Efficacy of clove oil and ethanol against Saprolegnia
sp. and usability as antifungal agents during
incubation of rainbow trout Oncorhynchus mykiss
(Walbaum) eggs
Petri Hoskonen1, Jouni Heikkinen2, P€aivi Eskelinen3 & Juhani Pirhonen1
1Department of Biological and Environmental Science, University of Jyv€askyl€a, Jyv€askyl€a, Finland2Department of Biosciences, University of Eastern Finland, Kuopio, Finland3Finnish Game and Fisheries Research Institute, Jyv€askyl€a, Finland
Correspondence: J Pirhonen, Department of Biological and Environmental Science, University of Jyv€askyl€a, P.O. Box 35, 40014
Jyv€askyl€a, Finland. E-mail: [email protected].
Abstract
Inhibitory concentrations of clove oil and etha-
nol against growth of Saprolegnia sp. hyphae
were screened by a modification of the hemp
(Cannabis sativa L.) seed MicroPlate (HeMP)
method and their usability as antifungal agents
during incubation of rainbow trout Oncorhynchus
mykiss eggs was tested. In vitro experiment
showed that in continuous static exposure, clove
oil at 100 mg L�1 significantly inhibited the
growth of Saprolegnia, whereas in bath expo-
sures, clove oil at 500 mg L�1 had no significant
effect at any exposure time tested (15, 60 and
240 min), but clove oil at 10 000 mg L�1 signif-
icantly inhibited growth at all exposure times.
Clove oil and ethanol treatments had no visible
effects on the onset or spread of the fungus
during incubation of rainbow trout eggs. Clove
oil at 1000 mg L�1 resulted in 95–100% mortal-
ity before the eyed stage was reached. Sublethal
concentrations of clove oil and ethanol had no
effects on the development rate of the embryo or
growth and yolk utilization efficiency after
hatching. This study suggests that clove oil and
ethanol may not be options in controlling aqua-
tic fungi infestations during incubation of rain-
bow trout eggs.
Keywords: water mould, aquatic fungi, euge-
nol, fungicide, hemp seed, yolk efficiency
Introduction
Aquatic fungi are causing problems and economic
losses during egg incubation in fish hatcheries
worldwide. In general, the disease is caused by
species belonging to genera Achlya, Aphanomyces
and Saprolegnia (Noga 1993). Fungal infections on
fish eggs begin when zoospores in water colonize
dead eggs, and nearby live eggs may be directly
infected or suffocated by the expanding mycelium
(Pottinger & Day 1999). Saprolegnia-infected eggs
are easily recognized by the fluffy, cotton-like,
white to greyish patches surrounding the eggs
(Stueland, Hatai & Skaar 2005). Different species
and strains of Saprolegnia differ significantly in
their pathogenicity (Hatai & Hoshiai 1993).
Fungal infestations can be controlled chemically
by treating the eggs with antifungal agents and
physically by removing dead eggs. Manual
removal of dead eggs at regular intervals is a labo-
rious procedure which can be safely performed
only after the eyed stage (Jensen & Alderdice
1989) and therefore the spreading of fungi before
the eyed stage must be prevented by other means.
In modern large-scale fish hatcheries, chemical
control of fungi is the preferred method, but
suitable cost-effective, easy to use, environmental-and user-safe chemical has not been found to
replace malachite green, which has been banned,
for example, in the European Union and USA
(Roberts 2002). Formalin is currently the only
© 2013 John Wiley & Sons Ltd 1
Aquaculture Research, 2013, 1–9 doi:10.1111/are.12200
chemical approved by the U.S. Food and Drug
Administration (FDA) to control fungus on salmo-
nid eggs (Winton 2001) but there are concerns
about its safety (Masters 2004; Wooster, Martinez,
Bowser & O’Hara 2005.
Clove oil is an essential oil derived from the
stems, leaves and buds of Eugenia caryophyllata and
E. aromatica (Griffiths 2000). The active ingredient
of clove oil is eugenol (4-allyl-2-methoxyphenol),
which comprise 70–90% of the oil (Usta, Krey-
diyyeh, Bajakian & Nakkash-Chmaisse 2002).
