Incidence of malformations in fish

embryos/larvae (review)

Prepared by Erik Prochazka

Smart Water Research Facility, Griffith University – Nathan/Gold Coast Campus

Friday, 18 September 2009

Incidence of malformations in fish embryos/larvae September 18, 2009

Contents
1 Introduction .................................................................................................................................... 3
2 Environmental conditions ............................................................................................................... 3
2.1 Temperature, salinity and pH ................................................................................................. 3
2.2 Chemical pollution .................................................................................................................. 4
2.2.1 Parental exposure ........................................................................................................... 4
2.2.2 Direct exposure ............................................................................................................... 5
3 Diet and nutrition............................................................................................................................ 7
4 Breeding conditions/protocols ....................................................................................................... 7
4.1 Overripening of oocytes (post-ovulatory ageing) ................................................................... 7
4.2 Induced spawning ................................................................................................................... 8
4.3 Photoperiod manipulation ...................................................................................................... 8
5 Conclusion ....................................................................................................................................... 8
6 Bibliography .................................................................................................................................... 9

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Incidence of malformations in fish embryos/larvae September 18, 2009

1 Introduction
Embryonic and larval malformations are recognised as a recurring problem in fish aquaculture and
represent both ethical and economical challenges for the industry (Takle et al., 2005). A variety of
natural environmental factors, such as temperature, salinity, pH, etc., have been identified as
responsible for inducing deformities in fish embryos and/or larvae (Kjørsvik et al., 1990). In addition
to these, human activities and breeding in hatcheries added a series of new parameters that may
exert considerable impact on developing fish (Von Westernhagen et al., 1988).

The natural background level of embryonic malformations is generally expected to be less than 10%
(Klumpp & Von Westernhagen, 1995). However, this frequency increases significantly with level of
environmental stress, and has been reported as high as 90% (Zoarces viviparus) in ecosystems
burdened with a high level of environmental pollution, such as in highly contaminated German
coastal and estuarine waters (Gercken et al., 2006).

The purpose of this review is to examine incidence of specific types of embryonic and larval
malformations and identify the conditions under which they may occur, such as natural
environmental stressors, anthropogenic pollution, and hatchery rearing practices. Only considered
were malformations that occur in the embryonic and early larval stages before the commencement
of individual feeding. Cases of abnormal development in late larval stage (ie. post-yolk-sac feeding
stage), the juveniles, or the adult fish were excluded from the review, even though they have been
described in the literature for a variety of species (eg. Fraser et al., 2004, Fraser & De Nys, 2005,
Gavaia et al., 2002) .

2 Environmental conditions

2.1 Temperature, salinity and pH

Owing to the ectotherms’ inability to metabolically regulate their own temperature, the ambient
environmental temperature is an all-pervasive regulator of their physiological processes. In addition
to temperature, salinity and pH also directly affect the fish physiology as well as influence the
physical nature of water, ie. its dissolved gas content, crystalline arrangement, solvent
characteristics, viscosity and fluid dynamics, which may have a direct impact on the fish or affect the
biochemical and toxicological characteristics of pollutants potentially present in the water (Finn,
2007).

A widely recognised effect of temperature on fish reproduction is its impact on hatch rate. A study
on hatch rate of Australian bass larvae (Macquaria novemaculeata) showed retarding and
accelerating effects of low and high temperatures respectively across the studied temperature range
of 12 to 24°C (Van Der Wal, 1985). Similar effects on the embryonic development rate were
observed in a marine species, striped trumpeter (Latris lineata) (Bermudes & Ritar, 1999).

A study involving striped bass (Morone saxatilis) and blueback herring (Alosa aestivalis) eggs
exposed to heat shock conditions demonstrated increased incidence of deformed larvae at hatching
(Koo & Johnston, 1978). The deformities included shortened body, enlarged finfold and curved or
twisted spine. The authors conclude that the severity and incidence of the deformities were related
to the temperature dose (ie. the product of the elevated temperature and exposure time) (Koo &

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Incidence of malformations in fish embryos/larvae September 18, 2009

Johnston, 1978). The effects of salinity on the eggs of black bream (Acanthopargus butcheri)
included high incidence (up to 93%) of deformities at salinities below 15‰, characterised mainly by
curvature of spine and bent tail (Haddy & Pankhurst, 2000).

