Efficacy of clove oil and ethanol against Saprolegnia sp. and usability as antifungal agents during incubation of rainbow trout Oncorhynchus mykiss (Walbaum) eggs

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



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.



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.



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.



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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Documents

Efficacy of clove oil and ethanol against Saprolegnia sp. and usability as antifungal agents during incubation of rainbow trout Oncorhynchus mykiss (Walbaum) eggs