over sterilized NaCl, because <strong>of</strong> the huge difference in prize, and because sea salt also
contains many other compounds in addition to NaCl, which might give a better effect than sterilized
NaCl.
1.3.3 Hydrogen peroxide
As Pyceze, hydrogen peroxide has also been included in the EU lists <strong>of</strong> approved substances
for which it is not necessary to establish a maximum residue limit for protection <strong>of</strong> public health,
Annex II <strong>of</strong> Council Regulation (EEC) 2377/90 (1990). According to the EMEA´s Committee for
Veterinary Medicinal Products it was included because it is a chemical that is a normal product <strong>of</strong>
aerobic metabolism, and it may result from a number <strong>of</strong> oxidase-catalase reactions in various

11
INTRODUCTION
organisms. Because <strong>of</strong> this, residues <strong>of</strong> hydrogen peroxide in fish and other products <strong>of</strong> animal
origin cannot be distinguished from the levels <strong>of</strong> hydrogen peroxide originating from the organism
itself. And in contact with oxidazable organic matter, and in contact with certain metals, hydrogen
peroxide rapidly decomposes to water and oxygen (EMEA´s Committee for Veterinary Medicinal
Products 1996). In the industy it is used as a bleaching and oxidizing agent. It is used to treat
sewage and industrial effluents, in cosmetics, as a disinfectant in human- and veterinary medicine,
and as an antimicrobial agent in the processing <strong>of</strong> some foods (Schreier et al. 1996, EMEA´s
Committe for Veterinary Medicinal Products 1996). There is no evidence that hydrogen peroxide is
carcinogenic, and it is proved not to be teratogenic (EMEA Committee for Veterinary Medicinal
Products 1996). Hydrogen peroxide is considered to be a low regulatory priority drug by the FDA
(2002) which means that it can be used in the USA at the approved treatment regimes without an
investigational new animal drug permit or a new animal drug application, eliminating the costly
registration process (Schreier et al. 1996, Gaikowsky et al. 1998). Hydrogen peroxide is a relatively
cheap chemical to use.
There are also some down sides to hydrogen peroxide. It is highly temperature dependent.
Below 8 °C it should be used in one dose and at temperatures higher than this the dose should be
20% lower. Moreover, hydrogen peroxide should not be used in fish-farming when the temperature
in the water is higher than 13 °C because <strong>of</strong> risk <strong>of</strong> serious gill-damage to the fish (Bovbjerg et al.
2000). When decomposing hydrogen peroxide releases O2 gas. If there is much over-saturation the
gas bubbles will rise to the surface (Hjelme 2000b). In a cylinder full <strong>of</strong> fungi-infected fish eggs the
bubbles may get trapped under the eggs building up a pressure before they can escape. When the
oxygen finally escapes the event will turn the eggs in the cylinder and this may kill the eggs if it
happens the first two to three weeks when the eggs are vulnerable to movement (pers. comm.
Wardum 2004). Moreover, hydrogen peroxide is affected by the amount <strong>of</strong> organic matter in the
water. In water with high organic content, the dosis <strong>of</strong> hydrogen peroxide needed is higher in order
to get the same effect as in cleaner water (Hjelme 2000b).
1.3.4 BioCare
BioCare is an oxidizing disinfectant and the active ingredient is hydrogen peroxide. Na2CO3
is basic when dissolved in water. It neutralizes acid organic compounds and carbonic acids. BioCare
is specially designed for use in outdoor fish pools, where it is supposed to reduce the amount <strong>of</strong>
pathogens, increase oxygen content, decompose organic matter, and stabilize the pH (Hjelme

12
INTRODUCTION
2000b). It is considered to be a very environmentally compatible chemical because it does not
produce any toxic biproducts when it decomposes (Hjelme 2000a).

2.1 PRELIMINARY STUDY
2 MATERIALS AND METHODS
MATERIALS AND METHODS
There were two practical parts <strong>of</strong> this study. The first was to identify the pathogenic fungi in
the water and the second was to get an indication <strong>of</strong> which concentrations and exposure durations <strong>of</strong>
the different substances were most effective in inhibiting the fungi from spreading. To get a sample
<strong>of</strong> the fungi, water was collected from a cylinder containing heavily infected salmon eggs in a
presterilized bottle. The water was brought to the laboratory where 0.1 mL <strong>of</strong> the water sample was
aseptically distributed on each <strong>of</strong> two Dichloran-rosbengal (DRBC) agar plates. DRBC-agar
contains antibiotics (chloramphenicol) which restricts bacterial growth and therefore prevents the
plates from getting overgrown by bacteria. The plates were incubated for 6 days at 25 ˚C, and then
sent to Ph.D -student on <strong>Saprolegnia</strong>-infections on salmonids Svein Stueland, National Veterinary
Institute, Oslo, Norway for identification <strong>of</strong> the fungi.
The next step was to treat fungus-infected egg-particles with the different test chemicals at
different concentrations and exposure durations. Dead fungus-infected salmon eggs were collected
and 5 equally sized particles were placed randomly in jars that already had treatments assigned to
them. The particles were treated two times with three days in between. The different treatments are
described in table 2.1. Between the treatments the particles were provided with fresh water on a
daily basis.
Ibiza salt
(ppt)
Table 2.1: Exposure durations, test chemicals, and concentrations used in the preliminary
study. (ppt = parts per thousand, ppm = parts per million)
*
H2O2 (hydrogen peroxide) concentrations were calculated by solution, not by active
ingredient.
60 minutes exposure time 30 minutes exposure time 10 min -
BioCare
(ppt)
H2O2 *
(ppm)
Ibiza salt
(ppt)
BioCare
(ppt)
H2O2 *
(ppm)
Positive
control
(ppt)
Negative
control
15 0.5 250 25 1.0 500 0.1 -
20 1.0 500 30 2.0 750
25 2.0 750 35 3.0 1000
30 40
All the treatments were carried out in triplicates, and when the water was changed in the jars
that were treated, the water in the negative controls was changed as well. After the particles were

