Subchronic Toxic Effects of Fluoride Ion on the Survivaland Behaviour of the Aquatic Snail Potamopyrgus antipodarum(Hydrobiidae, Mollusca)

Alvaro Alonso • Julio A. Camargo

Received: 26 March 2010 / Accepted: 7 June 2010 / Published online: 25 June 2010

� Springer Science+Business Media, LLC 2010

Abstract Short-term bioassays usually assess lethal

effects of pollutants in animals, whereas subchronic bio-

assays are more suited for assessing effects on animal

behaviour. Among them, videotaped bioassays are an

improvement in the behavioural monitoring because they

are easily and cheaply implemented. The present study

focuses on the assessment of subchronic (14-day) effects of

fluoride ion on the survival, proportion of dead plus

immobile animals, and velocity (monitored by a video-

taping and image analysis system) of the aquatic snail

Potamopyrgus antipodarum (Hydrobiidae, Mollusca). One

control and three nominal fluoride concentrations (5, 20,

and 40 mg F-/l [actual mean concentrations of 5.2, 17.5,

and 37.0 mg F-/l, respectively]) were used. Each treatment

(including the control) was replicated 12 times. Mortality,

number of dead plus immobile animals, and velocity were

monitored after 0, 7, and 14 days of exposure. After

14 days, animals exposed to 40 mg F-/l showed higher

mortality, number of dead, and immobile individuals than

control animals. Snails exposed to 5 and 20 mg F-/l were

not affected by fluoride ion regarding these endpoints. In

contrast, snails exposed to 20 mg F-/l for 7 and 14 days

showed lower velocity than control animals. Therefore,

velocity was sensitive to environmental fluoride concen-

trations and as such is a useful parameter for ecologic risk

assessment. In addition, videotaping allowed us to detect

behavioural patrons in velocity at very short exposures

(seconds) during the monitoring process by showing that

the velocity of snails must be monitored at least during

the course of several minutes. We conclude that in

P. antipodarum, velocity is a more sensitive endpoint than

the classic mortality and immobility endpoints.

Short-term bioassays usually assess lethal effects of pol-

lutants on animals; however, they are not suited for

detecting the effects of toxicants on animal behaviour

(Rand 1985; Roast et al. 2000; Cheung et al. 2002; Kane

et al. 2004; Scott and Sloman 2004). Short-term lethal

bioassays have the advantages of being easy to assess,

cheap, and fast; however, they fail to detect other adverse

sublethal effects that lower concentrations of toxicants can

exert on animals (De Lange et al. 2009). One suitable

balance between effort (i.e., shorter exposure) and extrap-

olating results (i.e., environmentally realistic concentra-

tions) can be attained with behavioural subchronic

bioassays, since behaviour is a good link between physio-

logical response and ecological processes (Scott and Slo-

man 2004; Wallace and Estephan 2004; Beketov and Liess

2005). In addition, sublethal endpoints can assess the

effects of lower toxic concentrations with exposure times

slightly longer than short-term bioassays. These lower

concentrations are more similar to those found in the field,

and therefore behavioural ecotoxicology allows a more

realistic ecologic risk assessment (ERA) of toxicants with

little cost (Amiard-Triquet 2009).

The use of automated procedures for recording animal

behaviour decreases bias due to operator subjectivity, thus

improving the repeatability of the results in ecotoxicology

(Kane et al. 2004; Mills et al. 2006; Alonso et al. 2009).

Among them, videotaped bioassays are often used because

they are relatively easy and cheap to implement (Myrick

2009). However, there are areas of uncertainty in the study

of behavioural responses to toxicant exposure (Kane et al.

2004). For instance, during the recording of behavioural

A. Alonso (&) � J. A. Camargo

Departamento de Ecologıa, Facultad de Ciencias, Universidad de

Alcala, 28871 Alcala de Henares, Madrid, Spain

e-mail: aafernandez1976@yahoo.es

123

Arch Environ Contam Toxicol (2011) 60:511–517

DOI 10.1007/s00244-010-9562-x

endpoints at very short exposures, different trends among

animals of the same population can be found (Peeters et al.

2009). However, the extent to which this fact represents a

handicap for the application of behavioural endpoints in

ecotoxicology needs further study.

