1. Reviews in Fish Biology
and Fisheries 7, 463±491 (1997)
Effects of pollution on reproductive behaviour
of ®shes
J A C K I E C . J O N E S and J O H N D. R E Y N O L D S
School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, United Kingdom
Contents
Abstract
Introduction
Fish behaviour in pollution research
Case study 1: mosquito®sh and paper mill ef¯uent
Case study 2: sun®sh and herbicide
Comparisons between case studies
Review of studies
Motivation for research
page 463
464
465
480

2. Selection of study species and pollutant
Laboratory or wild
Emphasis on reproductive behaviour
Effects on reproductive success
Experimental designs
Secondary effects of pollution
Prospects for the future: integrating pollution and reproductive behaviour
From individual behaviour to populations
Effects on future reproductive success
Population size
Genetic changes
Adaptation
Uses of reproductive behaviour studies
Behavioural assays for protecting other species and habitats
Conservation
Conclusion
Acknowledgements
References
483
483
Abstract
This review attempts to integrate pollution research with behavioural ecology by focusing
on reproductive behaviour of ®shes. A search of Aquatic Sciences and Fisheries
Abstracts and other sources showed that only 0.1% of 19 199 studies of aquatic pollution
Author to whom correspondence should be addressed (e-mail: Reynolds@uea.ac.uk).
0960±3166 # 1997 Chapman & Hall

3. and ®shes during the past 20 years have made this link. Effects on parental care and
courtship have 3been investigated using a variety of pollutants (e.g. acidi®cation, and
Jones
herbicide, thermal ef¯uent) in several ®sh families (e.g. Cichlidae, Poeciliidae,
Gasterosteidae, Cyprinidae).
Eleven of the 19 studies found a change in behaviour from the norm. Effects on
courtship included decreases or increases in frequency of displays, increased courtship
duration, or performance of male-like behaviour by masculinized females. Studies of
parental care have found decreased nest-building activity, decreased offspring defence,
or changes in division of parental care between the sexes. Few studies have measured
reproductive success or extrapolated their results to effects on populations.
We develop a framework for exploring links between pollution and behavioural
ecology which suggests potential impacts on life history trade-offs in reproduction, genetic
changes in populations, and population sizes. Many reproductive behaviours of ®sh
species are readily quanti®able and behaviours such as courtship by male guppies and
other members of the Poeciliidae show some promise for pollution monitoring and
behavioural toxicity tests. Choice of such assays would have to compete with the
sensitivity and practicality of
more traditional methods but may serve as useful complements. There is considerable
scope for further research into conservation. A synthesis between behavioural ecology
and toxicology should thus provide useful insights for both ®elds.
Introduction
Evidence of the state of many major British rivers during the reign of George III (1760
±
1820) was made apparent by a letter reportedly written by a member of Parliament to the
Prime Minister. The letter complained about the odour and appearance of the River
Thames. It was written not in ink, but with water from the Thames itself (Strandberg,
1971). Historically, the Thames had been a major salmon river but towards the end of the
18th century, ®sh species had declined dramatically and large areas of the river became
devoid of ®sh altogether (Wood, 1982). This was typical of many rivers in heavily
populated areas of Europe.
Fish populations are vulnerable because the aquatic environment is the recipient of
virtually every form of human waste (Moyle and Leidy, 1992). The IUCN (International
Union for Conservation of Nature) Red List (1996) records 734 ®sh species as
threatened and 92 species as extinct worldwide. Water pollution is one of several
contributors to such declines in ®sh populations (Clark, 1992; Moyle and Leidy, 1992;
Lawton and May, 1995; Maitland, 1995).
Pollution can be de®ned as ``the presence in the environment, or the introduction into
it, of products of human activity which have harmful or objectionable effects'' (Oxford
English Dictionary, CD-ROM). Studies of ®shes have ®gured prominently in pollution
research, particularly in sublethal physiological effects (review: Kime, 1995). Many
studies focus on reproduction because this is one of the most vulnerable periods in the
life cycle of ®shes (Gerking, 1980; Little et al., 1985).
Recently there has been development towards the use of behaviour in toxicological research (Little et al.,
1985; Dùving, 1991; Smith and Logan, 1997). This represents a fusion of the ®elds of behaviour,
ecology, toxicology and conservation biology. In this article, we present what might be termed a
`prospective review'. The `review' element concerns studies that have examined impacts of pollutants
on reproductive behaviour of

4. Effects of pollution on reproductive behaviour
465
®shes. The `prospects' that we are interested in concern the value of integrating
pollution research into the ®eld of behavioural ecology for gaining insights into water
quality management and conservation of ®shes.
Fish behaviour in pollution research
Before focusing on reproductive behaviour speci®cally, it is instructive to consider the
overall role of ®sh behaviour in pollution research. We searched Aquatic Sciences and
Fisheries Abstracts ( ASFA) on CD-ROM from 1978 to 1997 for references
containing various combinations of truncated search terms such as `behav ', to
include various spellings and forms of the key words (e.g. `behave',
`behaviour', `behavioral'). The result (Fig. 1) shows that only 13.8% of studies
involving ®sh and pollution included behaviour and of these, only 1%
involved reproductive behaviour as opposed to other behaviours such as
attraction and avoidance. Thus, of the original 19 199 studies of aquatic
pollution and ®shes, only 0.14% involve reproductive behaviour. This
con®rms the impression from previous reviews of the paucity of such
studies (Atchison et al., 1987; Beitinger,
1990; Dùving, 1991; Scherer, 1992).
The lack of studies of reproductive behaviour in pollution research can be attributed
in part to a perception that behaviour is dif®cult to measure and interpret. This makes
behavioural assays less appealing than lethal toxicity tests; death is a rather de®nitive
endpoint! However, behavioural responses may provide more sensitive early warnings
than standard test methods (Smith and Logan, 1997), and ethologists have been making
accurate measurements and analyses of many forms of animal behaviour for over 50
Reproduc*
Behav*
26
152
Feed*
Behav*
576
Predat*
or Prey
366
Avoid* or
Attract*
2641
Behav*
19 199
(No Extras)
0.1
1
10
100
% References With Pollut*, Fish* and...
Fig. 1. Results of a search of the Aquatic Sciences and Fisheries Abstracts CD-ROM from 1978 to
1997 for references on pollution and various types of ®sh behaviour. Numbers beside each bar show
the number of studies found for that category.