Fluctuations in pH away from the neutral range are known to have various negative effects on fish
development and physiology. These include effects of low pH on oogenesis (Oncorhynchus mykiss)
(Zelennikov, 1997), fertilisation rate (Coregonus lavaretus lavaretus) (Keinänen et al., 2003),
ionoregulation and acid-base balance (Goss et al., 1992), increased toxicity of metals as a result of
their solubilisation (Gensemer & Playle, 1999, Kane & Rabeni, 1987, Keinänen et al., 2003, Von
Westernhagen et al., 1988), and toxic effects of ammonia at high pH (Randall & Tsui, 2002).

Keinänen and co-workers (2003) observed an increased proportion of embryos of anadromous

whitefish (Coregonus lavaretus lavaretus) that exhibited curved tail tip and blood circulation failure
with decreasing pH in the presence of aluminium (Keinänen et al., 2003). Similarly, an earlier study
by Kane and Rabeni (1987) demonstrated pH dependent aluminium toxicity in smallmouth bass
larvae (Micropterus dolomieui) describing high mortality and sub-lethal effects including
morphological deformities and abnormal swimming behaviour (Kane & Rabeni, 1987).

This brings us to an issue of chemical pollution and its effects on the fish development.

2.2 Chemical pollution

Sub-lethal effects of chemical contamination on fish oocytes and spermatogonia (via parental
exposure), eggs, embryos, and larvae have been extensively documented and reviewed for a number
of species by Von Westernhagen and co-workers (1988) and more recently for salmonids by Finn
(2007).

The susceptibility of fish to pollutants appears to be largely species-specific (Klumpp et al., 2002) and
may be influenced by other environmental factors, such as temperature, salinity and pH (Finn, 2007,
Kane & Rabeni, 1987, Keinänen et al., 2003, Von Westernhagen et al., 1988).

The following sections briefly summarise the embryonic and larval malformations that may arise
from exposure to various forms of chemical pollution at various stages of development, from
parental exposure to late embryonic and early larval stages.

2.2.1 Parental exposure

Exposure of the female fish to lipophilic organic compounds (eg. organochlorines, organometals) and
heavy metals may result in their uptake and storage in the lipid-rich tissues (eg. liver). During
vitellogenesis these stored substances may be transported with the lipid reserves into the
developing gonads and potentially negatively impact succeeding generations through
bioaccumulation in the ovarian oocytes (Black et al., 1988, Cameron et al., 1992, Von Westernhagen
et al., 1988).

In case of male fish, the effect of pollutants on their sperm quality is mostly observed in terms of
reduced sperm motility. This has been observed, for example, in exposure to heavy metals (eg. Cd,
Zn) (Kime et al., 1996), or organochlorine pesticides (Singh et al., 2008). Given successful

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Incidence of malformations in fish embryos/larvae September 18, 2009

fertilisation, this suggests a potential for transfer of these pollutants (or their effects on the genetic
material) into the newly fertilised egg.

The observed consequences of maternal ovarian contamination on the progeny can range from
smaller size at hatch (Pseudopleuronectes americanus) (Black et al., 1988) to severe embryonic
malformations due to chromosomal aberrations and down-regulation of specific enzyme signal
pathways (Oryzias latipes) (Hano et al., 2007, Zhang et al., 2008).

Maternal exposure to antifouling agents, tributyltin (TBT) and triphenyltin (TPT), widely used in
paints for shipping and marine structures, and known to bioaccumulate in aquatic biota (Hano et al.,
2007), has been shown to adversely affect embryogenesis in Japanese medaka (Oryzias latipes)
(Hano et al., 2007, Zhang et al., 2008). The effects included abnormal eye development, shrunken
yolk sac, delayed development of the body and internal organs, and haemorrhage followed by fatal
heart rhythm abnormalities (Hano et al., 2007). In their study, Hano and co-workers (2007), injected
fertilised eggs with TBT concentrations previously seen in the eggs of several species of marine fish
collected in the wild and thought to be transferred there through parental exposure (Hano et al.,
2007). Similar results were observed after exposure of adult female medaka fish (O. latipes) to TPT
(Zhang et al., 2008). Here the exposure caused decrease in spawning frequency, spawned egg
number, egg quality and gonad development; and resulted in teratogenic effects in the embryos that
included haemorrhaging, eye defects, morphological malformations, incidence of conjoined twins,
and swim-up failure (Zhang et al., 2008).

