Effects of different bronopol treatments on final

survival rates in the artificial incubation of crayfish

eggs (Pacifastacus leniusculus, Astacidae)

Alvaro Gonzalez, Jesus D. Celada, Pedro M. Melendre, Jose M. Carral, Marıa Saez-Royuela,

Rocıo Gonzalez & Vanesa Garcıa

Dpto. Produccion Animal, Universidad de Leon, Leon, Spain

Correspondence: Alvaro Gonzalez Martın. Dpto. Produccion Animal, Universidad de Leon, Campus de Vegazana s/n, 24071, Leon,

Spain. E-mail: agonm@unileon.es

Abstract

Two experiments were conducted on the effects

of bronopol in the artificial incubation of crayfish

eggs (Pacifastacus leniusculus) with the aim to

search an alternative to formaldehyde. In the

first experiment, 50, 250, 500 and 1000 ppm

bronopol and 3000 ppm formaldehyde (control)

in periodical administrations were tested on a

density of 6.6 eggs cm�2. After 44 days of incu-

bation, the highest survival was obtained with

1000 ppm bronopol (81.9% to stage 2 juvenile,

with no significant difference from formaldehyde),

whereas lower bronopol concentrations resulted

in significantly lower survival. In the second

experiment, 1000, 3000 and 5000 ppm bronopol

and 3000 ppm formaldehyde (control) adminis-

tered for 15 min every second day were tested

on eggs at a density of 20 eggs cm�2. After

78 days of incubation, bronopol at 3000 ppm

allowed for a stage 2 juvenile survival rate of

65.0% (with no significant difference from form-

aldehyde), whereas significantly lower survival

was obtained with 1000 ppm or 5000 ppm. This

study shows that bronopol may constitute an

alternative to formaldehyde in the artificial incu-

bation of crayfish eggs. A concentration of

3000 ppm administered for 15 min every second

day may be adequate even on long incubations

at high densities (at least 20 eggs cm�2, one

complete layer).

Keywords: antifungal treatment, artificial incu-

bation, astacid crayfish, bronopol

Introduction

Recent knowledge on artificial incubation tech-

niques for crayfish eggs, along with studies on egg

storage and transport, provides reliable options to

intensify the reproductive phase of astacid culture

(see review by Gonzalez, Celada, Garcıa, Gonzalez,

Carral & Saez-Royuela 2009).

Dead crayfish eggs are a good substrate for

Saprolegnia spp. and other Oomycetes (Edgerton,

Evans, Stephens & Overstreet 2002), and hyphae

are capable of spreading from infected eggs to sur-

rounding healthy ones, which increases mortality

during incubation (Celada, Carral, Saez-Royuela,

Melendre & Aguilera 2004). Although periodical

removal of dead eggs has a positive effect on the

hatching rate as it reduces fungal growth (Carral,

Perez, Celada, Saez-Royuela, Melendre & Aguilera

2004; Policar, Kozak & Martın 2006), it is labori-

ous, and manipulation can cause damage to

healthy eggs (Saez-Royuela, Melendre, Celada, Car-

ral, Gonzalez, Gonzalez & Garcıa 2009). Thus, the

use of antifungal treatments has to be considered,

and several studies have been addressed to find

effective chemical agents. In the current state of

knowledge, among the permitted fungicides, only

formaldehyde at a dose of 3000 ppm administered

for 15 min every second day has shown satisfac-

tory results (Melendre, Celada, Carral, Saez-Royuela

& Aguilera 2006). There is concern about user

safety because of its suspected carcinogenicity and

its potential adverse effects on the aquatic environ-

ment (Arndt, Wagner & Routledge 2001; Gieseker,

Serfling & Reimschuessel 2006), thus making the

© 2011 Blackwell Publishing Ltd 1

Aquaculture Research, 2011, 1–5 doi:10.1111/j.1365-2109.2011.03036.x

search for an alternative chemical advisable.

Bronopol, an aliphatic halogenitro (2-bromo-2-

nitropropane-1,3-diol), is currently licensed world-

wide (Birkbeck, Reid, Darde & Grant 2006), being

widely used for the treatment and control of fun-

gal infections in farmed, fertilized salmonid

eggs (Cawley 1998; Aller-Gancedo & Fregeneda-

Grandes 2007), as it prevents sporulation and

hyphae growth (Pottinger & Day 1999). It is also

an efficient broad spectrum bactericide with a wide

range of applications in food production, water dis-

infection and healthcare products (Kumanova,

Vassileva, Dobreva, Manova & Kupenov 1989).

The aim of this study was to obtain data on

comparative effectiveness of bronopol in the artifi-

cial incubation of crayfish eggs.