Clove oil has been discovered as an effective anaes-
thetic for fish (Keene, Noakes, Moccia & Soto
1998; Cho & Heath 2000; Hoskonen & Pirhonen
2004) and because of its known antibacterial and
antifungal properties, a few studies have also tested
it as a fungicide against aquatic fungi. Hussein,
Wada, Hatai and Yamamoto (2000) found that the
minimum inhibitory concentration (MIC) and the
fungicidal concentration of eugenol mixed in
dimethyl sulfoxide against four different Saprolegnia
sp. were 500 and 1000 mg L�1 respectively. In
contrast, the MIC of eugenol in FA 100 (a commer-
cial, water-soluble product that contains 10%
eugenol in volume) varied between 125 and
250 mg L�1 and the fungicidal concentration was
1000 mg L�1 (Hussein et al. 2000). Tampieri,
Galuppi, Carelle, Macchioni, Cioni and Morelli
(2003) observed that the MIC and minimum lethal
concentration (MLC) of eugenol against S. parasitica
were 200 and 250 mg L�1 respectively. Bouchard,
Patel and Lahey (2001) found that clove oil con-
centrations over 20 g L�1 resulted in very high
death rates of eyed rainbow trout, Oncorhynchus
mykiss (Walbaum), eggs. The eggs treated with
clove oil at 1 g L�1 had the lowest rate of fungus
infection and the authors suggested that future
studies should examine lower doses, frequency of
application and suitability to green eggs (Bouchard
et al. 2001).
The present study included two experiments.
First, the effects of continuous clove oil exposure
and clove oil baths on growth of Saprolegnia sp.
hyphae were tested in vitro. The method used was
a modification of the Hemp (Cannabis sativa L.)
seed MicroPlate (HeMP), which was introduced by
Stueland, Heier and Skaar (2005). Second, efficacy
of clove oil and ethanol baths as antifungal
agents during incubation of rainbow trout eggs
from newly fertilized green eggs to hatching was
tested in vivo. The effects of treatments on the sur-
vival rate of the eggs, size and malformations at
hatching and growth and yolk utilization efficiency
after hatching were also studied.
Materials and methods
In vitro experiment
Continuous exposure
The Saprolegnia sp. strain (Fin 22; Bangyeekhun,
Pylkk€o, Vennerstr€om, Kuronen & Cerenius 2003;
Di�eguez-Uribeondo, Fregeneda-Grandes, Cerenius,
P�erez-Iniesta, Aller-Gancedo, Teller�ıa, S€oderh€all &
Mart�ın 2007. GenBank accession number
AM228805) was point-inoculated in the middle of
a Sabouraud dextrose agar (LAB M, Bury, UK)
plate and incubated at room temperature for
2 days. Whole hemp seeds with husk (Lassila
farm, Tuusula, Finland) were autoclaved at 121°Cfor 15 min, cooled and placed individually using
sterile forceps in a circle around the Saprolegnia
colony (Fig. 1a). Agar plate was incubated for
another 2 days at room temperature during which
the Saprolegnia colony spread over the hemp seeds.
Fifty millilitres of all test and control solutions
were mixed in sterile glass flasks. Densities of clove
oil and ethanol were 1.04 and 0.79 g mL�1
respectively. All final solutions contained 1% of
Sabouraud liquid medium (LAB M), desired
amounts of clove oil (distributor Oy Anders Meder
Ab, Helsinki, Finland) and 95% ethanol, with rest
of the volume adjusted by deionized water. Clove
oil is not soluble in cold water, and the most com-
monly used solvent is ethanol (Boyer, White, Stier
& Osenberg 2009). Because it is recommended
that a stock solution containing one part of clove
oil and nine parts of ethanol is mixed before usage
(Cho & Heath 2000; Javahery, Nekoubin & Mora-
dlu 2012), the effects of clove oil stock solution, as
well as ethanol and clove oil alone, were tested.
Stock solution of clove oil (one part clove oil and
nine parts ethanol) was tested at clove oil concen-
trations of 500 and 5000 mg L�1 (with ethanol
concentrations of 3555 and 35 550 mg L�1
respectively). Clove oil alone was tested at 100,
500, 1000, 5000 and 10 000 mg L�1. Ethanol
alone was tested at 3555 and 71 100 mg L�1.