14
MATERIALS AND METHODS
treated two times, the five particles from each jar were placed on petri dishes with DRBC-agar. The
agar plates were left for incubation at 8-9 ˚C for 7 days. This incubation-temperature was similar to
what was expected to be the temperature in the water at the hatchery. The plates were checked upon
daily with a stereoscope, for the following seven days, and the time it took for the fungi to spread
from the particles and infect the agar was noted.
2.2 MAIN STUDY AT THE HATCHERY
2.2.1 Test conditions
Temperature, pH, and dissolved oxygen in the intake water <strong>of</strong> the hatchery were monitored
through the treatment period. The temperature measurements were performed once every three
hours by three Tinytag Plus, Gemini Data Loggers which were placed in three different cylinders
throughout the treatment period. The measurements were performed at the same time every day,
times likely to include the warmest and coldest temperatures every day were chosen. The dissolved
oxygen in the water was measured three times a week by an OxyGuard, Handy Beta and pH was
measured three times a week by OxyGuard Handy pH. The amount <strong>of</strong> dissolved oxygen was
considered to be satisfying as long as it was above 80 %.
2.2.2 Treating eggs in the hatchery
In all trials it is necessary to gain as many samples as possible to get a safe base for statistical
analysis <strong>of</strong> the trials. 66 incubation cylinders were available for the project and therefore there was
a need to give preference to some <strong>of</strong> the tests. The negative control, positive control, and Ibiza salt
became the trial´s highest priorities. The controls were the tests that would be compared with all the
other tests and were therefore very important. Ibiza salt was preferred because <strong>of</strong> its very common
and environmentally safe appearance. Therefore, more incubation cylinders were assigned to these
three groups compared to the number <strong>of</strong> incubation cylinders assigned to BioCare and hydrogen
peroxide treatments. Hydrogen peroxide concentrations were calculated from active ingredient. The
negative controls did not get any treatment and were left undisturbed throughout the treatment
period. The positive controls were treated with Pyceze at the same concentration, exposure duration,
and time interval between treatments as they had been used earlier at the hatchery. The specific
treatments are given in table 2.2, and further informations about the substances are given in
appendix I.

15
MATERIALS AND METHODS
Table 2.2: The treatments assigned to each cylinder unit, and intervals between treatments.
* H2O2 concentrations were calculated from active ingredient.
Substance Concentration Exposure Cylinder unit Treatment
duration
(min)
no.
intervals
Ibiza sea salt
15 ppt 30 5
6
Twice a week
60 8
12
20 ppt 30 10
16
60 7
18
H2O2 * 500 ppm 15 11
30 19
1000 ppm 15 13
30 9
BioCare
1.0 ppt 15 17
30 15
1.5 ppt 15 3
30 20
Positive control 0.1 ppt 40 4
14
Thrice a week
Negative control - - 1
2
21
X
-
In the hatchery the 66 incubation cylinders were set up in three rows with 22 cylinders in
each. The cylinders were connected in a way so that one pump could supply three cylinders at the
same time (figure 2.1 and 2.2). This way <strong>of</strong> connecting the cylinders left one unconnected cylinder
at the end <strong>of</strong> each row. The three single cylinders were used as an extra set <strong>of</strong> negative controls, and
numbered X1, X8 and X15. Altogether there were 21 units with 3 cylinders in each plus the three
single ones. The connected cylinder units were numbered from 1 to 21 and treatments were
assigned to each <strong>of</strong> the units by a random draw. One cylinder unit was used to keep the temperature
loggers in, and because they had not received any treatment the eggs in them were used as negative
control as well. Now there were two extra units (= 6 cylinders) with negative controls. In every unit
the three connected cylinders got equal treatments through the entire treatment period.

16
MATERIALS AND METHODS
Figure 2.1: Three cylinders connected to the same intake water source. The substances were
pumped into the bottle from the bottom and equally distributed to the three cylinders.
Figure 2.2: Two rows <strong>of</strong> cylinders. The solutions were pumped from the grey bucket and into the
cylinder units. The pump is located in the black bucket.
The cylinders were filled with approximately 3 dL <strong>of</strong> overripe rainbow trout (Oncorhynchus
mykiss) eggs in each. The parental fishes were <strong>of</strong> the Faroese strain, and the eggs came from 16
different motherfishes and had three different fathers. The fertilized eggs were all mixed together

17
MATERIALS AND METHODS
before they were distributed to the cylinders. Poor quality (overripe) eggs were used in the
experiment because the high proportion <strong>of</strong> dead eggs among them would be a good substrate for the
fungi to colonize. 29 % <strong>of</strong> the eggs were dead at the beginning <strong>of</strong> the trials.
The two pumps used to pump the substances into the cylinders had different pumping
capacity and needed to be adjusted every once in a while. In average pump І had the capacity <strong>of</strong>
1.04 dL/min and pump П had the capacity <strong>of</strong> 0.75 dL/min, and the water flow in the hatchery was 5
dL/min. The concentrations needed to feed the pumps with in order to get the correct concentrations
into the cylinders were calculated.
2.2.3 Gathering data
For the first two weeks <strong>of</strong> the treatment period, the amount <strong>of</strong> dead eggs that could be seen in
the upper layer <strong>of</strong> eggs in the cylinders were counted twice. It was assumed that eggs would not die
<strong>of</strong> any other cause than being killed by fungus, therefore the dead eggs on the surface were not
counted again before fungus was confirmed in the cylinder. The cylinders were checked once a
week for fungus, and the degree <strong>of</strong> fungal infection in each cylinder was noted. The degrees <strong>of</strong>
fungal infection were divided into seven categories. The fungus had infected:
0- zero eggs
1- one egg, and had not developed much,
2- one or a few eggs, and had developed long hairs,
3- a whole ring <strong>of</strong> eggs around the initial egg,
4- eggs outside the first ring,
5- two whole rings <strong>of</strong> eggs around the initial egg,
6- eggs outside the two rings.
The treatment <strong>of</strong> the eggs had to last until the eggs reached the eyed stage. When they had
reached the eyed stage they were hardy enough to be handled, and from this stage the fungus could
be controlled by removing infected eggs. Rainbow trout eggs need 175 daily temperature units to
reach the eyed stage (Refstie 1993). With a water temperature <strong>of</strong> about 8˚C it would take three
weeks for the eggs to reach the eyed stage. Unfortunately the temperature in the intake water was
very low, which induced a prolonging <strong>of</strong> the treatment period with four additional weeks.

18
MATERIALS AND METHODS
After 7 weeks <strong>of</strong> treatment the eggs were removed from the cylinders and put into trays. The
eggs from one cylinder were put in one tray. By a mistake number 1.1, 1.2 and 1.3 were mixed in
one tray and 2.1, 2.2 and 2.3 in another tray. These were both negative controls. In the trays the
eggs were washed and treated roughly. The rough treatment allows proteins in dead eggs to
coagulate, which turns the eggs white (Kittelsen et al. 1993), and this makes them easier to
distinguish from the live eggs. The dead eggs were removed and measured in dL. To find the
amount <strong>of</strong> eggs in one litre, randomly picked eggs were lined up until the line was 25 cm long.
Knowing the number <strong>of</strong> eggs in this line made it possible to find the number <strong>of</strong> eggs per litre using
a table from Ingebrigtsen (1982).
After the eggs had hatched and the fry had lost their yolk-sacs, the dead eggs and fry left were
counted and removed. Cripples were counted and removed. The kind <strong>of</strong> abnormality they were
suffering from were noted within six categories, the first five categories were the most frequent
kinds <strong>of</strong> abnormalities, and the sixth category was a collecting category for the cripples with
abnormalities that did not fit into any <strong>of</strong> the five other categories. The categories were:
1. no caudal fin (tail)
2. snail formed
3. angled spine
4. Siamese twins
5. abnormality <strong>of</strong> yolk-sac
6. Others
When the cripples had been removed, samples <strong>of</strong> 100-300 fry from ten randomly picked trays
were weighed and the fry were counted in order to get an average weight <strong>of</strong> one single fry.
Afterwards, the fry in each tray were weighed, and this weight was divided with the average weight
<strong>of</strong> a single fry in order to find the number <strong>of</strong> fry in each tray.
2.2.4 Fungal spores in the intake water
To find out how many fungal spores were in the water and what fungal infection pressure the
eggs were dealing with, efforts were made to count the fungal spores in the intake water during the
treatment period. Once a week, a sample <strong>of</strong> the intake water in the hatchery was collected using a