2.2.2 Direct exposure

Direct exposure of embryos and larvae to chemical contaminants can occur at various stages of their
development. Experimentally induced impairment of cell division has been observed as early as in
the two- to eight-cell stages in plaice (Pleuronectes platessa) and cod (G. morhua) exposed to
aromatic compounds, such as benzene and xylene, as well as chlorinated hydrocarbons, such as
pesticides DDT or DDE (reviewed in Von Westernhagen et al., 1988).

The review by Von Westernhagen and co-workers (1988) summarises the reported early embryonic
stage deformities. These include unusual shape of blastodisc (heavy metals), deformations of
blastomeres (chlorinated hydrocarbons), and irregular cleavage of blastomeres (petroleum
hydrocarbons and chlorinated hydrocarbons). All of these aberrations have a potential to cause
morphological malformations in the later stages of embryonic development, however natural cell
repair system may prevent this from occurring (reviewed in Von Westernhagen et al., 1988). Other
described abnormalities include amorphous embryonic tissue (heavy metals, petroleum
hydrocarbons, chlorinated hydrocarbons), which usually results in early embryonic mortality; and
tissue necrosis (heavy metals, petroleum hydrocarbons, chlorinated hydrocarbons), which may
result in embryonic malformations and mortality (reviewed in Von Westernhagen et al., 1988).

To provide a couple of specific examples, Carls and Rice (1988) studied the sensitivity of walleye
pollock (Gadus morhua) eggs and larvae to the water soluble fraction of crude oil. The observed
embryonic morphological abnormalities included formation of membranous vesicles, deformations
of yolk, eye, brain, intestine, jaw, and pericardial sac (Carls & Rice, 1988). These were largely dose
dependent and strongly correlated with the mortality after hatch. The study also showed high and

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Incidence of malformations in fish embryos/larvae September 18, 2009

rapid mortality rate in exposed larvae without having developed any observable morphological
abnormalities (Carls & Rice, 1988). Similar effects were observed in gilthead sea bream (Sparus
aurata) (Glamuzina et al., 1990). More recently, eggs of winter flounder (Pleuronectes americanus)
exposed to a combination of diesel and various heating oils exhibited cellular and/or chromosomal
abnormalities (Hughes, 1999). Likewise, Debruyn and co-workers (2007) found an increase in
occurrence and severity of embryonic and larval deformities in lake whitefish (Coregonus
clupeaformis) exposed to crude oil-derived polycyclic aromatic hydrocarbons (PAHs). These included
several skeletal deformities, such as lordosis and scoliosis, and various ocular and jaw deformities
(Debruyn et al., 2007).

Experiments with fish embryos at later stages of development exposed to pesticides, such as
parathion, malathion, diazinon, carbaryl, fenitrotion, or phosphamidon, showed stunted growth, and
increased incidence of deformities such as curving of the tail, deformed head regions, enlargement
of the pericardial sac, circulatory failure, deformed vertebral and spinal column, and poorly
developed eye pigment and chromatophores (reviewed in Von Westernhagen et al., 1988).

Laboratory exposure of Australian crimson-spotted rainbow fish (Melanotaenia fluviatilis) eggs and
larvae to a synthetic pyrethroid esfenvalerate showed virtually no toxic effects on exposed fertilised
eggs. The larvae hatched without any gross abnormalities. The less than 1 week old larvae, however,
showed significantly increased mortality following exposure, which slowly decreased with age and
weight increase in the test animals (Barry et al., 1995). Pyrethrins and synthetic pyrethroids, used
widely as insecticides, are known to be highly toxic to fish as well as to bioaccumulate in aquatic
biota (Haya, 1989, Mauck et al., 1976).

Laboratory embryonic exposure of zebrafish (Danio rerio) to polybrominated diphenylethers

(PBDEs), ubiquitous environmental contaminants extensively used as flame retardants in a wide
range of products, produced developmental abnormalities including pericardial oedema, yolk sac
deformations, reduced pigmentation, lowered heart rate, and developmental delay at
concentrations as low as 25-50 nM (Boxtel et al., 2008).