Control solution contained only deionized water
and 1% of Sabouraud liquid medium. Within 1 h
of preparation, 1 mL of each solution was pipetted
into the wells in 24-well flat bottom tissue culture
plates. The 10 treatments were divided to the 10
© 2013 John Wiley & Sons Ltd, Aquaculture Research, 1–92
Saprolegnia prevention with clove oil and ethanol P Hoskonen et al. Aquaculture Research, 2013, 1–9
plates with two or three wells of each solution on
each plate. Altogether there were 21 wells of each
solution (n = 21).
Finally, one Saprolegnia colonized hemp seed was
transferred using sterile forceps into each well in
each of the plates and the plates were sealed with
laboratory film. After 48 h incubation in the dark
at room temperature, the wells were inspected
using a stereo microscope. Mycelia growth on the
surface of the hemp seeds was inspected and graded
from 1 to 3. No growth or strongly reduced growth
(hyphae very short and not covering the whole
seed) was graded as 1, reduced growth (short
hyphae covering the whole seed) was graded as 2,
and abundant hyphae growth (long hyphae cover-
ing the whole seed) was graded as 3 (Fig. 1b–d).
Bath exposure
Hemp seeds were colonized with Saprolegnia follow-
ing the same procedure as in continuous exposure
experiment. Fifty millilitres of each test solution
was mixed in sterile Erlenmeyer flasks. The test
solutions contained desired amounts of clove oil
and ethanol, with rest of the volume adjusted by
deionized water. Stock solution of clove oil (one part
clove oil and nine parts ethanol) was tested at clove
oil concentrations of 500 and 10 000 mg L�1
(with ethanol concentrations of 3555 and 71 100
mg L�1 respectively). Ethanol was tested at 3555
and 71 100 mg L�1 and deionized water was used
as control solution. A volume of 1% solution of
Sabouraud liquid medium was mixed, autoclaved,
cooled and 1 mL of the medium was pipetted into
the wells in 24-well flat bottom tissue culture
plates. About 50 Saprolegnia-colonized hemp seeds
were transferred using sterile forceps from agar
plates into each Erlenmeyer flask containing the
test and control solutions. After 15, 60 and
240 min, 10 hemp seeds from each Erlenmeyer
flask were transferred using sterile forceps into ster-
ile glass flasks with 500 mL of deionized water.
After 5 min of rinsing of the exposed hemp seeds in
deionized water, the seeds were transferred using
sterile forceps into the wells of tissue culture plates
containing liquid Sabouraud medium. The hemp
seeds with different treatments were mixed to the
tissue culture plates with each plate containing
seeds of every treatment, and sealed with labora-
tory film. After 48-h incubation in the dark at room
temperature, the wells were inspected using a
stereo microscope. Mycelia growth on the surface of
the hemp seeds was inspected and graded as in con-
tinuous exposure experiment.
In vivo experiment
On 25 April 2007, approximately 1000 newly
fertilized and unsterilized rainbow trout eggs from
five different parent fish pairs were taken from the
Savon Taimen fish farm in Rautalampi, Finland, to
the laboratory of the Department of Biological and
Environmental Science, University of Jyv€askyl€a.
From the eggs of each parent pair, seven batches of
100 eggs were randomly transferred to 35 incuba-
tion vessels, so that in each of the seven treatments
there was one batch of eggs from each parent
pair. Incubation vessels were plastic round bottom
cups (bottom diameter ca. 7.5 cm, water depth and
volume adjusted to 4 cm and 200 mL, respectively,
(a)
(c)
(b)
(d)
Figure 1 (a) Saprolegnia sp. colony
surrounded by hemp (Cannabis sati-
va L.) seeds (b) grade 1 = no
hyphae growth or strongly reduced
growth (hyphae very short and
not covering the whole seed) (c)
grade 2 = reduced hyphae growth
(short hyphae covering the whole
seed) and (d) grade 3 = abundant
hyphae growth (long hyphae cov-
ering the whole seed).
© 2013 John Wiley & Sons Ltd, Aquaculture Research, 1–9 3
Aquaculture Research, 2013, 1–9 Saprolegnia prevention with clove oil and ethanol P Hoskonen et al.
with an overflow tube through the brim). The 100
eggs fitted loosely on the bottom of the vessels in
one layer. All incubation vessels were supplied with
aerated flowing (ca. 0.15–0.2 L min�1) well water
(12.5°C, pH 7.04, total gas pressure 99–101%)
from one aeration tank, on the bottom of which
some 300 Saprolegnia sp.-infected rainbow trout
eggs in a glass jar were placed on 27 April 2007.