5. years. Furthermore, during the past 20 ± 30 years there has been a vigorous
development of 5 the ®eld of behavioural ecology, which is concerned with how and
Jones
behaviour affects life histories, reproductive success of individuals and their interactions
with the surrounding environment (Krebs and Davies, 1997). In linking the
reproductive behaviour of individuals to their environment, one can explore how
stressful conditions such as those caused by pollution will affect offspring production,
survival, and population dynamics.
We begin by reviewing the studies that have integrated the ®elds of reproductive
behaviour and pollution research. The behaviours considered include courtship,
spawning interactions and parental care. Studies that investigated effects of pollution
on fecundity or reproductive success, or presence ± absence of spawning only,
without measuring associated reproductive behaviours, are not included in this review.
Based on the above ASFA search and an additional literature survey, 19 studies
were found that investigated the effects of pollution on reproductive behaviour
(Table 1). This total excludes a few studies revealed by the initial ASFA keywords that
proved not to be relevant to this review. Courtship was studied most often, followed
by parental care. The studies varied widely in selection of ®sh species and pollutant,
and in the
relative contribution that reproductive behaviour made to the research.
Eleven of the 19 studies found a change in reproductive behaviour from the norm. It
should be borne in mind that there may be a publication bias towards signi®cant effects
of pollutants. Studies investigating parental care have found either no effect, decreased
nest-building or egg-directed activity, decreased protection of the young, disruption to
care or changes in division of parental care between the sexes. Effects on courtship
ranged from no effect to either decreases or increases in display frequency, increased
duration of courtship and performance of male-like display behaviour by masculinized
females. Additional effects reported, but without strong supporting evidence, are
described in Table 1. Two contrasting cases from Table 1 illustrate the range of
approaches used.
C A SE S T U DY
F F LU E N T
1 :
M O SQ U I T O FI S H
AND
PA P ER
M ILL E
Howell et al. (1980) investigated the impact of paper mill ef¯uent on a local population
of mosquito®sh (Gambusia af®nis holbrooki, Poeciliidae), in Elevenmile Creek,
Escambia County, Florida, USA. This research was precipitated by observations that
some females in the study area exhibited morphological signs of masculinization. Many
females had developed a gonopodium, the modi®ed anal ®n used by males for internal
fertilization in this family of ®shes (Figs 2 and 3). Fish from above and below the
ef¯uent out¯ow were compared using histological examinations of gamete abnormalities
and karyotypes. Comparisons of reproductive behaviour between the two ®sh groups
were also made in the laboratory. Fish were observed in each of the following
combinations: (1) three pregnant masculinized females and three normal females, (2) two
masculinized females, (3) a precociously masculinized male and a normal female, (4) a
precociously masculinized male, a normal male and three normal females, and (5) a
normal male and a normal female. No quantitative data were recorded, but descriptions
of morphology and reproductive behaviour were provided.
Howell et al. (1980) showed that ®sh from above the ef¯uent discharge exhibited normal sexual
expression. However, all females 13 ± 45 mm standard length, collected from below the ef¯uent
out¯ow, showed some degree of gonopodial development. There was no evidence of testicular tissue,
and heteromorphic sex chromosomes were absent.