Materials and methods

Eggs of Pacifastacus leniusculus (Dana, 1852) ber-

ried females from a crayfish farm were detached

by sliding blunt forceps smoothly from the base to

the tip of the pleopods. The eggs were pooled and

artificially incubated in the experimental devices

described by Carral, Celada, Gonzalez, Gaudioso,

Fernandez and Lopez-Baisson (1992) up to stage 2

juvenile production. Embryonic phases were identi-

fied following Celada, Paz, Gaudioso and Fernandez

(1987).

Artesian well water was supplied in an open

system at a flow rate of 0.5 L min�1 (12.5 mL

cm�2 min�1). The parameters of incoming water

quality were: pH 7.9, hardness 5.2ºdH (calcium:

32.3 mg L�1), dissolved oxygen about 8 mg L�1,

nitrite < 0.015 mg L�1, ammonium < 0.02 mg

L�1, total dissolved solids 110.5 mg L�1 and total

suspended solids < 0.5 mg L�1. Temperature was

10 ± 1°C until eggs reached the eyed stage (phase

XIII). Thereafter, it was raised to 15.5 ± 1°C up to

final stage 2 juvenile production.

During the incubation, different treatments were

administered up to the beginning of hatchings by

peristaltic pumps, and discharging antifungical

solution to the incoming water flow. Chemicals

used were formaldehyde (Proquiman S.L.) and

bronopol (as Pyceze, 50% W/V bronopol, Novartis

Animal Vaccines Ltd.).

Eggs were checked daily by careful ocular

inspection throughout incubation and any pres-

ence of fungal growth was noted. Dead eggs were

not removed. The number of stage 1 (after hatch-

ing) and stage 2 (after first moult) juveniles was

quantified. Collection of stage 2 juveniles was

carried out daily, as final survival is reduced, the

longer they remain in the incubators (Melendre,

Celada, Carral, Saez-Royuela & Aguilera 2007).

Survival rates were calculated as the percentage of

juveniles from the initial number of eggs.

Prior to statistical analysis, arc-sine transforma-

tion of percentages was performed. Results were

examined by analysis of variance (one-way ANOVA)

using the SPSS 16.0 computer program (SPSS Inc.,

Chicago, IL, USA). Mean comparison was tested

using the Duncan′s test. The significance level was

P < 0.05.

Experiment 1

On January 29, 2250 eggs were stripped from

maternal pleopods of 14 females at phases X–XII

(embryo with masticatory appendages rudiments-

embryo with pulsating heart), pooled and artifi-

cially incubated at a density of 6.6 eggs cm�2

(one incomplete layer of eggs with scarce contacts,

450 eggs per treatment, 75 eggs per replicate).

Five treatments were tested: bronopol at 50 ppm

for 30 min daily (manufacturer’s recommendation

for salmonid eggs), bronopol at 250, 500 and

1000 ppm for 15 min every second day, and

formaldehyde at 3000 ppm for 15 min every sec-

ond day used as control, according to Melendre

et al. (2006). Each treatment was tested on six

replicates.

Experiment 2

On December 12, 3632 eggs were stripped from

maternal pleopods of 23 females at phases VIII–IX

(embryo with mandibular rudiments-embryo with

naupliar appendages), pooled and artificially incu-

bated at a density of 20 eggs cm�2 (one complete

layer, eggs contacting, 908 per treatment, 227 per

replicate. Four treatments were tested for 15 min

every second day: bronopol at 1000, 3000 and

5000 ppm, and formaldehyde at 3000 ppm (con-

trol). Each treatment was tested on four replicates.

Results and discussion

Experiment 1

Artificial incubation lasted for 44 days. Hatchings

began on day 24, stage 2 juveniles were obtained

from day 31, and the first moult period lasted for

© 2011 Blackwell Publishing Ltd, Aquaculture Research, 1–52

Bronopol in artificial incubation of astacid eggs A Gonzalez et al. Aquaculture Research, 2011, 1–5

14 days. First observation of fungal growth took

place as follows: day 6 (50 ppm and 250 ppm

bronopol), day 17 (500 ppm bronopol) and day

17 (1000 ppm bronopol, slight hyphae that did

not affect all dead eggs throughout the incuba-

tion). No fungal growth was observed on eggs

receiving formaldehyde.

The highest survival rate to stage 2 (81.9%)

was obtained with 1000 ppm bronopol for

15 min every second day, with no significant dif-

ference from the formaldehyde control (Table 1).

As bronopol concentrations were lowered, results

decreased significantly to 18.1% survival, obtained

with 50 ppm for 30 min daily. Several authors

have pointed out that antifungal agents success-

fully used in artificial incubation of fish eggs (e.g.

sodium chloride, hydrogen peroxide, formaldehyde

or isopropyl alcohol) are not effective in astacid

eggs at the same concentrations and administra-

tion frequencies (Celada et al. 2004; Melendre

et al. 2006; Policar et al. 2006; Saez-Royuela

et al. 2009). This can also be applied to bronopol,

as the dose recommended by the manufacturer

for salmonid eggs was ineffective on crayfish

eggs.