The water mould was acquired from Savon Taimen
fish farm. The photoperiod was variable because of
other experiments in the laboratory, but the eggs
were shielded from bright lights and disturbance.
The bathings were started on Monday, 30 April
2007 (experimental day 5), and continued until the
onset of hatching (Friday, 18 May 2007; day 23).
For the treatments, three stock solutions were
mixed using clove oil, 95% ethanol and deionized
water so that the concentration of ethanol
(7110 mg L�1) and the volume of stock solution
(2 mL) administrated to incubation vessels was the
same in all treatments. The 15-min baths of clove
oil at 1000 mg L�1 (stock solution containing one
part of clove oil and nine parts of ethanol), clove oil
at 500 mg L�1 (stock solution containing one part
of clove oil, one part of deionized water and 18 parts
of ethanol) and ethanol (stock solution containing
one part of deionized water and nine parts of etha-
nol) were repeated one (Monday) or three (Monday,
Wednesday, Friday) times per week. The control
was bathed with the well water from the aeration
tank three times per week. The bathings were per-
formed one treatment at a time, by stopping the
water flow to the vessels, adding 2 mL of stock solu-
tion (or water in control treatment) with a syringe,
waiting for 15 min and then carefully sucking out
about two-thirds of the water in the vessels with a
large syringe to speed up the dilution as the water
flow was turned on.
During incubation until the eyed-egg stage
(10 May 2007, day 15; 201 day-degrees), dead
eggs were counted from photographs once a week
(on Fridays). After the eyed stage, the dead eggs
were manually removed from the incubation ves-
sels and counted once a week (on Fridays). First
hatching was observed on Friday, 18 May 2007,
and the exposure to Saprolegnia zoospores and all
treatments were ended. The next Monday, 21 May
2007, most of the eggs were hatched (day 26;
333 day-degrees). On 22 May, all unhatched eggs
were counted and all hatched fry were visually
classified as healthy, abnormal or dead. Healthy
fry from the incubation vessels of each treatment
were combined and transferred into larger buckets
(volume 2 L, water depth 15 cm and water flow
ca. 0.5 L min�1). Fry that hatched after 22 May
(a few fry hatched on 25 May) were calculated,
but not combined with the ones hatched earlier.
After 8-day intervals (22 May, 30 May and 7 June
2007, i.e. experimental post-hatch days 1, 9 and
17), the dead fry were counted and 15 healthy fry
from each treatment were weighed (to 0.1 mg),
measured in length (to 0.5 mm) and preserved in
formalin (10% formalin in deionized water) for
1 day. Formalin hardened the yolk sac and
allowed its separation from the body. The yolk sac
and body of fry were dried to constant weight
(75°C for 1 day) and weighed (to 0.1 mg). From
the dry weights of body and yolk sac, the average
yolk utilization efficiency (E%) for two consecutive
8-day periods was calculated with the formula:
E% ¼ 100ðB2� B1Þ=ðY1� Y2Þ
where B1 and B2 are average body dry weights,
and Y1 and Y2 are average yolk sac dry weights
at the start and end of the time period.
Statistics
All statistical tests were performed with SPSS soft-
ware (SPSS Inc., Chicago, IL, USA), version 12.0.1,
and in all statistical tests P = 0.05 was taken as the
level of significance. In the in vitro experiment, the
growth grades of Saprolegnia hyphae were analysed
with non-parametric Kruskal–Wallis test. In cases
where Kruskal–Wallis test showed significant differ-
ences between treatments, pairwise comparisons
were performed with Mann–Whitney test. In the in
vivo experiment, the numbers of hatched healthy
and abnormal fry, wet body weight, length and dry
weight of body and yolk sac were analysed with
one-way ANOVA, and post hoc comparisons were per-
formed with Fisher’s LSD test.