Acute exposure to copper (Cu) at a concentration 0.3 µmol/L did not cause any disturbances in the
embryonic/larval development in carp (Cyprinus carpio) (Flik et al., 2002), however exposure of mahi
mahi (Coryphaena hippurus) embryos to a concentration of 1 µg/L of copper chloride (CuCl2)
resulted in significantly increased mortality rates and development of yolk-sac oedema and skeletal
deformities (Adema-Hannes & Shenker, 2008).

Westernhagen and co-workers (1988) strongly assert that the observed morphological deformities
are not particularly pollutant-specific and virtually all can be also observed when fish embryos
develop under naturally stressed conditions, such as the extremes of temperature and salinity (Von
Westernhagen et al., 1988). They also emphasize that it might be difficult if not impossible to
identify any particular substances responsible for one or several sub-lethal morphological effects
and even clearly distinguish between morphological, physiological, or behavioural abnormalities,
since one may result from the other, as they are frequently related (Von Westernhagen et al., 1988).

Other environmental factors, such as diet, and breeding protocols in hatchery-reared fish may also
impact on their embryonic and larval development. These are briefly discussed in the following
sections.

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Incidence of malformations in fish embryos/larvae September 18, 2009

3 Diet and nutrition

The effects of diet composition on reproduction in fish have been previously reviewed by Izquierdo
and co-workers (2001). It appears that certain components of broodstock diet are prerequisite for
normal embryonic development (Izquierdo et al., 2001).

The lipid soluble vitamins, namely the vitamin A and E, are known to be recruited from the
peripheral tissues and muscles in the adult female fish and transported to the developing oocytes
(Lubzens et al., 2009). Here they serve as antioxidants, however vitamin A’s main role in developing
embryos is in regulating gene transcription (Balmer & Blomhoff, 2002). Suboptimal levels of vitamin
E has been shown to result in reduced larval survival and increased developmental abnormalities
(reviewed in Izquierdo et al., 2001). Vitamin C in the gonads of fish has been linked to the process of
vitellogenesis and its content in eggs can be directly correlated to the hatching rate in rainbow trout
(Oncorhynchus mykiss) (Kjørsvik et al., 1990).

Other dietary components required for normal embryonic and larval development include essential
fatty acids, deficiencies of which generally result in reduced egg viability, low embryonic survival,
and increased occurrence of embryonic malformations (reviewed in Izquierdo et al., 2001).
Furthermore, there is a limited evidence that the relative composition of the diet of parent fish in
terms of its protein, lipid and carbohydrate content may have a significant impact on the
reproductive performance and egg quality of the fish (Dicentrarchus labrax) (Cerdá et al., 1994). The
authors tested two dietary formulations, one with relatively high protein, low lipid, and low
carbohydrate content, and the second one with equal proportions of protein and carbohydrate
content and low lipid content. The larvae hatched from the latter feeding group showed
morphological deformities in more than 50% of cases (Cerdá et al., 1994).

4 Breeding conditions/protocols
Hatchery-reared fish have been commonly associated with an increased incidence of spinal
deformities (Andrades et al., 1996). The authors list axial deviations, operculum atrophies and
cranial abnormalities as frequently observed deformations in the gilthead sea bream (Sparus
aurata), however without offering an explanation as to the cause of these (Andrades et al., 1996).

The possible causes may include post-ovulatory ageing of oocytes, induced spawning, and
photoperiod manipulation.

4.1 Overripening of oocytes (post-ovulatory ageing)

Overripening, or post-ovulatory ageing, of oocytes has been shown to reduce the ability of the eggs
to produce a healthy fish (Aegerter & Jalabert, 2004, Bonnet et al., 2007). Excessive ageing results in
reduced egg viability, which may be interpreted in terms of proteolytic breakdown of the yolk
protein, and a loss of small organic molecules such as amino acids and small peptides through the
egg membranes (Kjørsvik et al., 1990). The time period in which the eggs remain viable after
ovulation is highly species-specific and may range from 1 hour in striped bass (Roccus saxatilis) to a
couple of weeks in Pacific herring (Clupea harengus pallasi) (Kjørsvik et al., 1990). Additionally, this
time period, as reviewed by Kjørsvik and co-workers (1990), also appears to be dependent on the
ambient temperature (Kjørsvik et al., 1990).

