Chemosphere 84 (2011) 533–537

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Chemosphere

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The freshwater planarian Polycelis felina as a sensitive species to assessthe long-term toxicity of ammonia

Álvaro Alonso ⇑, Julio A. CamargoDepartamento de Ecología, Facultad de Ciencias, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain

a r t i c l e i n f o

Article history:Received 9 March 2011Accepted 7 April 2011Available online 4 May 2011

Keywords:BehaviourSub-lethal effectsUnionized ammoniaPlanarianFreshwater

0045-6535/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.chemosphere.2011.04.030

⇑ Corresponding author.E-mail address: aafernandez1976@yahoo.es (Á. Alo

a b s t r a c t

Behavioural endpoints are a good link between physiological and ecological effects. However long-termbehavioural endpoints are not uniformly studied over all different organism groups. For example behav-iour has been scarcely studied in planarians. Unionized ammonia (NH3) is one of the most widespreadpollutants in developed countries, and is known to alter animal behaviour. In this study a long-term(30 d) bioassay was conducted to assess the effect of this pollutant on survival and behavioural activity(e.g. locomotion activity) of the freshwater planarian Polycelis felina. One control and three environmen-tally-realistic concentrations of unionized ammonia (treatments of 0.02, 0.05, and 0.09 mg N–NH3 L�1)were used in quintuplicate. The behaviour of planarians was measured after 0, 10, 20 and 30 d of ammo-nia exposure. Mortality was recorded every 2 d. Unionized ammonia increased mortality in the two high-est NH3 concentrations and the locomotory activity was depressed in all treatments after 20 d ofexposure. Behavioural effect was observed at concentrations 20 times lower than the short-term LC50for this species. Previous studies proposed safe concentrations of unionized ammonia of 0.01–0.10 mg N–NH3 L�1 to aquatic ecosystems, but our study has shown that these concentrations will affectplanarians. Because planarians play a key role in streams (as predator/scavenger), safe concentrationsshould be below 0.02 mg N–NH3 L�1 to protect this species in the freshwater community. Our resultscan contribute to improve the knowledge about ammonia toxicity to freshwater ecosystems, we recom-mend that safe concentrations of unionized ammonia should be based on very sensitive species.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

The aim of ecotoxicology is to assess the deleterious effects ofcontaminants on populations, communities and ecosystems(Walker et al., 2001). Bioassays with sensitive species and end-points are often used as first step to reach this aim. Several (inver-tebrate) animal groups from freshwater ecosystems have beenshown to be sensitive to a wide range of pollutants; among themplanarians have been shown to be suitable for toxicological bioas-says. Planarians play a key role in freshwater lotic ecosystems, theyare one of the most abundant predators in streams and upperreaches of rivers (Thorp and Covich, 2001). They have been em-ployed to assess several toxicants (i.e. ammonia, nitrite, copper,herbicides, aluminium, acidification, etc.) using different endpoints(i.e. mortality, regeneration, fecundity, fertility, movement, preda-tion rate, etc.) (Camargo and Ward, 1992; Horvat et al., 2005; Alon-so and Camargo, 2006, 2008; Knakievicz and Ferreira, 2008;Kovacevic et al., 2009).

Behavioural endpoints take into account the deleterious effectsof pollutants in an ecological context (Scott and Sloman, 2004),

ll rights reserved.

nso).

linking physiological and ecological effects. Behavioural endpointshave been studied in other animal groups (i.e. insects, crustaceans,molluscs, oligochaeta, fish, and amphibians) (Kramer et al., 1989;Gerhardt et al., 1994; Richardson et al., 2001; Alonso and Camargo,2004a, 2009; Craig and Laming, 2004; Scott and Sloman, 2004;Schriks et al., 2006; Gerhardt, 2007a,b; Beketov and Liess, 2008).Many studies have shown that behaviour is a more sensitive end-point than other classical endpoints (e.g. mortality, immobility);e.g. Beketov and Liess (2008) showed that behavioural endpointsin invertebrates were 7–22 times more sensitive than acute LC50values for several pesticides and Alonso and Camargo (2011)showed that velocity of a freshwater snail was affected by fluorideion at lower concentrations than mortality. However, the use ofbehavioural endpoints is still limited in ecological risk assessment.