19
MATERIALS AND METHODS
presterilized bottle. From this sample 10 mL were filtrated aseptically, using a sterilized Sartorius
500 mL filtration system, through a Millipore membrane filter with
0.45 µm pores and a diameter <strong>of</strong> 47 mm. These filters were
aseptically placed on sterile DRBC-agar plates and incubated at 25
˚C for 4 days (figure 2.3). Filter pores <strong>of</strong> 0.45 µm are able to stop
both fungal spores and coli<strong>of</strong>orm bacteria (Thougaard et al. 1996;
pers. comm. Niclasen 2004). It was therefore important to use an
agar with antibiotics, that inhibits bacterial growth. The filtration
was repeated two more times in order to get triplicate samples.
After incubation the fungal colonies on each plate were studied
and counted through a stereoscope.
Figure 2.3: The growth <strong>of</strong> fungal spores from 10 mL <strong>of</strong> intake water after four days <strong>of</strong> incubation.
2.2.5 Statistical analysis
The final results in the project were final fungal infection, percent hatch, and percent cripples.
In order to find out if there were differences within exposure durations and/or concentrations <strong>of</strong> one
substance, or if there was an interaction between the two variables, a two-way ANOVA was used.
To find out if there were any differences between the different substances a one-way ANOVA was
used. The null-hypothesis, which stated that there was no difference between the variables, was
rejected at p

20
MATERIALS AND METHODS
This would obstruct the data from fitting a normal curve which is a condition that needs to be
fulfilled in order to make ANOVA tests. Because <strong>of</strong> uncertainty regarding what made the hatching
rate in cylinder 16.1 so low, the cylinder was also excluded when comparing cripple rates and
degrees <strong>of</strong> fungal dispersion
Also in the BioCare treatments there was one cylinder which was excluded from the statistical
tests. This was cylinder 15.3 (1.0 ppt for 30 minutes). All the eggs in this cylinder were dead at the
end <strong>of</strong> the incubation period. It is possible that the water supply tube has been clogged for a while
resulting in the eggs dying due to lack <strong>of</strong> new water containing oxygen, or that air bubbles from the
released hydrogen peroxide have turned the eggs while they were in the vulnerable state.

3.1 PRELIMINARY STUDY
3.1.1 Identification <strong>of</strong> the fungi
3 RESULTS
RESULTS
There was not observed any sexual reproduction in vitro (under laboratory conditions) <strong>of</strong> the
fungus isolated from the water <strong>of</strong> a cylinder with heavily infected salmon eggs in it. There were,
however, discovered long hairs in bundles on the surface <strong>of</strong> secondary zoospore cysts and on a 25
% Glucose-Yeast solution about 12 % indirect germination was observed (work done by Stueland
2004). From these results the most correct classification <strong>of</strong> the fungus was <strong>Saprolegnia</strong> sp. (Hughes
1994).
3.1.2 Testing the candidate substances
In the laboratory study Ibiza salt was the one <strong>of</strong> the three candidate substances with the best
antifungal effect (figure 3.1). Ibiza salt treatments inhibited the fungus for a considerable longer
period <strong>of</strong> time than the other treatments, including Pyceze which was used as positive control. The
statistical tests showed a significant difference between the treatments (p

Infection time (days)
8
7
6
5
4
3
2
1
0
60 min.
Ibiza salt
1 5 ppt
20 ppt
25 ppt
30 ppt
22
RESULTS
Figure 3.1: Results from the preliminary study. The fungistatic properties <strong>of</strong> the substances
expressed as infection time (days until the fungus had infected the agar).
3.2 MAIN STUDY AT THE HATCHERY
3.2.1 Test conditions
60 min.
Ibiza salt
25 ppt
30 pp t
35 pp t
40 pp t
30 min.
BioCare
0 ,5 pp t
1 ppt
2 pp t
15 min.
BioCare
1 p pt
2 p pt
3 p pt
Concentration
30 min.
2 50 ppm
5 00 ppm
7 50 ppm
During the treatment period mean pH in the intake water was between 6.15 and 7.23, with a
mean value <strong>of</strong> 6.9, and mean oxygen saturation was 95.5 %, ranging between 86 % and 105 %.
The temperature in the water during the treatment period was between 0.5 °C and 11.3 °C,
with a mean value <strong>of</strong> 5.2 °C (figure 3.2). Not until the 40 th day the temperature reached 8 °C for the
first time. With a mean temperature <strong>of</strong> 8 °C it would take 3 weeks for the eggs to reach the eyed
stage and, hence the treatment would only have lasted for 3 weeks. However, the low mean
temperature resulted in a treatment period <strong>of</strong> 7 weeks, much longer than originally expected.
H2O2
500 ppm
15 min.
H2O2
750 pp m
100 0 p pm
Controls
Neg . Con t.
Po s. Con t.

Temperature (ºC)
12,00
10,00
8,00
6,00
4,00
2,00
0,00
1
4
7
Day <strong>of</strong> treatment
9
12
15
18
20
23
26
29
31
34
37
40
42
45
23
RESULTS
Figure 3.2: Temperature <strong>of</strong> intake water at the hatchery. The Temperature was measured eight
times daily throughout the treatment period.
3.2.2 Comparison <strong>of</strong> all substances
Based on the results from the laboratory study one could expect that Ibiza salt would have
good results at the hatchery as well and that hydrogen peroxide have rather poor results. But this
certainly was not the case. Hydrogen peroxide had very promising results while Ibiza salt, together
with BioCare, had very poor results (sometimes worse than negative control) (figures 3.3, 3.4, 3.5).
Regarding fungal dispersion there were statistically highly significant differences between the
treatments (p= 0.000) (figure 3.3). Positive control had the lowest mean fungal degrees, 0.7, while
Ibiza salt and negative control had the highest mean fungal degrees, 5.4 and 5.3 respectively.
Also when comparing hatching rates there were highly significant differences (p=0.000)
between the treatments (figure 3.4). Positive control and hydrogen peroxide had the highest mean
hatching rates, 51.7 % and 51.0 % respectively, while BioCare had the lowest hatching rate with a
mean value <strong>of</strong> 24.2 %.
These hatching rates seem very low but when excluding the 29 % <strong>of</strong> the eggs that were dead
at the beginning <strong>of</strong> the experiment the hatching rate for positive control was 72.8 %, the hatching
rate for hydrogen peroxide 71.8 %, and the hatching rate for BioCare 32.4 %.
Regarding cripple rates there were, however, not detected any statistically significant
differences between the substances (figure 3.5).