6. Table 1. Summary of effects of pollution on ®sh reproductive behaviour
Behavioural category
Species
Courtship
Guppy
Poecilia
reticulata
Courtship
Guppy
Poecilia
reticulata
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7. 4
Table 1. (continued)
Behavioural category
Species
Courtship
Guppy
Poecilia
reticulata
Courtship
Fathead
minnow
Pimephales
promelas
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8. Courtship
Courtship
Orange
chromide
Etroplus
maculatus
Monochloramine
Either 0.025 or
0.05 mg lÿ1
from within
24 hours of
pair being
placed
together
until
spawning.
Mosquito®sh
Gambusia
af®nis
holbrooki
Paper mill
ef¯uent.
Fish collected
6.5 km downstream of the
ef¯uent output.
Little courtship and no
spawning occurred at
0.05 mg lÿ1 , with
®sh
dying or becoming ill.
Comparisons between
0.025 mg lÿ1 and
No spawning
at higher
concentration.
Hatching data
not given.
con-
trol showed: 64% increase in length of
courtship period, 34%
reduction in clutch
size, increased ®n¯icker by males,
reduced quivering
and ®n-¯icker by
Not
females.
determined.
Exposed females
became masculinized.
Masculinized pregnant
females displayed only
typical male reproductive behaviour (chasing
normal and masculinized females with
gonopodial swings and
thrust attempts). Exposed males matured
precociously and
exhibited typical but
more aggressive courtship. A masculinized
male was dominant
over a normal male.
Increased
courtship
length may
increase predation risk. An
additional behaviour which
increased was
gill-purging.
This might be
misinterpreted
by conspeci®es
as aggression
and hamper
pair formation.
None
suggested.
A comprehensive study,
primarily aimed at
determining suitability
of behavioural toxicity
for pollution monitoring.
Stafford
and Ward
(1983)
No data or analyses
presented. One replicate
for each combination
involving 1 ± 3 masculinized or normal ®sh.
Interesting ®ndings from
wild-exposed ®sh merit
further investigation.
Howell
et al. (1980)
continued overleaf
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9. Table 1. (continued)
Behavioural category
Species
4
Courtship
and
aggression
Mosquito®sh
Gambusia
af®nis
Courtship
and
aggression
Mosquito®sh
Gambusia
af®nis
af®nis
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10. Male
courtship
Cichlid
Sarotherodon
mossambicus
Endosulfan
insecticide.
Three treatments: (A)
adults exposed
to 0.5 ìg lÿ1
for
4 weeks;
(B) juveniles
exposed to
0.6 ìg lÿ1 for
9
weeks (with
1.5 ìg lÿ1 for
®rst 3 days);
(C) juveniles
exposed to
either 0.6 or
0.2 ìg lÿ1 for
6
weeks.
(A) No effect on adult
male sexual behaviour
of female clutch
production or mouthbrooding time.
(B) Onset of juvenile
male sexual behaviour
and female clutch
production delayed.
Females had higher
clutch production rates
but lower retention
times than controls,
suggesting that clutch
abortion was
occurring.
(C) More control
males showed normal
breeding behaviour
between days 36 and
42 than exposed males.
Many fry died
at low exposure concentration (24 hour
LC50 was
0.5 ìg lÿ1 ).
Delayed breeding displays in
newly maturing males may
explain a
reduction of
approximately
75% in nests
of related
Tilapia rendalli in the
Okavango
Delta,
Botswana.
Concentrations were
similar to those found in
the wild soon after
spraying. Additional
direct physiological
effects noted on ®sh.
Matthiessen
and Logan
(1984)
continued overleaf
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11. 4
Table 1. (continued)
Behavioural category
Species
Nest
building
Cichlid
Tilapia
rendalli
Parental
care
Threespine
stickleback
Gasterosteus
aculeatus
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12. Parental
care
Naked goby Episodic
Gobiosoma hypoxia.
bosc
Field; down to
,0.5 mg l
Laboratory;
down to
Males guarded their
eggs and nests until
the dissolved oxygen
level reached almost
lethal levels (0.15 ±
0.6 mg lÿ1 ), when
0.15 mg l
abandoned.
ÿ1
ÿ1
Parental
care
Bluegill
sun®sh
Lepomis
macrochirus
Redear
sun®sh
Lepomis
microlophus
Parental
care
Fathead
minnow
Pimephales
promelas
Herbicides
2,4-dichlorophenoxyacetic acid
and Aquathol-K
sprayed on surface of lake to
obtain a concentration of
4 mg lÿ1 .
Sprayed water
was used as a
control.
they
Although some males
abandoned nests temporarily in each treatment, there was no
signi®cant difference
between them. Congeners usually intruded
during male absence
and some egg predation was observed in
all treatments. There
was no signi®cant effect on male parental
care (e.g. displays towards intruding males,
Waterborne lead. egg fanning).
Males exposed to lead
0.5 mg lÿ1
spent signi®cantly less
lead for 30
days while
time in activities
®sh
directed towards ceilspawned.
ing of nest, including
preparation of substrate for eggs, and
touching ceiling with
dorsal pad.
The eggs died
if males left
the nest. If the
male does not
leave the nest
when lethal
limits are
being reached
he will die.
Not
determined.
Even if there
are no eggs in
the nest, the
male will lose
mating opportunities if he
abandons
unnecessarily.
Drastic
decrease in
egg production
by exposed females. Role of
reduced parental behaviour
in reproductive
output unclear.
Reproductive
success may
be reduced
due to less
cleaning of
nest ceiling
and egg mass
surface.
Thorough study, unusual Breitburg
in incorporating both
(1992)
®eld observations and
laboratory experiments.
None expected Sample sizes were low
from herbicide (5 ± 9 males per treatexposure.
ment). A possible trend
towards higher rates of
temporary desertion in
both species and consequently effects on egg
predation might prove
signi®cant with larger
sample sizes.
Dif®cult to interpret the
cause of reduced nest
maintenance behaviours,
i.e. an effect of lead
exposure versus fewer
eggs received.
Bettoli and
Clark
(1992)
Weber
(1993)
continued overleaf
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13. Table 1. (continued)
Behavioural category
Species
4
Parental
care and
aggression
Convict
cichlid
Cichlasoma
nigrofasciatum
Parental
care
Convict
cichlid
Cichlasoma
nigrofasciatum
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14. Parental
care
Bluegill
sun®sh
Lepomis
macrochirus
Di¯ubenzuron; a
pesticide
2.5 ìg lÿ1 and
ÿ1
30 ìg l
applied twice,
®rst application
into the designated ®sh enclosures 2 months
after ®sh introduced and second application
1 month later.
No effects on male
reproductive behaviour
observed.
None due to
behaviour.
Only one
spawning event
was observed
in all exposure
treatments
(30 ìg lÿ1 )
but
most spawning
events (86%)
took place
prior to exposure. Spawning
appeared to be
more in¯uenced by water
temperature
than di¯ubenzuron exposure. Reduced
juvenile
growth rates.
Reduced juvenile growth
could have
major impolications for ®sh
survival during
winter through
starvation and
increased predation risk.
No statistical analysis
Tanner and
was performed and only Hoffett
2 replicates per treat(1995)
ment used. Reproductive
behaviour effects not
quanti®ed and hatching
success based on one
spawning event at
30 ìg lÿ1 . Most
spawn-
ing events took place
before exposure. However, focused on ecological context of effect of
pesticide exposure on
natural food supply, and
effects on reproductive
behaviour were not intended as a main focus.
continued overleaf
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15. 4
Table 1. (continued)
Behavioural category
Species
Parental
care
Bluegill
sun®sh
Lepomis
macro- chirus
study took place in littoral enclosures and investigated effects on
reproductive success
from spawning to juvenile production.
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16. Parental
care
Bluegill
sun®sh
Lepomis
macrochirus
Esfenvalerate: a
pesticide
0.01, 0.08, 0.2,
ÿ1
1.0 and 5 ìg l
applied twice,
®rst application
1 month after
®sh introduced
and second application another
month later.
At 1 ìg lÿ1 ,
one
male guarding a
nest with
embryos rimcircled rapidly.
In all enclosures except 5
ìg lÿ1 , where the
adults died, rimcircling occurred
both before and
after exposure.
None due to
behaviour.
Adults died at
5 ìg lÿ1 .
Spawning
ceased for 15
days in
1 ìg lÿ1
enclo-
sure following
both applications and larval mortality
was observed
following the
second application. A
reduction in
juvenile
growth rate
was measured
at 1.0, 0.2 and
0.08 ìg lÿ1 .
If esfenvalerate
were to be applied repeatedly biweekly
at concentrations greater
than
0.44 ìg lÿ1 ,
spawning and
hence juvenile
production
would cease.
Reduced juvenile growth
could have
major implications for ®sh
survival over
winter through
starvation and
increased
predation risk.
No statistical analysis
was performed and 1
replicate per treatment
was used. Reproductive
behaviour effects not
quanti®ed but played a
minor role. The rapid
rim-circling that occurred in the 1 ìg lÿ1
treatment was
observed in only
one male. Combined data from
before and after
pesticide aplication were used
to calculate
average hatch- ing
success.
Tanner and
Knuth
(1996)
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17. 1
Effects of pollution on reproductive
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Fig. 2. Normal specimens of Gambusia af®nis holbrooki collected above paper-mill ef¯uent in
Elevenmile Creek, Escambia County, Florida (Howell et al., 1980; reproduced by permission of
Copeia). The top ®sh is a pregnant female (31 mm standard length). The bottom ®sh is a male
(25 mm standard length) showing gonopodial differentiation of the anal ®n.
Masculinized females exhibited some male reproductive behaviour and males exhibited
precocious development of secondary sex characters and reproductive behaviour. A
physiologically normal male and female when placed together exhibited normal
behaviour (see Table 1 for detailed behavioural ®ndings).
C A SE S T U DY 2 : S U N FI S H A N D H E R B IC ID E
Bettoli and Clark (1992) investigated the effects of herbicide exposure on nesting
behaviour in bluegill sun®sh, Lepomis macrochirus and redear sun®sh, L. microlophus
(Centrarchidae) in Old Monterey Lake, Monterey, Tennessee, USA. Underwater video
cameras were set up near nests in the wild and ®sh were ®lmed prior to, during and after
herbicide application (Aquathol-K or 2,4-dichlorophenoxyacetic acid). Some of the
redear sun®sh observations were also made from the lakeside using binoculars. Speci®c
behaviours such as display, spawning and egg fanning were recorded, along with
incidence and timing of nest abandonment. Statistical comparisons were made between
males with nests in test plots sprayed with Aquathol-K, 2,4-D or with water (control).
There was no difference in the number of nests abandoned nor in the actual time of abandonment
between treatments (herbicide or water sprayed). When abandonment did occur, congeners usually
entered the nest to feed on eggs or fry until the male returned. Finally, frequencies of rim circling,
egg fanning and agonistic behaviours of the guarding males did not differ between treatments.
Bettoli and Clark (1992) concluded that these herbicides, if applied properly, cause no
pronounced shift in sun®sh reproductive behaviour.
1