Human activities produce many kinds of pollutants,

which end up in continental water ecosystems; however,

most of their effects on the behaviour of aquatic animals

are still unknown. One of the chemicals whose concen-

tration can be increased by human activities is inorganic

fluoride, being phosphate fertilizer production, chemical

production, aluminum smelting, and the production of

bricks, ceramics, and glass the main human sources

(Environment Canada 2001; WHO 2002; Camargo 2003;

Metcalfe-Smith et al. 2003). These human activities can

increase [100 times the natural background level of inor-

ganic fluorides (Camargo 2003). Field fluoride levels

ranging from 4.6 to 16.2 mg F-/l have been found in

fluoride-polluted ecosystems, either by natural or anthro-

pogenic causes (Environment Canada 2001; World Health

Organization 2002; Camargo 2003). It appears in fresh-

water ecosystems as dissolved anion fluoride (F-) in waters

of relatively low hardness (Environment Canada 2001).

Therefore, aquatic invertebrates living in soft waters are

more sensitive to fluoride pollution than those living in

hard waters because the bioavailability of fluoride ion

decreases with increasing water hardness (Camargo 2003).

Fluoride ion inhibits enzymatic activities, causing delete-

rious effects on freshwater animals (Reddy et al. 1989;

Environment Canada 2001; Camargo 2003). These altera-

tions can modify the behaviour of animals; however, only

short-term effects have been studied thus far (Camargo

2003).

This study aims to assess the subchronic (14-day) effects

of fluoride on three different endpoints of the aquatic snail

Potamopyrgus antipodarum (Hydrobiidae, Mollusca) as

follows: (1) mortality (2) immobility, and (3) velocity of

movement assessed during videotaped monitoring (i.e.,

short observations) and during the entire subchronic bio-

assay (i.e., first and second weeks). This study can con-

tribute to understanding the effects of fluoride on aquatic

molluscs and to identify the most suitable endpoint by

which to assess them.

Materials and Methods

Test Organisms

P. antipodarum is native to New Zealand (Winterbourn

1970), but it has reached different aquatic ecosystems

around the world (Alonso and Castro-Diez 2008).

Although, in its natural range, both sexual and asexual

reproduction coexists, nonnative populations are almost

exclusively parthenogenetic female snails (Alonso and

Castro-Diez 2008). This sensitive species has been rec-

ommended in both reproduction (Duft et al. 2007) and

behavioural tests (Alonso and Camargo 2009). Animals for

the study were obtained from our laboratory culture

(Department of Ecology, University of Alcala). This cul-

ture was initiated with snails collected from an upper reach

of the Henares River (Guadalajara, Spain) during January

2009. Animals were kept in 60-l glass aquaria with United

States Environmental Protection Agency (USEPA) mod-

erately hard water (96 mg NaHCO3, 60 mg CaSO4*2H2O,

4 mg KCl, and 122.2 mg MgSO4*7H2O/l deionised water)

(USEPA 2002) enriched with calcium carbonate (10 mg

CaCO3/l deionised water). The culture was kept at room

temperature (20–22�C), and animals were fed with fish

food (Tetra GmbH, Germany) and dry algae. The dry food

fed per individual per day was approximately 0.10 mg. The

culture was aerated with an air pump and an aquarium

filter. Animals are capable of growing and reproducing

under these conditions. Forty-eight animals (mean shell

length 3.14 ± 0.47 mm) were selected for the bioassay,

and they were acclimatized to the experimental conditions

(15�C) in a climatic chamber for 1 week before the bio-

assay. Animals were regularly fed during the acclimatiza-

tion period.