Fungal degrees
7
6
5
4
3
2
1
0
20 ppt 15 ppt 20 ppt 15 ppt 1000
ppm
500
ppm
24
1000
ppm
Concentrations
500
ppm
1.5
ppt
1.0
ppt
1.5
ppt
RESULTS
Figure 3.3: Mean fungal degrees <strong>of</strong> the treatments. The blue (upper) line represents the negative control and
the green (lower) line represents the positive control. The upper stars represent statistical differences
between negative control and each individual treatment, and the lower stars represent statistical differences
between positive control and each individual treatment.One star (*) represents a significant difference
(p

Cripple rate
2,5
2
1,5
1
0,5
0
*
20
ppt
15
ppt
20
ppt
15
ppt
1000
ppm
500
ppm
25
1000
ppm
Concentrations
500
ppm
1.5
ppt
1.0
ppt
1.5
ppt
RESULTS
Figure 3.5: Mean cripple rate <strong>of</strong> the treatments. The line in the graph represents both negative and positive
control which had the same mean cripple rate. The stars represent statistical differences between negative
and positive control and each individual treatment. One star (*) represents a significant difference (p

26
RESULTS
Fungal degrees - There was no significant difference between concentrations. But at the
concentration 20 ppt there was a statistically highly significant difference between exposure
durations (p

27
RESULTS
(table 3.1). These results show that the bigger the amount <strong>of</strong> Ibiza salt used for treating the eggs the
more cripples were found amongst the fry.
3.2.4 Hydrogen peroxide
Table 3.2: Results from the hydrogen peroxide treatments. Means and standard deviations (SD) for the
hydrogen peroxide treatments. N is the number <strong>of</strong> replicates within each treatment.
* N for negative control was eight replicates at hatching rate and cripple rate, and nine replicates for fungal
degrees.
Treatment
Substance Time/min
Hydrogen
peroxide
30
15
Fungal
degrees Hatching rate Cripple rate
Konc/pp
m N Mean SD Mean SD Mean SD
1000 3 2.0 0.0 50.6 5.9 1.2 0.4
500 3 3.0 1.0 49.0 13.5 0.7 0.5
1000 3 2.3 0.6 50.7 1.9 0.7 0.3
500 3 1.0 1.7 53.5 3.5 0.9 0.2
Positive control - - 6 0.7 1.0 51.7 3.9 0.8 0.3
Negative control - - 8 (9)* 5.3 1.1 40.2 13.9 0.8 0.3
Fungal degrees - There were no significant differences between either exposure durations or
concentrations. Although not significant there was a tendency for interaction (p=0.088). This
interaction manifested itself in mean fungal degrees. At 1000 ppm mean fungal degrees were
slightly higher at 15 than at 30 minutes (2.3 and 2.0 respectively). But at 500 ppm mean fungal
degrees were much higher at 30 than at 15 minutes (3.0 compared to 1.0) (table 3.2). There were
statistically significant differences between both 500 ppm for 30 minutes (p=0.015) and 1000 ppm
for 15 minutes (p=0.038) and positive control (figure 3.3). Mean fungal degrees for these treatments
were 3.0 and 2.3 respectively compared to 0.7 for positive control (table 3.2). Compared to negative
control all treatments had considerable lower mean fungal degrees. The differences were highly
significant (p

28
RESULTS
for hydrogen peroxide treatments was 51.0 % (ranging between 49.0 % and 53.5 %) and mean
hatching rate for negative control was 40.2 % (table 3.2). It is worth mentioning that hydrogen
peroxide at the concentration 500 ppm for 15 minutes had a higher hatching rate than positive
control although the difference was not statistically significant. If the dead eggs at the beginning
were not included the hatching rate for 1000 ppm for 30 minutes was 71.3 %, the hatching rate for
500 ppm for 30 minutes 69.0 %, the hatching rate for 1000 ppm for 15 minutes 71.4 %, and the
hatching rate for 500 ppm for 15 minutes 75.4 %.
Cripple rate - There were no significant differences between neither exposure durations nor
concentrations. Although not significant there was a tendency for interaction (p=0.078). This
interaction manifested itself in mean cripple rates. At 1000 ppm there was a higher cripple rate at 30
than at 15 minutes (1.2 % compared to 0.7 %) whereas at 500 ppm the cripple rate at 15 minutes
was higher than the one at 30 minutes (0.9 % compared to 0.7 %) (table 3.2). There were no
significant differences in cripple rates neither when compared to positive nor to negative control
(figure 3.5).
Compared to Ibiza salt and BioCare hydrogen peroxide gave the best results (figures 3.3, 3.4
and 3.5). But it was not better than positive control (table 4). When comparing fungal degrees there
were significant differences between two <strong>of</strong> the hydrogen peroxide treatments and positive control,
namely 500 ppm for 30 minutes and 1000 ppm for 15 minutes which both had higher mean fungal
degrees than positive control (figure 3.3).
500 ppm for 15 minutes had the best mean hatching rate and the best mean fungal degrees
(table 3.2). This treatment could be considered as good as positive control. When this treatment
only was compared to positive control there were no significant differences between either hatching
rates, criple rates, or fungal degrees (figures 3.3, 3.4 and 3.5).

3.2.5 BioCare
29
RESULTS
Table 3.3: Results from the BioCare treatments. Means and standard deviations (SD) for the BioCare
treatments. N is the number <strong>of</strong> replicates within each treatment.
* N for negative control was eight replicates at hatching rate and cripple rate, and nine replicates
for fungal degrees.
Treatment
Fungal
degrees Hatching rate Cripple rate
Substance Time/min Konc/ppt N Mean SD Mean SD Mean SD
BioCare
30 1.5 3 6.0 0.0 5.8 2.1 0.4 0.5
1.0 2 4.5 0.7 28.4 3.7 0.9 0.2
15
1.5 3 3.0 1.0 37.4 13.0 2.1 0.4
1.0 3 5.0 0.0 22.4 18.6 1.4 0.5
Positive control - - 6 0.7 1.0 51.7 3.9 0.8 0.3
Negative control - - 8 (9)* 5.3 1.1 40.2 13.9 0.8 0.3
Fungal degrees - There was no significant difference between concentrations but there was a
statistically highly significant difference between exposure durations at the concentration 1.5 ppt
(p

30
RESULTS
negative control had a mean hatching rate <strong>of</strong> 40.2 % (table 3.3). If the dead eggs at the beginning <strong>of</strong>
the experiment were not included the hatching rate for 1.5 ppt for 30 minutes was 8.1%, the
hatching rate for 1.0 ppt for 30 minutes 39.9%, the hatching rate for 1.5 ppt for 15 minutes 52.6%,
and the hatching rate for 1.0 ppt for 15 minutes 31.5%.
Cripple rate - There was no significant difference between concentrations. However, there
was a highly significant difference between exposure durations at the concentration 1.5 ppt
(p

