reviewing the serotonin reuptake inhibitors (ssris) footprint in the aquatic biota: uptake,...

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Environmental Pollution 197 (2015) 127e143

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Environmental Pollution

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Review

Reviewing the serotonin reuptake inhibitors (SSRIs) footprint in theaquatic biota: Uptake, bioaccumulation and ecotoxicology

Liliana J.G. Silva a, *, Andr�e M.P.T. Pereira a, Leonor M. Meisel b, c, Celeste M. Lino a,Angelina Pena a

a REQUIMTE, Group of Bromatology, Pharmacognosy and Analytical Sciences, Faculty of Pharmacy, University of Coimbra, Polo III, Azinhaga de Stª Comba,3000-548 Coimbra, Portugalb INFARMED, I.P. e National Authority of Medicines and Health Products, Parque de Saúde de Lisboa e Avenida do Brasil, 53, 1749-004 Lisboa, Portugalc Department of Pharmacology, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal

a r t i c l e i n f o

Article history:Received 22 July 2014Received in revised form28 November 2014Accepted 1 December 2014Available online

Keywords:Environmental contaminantsSelective serotonin re-uptake inhibitorsUptakeBioaccumulationEcotoxicology

* Corresponding author. Group of Health SurveillanStudies, Faculty of Pharmacy, University of Coimbra,Azinhaga de Santa Comba, 3000-548 Coimbra, Portug

E-mail address: [email protected] (L.J.G. Silva).

http://dx.doi.org/10.1016/j.envpol.2014.12.0020269-7491/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Selective serotonin re-uptake inhibitors (SSRIs) antidepressants are amongst the most prescribedpharmaceutical active substances throughout the world. Their presence, already described in differentenvironmental compartments such as wastewaters, surface, ground and drinking waters, and sediments,and their remarkable effects on non-target organisms justify the growing concern about these emergingenvironmental pollutants. A comprehensive review of the literature data with focus on their footprint inthe aquatic biota, namely their uptake, bioaccumulation and both acute and chronic ecotoxicology ispresented. Long-term multigenerational exposure studies, at environmental relevant concentrations andin mixtures of related compounds, such as oestrogenic endocrine disruptors, continue to be sparse andare imperative to better know their environmental impact.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The potential for negative effects of pharmaceuticals even atsublethal concentrations, in the aquatic environment, namely formarine organisms, has been of concern since the issue was firstbrought to attention in 1985 (Silva et al., 2012). Nonetheless, theecotoxicological risks associated to the ubiquitous occurrence ofpharmaceuticals in aquatic ecosystems are far from known(Gonzalez-Rey and Bebianno, 2013). Their environmental presenceis a growing problem that must be tackled to meet the Directive2013/39/EU, in order to minimize their aquatic environmentalcontamination and support future prioritization measures.

Selective serotonin re-uptake inhibitors (SSRIs) antidepressantshave been widely marketed since the mid-1980s (Schultz andFurlong, 2008). The members of this class include fluoxetine, cit-alopram, paroxetine, sertraline, and fluvoxamine. Citalopram is aracemic mixture of R-citalopram and S-citalopram enantiomers

ce, Center of PharmaceuticalP�olo das Ciencias da Saúde,al.

with different potencies, but since S-citalopram is more potent it isalso marketed as the single S-enantiomer formulation, escitalo-pram (Kosjek and Heath, 2010). As other pharmaceuticals, SSRIsenter wastewater treatment primarily through domestic wastefrom human excretion or by direct disposal of unused or expireddrugs in toilets, with wastewater treatment plants (WWTPs) beingconsidered the major environmental source to the surroundingwater bodies (Schultz et al., 2010). Antidepressants are also clini-cally administered within healthcare facilities, namely, hospital,nursing, assisted living and independent living healthcare facilities,but these are a particularly under characterized source of phar-maceuticals in municipal wastewaters (Nagarnaik et al., 2011).

As a result, their presence in different environmental matrices isubiquitous and they are amongst the most commonly detectedhuman pharmaceuticals in the aquatic environment. As far as weknow the presence of SSRIs in the environment, specificallyfluoxetine, was first reported by Kolpin et al. (2002) in the UnitedStates of America (USA) surface waters, and by Metcalfe et al.(2003) in Canada WWTP effluents. Later on, in 2005, a study re-ported the presence of two SSRIs and their metabolites (fluoxetine,sertraline, norfluoxetine, and desmethylsertraline) in different fishtissues residing in a municipal effluent-dominated stream (Brooks

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L.J.G. Silva et al. / Environmental Pollution 197 (2015) 127e143128

et al., 2005). Since then, as the detection technology improved(Silva et al., 2014), several publications, from different countries,referred the presence of these residues in a wide range of watersamples, including wastewaters, in concentrations ranging from0.15 to 32,228 ng L�1, surface and groundwaters, ranging between0.5 and 8000 ng L�1, and drinking waters, from 0.5 to 1400 ng L�1

Table 1Physico-chemical properties of SSRIs and their main metabolites (adapted from Silva et

Compound Metabolite CAS number Structural formula

Citalopram 59729-33-8

Desmethylcitalopram 62498-67-3

Fluoxetine 54910-89-3

Norfluoxetine 83891-03-6

Fluvoxamine 54739-18-3

Paroxetine 61869-08-7

Sertraline 79617-96-2

Desmethylsertraline 87857-41-8

� Unavailable data.a Measured on salt form (HCl) of each SSRI.b Average calculated from experiments with five different soils and sediments at pH 5

(Silva et al., 2012). Their presence has also been reported in sedi-ments and soils, up to 1033 ng g�1 (Lajeunesse et al., 2012; Schultzet al., 2010).