The monitoring of invertebrate movements (e.g. swimming,sliding, time to start normal activity, etc.) is relevant under an eco-logical point of view, as they permit animals to predate, to escapefrom predators and pollution, to reproduce, to get food, etc. (Burriset al., 1990; Camargo and Ward, 1992; Golding et al., 1997; Alonsoand Camargo, 2004b; Cold and Forbes, 2004). Therefore, any effecton the behavioural activity caused by a toxicant may potentiallyalter the ecological function of the individual and ultimately thefunction of the whole aquatic ecosystem, as those alterations can

534 Á. Alonso, J.A. Camargo / Chemosphere 84 (2011) 533–537

reduce the fitness of invertebrate populations (Jones et al., 1991;Cold and Forbes, 2004).

Ammonia is one of the most widespread toxicants in developedcountries (Abel, 2000; Spencer et al., 2008). Its origin is both natu-ral (decomposition of organic matter and animal excretions) andanthropogenic (e.g. farming runoff, atmospheric deposition, indus-trial wastes, and urban effluents). Urbanization has increased itsconcentration in many aquatic ecosystems (Vitousek et al., 1997;Camargo and Alonso, 2006; Passel et al., 2007; Spencer et al.,2008). Ammonia has been tested as a toxicant with deleterious ef-fects on behaviour (Alonso and Camargo, 2004a,b, 2006, 2009;Kirkpatrick et al., 2006). There are two ammonia species, unionized(NH3) and ionized (NHþ4 ), the chemical equilibrium is controlled bypH and water temperature, the higher is pH and temperature thehigher is the unionized ammonia concentration (Emerson et al.,1975). The unionized form is toxic to aquatic animals, whereasthe ionized form has very low toxicity (Abel, 2000; Camargo andAlonso, 2006; Passel et al., 2007). The potential effects of unionizedammonia on the behaviour of planarians have been scarcely stud-ied at long-term exposures. A previous study has shown thatammonia and other nitrogen nutrients can alter natural popula-tions of invertebrates, and that laboratory species sensitivitiesand field tolerances are positively correlated (Beketov, 2004). Asimilar result was shown in a stream in Central Spain by Alonso(2005), showing that the absence of planarians in polluted reachescan be partially explained by its high sensitivity to unionizedammonia. Previous studies have demonstrated that unionizedammonia has a high short-term toxicity to one species of planarian(Alonso and Camargo, 2006). As planarians play a key role in fresh-water ecosystems – they are one of the most abundant predators instreams and upper reaches of rivers (Thorp and Covich, 2001) – thestudy of unionized ammonia effects on this group are of concern inan ecotoxicological perspective.

Mortality, growth and reproduction are the most often em-ployed endpoints in ecological risk assessment and regulatoryactivities, as they can be objectively quantified. By contrast,behavioural endpoints have been avoided, as they have been con-sidered to depend on the observer’s subjectivity, which limitsrepeatability. Behaviour is normally altered before death, growthand reproduction, so it has been suggested as a more sensitiveecological warning signal (Roast et al., 2000; Mills et al., 2006;Pestana et al., 2007). Therefore, development of quantitativenon-subjective behavioural endpoints is considered a priority inaquatic ecotoxicology. In the last years several studies have beenconducted with this aim (Gerhardt et al., 1994; Gerhardt, 2007a;Alonso and Camargo, 2009; Alonso et al., 2009), but in spite of theecological importance of freshwater planarians, they have notbeen employed in these studies. The aim of this study is to assessthe long-term effects of environmentally-realistic unionizedammonia concentrations on the behavioural activity and survivalof the freshwater planarian Polycelis felina (Planariidae, Turbellar-ia) at long-term (30 d) exposure. Our results may contribute tounderstand the utility of planarians for long-term behaviouralbioassays.

2. Materials and methods

Planarians were collected in a non-polluted upper reach of theHenares River (Central Spain). Individuals were separated fromstones using a soft paintbrush and were kept in plastic containerswith river water. Once in the laboratory, planarians were placed inglass aquaria (1.5 L) and sequentially acclimated to test water(bottled drinking water without chlorine) for a week prior to thebioassay. Mean (±SD) physical–chemical properties of the testwater were: conductivity = 734 ± 11.6 lS cm�1, pH = 8.11 ± 0.3,

calcium = 76.1 ± 3.3 mg Ca2+ L�1, and unionized ammonia<0.002 mg N–NH3 L�1.