Table 3.4: Number <strong>of</strong> <strong>Saprolegnia</strong> sp. spores per litre <strong>of</strong> intake water.
Week Spores / L
<strong>Saprolegnia</strong> sp. spores / L
1 1150
2 67
3 0
4 200
5 33
6 33
1400
1200
1000
800
600
400
200
0
1 2 3 4 5 6
Week
Figure 3.6: Fungal spores per litre intake water at the hatchery
31
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
All fungal spores / L
RESULTS
<strong>Saprolegnia</strong> sp.
All fungi

4 DISCUSSION
DISCUSSION
Present study was preformed in a hatchery using the equipment they normally use to hatch
and treat eggs, and is therefore similar to the way eggs would be treated in a normal hatchery.
However there were not as many eggs in the incubation cylinders as is usual in a hatchery. There
were about 3 dL <strong>of</strong> eggs in each cylinder, while one cylinder could contain 25 L <strong>of</strong> eggs. The eggs
in present study have therefore not been exposed to the same pressure and density as eggs would in
normal conditions in a hatchery. The quality <strong>of</strong> eggs affect infection rates (Schreier et al. 1996) .
Poor quality (overripe) eggs were used in the present study to make sure that there was a good basis
for natural infection <strong>of</strong> the eggs and it probably gave a smaller hatching rate than good quality eggs
would give.
Also worth noting is that the substances that were in liquid form made the work both easier
and less time demanding. There are presumably many different systems that take care <strong>of</strong> the
treatment <strong>of</strong> the eggs in hatcheries, but done by handcraft, a liquid substance is absolutely to prefer
above a solid substance on condition that they are both equally qualified as fungistatic agents or if
the liquid is better.
4.1 PRELIMINARY STUDY
4.1.1 Identification <strong>of</strong> the fungi
In present study groups <strong>of</strong> long hairs on secondary zoospore cysts and indirect germination
were observed in vitro (in laboratory conditions) <strong>of</strong> fungus isolated from salmon eggs. But no
sexual reproduction was observed. Willoughby (1985) described two vegetative characteristics <strong>of</strong>
the <strong>Saprolegnia</strong> diclina-<strong>Saprolegnia</strong> parasitica complex. They were groups <strong>of</strong> long, hooked hairs
on secondary zoospore cysts and indirect germination <strong>of</strong> secondary zoospore cysts. Even though the
fungus isolated from salmon eggs had these characteristics the conclusion was not to group it in this
complex, but to name it <strong>Saprolegnia</strong> sp. This was due partly to different opinions among scientists
regarding the naming <strong>of</strong> pathogenic <strong>Saprolegnia</strong> species, and partly to a recent conclusion by
scientists that all non-sexual isolates <strong>of</strong> <strong>Saprolegnia</strong> from fish should be named <strong>Saprolegnia</strong> sp.
(Hughes 1994). In present study rainbow trout eggs were treated against saprolegniasis. However,
due to lack <strong>of</strong> time, no identification was done on fungus isolated from rainbow trout eggs, only on
fungus from salmon eggs which were incubated earlier than the rainbow trout eggs. But it seems
likely that the fungus infecting the rainbow trout eggs was the same as the fungus that infected the

33
DISCUSSION
salmon eggs, namely <strong>Saprolegnia</strong> sp. This assertion is built on two assumptions. The first
assumption is that there seems to be no difference in how saprolegniasis affects salmon eggs and
how it affects rainbow trout eggs. Secondly the white, fluffy colonies on agar, isolated from the
intake water were assumed to be <strong>Saprolegnia</strong> sp. because <strong>of</strong> the similar appearance to the colonies
earlier identified to be <strong>Saprolegnia</strong> sp. When this pathogenic fungus was found in the water it
seems very likely that it was this fungus which also infected the rainbow trout eggs.
4.1.2 Testing the candidate substances
In the laboratory study all three <strong>of</strong> the candidate substances demonstrated fungistatic
properties.
Ibiza salt had the best results, but one may add that most <strong>of</strong> the concentrations <strong>of</strong> Ibiza salt
used (table 2.1) could not be applied to treat living eggs, because salt is toxic to eggs at too high
concentrations: Kitancharoen et al. (1997) reported that 25 ppt salt for 60 minutes twice a week was
best for fungal control without affecting the egg condition, and Marking et al. (1994) reported that
30 ppt salt for 60 minutes every other day increased hatching rate and inhibited fungal infection, but
concentrations above 30 ppt were toxic to eggs. Edgell et al. (1993) reported that although fungus
control is more effective at higher salt concentrations, salt solutions may cause egg deaths at levels
<strong>of</strong> 25 ppt or higher. So if the treatments in the laboratory that had a salinity <strong>of</strong> or above 25 ppt were
removed, only two treatments would be left, 15 ppt and 20 ppt salinity. Both had inhibited the fungi
from growing for more than four days, (which was the longest period <strong>of</strong> time between treatments at
the hatchery) so they were both acceptable to use at the hatchery.
The only one <strong>of</strong> the hydrogen peroxide concentrations that made any difference from negative
control was the 750 ppm concentration (figure 3.1). Compared to negative control it inhibited the
fungus for one day more at 60 minutes exposure, and for 0.4 day longer at 30 minutes exposure.
The hydrogen peroxide treatments were not very useful because the concentrations were calculated
from solution instead <strong>of</strong> active ingredient. The concentrations used in the laboratory study <strong>of</strong> 250
ppm, 500 ppm, 750 ppm, 1000 ppm <strong>of</strong> the solution correspond respectively to 87.5 ppm, 175.0
ppm., 262.5 ppm, and 350.0 ppm <strong>of</strong> active ingredient.
The BioCare treatments demonstrated increasing fungistatic properties with increasing
concentrations at both <strong>of</strong> the two exposure durations.