18. Fig. 3. Masculinized specimens of Gambusia af®nis holbrooki collected below a paper-mill ef¯uent in
Elevenmile Creek, Escambia County, Florida (Howell et al., 1980; reproduced by permission of
Copeia). (A) Immature male (15 mm SL) showing precocious anal ray elongation. (B) Mature male
(17 mm SL) showing precocious gonopodium. (C) Pregnant female (23 mm SL) with a gonopodium.
(D) Pregnant female (28 mm SL) with a gonopodium. (E) Pregnant female (34 mm SL) with a
gonopodium. (F) pregnant female (36 mm SL) with a gonopodium.
C O M PA R IS O N S BE T W E E N C AS E S T U D I E S
These studies show the diversity of approaches to the study of effects of pollution on ®sh
reproductive behaviour. Whereas the mosquito®sh study focused primarily on morphology, with some unquanti®ed behavioural observations, the sun®sh study was restricted
entirely to reproductive behaviour. Many mosquito®sh were put into each observation
tank and only one replicate of each ®sh combination was used. The sun®sh study used
between ®ve and nine individually sprayed areas per treatment to study effects of
herbicide exposure on ®sh in the wild. Owing to the nature of the data collected in the
®rst study, no analysis could be undertaken, while in the second study a rigorous analysis
of the data was performed. However, the mosquito®sh study was concerned primarily
with morphological effects, with behavioural observations comprising a small part of the
research. Although the behavioural data were non-quantitative, they provide a starting
point for further work.