Experimental Design and Monitored Endpoints

A subchronic bioassay (14 days) with toxic renovation

every 7 days was conducted. One control and three nomi-

nal fluoride concentrations were used (5, 20, and 40 mg

F-/l). Each treatment consisted of 12 randomly selected

individuals that were individually placed on glass vessels

(0.1 l). Therefore, each treatment (including control) was

replicated 12 times. During the bioassay, all animals were

fed with fish food (Tetra GmbH, Germany) ad libitum

every 7 days. Each feeding event lasted for 3 h, at the end

of which control and toxicant solutions were renewed. All

experimental vessels (0.1 l) were covered with perforated

plastic foil to decrease water evaporation. No aeration was

provided during the experiment. Nominal fluoride con-

centrations were prepared by weighting the required

amount of sodium fluoride (minimum 99%, lot 47H1049;

Sigma, Germany), and subsequently dissolving it in

USEPA water. The bioassay was conducted in a climate

chamber at 15�C. Dissolved oxygen, water temperature,

and pH were measured in each water renovation. Mean

(n = 5–9) (±SD) bioassay physicochemical parameters

were pH 8.2 ± 0.1, dissolved oxygen (mg O2/l) 8.1 ± 0.4,

ammonia (mg N-NH4/l)\0.05, and water temperature (�C)

15.5 ± 0.5. Actual fluoride concentrations were measured

before and after each water renovation through a

512 Arch Environ Contam Toxicol (2011) 60:511–517

123

spectrophotometric method (Hanna). Actual total hardness

(as mg CaCO3/l) and chloride concentrations (Cl-) were

measured using the ethylenediaminetetraacetic acid

(EDTA) titrimetric and argentometric methods, respec-

tively (APHA 1995). Mean hardness (as mg CaCO3/l) was

90.7 ± 7.5 (n = 9), and mean chloride was \5 mg Cl-/l

(n = 8) for USEPA water used during the bioassay.

Three endpoints were monitored during the bioassay:

mortality, number of dead plus immobile animals, and

velocity of movement. These endpoints were monitored

after 0, 7, and 14 days. Snail activity was monitored using

a videotaping and image analysis system according to

methodology described by Myrick (2009). After 0, 7, and

14 days, a 3-min video was recorded for each animal and

each treatment (12 animals/treatment and recording per-

iod). Each animal was placed on a Petri dish (9.5 cm

diameter) with test water (40 ml); above the Petri dish, a

video camera (Werlisa DV570HD) was installed. Each

videotaping session was started 20 s after placing the ani-

mal on the Petri dish. Snails that had not moved after 3 min

of videotaping were observed under a binocular micro-

scope to observe their reaction after a gentle touch with

forceps on the operculum: the animal was considered

immobile if it retracted its soft body into the shell, and was

considered dead if no reaction was observed. The free

software ImageJ 1.41 (Wayne Rasband, National Institutes

of Health United States) (http://rsb.info.nih.gov/ij/) was

used to analyze the videotapes of mobile snails. The

Manual Tracking plugin was used as complement of the

software. Each video file was converted into a group of

images (5395 frames/video) using the eight-bit grey-scale

option of ImageJ and subsequently scaled (32 pix-

els = 7.5 mm). The position of each animal was manually

marked every 540 frames (every 18 s), thus obtaining a

total of 10 positions in 180 s. ImageJ was used to estimate

the velocity between two marked positions. Therefore, for

each animal, a total of 10 velocities were estimated for

each recording time point (0, 7, and 14 days).

Statistical Analysis

The cumulative percentage of dead animals and for dead

plus immobile animals was estimated for each fluoride

treatment at 7 and 14 days of bioassay. To elucidate dif-

ferences between numbers of dead animals or dead plus

immobile animals, Kaplan–Meier test was conducted

between each treatment and control (Bland and Altman

1998). To decrease the probability of committing type I

errors, a level of significance of p \ 0.001 was chosen.

Afterward, treatments without significant differences among

controls were selected for behaviour analysis. Repeated-

measures two-way analysis of variance (ANOVA) was

conducted for each recording time point (0, 7, and 14 days),

using fluoride concentration (control and 5 and 20 mg F-/l)

as intersubject factor, time (from 18 to 180 s in 10-s steps) as

intrasubject factor, and mean velocity of active snails as

dependent variable. When repeated-measures two-way

ANOVA was significant, a post hoc test was conducted to

assess differences between each treatment and control at

each recording time point (Dunnett test). A significance

level of p \ 0.05 was chosen. All statistical analyses were

conducted with SPSS 15.0 software (SPSS, Chicago, IL).

For each endpoint (i.e., mortality, number of dead

plus immobile animals, and velocity), the fluoride treat-

ment with no significant differences compared with the

control was considered as the NOEC (no observed effect

concentration) and the one with the least significant dif-

ference was considered the LOEC (lowest observed effect

concentration).