34
DISCUSSION
Treatment with 0.1 ppt Pyceze for 10 minutes was equal to the negative control. Pyceze
treatments lasted for 10 minutes, which was lower than the recommended exposure duration <strong>of</strong> 40
minutes.
4.2 MAIN STUDY AT THE HATCHERY
4.2.1 Test conditions
The water quality (hardness, pH, temperature, organic matter etc.) can increase or decrease
the toxicity <strong>of</strong> some chemicals (Piper et al. 1982; Howe et al. 1994 quoted from Schreier et al.
1996). In this study the alkalinity (hardness) <strong>of</strong> the water was not measured, but the water in the
Faroe Islands is generally not hard. In a study <strong>of</strong> drinking water in the Faroe Islands the lowest
alkalinity was 7.3 mg/L HCO3 and the highest was 21.0 mg/L HCO3 (Larsen 2000).
4.2.2 Ibiza salt
In present study 20 ppt for 30 minutes was the best treatment. Although it had the highest
fungal degrees it also had the highest hatching rate and a low cripple rate (figures 3.3, 3.4 and 3.5).
The hatching rate was 44.3 %, 4.1 % higher than for negative control, and the cripple rate was 0.7,
0.1 % lower than both negative and positive control (table 3.1). In the study conducted by Edgell et
al. (1993), 20 ppt for 60 minutes was the best <strong>of</strong> various salt treatments to prevent mortality
amongst salmon eggs. However a 26:1 mixture <strong>of</strong> sodium chloride and calcium chloride yielded
better results than sea salt which was second best. In their study the eggs were treated three times a
week.
Fungal degrees - At 20 ppt there was a statistical highly significant difference between
exposure durations regarding fungal degrees. 60 minutes yielded lower fungal degrees than 30
minutes, mean values <strong>of</strong> 4.3 and 5.8 respectively (table 3.1). Negative control had mean fungal
degrees <strong>of</strong> 5.3, 1.0 degree higher than the best Ibiza salt treatment. But at the same time negative
control had the same or lower mean fungal degrees than the rest <strong>of</strong> the treatments. Hence it can be
speculated that a threshold <strong>of</strong> the amount <strong>of</strong> Ibiza salt has to be reached before it functions as a
fungistatic.

35
DISCUSSION
Hatching rate – There was no significant difference between the hatching rates neither within
the Ibiza salt treatments nor between Ibiza salt and negative control (figure 3.4).
Cripple rate - Regarding cripple rates there were highly significant differences between both
exposure durations and concentrations, with the higher concentrations yielding more cripples than
the lower and the longer exposure durations yielding more cripples than the shorter. However,
negative control had a slightly higher cripple rate than the 30 minutes treatments (figure 3.5). These
findings are in agreement with Edgell et al. (1993) who found that the fungistatic effect <strong>of</strong> salt was
better at higher concentrations but to high concentrations could cause egg death. To conclude it
would be important to find some kind <strong>of</strong> compromise where the amount <strong>of</strong> salt was high enough to
be fungistatic and at the same time not so high that it would damage the eggs.
The outcome for Ibiza salt and negative control was almost the same (figures 3.3, 3.4 and
3.5). There was one treatment where there was a significant difference compared to negative
control, namely 20 ppt for 60 minutes which had a significant difference in cripple rates, the salt
having more cripples than negative control (table 2). At the same time this treatment had the lowest
fungal degrees, which was highly significant different from 15 ppt for 60 minutes.
Compared to positive control Ibiza salt treatments were worse in all aspects excluding cripple
rates where 30 minutes treatments yielded lower cripple rates than positive control (figure3.5).
Ibiza salt does have a certain fungistatic effect, but it was not a good choice for controlling
fungal infections because it was too contaminated. However, there have been many experiments
with other salts which have turned out well. Although less effective than hydrogen peroxide, it does
have a fungistatic effect which many authors have found to improve the hatching rate (Marking et
al. 1994; Schreier et al. 1996; Kitancharoen et al. 1997; Kitancharoen et al. 1998). Furthermore
studies have been done on other salts than sodium chloride, for example sea salt and a 26:1 mixture
<strong>of</strong> sodium chloride and calcium chloride (Edgell et al. 1993; Waterstrat & Marking 1995; Schreier
et al. 1996; Kitancharoen et al. 1997).
Although the results for the Ibiza treatments in this study were not favourable compared to the
controls they were not so poor compared to other studies. Kitancharoen et al. (1997) used sodium
chloride to treat rainbow trout eggs against saprolegniasis and 20 ppt for 60 minutes two times a

36
DISCUSSION
week yielded a hatching rate <strong>of</strong> 46.0 %. The same treatment in present study yielded a mean
hatching rate <strong>of</strong> 42.8 % (table 3.1). But if the dead eggs at the beginning <strong>of</strong> present study were not
included, the mean hatching rate for this treatment was 60.2 %, considerably higher than for the
other study.
It has been pointed out that salt treatment may be impractical in treating big amounts <strong>of</strong> eggs
because it is inconvenient to handle and mix the large volume <strong>of</strong> salt required (Waterstrat &
Marking 1995). This also applies to the Ibiza salt treatments used in present experiment.
Furthermore, the Ibiza salt was rather impure and it was probably the impurities which clogged the
water supply to the cylinders where all the eggs died during the treatment period.
The general conclusion for Ibiza salt treatment is that if it should be used as a fungal control
agent, it should be at the concentration 20 ppt for 30 minutes. But at the same time one could use no
treatment at all, in stead <strong>of</strong> Ibiza salt, save a lot <strong>of</strong> money and work, still get the same hatching rate
and there are possibilities for that there would be less cripples as well. Using the positive control
(Pyceze) instead <strong>of</strong> Ibiza salt would gain a higher hatching rate and also save some work because it
is a liquid.
4.2.3 Hydrogen peroxide
In present study there were no statistical significant differences between the hydrogen
peroxide treatments, which all had overall low fungal degrees, high hatching rate and low cripple
rates (figures 3.3, 3.4 and 3.5). Yet, the results revealed a tendency for the 500 ppm for 15 minutes
treatment to be slightly more effective than the other hydrogen peroxide treatments.
Hydrogen peroxide came out <strong>of</strong> this test as the best candidate substance for fungal control. It
is considered to be one <strong>of</strong> the most effective antifungal agents available for use on fish eggs at
present time (Gaikowsky et al. 1998). Kitancharoen et al. (1998) concluded that 1000 ppm
hydrogen peroxide for 60 minutes twice a week was equally effective as 2 ppm malachite green
with the same exposure durations. Marking et al. (1994) reported that hydrogen peroxide was
fungicidal and non-toxic to eggs at 500 ppm for 60 minutes every other day, and that it is
considered safe in the environment.