19. To obtain a clearer picture of effects of pollution on reproductive behaviour, we
assess various aspects of the studies carried out so far.
Review of studies
M O T I VAT I O N F O R R E S E A
RCH
Some studies were aimed towards development of a behavioural assay for toxicity testing,
based on changes in behaviour such as courtship (Stafford and Ward, 1983; SchroÈ der
and Peters, 1988a; Bortone et al., 1989). This information could be used in a similar vein
to lethal toxicity tests, but with behavioural responses as an end point (Stafford and
Ward,
1983; Little et al., 1985; Bortone et al., 1989). SchroÈ der and Peters (1988a)
contended that as a behavioural assay, courtship by guppies ( Poecilia reticulata,
Poeciliidae) is extremely sensitive to very low concentrations of aquatic
contaminants. Stafford and Ward (1983) found orange chromides ( Etroplus
maculatus, Cichlidae) also to be a suitable test organism and proposed that
behavioural assays are liable to be more sensitive measures of effects of toxicants
than previous methods.
The aims of the other studies were less clear, but many were concerned with effects
on reproductive behaviour and reproductive success (Howell et al., 1980; Matthiessen
and Logan, 1984; Ryabov, 1985; SchroÈ der and Peters, 1988b; Pyron and
Beitinger,
1989; Bettoli and Clark, 1992; Lorenz and Taylor, 1992a,b; Weber, 1993; Tanner and
Knuth 1995, 1996; Tanner and Moffett, 1995). For example, Lorenz and Taylor (1992b)
tested the hypothesis that if offspring survival is reduced in an unfavourable
environment (in this case due to acid stress), then parents may reduce their care to
conserve energy for future reproduction (Carlisle, 1982). This was supported by tests
with convict cichlids (Cichlasoma nigrofasciatum, Cichlidae), which suffered a decrease
in reproductive success. Some studies went further in hypothesizing that reduced
reproductive output owing to changes in behaviour may have a detrimental effect on
population numbers (Douthwaite et al., 1981; Colgan et al., 1982; Krotzer, 1990;
Breitburg, 1992). Krotzer (1990) studied the effect of masculinization on the behaviour
of female mosquito®sh (Gambusia af®nis af®nis, Poeciliidae). It was suggested that if
females became more aggressive and less receptive to males, exposed populations in
the wild could suffer. The primary aim of three studies of bluegill sun®sh by one
research group was to measure effects of pesticides on reproductive success from
spawning to juvenile production, with reproductive behaviour playing a very minor role
(Tanner and Knuth, 1995, 1996; Tanner and Moffett, 1995). Although some studies
went into greater depth on the consequences of changes in behaviour than others, the
information provided by most studies could be used as a starting point towards further
research.
S E L E C TI O N O F S T U DY S P E C IE S A N D P O L L U
TAN T
Studies varied widely in species and pollutant investigated. Most of those that focused on
effects on the reproductive behaviour of the ®sh chose a ®sh ± pollutant
combination occurring in the wild at a site of speci®c concern. Examples include
®sh ± pollutant combinations such as mosquito®sh exposed to paper mill or kraft mill
ef¯uent (Howell et al., 1980; Bortone et al., 1989), cichlids (Tilapia rendalli,
Cichlidae) exposed to endosulfan insecticide (Douthwaite et al., 1981), threespine
sticklebacks (Gasterosteus

20. aculeatus, Gasterosteidae) exposed to power station thermal ef¯uent (Ryabov, 1985),
naked gobies (Gobiosoma bosc, Gobiidae) exposed to hypoxic water conditions
(Breitburg, 1992) and bluegill sun®sh exposed to herbicide contamination (Bettoli and
Clark, 1992). Although not all of these studies were carried out in the ®eld, such
combinations were known to occur in the wild. This gives the ®ndings direct relevance to
such affected areas.
Other criteria may affect selection of ®sh species. For example, Weber (1993) used
fathead minnows ( Pimephales promelas, Cyprinidae) because this species is a standard
test species for use in aquatic toxicity studies (Denny, 1987). Fathead minnows were
also used by Pyron and Beitinger (1989) to investigate the in¯uence of selenium
exposure.
Other ®sh may be readily available, have well-documented reproductive behaviour,
survive well in the laboratory, or breed readily. These were used speci®cally in the
behavioural toxicity tests. For example, the suitability of orange chromide cichlids for
toxicity tests was investigated under exposure to monochloramine (Stafford and Ward,
1983) and guppies were exposed to treatment-plant wastewater (SchroÈ der and
Peters,
1988a). However, these ®sh species may not encounter such pollutants in the wild.
Researchers have thus sacri®ced direct relevance of the environmental situation
normally faced by the ®sh for the sake of practicality and standardization.
L A B O R AT
WILD
O
RY
OR
Studies have differed greatly in the origin of the ®sh used and the type of exposure
received. Nine of the 19 studies in Table 1 used laboratory-bred and laboratory-exposed
®sh (Colgan et al., 1982; Stafford and Ward, 1983; Matthiessen and Logan, 1984;
SchroÈ der and Peters, 1988a,b; Pyron and Beitinger, 1989; Lorenz and Taylor,
1992a,b; Weber, 1993). One study used wild mosquito®sh, exposed and observed in the
laboratory (Krotzer, 1990) while three used hatchery-reared bluegill sun®sh and
exposed them in outdoor littoral enclosures (Tanner and Knuth, 1995, 1996; Tanner
and Moffett, 1995). Five studies used wild ®sh that were exposed to the pollutant in the
®eld. Of these, three observed the ®sh in the wild: sun®sh, Lepomis macrochirus and L.
microlophus (Bettoli and Clark, 1992), Tilapia rendalli (Douthwaite et al., 1981), and
threespine stickleback
(Ryabov, 1985). The other two observed mosquito®sh under non-polluted conditions in
the laboratory (Howell et al., 1980; Bortone et al., 1989) after exposure in the ®eld. Only
one study used wild ®sh (naked gobies) in both wild and laboratory exposure
observations (Breitburg, 1992). Differences in the results of these studies could, in part,
have arisen because of the varied background and exposure conditions of experimental
®sh.
Although results may be less directly relevant to the wild, there is a bene®t in using
laboratory ®sh strains compared with wild ®sh because one can eliminate the potential
effects of tolerance through genetic adaptation or physiological acclimation. Tolerance
to pollutants has been shown in several ®sh species, including mosquito®sh, Gambusia
holbrooki (Poeciliidae) (Benton et al., 1994), rainbow trout, Oncorhynchus
mykiss
(Salmonidae) (Pascoe and Beattie, 1979) and central mudminnow, Umbra limi
(Umbridae) (Kopp et al., 1992), as well as in other taxa (Bryan and Langston, 1992;
Gustavson and Wangberg, 1995). There is considerable scope for further research into
differences among wild populations in pollution tolerance.