A long-term (30 d) bioassay with mortality and behaviour asendpoints was conducted with daily renewal of the toxic solutionand control water. Toxic solutions were prepared from a stockammonia (N–NH4 + N–NH3) solution of 100 mg L�1. Stock ammo-nia solution was prepared with ammonium chloride (NH4Cl,PANREAC, Spain, Lot No. 149959380 purity of 99.5%), and it wasfreshly made every time for preparing the daily renewal ofammonia solutions. A control (<0.002 mg L�1 N–NH3) and threenominal concentrations of unionized ammonia (0.02, 0.05, and0.09 mg L�1 N–NH3) were used in quintuplicate. These concentra-tions were selected as environmentally-realistic concentrations(Alonso, 2005; Passel et al., 2007). Glass vessels with a final vol-ume of 0.1 L were covered with a perforated plastic foil to reducewater evaporation. No extra air supply was employed to avoidammonia oxidation. The bioassay was conducted in a climaticchamber with a controlled temperature of 15 �C. Water tempera-ture, pH, dissolved oxygen, and actual concentrations of totalammonia were daily measured using standardized methods de-velop by APHA (1995). The mean pH and water temperature mea-sured during the bioassay were used to calculate the actualconcentrations of unionized ammonia in each treatment (All-eman, 1998). Before the bioassay, body lengths of planarian weremeasured using a Delta-T area meter (Cambridge, UK); eachplanarian was placed in a Petri dish with test water and its imagewas recorded in a computer through a camera, and its bodylength was measured using the Delta-T software. For each repli-cate four individuals of P. felina (4.4–10.2 mm in length) wereplaced in each vessel. During acclimatization and bioassay planar-ians were fed with chicken liver (fragments of 1 cm length). Thefood was supplied during 1 h every 2 d. After feeding planarianswere transferred to new test water.

The endpoint mortality was recorded every 2 d, and the cumu-lative mortality in each treatment was calculated at the end of 30 dof exposure. Dead animals were removed in each observation. Aplanarian was considered to be dead when the body tissue startedto degenerate. This mortality criterion has been successfully con-sidered in previous studies with planarians (Camargo and Ward,1992; Alonso and Camargo, 2006).

The behavioural activity of each planarian was measured after0, 10, 20 and 30 d of continuous exposure to unionized ammonia.The behavioural endpoint was based on the locomotion activitymethodology of Raffa et al. (2001) with some modifications.Briefly, each individual (e.g. a maximum of four measurementsper vessel at each time) was placed into a transparent glass aqua-ria (1.5 L) (diameter 16 cm) containing test water (100 mL) at15 �C. Each planarian was placed with a soft paintbrush in thecentral part of the aquaria bottom. The aquarium was locatedover a paper with a 0.5 cm grid. Locomotion was quantified asthe number of squares crossed or re-crossed by each planarianduring 60 s of free movement or until it reached the lateral wallsof the aquarium. Locomotion was expressed as number of squaresper second. If after 60 s the animal did not move, it was consid-ered immobile. Dead or immobile animals were not used to esti-mate locomotion in each treatment. The water of the aquariumwas renewed after every ten measures. After the locomotion mea-surement, the individuals were placed back in their respectivetreatment vessel.

The effect of unionized ammonia on the locomotion of P. felinaafter each exposure time (0, 10, 20 and 30 d) was assessed througha one-way analysis of variance (ANOVA), where the dependent var-iable was locomotion of active planarians. For each vessel, thenumber of squares crossed per second was calculated per individ-ual and then averaged to obtain a value for each vessel. The heter-ogeneity of variance across treatments was tested through a

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Levene’s test. When variances were heterogeneous, data were log-transformed and retested to ensure the homogeneity of variance.Differences between each unionized ammonia treatment and con-trol was assessed through a Dunnett test for each exposure time.The cumulative mortality at the end of the bioassay (30 d) was as-sessed in the same way. P-values lower than 0.05 were consideredas significant for all statistical analysis. For both locomotion andmortality the highest concentration with no significant differencesfrom the control was considered as NOEC (no observed effect con-centration) for each time of exposure, and the lowest concentrationwith significant difference as LOEC (lowest observed effect concen-tration) for each time of exposure. SPSS 15.0 software was used forall statistical analyses.