Results
In vitro experiment
Continuous exposure
Kruskal–Wallis test showed significant differences
between treatments (P < 0.001). The control treat-
ment and ethanol alone at 3555 mg L�1 did not
© 2013 John Wiley & Sons Ltd, Aquaculture Research, 1–94
Saprolegnia prevention with clove oil and ethanol P Hoskonen et al. Aquaculture Research, 2013, 1–9
affect the growth of Saprolegnia hyphae and hence
these two treatments differed from all other treat-
ments (P < 0.001) (Table 1). Hyphae on most seeds
exposed to clove oil at 100 mg L�1 were short, but
covered evenly the whole seed (grade 2) and the
effect was statistically different from all other treat-
ments (P < 0.001). In all other treatments, the
hyphae did not grow at all or only a few short
hyphae were visible on the hemp seeds (grade 1).
Bath exposure
Exposure of 15, 60 and 240 min in stock solution
of clove oil at 10 000 mg L�1 with ethanol at
71 100 mg L�1 resulted in significantly reduced
growth of Saprolegnia hyphae compared with con-
trol treatment (P < 0.01) (Table 2). In most cases,
this treatment fully or strongly reduced the growth
of hyphae and the effect was significantly different
from all other treatments. The same concentration
of ethanol without clove oil had no significant
effect. Ethanol at 71 100 mg L�1 was the only
treatment in which the growth of Saprolegnia
hyphae was significantly affected by exposure
time. There were significantly more seeds with
reduced hyphae growth in the 240 min treatment
than either in 15 or 60 min treatments (P =0.013) (Table 2).
In vivo experiment
All the eggs of one fish pair died very early in the
experiment in every treatment group and thus
were excluded from the data. First visible fungal
growth in all incubation vessels was observed
9–10 days after introduction of Saprolegnia sp.
into the incubation system. The bathings did not
have any effects on the growth of fungus on the
eggs. All the dead eggs were surrounded by abun-
dant fluffy fungal growth, which also covered a
few living eggs even though the eggs were rela-
tively loosely fit on the bottom of the incubation
vessels.
The treatments did not influence the timing of
the eyed stage or hatching. There were significant
differences (P < 0.001) in the survival of the
embryos between the treatments (Fig. 2). Mortali-
ties of eggs exposed to mixture of 1000 mg L�1
clove oil and 7110 mg L�1 ethanol one or three
times per week were 95 and 100%, respectively,
before the eyed stage. In the other treatments,
there were no significant differences in the survival
to hatch or in the amount of visibly abnormal fry
which varied from 1.0 � 0.8% to 3.7 � 2.7%
(mean � SD) between the treatments.
Statistically significant differences were detected
between the treatments in the body dry weight
(P = 0.034) and body length (P = 0.005) on the
Table 1 The growth of Saprolegnia sp. hyphae on the
surface of hempseeds after 48 h incubation in continu-
ous exposure to 10 different treatments. See Fig. 1 for
explanation of grades 1–3
Treatment &
concentration mg L�1
Proportion of grades (%)
n 1 2 3
ControlA 21 0.0 0.0 100.0
Clove oil 100B 21 23.8 76.2 0.0
Clove oil 500C 21 100.0 0.0 0.0
Clove oil 1000C 21 100.0 0.0 0.0
Clove oil 5000C 21 100.0 0.0 0.0
Clove oil 10 000C 21 100.0 0.0 0.0
Clove oil 500 + ethanol 3555C 21 100.0 0.0 0.0
Clove oil 5000 + ethanol 35 550C 21 100.0 0.0 0.0
Ethanol 3555A 21 0.0 0.0 100.0
Ethanol 71 100C 21 100.0 0.0 0.0
Superscripts that are different indicate significant differences
between treatments.
Table 2 The effects of 15, 60 and 240 min exposures in
five different treatments on the growth of Saprolegnia sp.
hyphae on the surface of hempseeds after 48 h incuba-
tion in liquid Sabouraud medium. See Fig. 1 for explana-
tion of grades 1–3
Treatment &
concentration
mg L�1
Bath
time (min)
Proportion of grades (%)
n 1 2 3
Control 15Aa 10 0.0 0.0 100.0
60Aa 10 0.0 0.0 100.0
240Aa 10 0.0 0.0 100.0
Clove oil 500 +
ethanol 3555
15Aa 10 0.0 0.0 100.0
60Aa 10 0.0 0.0 100.0
240Aa 10 0.0 0.0 90.0
Clove oil 10 000 +
ethanol 71 100
15Ab 10 70.0 10.0 20.0
60Ab 10 60.0 20.0 20.0
240Ab 10 100.0 0.0 0.0
Ethanol 3555 15Aa 10 0.0 0.0 100.0
60Aa 10 0.0 0.0 100.0
240Aa 10 0.0 0.0 100.0
Ethanol 71 100 15Aa 10 0.0 0.0 100.0
60Aa 10 10.0 0.0 90.0
240Ba 10 30.0 20.0 50.0
Different upper case superscripts indicate significant differences
between bath times within a treatment. For each bath time
different lower case superscripts indicate significant differences
between treatments.