21. E MP H A S I S ON R EP R O D U C T IV E B E H AV I
O UR
Some studies were purely behavioural (e.g. SchroÈ der and Peters, 1988a; Pyron
and Beitinger, 1989; Lorenz and Taylor, 1992a). Others contained combinations
of reproductive physiology, morphology and behaviour (e.g. Howell et al., 1980;
Weber,
1993) while others dedicated little to effects on reproductive behaviour (Tanner and
Knuth, 1995, 1996; Tanner and Moffett, 1995). Usually, only a small part of the
reproductive behaviour repertoire was studied, such as courtship, spawning behaviour,
nest building, parental care of eggs, or defence of newly hatched fry. Ideally, the entire
reproductive behaviour repertoire should be investigated (from mate choice and courtship
to egg or larval independence) to see at what stages pollution exerts an effect. Time and
expense will hinder such an approach, but if the purpose is to develop a toxicity test for
water quality management, full reproductive cycle studies could serve as an important
®rst step, to be re®ned by simpler, focused behavioural
assays.
E F FE C TS O N R E PR O D U C T IV E S U C C E
SS
Eleven of the studies in Table 1 considered the possible effects of a change in reproductive
behaviour on reproductive success. Only three studies actually measured this directly: two
found a negative effect (Breitburg, 1992; Lorenz and Taylor, 1992b) and one reported an
increase in reproductive success (SchroÈ der and Peters, 1988b). The latter result should
be treated with caution owing to a small sample size and inappropriate experimental
design (see Table 1 and general comments under `Experimental designs', below). A
possible decrease in reproductive success was inferred by Douthwaite et al. (1981), who
measured the age distribution of juvenile Tilapia rendalli in the Okavango Delta,
Botswana, comparing sites that had been sprayed with insecticide 2 years previously
with control sites. Although few studies have measured impacts on reproductive output
via behavioural changes, in some cases it was likely that any such effects would have
been overshadowed by physiological problems. For example, Weber (1993) found
reduced egg production in female fathead minnows upon exposure to lead. Stafford and
Ward (1983) investigated the effect of monochloramine on orange chromide. At levels of
0.05 mg lÿ1 , some ®sh became ill and courtship and spawning stopped, while
at levels of 0.025 mg lÿ1 , female egg production was reduced. Finally, some
studies estimated effects on reproductive success without trying to establish a
causal link to changes in reproductive behaviour (Tanner and Knuth, 1995,
1996; Tanner and Moffett, 1995).
E X PE R I M E N TAL D ES I G N S
In Table 1 most of our comments concerning experimental designs are rather critical. In
fairness to the authors, we wish to emphasize two points strongly. First, many of the
studies were done before biologists were fully aware of the problems of nonindependence of samples within treatments (`pseudoreplication', Hurlbert, 1984). Second,
as noted earlier, a combination of physiological and behavioural effects of pollutants was
investigated in some studies, sometimes with effects on reproductive behaviour appearing
as a secondary objective (e.g. Howell et al., 1980).
Nevertheless, to evaluate our current state of knowledge in this ®eld, it must be borne
in mind that many of the studies lacked adequate controls, replication, or statistical
analyses. Multiple observations of the same ®sh under the same treatment cannot be
counted as separate statistical data points. One ®sh may be watched 10 times when
exposed to a pollutant and another ®sh may be watched 10 times in a control situation,

22. but effectively this still leaves one data point (e.g. mean behaviour per ®sh) under each
treatment (Hurlbert, 1984). Furthermore, 10 ®sh in one aquarium exposed to a pollutant
should not be compared as 10 independent samples to ®sh in a control tank. Fish
within aquaria are not independent because individuals may affect each other both
behaviourally and physiologically. For example, there may be in¯uences from each
other's activity levels, dominance, pheromones, or metabolic wastes. These problems
apply to more traditional non-behavioural tests as well. Furthermore, aquaria are bound
to differ by more than the presence or absence of the pollutant concerned. For example,
levels of nutrients, light intensities, or other uncontrolled and unknown factors may
confound treatments. This necessitates the use of multiple aquaria per treatment in
randomized or alternating spatial patterns. If multiple ®sh or observations are used per
aquarium, each aquarium should yield a single mean data point in a t-test or standard
ANOVA design, or observations from individual ®sh within tanks can be used in a nested
ANOVA-type design.
Our general impression from the information summarized in Table 1 is that many of
these early studies report tantalizing results, but the use of ®sh reproductive behaviour
in pollution research is still very much in its infancy.
Secondary effects of pollution
Additional studies are available in the literature which measure secondary effects of
pollutants on reproductive behaviour. For example, silting arises from high sediment
loading, and lowered oxygen levels often result from bacterial degradation under high
organic pollution (Clark, 1992; Mason, 1996). Although such studies were not included
in Table 1 because they did not use pollutants directly, their results could still be useful
in water quality management and conservation.
Silting was found to affect parental care in the ®fteenspine stickleback (Spinachia
spinachia, Gasterosteidae) (Potts et al., 1988). During parental care, males use their ®ns
to fan water over their egg mass. Silting increased the number of times males fanned
their eggs. Nest inspection and nest pushing also became more frequent. Lowered
oxygen has also been found to affect paternal care in the threespine stickleback (Reebs
et al., 1984), with an increase in the length of egg-fanning bouts. When oxygen levels
fell below 2.8 mg lÿ1 , males spent more time swimming outside the nest but
still fanned at intervals. In the guppy, lowered oxygen levels increased the
frequency of breathing at the surface by males at a cost of decreased
courtship (Kramer and Mehegan, 1981). To date, such studies have not
examined consequences for reproductive success.
Prospects for the future: integrating pollution and reproductive behaviour
There is now a growing interest in understanding sublethal effects of pollutants on
aquatic organisms (Alabaster and Lloyd, 1982; MuÈ ller and Lloyd, 1994). To evaluate
the usefulness of developing this ®eld further, it is helpful to integrate general theory
from behavioural ecology to clarify the biological links between behavioural
responses of individuals and population changes. We can then use this information to help
decide the prospects for using behavioural studies to help protect populations of the
®shes themselves, as well as developing assays for protecting other species in the
habitat.