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Incidence of malformations in fish embryos/larvae September 18, 2009

Overripening of eggs may cause specific embryonic malformations that can result in hatching of
deformed fry as well as increased mortality. Bonnet and co-workers (2007) report a significantly
increased incidence of malformations in rainbow trout (Oncorhyncus mykiss) embryos in the post-
ovulatory ageing experimental group (16-day). These experimental conditions induced the most
negative effects in comparison to the remaining groups. The observed malformations included
mainly cyclopia (34%), followed by ‘other’ defects consisting of, for example, occurrence of
conjoined twins (29%), yolk-sac resorption abnormalities (25%), spinal torsion (10%) and prognathia
(2%) (Bonnet et al., 2007). The survival of the embryos in this group was only 37% as opposed to
93% in the control group (Bonnet et al., 2007).

4.2 Induced spawning

Induced spawning of adult fish by hormonal stimulation is a common practice in today’s aquaculture
(Bonnet et al., 2007, Finn, 2007, Rottman, 1991). The substances used to induce spawning include
crude pituitary extracts and purified gonadotropins (eg. human chorionic gonadotropin - HCG) to
directly stimulate the ovaries and testes; luteinizing hormone-releasing hormones (LHRH) or their
analogs (LHRHa also referred to as GnRHa), alone or in combination with dopamine blockers (eg.
haloperidol) to enhance the potency of LHRHa; and finally steroids to stimulate the gametes directly
(Rottman, 1991).

Research has shown that spawning hormonal treatment may induce limited but significant egg
quality defects (Bonnet et al., 2007). Bonnet and co-workers (2007) describe that these defects in
rainbow trout (O. mykiss) are characterised by late embryonic mortalities occurring after eyeing and
possibly after hatching. Contrary to the results from other experimental groups described in the
same study (ie. post-ovulatory aging and photoperiod manipulation groups), where a specific
malformation was associated to a specific experimental manipulation, induced spawning increased
malformations of all the observed types equally (Bonnet et al., 2007).

4.3 Photoperiod manipulation

Photoperiod is an important parameter determining successful oogenesis in a variety of fish species,
although most markedly in salmonids (Bonnet et al., 2007, Finn, 2007). Some evidence suggests that
photoperiod manipulation may have negative impact on egg quality (O. mykiss) resulting in
increased incidence of morphological deformities in newly hatched larvae, especially abnormal yolk-
sac resorption, spinal torsion and other, including conjoined twins (Bonnet et al., 2007). Additionally,
it was also shown that application of an incorrect photoperiod may have a potential for later larval
mortality via non-inflation of the swimbladder (Pagrus auratus) (Fielder et al., 2002).

5 Conclusion
The early stages of embryonic development in fish generally exhibit a high incidence of
malformations. These commonly include deformities of head and spinal column, ocular deformities,
occurrence of conjoined twins, or yolk-sac resorption abnormalities. Despite extensive research into
the incidence of these abnormalities, it is not known to what extent they are dependent on
environmental variables such as temperature and salinity, parental status (gonad contamination),
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Incidence of malformations in fish embryos/larvae September 18, 2009

direct impacts of environmental contamination, or even natural factors like natural mutations
(Kjørsvik et al., 1990, Von Westernhagen et al., 1988). Furthermore, breeding protocols widely used
in fish farming industry and regarded as safe, such as photoperiod control and spawning induction,
may potentially have a negative impact on egg quality and can result in an increased frequency of
embryonic malformations and/or mortality (Bonnet et al., 2007).

With an exception of one study (Bonnet et al., 2007), where higher incidence of a specific
malformation (cyclopia) could be positively linked to a specific treatment (post-ovulatory ageing),
the types of malformations seem consistent following exposure to a variety of adverse natural or
anthropogenic conditions. This suggests that increased incidence of embryonic and/or larval
malformations in fish may be explained as a natural response to environmental stress and in most
cases cannot be simply attributed to a single specific factor.

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Incidence of malformations in fish embryos/larvae September 18, 2009

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