© 2013 John Wiley & Sons Ltd, Aquaculture Research, 1–9 5
Aquaculture Research, 2013, 1–9 Saprolegnia prevention with clove oil and ethanol P Hoskonen et al.
first post-hatch day (Table 3). The average body
dry weight of fry in the control group was signifi-
cantly smaller than in groups treated with ethanol
three times a week (P = 0.003) and in groups
treated with clove oil 500 mg L�1 once a week
(P = 0.016). The fry exposed to ethanol three
times per week during incubation were signifi-
cantly longer compared with the fry in the control
(P = 0.008) and the group treated with clove oil
with a concentration of 500 mg L�1 three times
per week (P = 0.003). Measurements of the
9-day-old fry showed no significant differences, but
at the termination of the yolk-sac stage, day 17,
the average wet weight of fry exposed to mixture
of clove oil at 500 mg L�1 and ethanol at
7110 mg L�1 once a week during incubation was
significantly smaller than in all the other treat-
ments (0.001 < P < 0.017) (Table 3).
The average yolk utilization efficiency during
the first 8-day period after hatching was lowest in
fry exposed to ethanol three times per week during
incubation and highest in fry exposed to clove oil
mixed in ethanol once a week and vice versa
during the latter 8-day period (Table 3). The aver-
age yolk utilization efficiency during the whole
16-day time period after hatching varied from
69.3–70.2% in clove oil treatments to
73.9–76.5% in ethanol and control treatments.
Mortality during this time period from hatching to
near the end of yolk-sac stage was 5.8% in control
and 1.1–3.1% in the other treatments.
Discussion
HeMP experiment demonstrated that in continu-
ous static exposure, clove oil effectively inhibited
the growth of Saprolegnia sp. hyphae at all test
concentrations (100–10 000 mg L�1) and all con-
centrations above 500 mg L�1 either fully or very
strongly reduced the growth of hyphae. However,
in bath exposures, clove oil at 500 mg L�1 had no
effect, whereas clove oil at a concentration of
10 000 mg L�1 significantly reduced the growth
of Saprolegnia hyphae for at least 48 h after all
exposure times tested. Bouchard et al. (2001)
observed that there was no significant difference
between the inhibitory effects of clove oil concen-
trations of 1000, 10 000 and 100 000 mg L�1
against saprolegnid fungus in continuous expo-
sure, which concurs with present findings. Previ-
ously reported MICs of eugenol against Saprolegnia
sp. have been in the range of 125–500 mg L�1
and MLCs between 250 and 1000 mg L�1
(Hussein et al. 2000; Tampieri et al. 2003). The
clove oil used in HeMP experiment has an eugenol
concentration of a minimum of 75% according to
the manufacturer. Thus, the eugenol concentra-
tion in the 100 mg L�1 clove oil treatment is
likely between 75 and 90 mg L�1, which is
somewhat lower than previously reported MICs.
Although only one strain of Saprolegnia sp. was
used in this study, the results are likely applicable
to other Saprolegnia sp. also because fungicidal
0
10
20
30
40
50
60
70
80
90
100
0 7 14 21 28
Cum
ulat
ive
mor
talit
y (%
)
Experimental day
Control 3×Ethanol 1×Ethanol 3×Clove 500 + Ethanol 1×Clove 500 + Ethanol 3×Clove 1000 + Ethanol 1×Clove 1000 + Ethanol 3×
Figure 2 Average (n = 4) cumulative mortality during incubation of rainbow trout Oncorhynchus mykiss
(Walbaum) eggs in well water (c. 12.5°C) containing Saprolegnia sp. zoospores and bathed one or three times per
week with pure water (control), ethanol at 7110 mg L�1, mixture of clove oil at 500 and ethanol at 7110 mg L�1
or mixture of clove oil at 1000 and ethanol at 7110 mg L�1. Day 0 (25 April): incubation started, day 2: exposure
to Saprolegnia sp. zoospores started, day 5: first bathings in all treatment groups, days 11–12: first visible fungal
growth in all incubation vessels, day 15: eyed stage (201 day-degrees), day 23: first hatching, day 26: most eggs
hatched (333 day-degrees). Erros bars omitted for clarity.