Results

The mean (n = 8) (±SD) actual fluoride concentrations

were 5.2 ± 1.0, 17.5 ± 2.2, and 37.0 ± 4.1 mg F-/l. The

cumulative percentage of mortality and dead plus immobile

animals are shown in Fig. 1. No animals died in the control

at the end of the bioassay. The treatment with the highest

fluoride concentration produced a higher number of dead

animals and dead plus immobile animals than the control

(Kaplan–Meier test; p \ 0.001). No animal died or was

immobile in the lowest fluoride concentration. The inter-

mediate treatment did not differ from the control with

regard to the percentage of dead plus immobile animals and

dead animals, and there were no significant differences

compared with the control (Kaplan–Meier test; p [ 0.001).

Therefore, animals exposed to 5.2 and 17.5 mg F-/l were

selected for subsequent behaviour analysis.

The mean velocity of active snails (mm/s) in each time

interval (10 periods of 18 s each) and each treatment are

shown in Fig. 2. Repeated-measures two-way ANOVA

indicated that snail velocity varied along the recorded time

intervals (from 18 to 180 s) for both control and fluoride

treatments and for all recording time points (at 0, 7, and

14 days) (p \ 0.001; ANOVA, Table 1). This trend was

similar across treatments because no significant effects

were found for interaction between time and treatment

(p [ 0.05, ANOVA, Table 1). Fluoride effect on velocity

was significant for taping at days 7 and 14 (Table 1), with

the highest fluoride treatment (17.5 mg F-/l) slowing

decreased snail velocity with respect to controls (p \ 0.05;

Dunnet test) (Fig. 2). Therefore, regarding mortality and

dead plus immobile animals, the NOEC was 17.5 mg F-/l,

and the LOEC was 37.0 mg F-/l; for velocity the NOEC

was 5.2 mg F-/l, and the LOEC was 17.5 mg F-/l. In our

study, the endpoint of velocity was approximately two

Arch Environ Contam Toxicol (2011) 60:511–517 513

123

times more sensitive than mortality or dead plus immobile

animals.

Discussion

Our study has clearly shown that fluoride ions cause

mortality, immobility, and/or decrease of snail velocity at

subchronic exposures (7 and 14 days), with the latter

endpoint being the most sensitive. One of the aquatic

invertebrates more widely used in ecotoxicological bioas-

says, the water flea (Daphnia magna), shows a relatively

high tolerance to acute toxicity of fluoride, with 48 hours

LC50 values ranging from 98 to 385 mg F-/l when the

endpoint was immobilization (LeBlanc 1980; Dave 1984;

Fieser et al. 1986; Metcalfe-Smith et al. 2003). Other

species of freshwater invertebrates (caddisfly larvae) pre-

sents lower LC50 values (B11.5 mg F-/l to 6 days) than

0

10

20

30

40

50

60

70

80

90

100

% c

umul

ativ

e de

ad p

lus

imm

obile

ani

mal

s17.5 mg F-/L 37 mg F-/L

*

*

0

10

20

30

40

50

60

70

80

90

100

14d7dTime

% c

umul

ativ

e m

orta

lity

*

A

B

Fig. 1 Cumulative percentage of dead plus immobile animals (a) and

dead animals (b) in the two treatments with higher fluoride

concentrations (mean concentrations of 17.5 and 37.0 mg F-/l) for

each recording time point (7 and 14 days). The percentage of dead

plus immobile animals and dead animals was zero in controls and at

the lowest fluoride concentration. * Significant difference between

each fluoride concentration and controls for each recording time point

(Kaplan–Meier test, p \ 0.001)

0

0.1

0.2

0.3

Velo

city

(mm

/s)

Control 5.2 mg F-/L 17.5 mg F-/L0 Days

0

0.1

0.2

0.3

Velo

city

(mm

/s)

7 Days

*

0

0.1

0.2

0.3

18 36 54 72 90 108 126 144 162 180

Velo

city

(mm

/s)

14 Days

*

Time (seconds)