37
DISCUSSION
Compared to negative control hydrogen peroxide treatments had 10.8 % higher mean hatching
rate, and if only 500 ppm for 15 minutes was compared with the negative control, it had 13.2 %
higher hatching rate (table 3.2). When the 29 % eggs that already were dead before the trials started
were subtracted from the initial lot <strong>of</strong> eggs, the mean hatching rate for the hydrogen peroxide
treatments was 20.3 % higher than for negative control, and the hatching rate <strong>of</strong> 500 ppm for 15
minutes was 23.9 % higher than for negative control.
Hydrogen peroxide treatments were equally effective as positive control in terms <strong>of</strong> hatching
rate and cripple rates (figures 3.4 and 3.5). There was however a significant difference in fungal
degrees, where hydrogen peroxide had more fungal infection than positive control (figure 3.3).
The treatments with 1000 ppm had lower fungal degrees than the 500 ppm for 30 minutes, but
the hatching rates for the three treatments were similar (figure 3.3). This could mean that those eggs
not killed by fungi in the 1000 ppm treatments were killed by the treatment, however, the statistical
analysis does not demonstrate any significant differences between the treatments.
Hydrogen peroxide is considered to be temperature dependent (Bovbjerg 2000; Hjelme
2000b). When treated with hydrogen peroxide, fish toxicity significantly increases with water
temperature (Pers. comm. Rach 1995 quoted from Schreier et al. 1996). Gaikowsky et al. (1998)
found a significant decrease in the probability <strong>of</strong> hatch related to hydrogen peroxide treatment, and
treatment with 1000 ppm for 15 minutes five times a week gave 7 % less hatch than the same
exposure durations with 500 ppm. In that study the temperature was 13.1 ºC – 13.5 ºC. In present
study the hatching rate <strong>of</strong> 1000 ppm for 30 and 15 minutes were respectively 2.9 % and 2.8 % less
than those for 500 ppm for 15 minutes (table 3.2). Without the 29 % dead eggs at the beginning, the
hatching rates <strong>of</strong> the 1000 ppm treatments were 4.1 % and 4.0 % less than the hatching rate for 500
ppm for 15 minutes. The smaller differences in present study can possibly be explained by a lower
temperature (mean 5.2 ºC, range 0.5 ºC – 11.3 ºC) and less frequent treatments.
The general conclusion for hydrogen peroxide is that it is very effective for fungal control at
all exposure durations and concentrations used in present study. It is definitely better than the
negative control, and there were no significant differences from positive control. Also hydrogen
peroxide is a liquid, which makes it easy to work with.

4.2.4 BioCare
38
DISCUSSION
BioCare turned out to be the worst candidate substance in this study. Compared to negative
control there was no statistically significant difference between fungal degrees, though there was a
tendency for BioCare to have lower fungal degrees (figure 3.3). There was a significant difference
between the hatching rates, where the hatching rates <strong>of</strong> BioCare treatments were lower than for
negative control (figure 3.4). BioCare had a mean hatching rate that was 16 % lower than the
hatching rate for negative control (table 3.3). There was no significant difference in the cripple rates
between BioCare treatments and negative control (figure 3.5). However there were more cripples
among the BioCare treated fry than there were in any other treatment group. These observations
indicate that BioCare treatments have been toxic to the eggs.
In all the different BioCare treatments there was a statistically significant difference between
the exposure durations at the concentration 1.5 ppt. The results from the treatment with 1.5 ppt for
30 minutes, which is the strongest concentration with the longest exposure duration, were the most
puzzling. This concentration was expected to inhibit the fungus but the treatment gave very high
mean fungal degrees, a very low hatching rate, only 5.8 %, and also the lowest cripple rate in the
whole study (figure 3.5). This hatching rate was 34.4 % lower than that for negative control
(hatching rate 40.2 %). The low hatching rate was probably a reason for the cripple rate to be so
low, if most <strong>of</strong> the eggs/fry were dead, also most <strong>of</strong> the cripples were dead. Therefore this specific
treatment was very likely to have been toxic to the eggs. If this treatment was to be removed from
the other BioCare treatments the mean values <strong>of</strong> them would be different. The mean fungal degree
would be 4.2, mean hatching rate would be 29.4 % and the mean cripple rate would be 1.5 %.
Compared to negative control they would have 1.1 lower mean fungal degrees, 10.8 % lower
hatching rate and 0.7 % higher cripple rate.
There was not much literature to find about BioCare, and the information that was found did
not contain any experiments similar to these.
The general conclusion for BioCare is therefore that at the exposure durations and
concentration used in this study, BioCare is not recommended for fungal control on rainbow trout
eggs. Actually, not using any substance for fungal control at all, instead <strong>of</strong> using BioCare
treatments, can give 31.7 % higher hatching rate, fewer cripples and save a lot <strong>of</strong> work and money.

4.2.5 Fungal spores in the intake water
39
DISCUSSION
In quantitative studies water mould propagule (any spore or other part <strong>of</strong> the fungi capable <strong>of</strong>
producing a new organism and used as a means <strong>of</strong> dispersal) numbers in most natural water bodies
typically seem to range from 10 1 to 10 3 spores/L (Beakes et al. 1994). The intake water at P/F
Fiskaaling comes from a river, and is treated with UV-rays, still, the number <strong>of</strong> fungal spores in
present study was between 17,000 and 2,200 spores/L, and for <strong>Saprolegnia</strong> sp. spores alone the
range was between 1,150 and 33 spores/L (figure 3.6 and table 3.4). It is possible that there has
been a water mould bloom in February, like the <strong>Saprolegnia</strong> spring and autumn blooms described
by Langvad (1999), where the normal amount <strong>of</strong> <strong>Saprolegnia</strong> spores (50-200 spores/L) increased by
a factor 20. There was no correlation between the amount <strong>of</strong> fungal spores or <strong>Saprolegnia</strong> sp. spores
and fungal infection degree on the eggs. In the first week there were most spores in the ingoing
water, but there was not observed any fungal infection until three weeks later, when one cylinder <strong>of</strong>
the negative controls was infected. In the fourth week fungal infection was observed in seven
cylinders. This was about the same time as the water became warmer, and the number <strong>of</strong> fungal
spores in the water had decreased as regards to the first sample. It is possible that the infections had
been there all the time, but they had been so small and developed so slowly that they had not been
observed by the naked eye until later in the period.
4.3 IDEAS FOR FUTURE EXPERIMENTS
It would be interesting to do more investigations with pure salt, sodium chloride, where the
limits for when the different concentrations become toxic are found, and perhaps monitor the water
quality to see if it has an effect on the efficacy <strong>of</strong> salt. It would also be interesting to use very high
concentrations for very short exposure durations. This is not possible in the treatment system used
in present study, so perhaps a system where eggs in the eyed stage were dipped in the solutions
would be an idea.
Hydrogen peroxide investigations could be preformed with more replicates for each treatment
to get a better statistical base for the results. Also there would be a bigger chance to establish if
there are any differences between exposure durations or concentrations. Based on the results from
present study, especially 500 ppm for 15 minutes twice a week is recommended to study further,
perhaps also at other treatment intervals than those used here. It would also be interesting to find out

40
DISCUSSION
how big the danger <strong>of</strong> oxygen bubbles which may kill the eggs is, and at which concentrations and
exposure durations the risk is at a minimum.
The method used to treat the eggs, was to pump the different solutions through the cylinders.
A disadvantage with that method is that the solution might be uneven distributed in the cylinders,
and that the solution will be diluted by the water already in the cylinder when the treatment starts.
This results in an uncertainty around the exact exposure duration and concentrations. One way to
get around this problem could be to carry out an experiment where a coloured solution was pumped
into cylinders made <strong>of</strong> a transparent material. Then one could see how the solution was distributed,
and make adjustments (cylinder shape, water flow etc.) which can make the system more reliable.