23. F R OM I N D I V I DUA L B E H AV IO U R T O P O P U L AT I
O NS
2
Jones and
Figure 4 is a schematic diagram of behavioural testing procedures, responses of the
animals, and potential uses for this information. Nearly all of the studies reviewed are
concerned with step 1, which links reproductive behaviour of ®shes and their exposure to
sublethal doses of the contaminant in question. As we have seen, few of the studies have
gone on to step 2, from reproductive behaviour to reproductive success, and these have
been concerned only with current broods rather than impacts on subsequent reproductive
bouts. We have not found any studies that take the `®nal' step (step 3) of testing for
population responses, although there was circumstantial evidence of a decrease in the
numbers of Tilapia rendalli in the Okavango Delta in two areas sprayed with endosulfan
insecticide (Douthwaite et al., 1981). Below, we discuss the logic and potential of these
under-developed steps.
Effects on future reproductive success
Life history theory, supported by a large body of empirical research on a variety of taxa,
suggests that commitment of resources to a given reproductive bout should have costs for
survival, growth, or reproduction in subsequent bouts (Williams, 1966; Roff, 1992;
Stearns, 1992; Sargent and Gross, 1993). If this were not true, animals could make huge
investments in reproduction forever (Partridge and Harvey, 1985). An example from
®shes concerns male bluegill sun®sh, which have been induced to ventilate their eggs by
fanning water across them more frequently when given larger broods experimentally
(Coleman et al., 1985). Increased fanning reduces the time available to males for
courting new females, and it reduces energy reserves through the loss of non-polar lipids
(Coleman and Fischer, 1991). This may impair survival and future reproduction. Such
reproductive trade-offs may be most evident under conditions of stress (Roff, 1992). For
example, in the sand goby ( Pomatoschistus minutus, Gobiidae), females that initially
produced large clutches before receiving a low food ration produced smaller clutches in
the next bout (Kvarnemo, 1997). This trade-off did not occur with high food. Thus,
environmental stressors such as pollutants could have delayed effects on reproduction
which would be overlooked if only a single reproductive cycle were studied.
Population size
Links between individual behaviour and population biology have been much sought-after
by ecologists (Sibly and Smith, 1985; Sutherland, 1996). In theory, any external force that
alters behaviour of individuals from the optimum under natural and sexual selection
could lead to reduced population sizes. But this effect may be far from straightforward,
depending on the nature of density dependence in the population. For example, Lorenz
and Taylor (1992b) showed that under conditions of low pH, convict cichlids were less
able to rear their young in the presence of conspeci®cs. But if population densities of
these ®sh were restricted by food or predation at a different stage of life, reduced survival
of young being guarded by parents may have no impact on population size. The dif®culty
of showing relationships between reproduction and population size is well known to
®sheries biologists who rarely ®nd clear patterns between stock size and recruitment (Hilborn and
Walters, 1992; King, 1995). Thus, if one is concerned with conservation, a major challenge is to
demonstrate clear effects of sublethal doses of pollutants on population size.

24. TESTS
EXPOSURE
USES
Fish exposed to sublethal dose
1
Monitoring water quality
4
Protection of
other species
and habitat
Effect on reproductive behaviour?
ORGANISM
RESPONSE
2
5
Effect on current and future
reproductive success?
6
3
7
Conservation of
the test fish
species
Effect on population?
POPULATION
RESPONSE
Number of changes
Genetic individuals
Adaptation
Fig. 4. Schematic diagram showing hierarchy of pollution effects on reproductive behaviour and the potential uses for such information. Circled
numbers are reference points for discussion in the text.
E
f
f
e
c
t
s
o
f
p
o
l
l
u
t
i
o
n
4

25. Genetic changes
2
Effects that change on reproductive
Jones
Environmental impacts of pollution the reproductive behaviour of individuals (e.g. choice and
of mate or oviposition site) could alter the genetic composition of populations. An
example from a natural environmental parameter concerns the effects of light levels on
behaviour of Trinidadian guppies (Reynolds, 1993). Under low light levels, large-bodied
males have higher mating success than smaller ones, because of female choice. This
pattern is reversed under higher light levels, when males may be hampered by greater
risk of predation. Because male body size has a genetic basis (Reynolds and Gross,
1992), such changes in mating behaviour in response to an environmental change may
have genetic consequences for the population. Genetic changes such as these have yet to
be shown in the context of pollution, but given the numerous demonstrations of
heritability of traits under natural selection (Falconer, 1989) and sexual selection
(Pomiankowski and Mùller, 1995; cf. Alatalo et al., 1997), researchers would be well
advised to bear these possibilities in mind.
Adaptation
Long-term selection may lead to the evolution of resistance. This depends on the
intensity of selection, additive genetic variance in resistance and the population size.
Benton et al. (1994) provided an example in a comparison of populations of freshwater
snails ( Helisoma trivolvis, Planorbidae) and mosquito®sh from a relatively clean site and
a site contaminated with a variety of heavy metals and other elements. They found that in
the populations exposed to the pollutants, both species may have evolved increased
tolerance. In mosquito®sh, this tolerant genotype seems to be linked to small body size.
Particularly relevant here is the possibility that altered mate choice, courtship or
parental care could be adaptive, leading to a buffering against environmental change.
This could occur either through short-term, facultative changes in behaviour or through
genetic responses to selection. This is a risky proposition, because changes in behaviour
could be non-adaptive, and genetic adaptations may not evolve quickly enough.
Furthermore, genetic bottlenecks and inbreeding are risks when the effective population
size is reduced owing to fewer individuals reproducing (Meffe, 1986).
USES
OF
STUDIES
R E P R O D U C TI VE
BE H AVI O U R
Aside from gaining a better understanding of pollution in an ecological context, what
direct practical uses can be made from the framework in Fig. 4? We believe the answers
depend on whether one is concerned with development of general assays for protecting
water quality for other species and the habitat, or with conservation of the particular
species under study.
Behavioural assays for protecting other species and habitats
Toxicity tests may be used to screen new chemicals, and to formulate water quality
criteria and standards (Council of the European Communities, 1978; Alabaster and Lloyd,
1982; Abel, 1989; Lloyd, 1992; Howells, 1994). Standardized protocols for toxicity tests, for example
LC50 tests using guppies or Daphnia magna (Daphniidae), or life-cycle tests using
salmonids, are often used (Alabaster and Lloyd, 1982; Lloyd, 1992; Howells, 1994; Mason,
1996). Toxicity tests are also used to assess hazards presented by ef¯uents, for example
from industry or sewage treatment works. Such monitoring of ef¯uent is
2