Fig. 2 Mean velocity (mm/s) (n = 8–12) of control active snails and

those in each fluoride treatment (mean concentrations of 5.2 and

17.5 mg F-/l) for each time interval (seconds) at each recording time

point (0, 7 and 14 days = top, middle, and bottom panels, respec-

tively). SD has been removed for clarity. Time intervals are 10

periods of 18 s each. * Significant differences between each treatment

and control (Dunnett test p \ 0.05)

514 Arch Environ Contam Toxicol (2011) 60:511–517

123

D. magna; however, these studies were conducted at lower

water hardness (see Camargo 2003 for review). Therefore,

acute effects were reported for other freshwater species at

concentrations lower than those used in our study (Cam-

argo 2003), but the lower-hardness water used in these

studies makes difficult the direct comparison with our

results. In general, behavioural endpoints show higher

sensitivity than mortality in the same species, However

they are still scarcely used in regulatory activities and ERA

(Scott and Sloman 2004). Traditional measures of behav-

ioural endpoints have been criticized because they have

included an important component of subjectivity and

therefore a variable response between and within individ-

uals (Gerhardt et al. 1994; Alonso et al. 2009). However,

during past years, several nonsubjective and quantitative

behavioural endpoints and bioassays have been developed

(Charoy and Janssen 1999; Kane et al. 2004; Mills et al.

2006; Alonso and Camargo 2009; Alonso et al. 2009).

Among them, invertebrate movements (e.g., swimming,

sliding, time to start normal activity, drift) are ecologically

relevant endpoints because they permit animals to compete

for resources, to avoid predators and pollution, to repro-

duce, to obtain food, etc. (Burris et al. 1990; Golding et al.

1997; Alonso and Camargo 2004, 2009; Cold and Forbes

2004; Beketov and Liess 2008). Dodson et al. (1995)

showed that D. pulex exposed to carbaryl exhibit erratic

swimming, which increases its vulnerability to predators.

Unionized ammonia, at both subchronic and chronic

exposures, has been found to affect time to start normal

movement in P. antipodarum (Alonso and Camargo 2004,

2009). Cadmium has been shown to cause decreased

swimming velocity in the marine shrimp Hippolyte inermis

at short-term exposures (Untersteiner et al. 2005). Several

pesticides caused drift-initiating action on three freshwater

arthropods at concentrations 7–22 times lower than the

reported LC50 values (Beketov and Liess 2008). Our study

shows that fluoride decreases the velocity of P. antipoda-

rum at concentrations lower than those causing mortality.

Therefore, fluoride may decrease its fitness under naturally

polluted scenarios (Jones et al. 1991; Cold and Forbes

2004). This behavioural impairment at the population level

may potentially cause alterations in the structure and

function of natural ecosystems through shifts in modifica-

tions of predation activity, food consumption, or competi-

tive relations (Dodson et al. 1995; Charoy and Janssen

1999; Evans-White and Lamberti 2009), since behaviour

links individual and community effects of environmental

toxicants (Kane et al. 2004; Scott and Sloman 2004).

Therefore, these kinds of behavioural endpoints can not be

considered less important than growth or reproduction;

however, they have the additional advantage of needing

shorter exposures (Alonso et al. 2009).

Fluoride concentrations in polluted freshwater ecosys-

tems can range from 0.5 to 20 mg F-/l either by natural or

anthropogenic causes (Sigler and Neuhold 1972; Environ-

ment Canada 2001; Camargo 2003). Our study showed that

a fluoride concentration within this environmental range

(17.5 mg F-/l) decreased the movement velocity of

mudsnail, showing that behavioural endpoints can detect

deleterious effects of subchronic exposures at concentra-

tions of polluted ecosystems. Given that behavioural end-

points usually requires less exposure time than growth or

reproduction endpoints, they can improve the ecological

risk assessment of aquatic ecosystems.

In our study, the velocity of P. antipodarum showed a

similar pattern across different treatments in each recording

day: at the beginning of the recording interval, velocity

increased up to B54–72 s of recording and remained quite

stable until the end of the recording period (180 s). The

highest fluoride concentration decreased the average

velocity of snails, but that pattern was similar between

exposed and nonexposed animals. This result highlights the

importance of recording animal behaviour for a sufficient

length of time, since behaviour can change in short periods.