5 CONCLUSION
CONCLUSION
Together with positive control which was Pyceze only hydrogen peroxide can be
recommended as a treatment option against saprolegniasis on rainbow trout eggs. Three <strong>of</strong> the
hydrogen peroxide treatments had slightly lower hatching rates while one treatment (500 ppm for
15 minutes) had a slightly higher hatching rate than positive control. Regarding both fungal degrees
and cripple rates there were no significant differences between this last treatment and positive
control. Hence further investigations on hydrogen peroxide would be interesting.
Based on the experiences from this study Ibiza salt is unfit for treatment <strong>of</strong> salmonid eggs. It
had almost the same results as negative control. It had an antifungal affect only at high
concentrations but at the same time the higher concentrations and longer exposure durations
resulted in more cripples.
Whereas Ibiza salt treatments had almost the same results as no treatment BioCare in many
ways had worse results than no treatment at all. All <strong>of</strong> the BioCare treatments had lower hatching
rates than negative control and, furthermore, BioCare seemed to have a toxic effect on the eggs.
<strong>Saprolegnia</strong> sp. was identified on salmon eggs. Although the fungus on the rainbow trout
eggs was not identified it was most likely this same species which also infected the rainbow trout
eggs.

Aknowledgements
AKNOWLEDGEMENTS
We wish to thank all the persons who have been involved in this project. Special thanks to
Astrid Hansen, Svein Stueland, Beinta Niclasen, Marjun í Túni Mortensen, and external supervisor
Peter Østergård for their help and for sharing their experience with us, to Petur Steingrund and
Petur Zachariassen for their help with the statistics, and to Roar Olsen and internal supervisor
Eyðfinn Magnussen for providing helpful comments on the manuscript.

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Personal communication
46
REFERENCES
Gregersen A. (2004): Managing director. P/F Fútaklettur. Sandavágur, Faroe Islands.
Hansen A. (2004): Production manager. P/F Fiskaaling, við Áir, Hvalvík, Faroe Islands.
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the Faroe Islands.
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Wardum, H. (2004): Former Managing director. P/F Fiskaaling, við Áir, Hvalvík, Faroe Islands.

APPENDIX 1
Test chemicals
APPENDIX
APPENDIX
Hydrogen peroxide - (H2O2) 35% active ingredient w/w from Brenntag Nordic, Gl. Strandvej
16, DK-2990 Nivaa,Tlf. 43-29-28-88
Ibiza salt - Ibiza Salt, Food Grade Salt from P/F M.J. Saltsøla
BioCare SPC - (Na2CO3 · 1.5 H2O2) Sodium Carbonate Peroxyhydrate, Brenntag
Nordic, Gl. Strandvej 16, DK-2990 Nivaa, Tlf. 43-29-28-88 (26.09.02)
Pyceze - 500 mg/mL active ingredient (2-bromo-2-nitropropane-1.3-diol
(bronopol)), Novartis Animal Vaccines, 4 Warner Drive, Springwood
IND EST, Braintree, Essex, England

APPENDIX
48
APPENDIX II
Ibiza salt
Minutes ppt
Cyl.
No.
Fungal
degrees
after 7
w.
Hatching
rate (%)
Cripple
rate (%)
Hatching rate
(%)
excluding dead
eggs at start
7.1
7.2
7.3
4
6
5
0
0
0
-
-
-
-
-
-
20
18.1
18.2
18.3
5
4
4
36.3
46.0
46.0
1.1
1.2
1.3
51.2
64.7
64.7
8.1
8.2
8.3
5
6
5
48.6
50.8
46.8
1.0
0.9
0.6
68.4
71.6
65.9
60
15
12.1
12.2
12.3
6
5
5
32.2
31.7
34.2
0.9
1.0
0.7
45.4
44.6
48.1
10.1
10.2
10.3
6
6
6
46.0
42.6
43.6
0.8
0.7
0.8
64.7
60.0
61.4
20
16.1
16.2
16.3
6
6
5
6.1
41.8
47.4
0.0
0.4
1.0
8.6
58.9
66.8
5.1
5.2
5.3
6
6
6
34.2
36.8
34.4
0.8
0.4
0.7
48.2
51.8
48.5
30
15
6.1
6.2
6.3
6
4
5
29.5
32.0
41.4
0.6
0.7
0.3
41.5
45.1
58.3

Hydrogen peroxide
Minutes ppm
30 1000
BioCare
15
500
1000
500
Minutes ppt
30 1,5
15
1,0
1,5
1,0
Cyl.
No.
9.1
9.2
9.3
19.1
19.2
19.3
13.1
13.2
13.3
11.1
11.2
11.3
Cyl.
No.
20.1
20.2
20.3
15.1
15.2
15.3
3.1
3.2
3.3
17.1
17.2
17.3
Fungal
degrees
after 7
w.
2
2
2
4
2
3
3
2
2
3
0
0
Fungal
degrees
after 7
w.
6
6
6
5
4
6
4
2
3
5
5
5
Hatching
rate (%)
49.3
57.1
45.5
33.5
57.7
55.9
48.6
51.1
52.4
49.8
56.6
54.2
Hatching
rate (%)
0.4
9.9
7.0
25.7
31.0
0.0
22.4
43.4
46.3
7.0
43.0
17.2
49
Cripple
rate (%)
0.9
1.6
1.1
0.4
1.2
0.5
1.0
0.7
0.4
0.8
1.0
1.1
Cripple
rate (%)
0.0
0.8
0.0
0.7
1.0
0.0
2.5
1.8
2.0
1.0
1.2
1.9
Hatching rate
(%)
excluding dead
eggs at start
69.4
80.5
64.1
47.2
81.2
78.7
68.4
71.9
73.8
70.1
79.7
76.4
Hatching rate
(%)
excluding dead
eggs at start
0.6
13.9
9.9
36.2
43.6
0.0
31.5
61.1
65.3
9.9
60.5
24.2
APPENDIX

Pyceze=pos. control
Minutes ppt
40
Neg. Control
0,1
Cyl.
No.
4.1
4.2
4.3
14.1
14.2
14.3
Cyl.
No.
1.1
1.2.
1.3.
21.1
21.2
21.3
2.1
2.2
2.3
1 x
8 x
15 x
Fungal
degrees
after 7
w.
0
2
0
2
0
0
Fungal
degrees
after 7
w.
5
6
6
6
6
4
3
6
6
-
-
-
Hatching
rate (%)
50.3
52.2
45.2
51.2
54.3
56.8
50
Cripple
rate (%)
1.2
0.4
0.7
0.6
0.8
0.8
Hatching Cripple
rate (%) rate (%)
23.0 0.8
(mean (mean
for 3 for 3
cyl.) cyl.)
47.4
15.7
49.4
35.7
(mean
for 3
cyl.)
48.4
50.2
51.7
0.7
0.7
0.4
0.7
(mean
for 3
cyl.)
1.3
0.7
1.0
Hatching rate
(%) excluding
dead eggs at
start
Hatching rate
(%)excluding
dead
eggs at start
70.8
73.5
63.7
72.2
76.5
80.0
32.4
(mean for 3 cyl.)
66.7
22.1
69.6
50.2
(mean for 3 cyl.)
68.2
70.7
72.8
APPENDIX