© 2013 John Wiley & Sons Ltd, Aquaculture Research, 1–96
Saprolegnia prevention with clove oil and ethanol P Hoskonen et al. Aquaculture Research, 2013, 1–9
concentrations of eugenol have been shown to be
same for different Saprolegnia sp. (Hussein et al.
2000). Differences in MICs or MLCs observed in
various studies may reflect different testing
methods rather than actual biological differences
(Pauli & Kubeczka 1996). The modified HeMP
method (Stueland, Heier et al. 2005) used in this
study proved to be a simple, inexpensive and reli-
able in vitro screening method for testing inhibi-
tory effects of chemicals against Saprolegnia
growth.
The combinations of clove oil and ethanol con-
centrations and frequencies of application used
during incubation of rainbow trout eggs had no
significant effects on the time of appearance or rate
of spreading of visible fungal growth. The clove oil
concentrations 500 and 1000 mg L�1 were
selected on the basis of literature (Hussein et al.
2000; Bouchard et al. 2001; Tampieri et al. 2003)
and results of the HeMP experiment. Bouchard et al.
(2001) found that 15-min baths in clove oil at
1000 mg L�1 every second day had no significant
effect on death rate of eyed rainbow trout eggs and
the concentration of 10 000 mg L�1 only slightly
increased mortality. However, in the present experi-
ment, both the one and three times per week baths
with clove oil at 1000 mg L�1 resulted in very high
death rates (95–100%) before the eyed-egg stage
was reached. This indicates that high concentra-
tions of clove oil are toxic to rainbow trout embryos
before the eyed stage. Holcomb, Woolsey, Cloud and
Ingermann (2004) reported a complete lack of
embryo development of O. mykiss eggs if they were
bathed in 1500 mg L�1 clove oil before fertilization,
but no effect was found with the clove oil concen-
tration of 150 mg L�1 supporting our finding of
clove oil toxicity at high concentrations to trout
eggs. Also, for example, hydrogen peroxide treat-
ments during blastopore formation increase rain-
bow trout egg mortality making the chemical better
suited to use after the eyed-egg stage (Gaikowski,
Rach, Olson, Ramsay & Wolgamood 1998) or the
concentration should be decreased during estimated
blastopore formation (Barnes & Gaikowski 2003).
Table 3 Average � SD (n = 15)
body wet weight and length, and
dry weight of body and yolk of c.
1-, 9- and 17-day-old rainbow trout
Oncorhynchus mykiss (Walbaum) fry
and yolk utilization efficiency during
two consecutive 8-day time periods
following hatching. Treatments
were 15 min baths repeated either
1 or 3 times per week during incu-
bation with a solution containing
clove oil and ethanol (clove oil at
500 mg L�1 and ethanol at
7110 mg L�1), ethanol alone at
7110 mg L�1 or control (water)
Day 1 Day 9 Day 17
Fry wet weight (mg)
Control 39 65.5 � 7.1 71.0 � 6.8 95.5 � 10.5A
Clove oil + ethanol 19 62.6 � 6.9 72.1 � 10.0 84.2 � 11.6B
Clove oil + ethanol 39 61.2 � 6.3 67.3 � 10.1 98.1 � 15.1A
Ethanol 19 60.8 � 8.1 75.6 � 10.8 95.0 � 10.2A
Ethanol 39 60.3 � 7.3 70.8 � 10.0 103.3 � 12.8A
Fry length (mm)
Control 39 13.1 � 1.7A 20.3 � 0.9 24.2 � 1.3
Clove oil + ethanol 19 14.1 � 1.6AB 20.5 � 1.0 23.5 � 1.2
Clove oil + ethanol 39 13.5 � 1.1A 19.7 � 1.1 23.5 � 0.9
Ethanol 19 13.2 � 2.1AB 20.7 � 1.0 23.7 � 0.5
Ethanol 39 15.1 � 0.9B 20.1 � 1.1 23.9 � 1.2
Body dry weight (mg)
Control 39 1.2 � 0.4A 6.0 � 1.0 12.4 � 2.2
Clove oil + ethanol 19 1.5 � 0.4B 6.8 � 1.3 11.1 � 2.1
Clove oil + ethanol 39 1.4 � 0.3AB 5.9 � 1.2 11.3 � 1.9
Ethanol 19 1.4 � 0.5AB 6.4 � 1.1 12.3 � 1.3
Ethanol 39 1.6 � 0.4B 6.3 � 1.2 12.3 � 2.0