Fig. 2. Cumulative mortality + SD at the end of the bioassay (30 d) for control andunionized ammonia treatments (0.02, 0.05, and 0.09 mg N–NH3 L�1). Asterisksshow significant differences between control (C) and each treatment (ANOVA;Dunnett test; P < 0.05).

3. Results

Mean ± SD (n = 56–112) values of the physical–chemical param-eters during the bioassay were: water temperature = 15.1 ± 0.7 �C,pH = 8.11 ± 0.3 and dissolved oxygen = 6.86 ± 0.5 mg O2 L�1. Themean ± SD (n = 30) actual unionized ammonia was 0.02 ± 0.001,0.05 ± 0.003 and 0.09 ± 0.006 mg L�1 N–NH3 for each unionizedammonia treatment. All concentrations cited in the results aremean actual concentrations of N–NH3 (mg L�1). The unionizedammonia concentration in control was less than 0.002 mg N–NH3 L�1. The mean mortality in the control at the end of the bioas-say was 5%.

Average values of locomotion of P. felina for each time and treat-ment are shown in Fig. 1. No significant differences between aver-age locomotion of planarians between control and treatments werefound at the beginning of the bioassay (P > 0.05; ANOVA). After10 d of continuous exposure to unionized ammonia, the highestconcentrations (0.05 and 0.09) decreased the locomotion of planar-ians with respect to the control (P < 0.05; Dunnett test). The sameoccurred in every concentration after 20 and 30 d of exposure(P < 0.05; Dunnett test). Thus, locomotion of planarians was signif-icantly reduced with increasing exposure time and ammonia con-centration (Fig. 1).

Cumulative mortality after 30 d of continuous exposure to un-ionized ammonia is shown in Fig. 2. The highest two unionizedammonia concentrations increased the number of dead planarianswith respect to control after 30 d. The NOEC for mortality after 30 dwas 0.02 mg N–NH3 L�1, and the LOEC was 0.05 mg N–NH3 L�1.The highest concentration caused a high mortality of planarians(mean mortality of 95%); the mortality in the lowest concentrationwas equal to that in the control (5%).

Fig. 1. Mean number of crossed or re-crossed squares per second + SD afterexposure to different concentrations of unionized ammonia (0, 10, 20 and 30 d).Asterisks show significant differences between control (C) and the treatment (0.02,0.05 and 0.09 mg N–NH3 L�1) for each exposure time (ANOVA; Dunnett test;P < 0.05). No SD is shown for 0.09 mg N–NH3 L�1 treatment at days 20 and 30because only individuals from one replicate were active after 20 d, and all animalsdied after 30 d.

4. Discussion

Our results show that P. felina is highly sensitive to the long-term lethal effect of unionized ammonia, as more than 60% ofthe exposed animals died after 30 d of continuous exposure to arelatively low concentration of 0.05 mg N–NH3 L�1. This corrobo-rates previous results of high sensitivity shown by this planarianin short-term bioassays (96 h LC50 = 0.39 mg N–NH3 L�1; Alonsoand Camargo, 2006). Behavioural activity measured as locomotionof P. felina was significantly reduced at an even lower concentra-tion of 0.02 mg N–NH3 L�1. The observed LOECs for both mortalityand behaviour are environmentally-realistic concentrations, and inthe range of several published quality criteria (0.01–0.10 mgN–NH3 L�1 depending on pH, water temperature or others) (USEnvironmental Protection Agency, 1986, 1999; Environment Can-ada, 2001; Alonso, 2005; Camargo and Alonso, 2006; Passel et al.,2007). Most of these criteria are calculated based on mortality,growth and reproduction bioassays, and behavioural endpointsare scarcely used. Given the low effect concentration for locomo-tion of P. felina, the current quality criteria are insufficient for P.felina to prevent deleterious effects. Wang et al. (2007) also founda high sensitivity to long-term effects of ammonia for two speciesof mussels (Lampsilis siliquoidea and Villosa iris, Unionidae), withtoxic effects at ammonia concentrations lower than the USEPAwater quality criteria.