Other studies have shown variations in behavioural patterns

during relatively short periods of time. Peeters et al. (2009)

Table 1 Summary of results of repeated-measures two-way ANOVAa

0 days 7 days 14 days

dfb F p dfb F p dfb F p

Source of variation

Within subject

Time 3.28 18.56 <0.001 4.48 13.55 <0.001 4.15 9.366 <0.001

Time 9 Treatment 6.56 0.944 0.472 8.95 0.960 0.507 8.31 0.431 0.906

Between subjects

Treatment 2 0.075 0.928 2 4.61 0.017 2 6.70 0.004

a Treatment (control and exposure to 5.2 and 17.5 mg F-/l) was the intersubject factor, time (10 observations each 18 s) was the intrasubject

factor, and velocity of was the dependent variable. The analysis was repeated at 0, 7, and 14 days of the experimentb Degrees of freedom have been corrected for sphericity using the Greenhouse–Geisser approach

Arch Environ Contam Toxicol (2011) 60:511–517 515

123

studied a natural population of Gammarus pulex using the

impedance method (Multispecies Freshwater Biomonitor),

showing that during the first hours animals modified their

activity (from active to less active or vice versa). Our

results support that velocity is relatively easy to assess as

an endpoint, which guarantees independence from human

subjectivity and repetitivity (Baatrup 2009). Therefore, this

behavioural endpoint can contribute to improve ERA

(including growth, reproduction, and behaviour) of aquatic

ecosystems.

Subchronic and chronic toxic effects of fluoride on

aquatic animals have been scarcely studied, and mostly

have been focused on the effects of fluoride on growth and

reproduction of D. magna (Dave 1984; Kuhn et al. 1989;

Reddy and Venugopal 1990; Metcalfe-Smith et al. 2003),

and on survival, growth, and food uptake of the freshwater

prawn Macrobranchium rosenbergii (Adhikari et al. 2006).

Fluoride effects on behaviour have been only assessed at

short-term exposures in freshwater invertebrates (Camargo

et al. 1992) and in fishes in natural conditions (Damkaer

and Dey 1989). Camargo et al. (1992) assessed the suble-

thal effect of fluoride on migration from the net of several

species of net-spinning caddisfly larvae, showing that

fluoride concentrations increased the number of larvae that

migrated from their capture nets (EC50 96 h ranging from

19.2 to 33.5 mg F-/l). Damkaer and Dey (1989) studied

the behaviour of upstream-migrating fish adults exposed to

fluoride ions in the Columbia River (very soft waters), and

they found that fluoride concentrations of approximately

0.5 mg F-/l adversely affected the migration of fish

(Oncorhynchus tshawytscha and O. kisutch). Fluoride

alters physiology, such as metabolism of carbohydrates and

proteins, and causes adverse effects in the nervous system

(Reddy et al. 1989; Environment Canada 2001; Camargo

2003). These malfunctions can explain behavioral altera-

tions because nervous system is directly related to

impairments in movement.

Conclusion

Fluoride ion caused mortality and immobility in the aquatic

snail P. antipodarum, but behavioural impairment (i.e.,

decreased velocity) was the most sensitive endpoint given

that this endpoint was sensitive to environmental fluoride

concentrations during subchronic exposures. Therefore,

velocity can be a useful parameter for a suitable ERA of

fluoride. Videotaping method allowed us to detect behav-

ioural patterns at very short exposures (seconds), thus

showing that the velocity of snails must be monitored

during the course of several minutes (at least 3) to provide

sound results. Our results suggest that changes in velocity

can be used as a sensitive sublethal endpoint in bioassays

with P. antipodarum instead of the less sensitive endpoints

of mortality and immobility.

Acknowledgments Funds for this research came from the Spanish

Ministry of Science and Innovation (CGL2006-06804/BOS), and the

University of Alcala provided logistical support. Alvaro Alonso is

currently supported by a Juan de la Cierva contract from the Spanish

Ministry of Science and Innovation. We extend our sincere gratitude

to Pilar Castro for comments and suggestions during the writing of the

manuscript. Special thanks to Enrique Gonzalez for help during the

experiments and to Pilar Castro for correcting the text. Finally, we are

grateful for two anonymous reviewers for their comments on this

manuscript.

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