26. especially useful when substances are dif®cult to analyse or where a concoction of
substances is present (Alabaster and Lloyd, 1982).
Whether the objective is to monitor ef¯uent water quality (arrow 4 in Fig. 4) or
formulate water quality criteria or standards (arrow 5), the ideal protocol involves a
species that can be reared quickly and easily, with a minimum of equipment and space,
and which responds quickly to the testing procedure, in a highly repeatable manner
(Alabaster and Lloyd, 1982; Smith and Logan, 1997). Given these requirements, it
seems unlikely that many of the ®sh species and reproductive behaviours reviewed in
Table 1 will be practical for general use. We suggest that the species that come closest
might be poeciliids such as the guppy (already a standard test species for traditional
toxicity tests) and mosquito®sh (Gambusia spp.). They are small and breed readily in
captivity, and male courtship is easily quanti®able and occurs under a wide variety of
circumstances (Farr, 1980; SchroÈ der and Peters, 1988a; Reynolds et al., 1993;
Houde,
1997). Rice®sh (Oryzias latipes, Cyprnodonitidae) and killi®sh ( Fundulus, Cyprnodonitidae) are also worth exploring, although their courtship behaviour is less elaborate
than in guppies. Female poeciliids have already been shown to undergo physical, and to
a less extent behavioural, masculinization as a result of exposure to paper mill ef¯uent
(Howell et al., 1980). It would be interesting to see whether the opposite effect ±
feminization ± occurs during exposure of males to oestrogenic compounds, and whether
changes in courtship could yield an early warning system for such pollutants (Purdom
et al., 1994; Sumpter et al., 1996).
Overall, it remains to be seen whether courtship by ®shes yields an improvement in
sensitivity, practicality, and cost over existing methods. This is well worth exploring,
particularly as automated behavioural monitoring techniques using videos and
computers become increasingly advanced.
Conservation
Studies of impacts of pollutants on reproductive behaviour may be more important for
understanding conservation of the test ®sh themselves (arrows 6 and 7 in Fig. 4) than as
general assays for monitoring water quality and recommending criteria on behalf of other
species. Such studies could complement, rather than replace traditional studies of effects
on physiology and survival.
The ecological framework presented (Fig. 4) suggests several reasons for focusing on
reproductive behaviour. For example, behavioural studies of the impacts of oestrogenic
compounds might yield insights into reproductive output in species where males provide
parental care. Indeed, parental care itself, including egg guarding and ventilation, places
high energetic demands on ®shes, which often lose weight during parental cycles
(Sargent and Gross, 1993; Smith and Wootton, 1995). Pollutants may exacerbate these
costs, as well as costs from sexual selection, including predation, courtship,
territoriality, mating competition, and mate choice (Magnhagen, 1991; Andersson,
1994; Reynolds and CoÃteÂ, 1995). One or more forms of such mating competition
are
widespread in ®shes. Although there are numerous routes towards reduced offspring
production in current and future breeding attempts (Fig. 4), we need more research on
these to explore properly their links to conservation.
Empirical justi®cation for focusing on reproductive behaviour comes from a recent
survey of conservation of ®shes (Bruton, 1995). Cross-species comparisons suggested
that ®sh with more complex reproductive behaviours are particularly at risk. Parental

27. care is found in 21% of teleost ®sh families (Gross and Sargent, 1985), and is
particularly common in species breeding in fresh water, estuaries, and coastal marine
environments. These are also the habitats that receive the greatest inputs of pollutants
(Clark, 1992; Mason, 1996).
Conclusion
The study of effects of pollution on reproductive behaviour of ®shes has yielded some
clear effects on courtship and partental care for several species, but few studies have
gone far enough to draw clear inferences for effects on populations. As a tool for
biomonitoring or use in water quality criteria, such studies are unlikely to be able to
compete with standard toxicity tests, although they may complement them effectively,
especially since there is evidence of strong sensitivity to pollutants. From the standpoint
of conservation of populations of the ®shes themselves, it would be useful to explore
further the links between alterations in reproductive behaviour, reproductive output, and
the mechanisms that govern population dynamics. Such an integration of behavioural
ecology with pollution research may provide useful insights for both ®elds.
Acknowledgements
We wish to thank W. Mike Howell for the photographs used for Figs 2 and 3 and for
comments on the manuscript. We also thank Mike Elliott, Alastair Grant and Peter
Matthiessen for helpful discussions and Nick Dulvy, Paul Hart, David Kime, Carl Smith
and two anonymous referees for comments on the manuscript.
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Accepted 3 July 1997