Unionized ammonia has a high solubility in lipids and is a neu-tral component. Both aspects allow a fast uptake across cellularmembranes, causing several physiological impairments in aquaticanimals, such as epithelial necrosis, increase in gill ventilation, col-lapse of gill lamellae, hyperexcitability, low oxygen uptake andconvulsions (Fromm and Gillette, 1968; Russo, 1985; Rebeloet al., 2000; Camargo and Alonso, 2006). One of the most importantroutes of ammonia uptake and excretion is via gills (Spicer andMcMahon, 1994; Harris et al., 2001; Weihrauch et al., 2002). How-ever, given that planarians have no specific respiratory structuresand perform gas exchange by simple diffusion through body walls(Ruppert and Barnes, 1994), their sensitivity can be attributed toan efficient ammonia uptake, even at low environmental concen-trations, by means of passive diffusion. Further, the mechanismsof ammonia detoxification of planarians may be less effective thanthose found in the gills of other aquatic species (Weihrauch et al.,2002), leading to a quick increase of the internal unionized ammo-nia. Both physiological causes can be acting at the same time,explaining why P. felina is so sensitive to the toxicity of unionizedammonia. However, most physiological studies on the effects ofammonia have been conducted with fish and crustaceans, so theeffects of ammonia on planarian physiology are still unknown.The reduction in the locomotion of P. felina can also be caused by

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the effect of ammonia on the two more external body layers (e.g.epithelial and muscular layer), impairing the sliding of planarians.This effect was recently found to decrease the behavioural activityof P. felina exposed to aluminium (Kovacevic et al., 2009).

The above mentioned water quality criteria might not be suffi-cient to protect this group of very sensitive freshwater inverte-brates. However, we must be cautious in our conclusions, as theextrapolation of laboratory results to realistic field community/population effects is still a complicated issue in ecotoxicology.Field studies have shown that ammonia and other nitrogen nutri-ents can alter natural populations of invertebrates, and that labora-tory species sensitivity and field tolerance are positively correlated(Beketov, 2004). The approach most often used to extrapolate lab-oratory data to field scenarios is the Species Sensitivity Distribu-tion (SSD). This method uses all available laboratory data of LC,EC or NOEC values to short/long term exposures for the speciesof the community to predict the adverse effect on the communityon the basis of a sensitivity distribution model (Newman and Cle-ments, 2008). The SSD model generally uses a cut-off value that al-lows a loss of the 5% most sensitive species in the community(Hazardous concentration = HC5) as acceptable for the ecosystemfunctioning (Newman and Clements, 2008). This is under theassumption that all species in a community have the same ecolog-ical importance in maintaining structure and function of theecosystem. However, planarians are usually one of the most abun-dant predators in streams and upper reaches of rivers (Thorp andCovich, 2001) so they are expected to perform as key-stone species.Given the high sensitivity of P. felina, HC5 for unionized ammoniaderived from SSD (based on long-term data) may probably not besufficient to protect P. felina. Therefore, in order to improve SSDmethods, key species in the community should have a higher con-tribution, i.e. HC concentration derived from SSD models should belower when these highly sensitive species are present in the stud-ied community. Additionally, the SSD approach can be improvedby means of the inclusion of behavioural endpoints to differentspecies, and in the case of planarians the locomotion activity hasdemonstrated to be a good candidate.

5. Conclusions

On the basis of our study, we conclude that the studied behav-ioural endpoint in the aquatic planarian P. felina (locomotion asmovement speed) is a more sensitive endpoint than mortality.Therefore, this type of endpoint should be considered in toxicityevaluations and also in SSD models. This endpoint can be quantita-tively monitored under laboratory conditions, and therefore it canbe applied in a non-subjective way for regulatory proposes. How-ever, further studies are needed to understand the physiologicalcauses of the observed high sensitivity in this planarian speciesand the likely sensitivity for other toxicants. Additionally, thedevelopment of new behavioural endpoints related to movementsof planarians (e.g. complexity, regularity, etc.) can improve theassessment of deleterious effects of toxicants.

Acknowledgements

This research was funded by the Spanish Ministry of Scienceand Technology (research project REN2001-1008/HID). The Uni-versity of Alcalá provided logistical support. Dr. Álvaro Alonsowas supported by predoctoral grants from the Council of Castilla-La Mancha, and the University of Alcalá, and by a postdoctoral con-tract from the Spanish Ministry of Science and Innovation (SMSI) atthe Aquatic Ecology and Water Quality Management Group of theWageningen University (The Netherlands). He is currently sup-ported by a Juan de la Cierva contract from the SMSI at University

of Alcalá. Our sincere gratitude to Dr. Pilar Castro and Dr. Mariekede Lange for correcting the English text and for their useful com-ments to improve this work.

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