Yolk dry weight (mg)
Control 39 17.0 � 1.9 10.3 � 3.3 18.2 � 2.2
Clove oil + ethanol 19 15.1 � 2.4 8.3 � 2.1 1.4 � 1.9
Clove oil + ethanol 39 16.3 � 1.9 9.5 � 2.5 1.9 � 0.9
Ethanol 19 16.5 � 3.0 9.5 � 1.9 2.2 � 1.8
Ethanol 39 16.4 � 3.0 9.6 � 2.2 2.2 � 2.6
Yolk utilization energy (%)
Control 39 71.1 76.1
Clove oil + ethanol 19 77.0 63.4
Clove oil + ethanol 39 67.8 70.7
Ethanol 19 71.9 80.9
Ethanol 39 69.3 81.1
Superscripts that are different indicate significant differences between treatments.
© 2013 John Wiley & Sons Ltd, Aquaculture Research, 1–9 7
Aquaculture Research, 2013, 1–9 Saprolegnia prevention with clove oil and ethanol P Hoskonen et al.
Eugenol is a lipophilic compound that can easily
permeate membranes and may possibly be a mito-
chondrial uncoupling agent (Usta, Kreydiyyeh,
Bajakian & Nakkash-Chmaisse 2002). Thus,
exposure to clove oil may interfere with metabolic
processes and development of the fish embryo.
However, sublethal concentrations of clove oil and
ethanol tested in this study did not seem to have
any dramatic effects on the developing embryos.
No differences in the time of reaching the eyed
stage of egg development and hatching (201 and
333 day-degrees, respectively) between treatments
were observed, which indicates that clove oil and
ethanol baths did not affect the development rate
of the rainbow trout embryo. There were no signif-
icant differences in the number of visibly mal-
formed fry at hatching between treatments. The
data suggest that exposure to 500 mg L�1 clove
oil once a week during incubation would affect
more the body dry weight of newly hatched larvae
and fry wet weight near the end of yolk-sac stage
than exposure three times a week.
Even if there has been recently relatively much
interest in the use of clove oil as a fish anaesthetic
(Javahery et al. 2012), it must be noted that there
are restrictions in the use of clove oil for fish aimed
to human consumption in some countries. For
example, FDA has banned its use as a food fish
anaesthetic in USA. According to US National
Toxicity Program (http://ntp-server.niehs.nih.gov;
see also The Carcinogenic Potency Project http://
potency.berkeley.edu), eugenol has not been found
to be carcinogenic in rats in long-term carcinoge-
nicity tests, but the results in mice are equivocal.
However, depending on the source of clove oil, it
may contain also small or, in some instances, negli-
gible amounts of isoeugenol and methyleugenol.
For isoeugenol, there is evidence, and for methyleu-
genol there is clear evidence of long-term
carcinogenicity in mice and rats. On the other hand,
formalin (accepted by the FDA) is known to be
human carcinogen (National Toxicity Program).
Conclusion
The results of this study together with literature
indicate that bath exposure of rainbow trout eggs
to clove oil and ethanol do not aid in controlling
aquatic fungi infestations during incubation. Etha-
nol alone is not effective in concentrations that
are usable in practice and fungicidal concentra-
tions of clove oil are lethal to embryos at least
before the eyed stage of egg development when
the need for chemical treatment would be greatest
Acknowledgments
We thank Dr. P. Pylkk€o who originally isolated
the Fin 22 strain. Finnish Food Safety Authority
Evira kindly provided the Saprolegnia strain and
Savon Taimen fish farm the rainbow trout eggs for
this study. We also thank J. Ahonen, M. Tiirola,
L-R. Sundberg and J. Tuikkanen for technical
assistance. The research was supported by a schol-
arship from the University of Jyv€askyl€a to PH.
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