Experiment 2

Artificial incubation lasted for 78 days. Hatchings

began on day 60, stage 2 juveniles were obtained

from day 67 and the first moult period lasted for

12 days. First observations of fungal growth took

place as follows: day 7 (1000 ppm bronopol) and

day 29 (3000 ppm bronopol, very slight hyphae

that did not affect all dead eggs throughout the

incubation). No fungal growth was observed on

eggs receiving formaldehyde or 5000 ppm bronopol.

As shown in Table 2, bronopol at 3000 ppm

allowed 65.0% of survivors to stage 2, with no sig-

nificant differences from the formaldehyde control.

A lower concentration of bronopol (1000 ppm)

resulted in a significantly lower survival (25.8%),

whereas the highest concentration (5000 ppm)

also resulted in significantly lower survival

(52.4%).

The concentration of 1000 ppm bronopol,

which was effective at a density of 6.6 eggs cm�2

(experiment 1), was not as effective at a density of

20 eggs cm�2, probably because of the longer

incubation period and contacts among eggs (due

to the higher density) facilitating hyphae propaga-

tion. Thus, a concentration of antifungal agent

can be efficient at a certain crayfish egg density,

but a higher density might require a higher con-

centration, as Celada et al. (2004) pointed out

using formaldehyde.

On a complete layer of eggs (20 cm�2), brono-

pol at 3000 ppm yielded good results, but with

5000 ppm, final survival was significantly lower.

Considering that survival to stage 1 was also sig-

nificantly lower (Table 2) and that fungal growth

was not observed, this result could be attributed to

a toxic effect, and thus the safety profile for cray-

fish eggs would have been surpassed.

Table 1 Survival rates in artificial incubation at a density of 6.6 eggs cm�2 receiving different antifungal treatments

Chemical agent

Concentration

(ppm)

Administration time/

frequency

Stage 1

(% ± SEM)

Stage 2

(% ± SEM)

Bronopol 50 30 min/daily 18.9 ± 4.0a 18.1 ± 3.9a

250 15 min/every other day 72.0 ± 6.3b 57.8 ± 6.1b

500 15 min/every other day 84.6 ± 6.2c 70.1 ± 8.0c

1000 15 min/every other day 95.3 ± 1.1c 81.9 ± 1.8d

Formaldehyde

(control)

3000 15 min/every other day 95.7 ± 0.4c 81.8 ± 3.2d

Values followed by different letters were significantly different (P < 0.05) from the others in the same column.

SEM, standard error of mean.

Table 2 Survival rates in artificial incubation at a den-

sity of 20 eggs cm�2 receiving different antifungal treat-

ments for 15 min every other day

Chemical

agent

Concentration

(ppm)

Stage 1

(% ± SEM)

Stage 2

(% ± SEM)

Bronopol 1000 30.2 ± 7.2a 25.8 ± 7.7a

3000 76.4 ± 3.2c 65.0 ± 3.4c

5000 62.3 ± 0.4b 52.4 ± 0.4b

Formaldehyde

(control)

3000 83.2 ± 0.5c 73.2 ± 0.7c

Values followed by different letters were significantly different

(P < 0.05) from the others in the same column.

SEM, standard error of mean.

© 2011 Blackwell Publishing Ltd, Aquaculture Research, 1–5 3

Aquaculture Research, 2011, 1–5 Bronopol in artificial incubation of astacid eggs A Gonzalez et al.

Previous works showed that 3000 ppm formal-

dehyde has no effects on the pace of embryonic

development, as eggs receiving this treatment

required the same time to hatch as those not

receiving any chemical treatment (Celada et al.

2004; Melendre et al. 2006; Saez-Royuela et al.

2009). In the present study, bronopol (even at

5000 ppm) did not change the hatching time

compared with formaldehyde, showing that the

doses tested do not affect the pace of embryonic

development either.

On the other hand, such bronopol doses would

not represent potential risks on users’ health

under the brief time and form of exposure entailed

here (Hahn, Scheider, Gartiser, Heger & Mangels-

dorf 2010).

This study shows that bronopol may protect

crayfish eggs from fungal growth (and perhaps

other pathogens) in artificial incubation, making it

an alternative to formaldehyde. A concentration of

3000 ppm administered for 15 min every second

day may be adequate, even on long incubations at

high densities (at least 20 eggs cm�2).

Acknowledgements

Funding of this study was the Plan Nacional de I

+D+i, Ministerio de Eduacion y Ciencia, Spain,

Research Project AGL2005-01127. We also thank

the Quinon S.A. crayfish farm for their collabora-

tion.

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