Since SSRIs mode of action is by modulating the neurotrans-mitter serotonin, aquatic organisms who possess physiologicalsystems regulated by transporters and receptors sensitive to

al., 2012).

MW pKa Log kowa Log kocb Solubility (mg L�1)a

324.16 9.59 1.39 5.63 31.1

310.15 10.50 e e e

309.13 10.05 1.22 4.65 60.3

295.12 9.05 e e e

318.16 9.39 1.21 3.82 e

329.14 10.32 1.37 4.47 35.3

305.07 9.47 1.37 4.17 3.52

291.06 9.41 e e e

.0e7.8.

L.J.G. Silva et al. / Environmental Pollution 197 (2015) 127e143 129

activation by these pharmaceuticals are potentially affected bythemwhen exposed, even to trace levels (Fong and Ford, 2014; Silvaet al., 2012). Their uptake and bioaccumulation in biotawas alreadyreported in concentrations ranging from 0.01 to 73 ng g�1 (Chu andMetcalfe, 2007; Ramirez et al., 2007; Schultz et al., 2011, 2010).Toxicological studies recently indicated that at ambient concen-trations SSRIs induce biological effects in fish, molluscs, and otheraquatic invertebrates, including delays in reproductive and physi-ological development, decreased aggressiveness, and inhibition offeeding responses (Demeestere et al., 2010; Fong and Ford, 2014).

Herewith a comprehensive review of the literature data withfocus on SSRIs footprint in the aquatic biota, namely their uptake,bioaccumulation, and both acute and chronic ecotoxicology ispresented. Finally, some final remarks and future needs are alsopointed out.

2. SSRIs characterization and mode of action

According to the latest Organisation for Economic Co-Operationand Development (OECD) report of 2011 (OECD, 2011), the con-sumption of antidepressants has grown by over 60% in all countriesover the past decade. SSRIs are one of the most commonly pre-scribed classes of pharmaceuticals worldwide (Schultz and Furlong,2008). Escitalopram, citalopram, fluoxetine, and paroxetine requirea lower daily dose, 0.01, 0.02, and 0.02 g, respectively, whencompared to sertraline and fluvoxamine that require 0.05 and 0.1 g,respectively (Christensen et al., 2007).

In humans, SSRIs, via inhibition of the serotonin reuptakemechanism, induce an increase in serotonin concentration withinthe central nervous system (Kreke and Dietrich, 2008). Followingoral ingestion, SSRIs are extensively metabolized, and the primarymetabolites released are generally N-desmethyl products. Most ofthese metabolites retain their pharmacological activity, but theyare, with the exception of norfluoxetine, less potent than the parentcompounds (Silva et al., 2012). Large discrepancies between SSRIsexcretion rates are reported in the literature and the amountsexcreted in the unchanged or conjugate forms vary considerably(Calisto and Esteves, 2009). Fluoxetine is mainly excreted in urine(with less than 10% excreted unchanged), as norfluoxetine or asfluoxetine N-glucuronide (Vaswani et al., 2003). Conversely, otherpharmacokinetics studies report that only approximately 20% isexcreted as norfluoxetine and its glucuronide (Nałecz-Jawecki,2007). Regarding citalopram, the excretion of the unchangedcompound ranges between 12 and 20%. Only 0.2% of an oral dose ofsertraline is excreted unchanged in the urine, but information on itsmetabolism is rather limited. Fluvoxamine suffers oxidative elimi-nation of a metoxil group to form a pharmacologically inactivecarboxylic acid, while paroxetine is transformed into an inactivecatechol intermediate, with only 2% being excreted as the parentcompound in urine and 1% in faeces (Calisto and Esteves, 2009).

The physico-chemical characteristics of SSRIs (Table 1) outlinetheir environmental behaviour. They are lipophilic, basic drugs,with pKa ranging between 9.05 and 10.5. As hydrochloride saltforms, all SSRIs have low octanolewater partition coefficients (Kow1.21e1.39) and relatively high water solubilities. Nonetheless, theypresent high sorption coefficients (presented as log Koc or organiccarbon normalized sorption coefficient) with soils and sediments,with log Koc values ranging from 3.82 to 5.63, for fluvoxamine(lowest degree of sorption) and citalopram (highest degree ofsorption), respectively, meaning that these are persistent com-pounds (Silva et al., 2012). The polarity of ionisable compounds,including many pharmaceuticals, will vary with site-specificreceiving system pH and is subject to change in different biolog-ical compartments, thus modifying their expected and actual bio-accumulation, partitioning behaviour, and toxicity (Ramirez et al.,

2009). Accordingly, SSRIs, as weak bases, are present in water pri-marily in the neutral form at high pH and in the ionic form at lowpH (Metcalfe et al., 2010).

In the past decade, there have been increasing concerns over theeffects of pharmaceutical compounds in the aquatic environment,however very little is known about the effects of antidepressantssuch as SSRIs (Bossus et al., 2014). The phylogenetically ancient andhighly conserved neurotransmitter and neurohormone serotoninhas been found in invertebrates and vertebrates, although its spe-cific physiological role and mode of action is unknown for manyspecies (Kreke and Dietrich, 2008). Many biological functionswithin invertebrates, such as reproduction, metabolism, moultingand behaviour, are under the control of serotonin (Bossus et al.,2014). In fish, serotonin is involved in the regulation of manyphysiological processes including branchial nitrogen excretion andintestinal osmoregulation (Morando et al., 2009). Another plausiblehypothesis for the mechanism of toxicity to the fish is that SSRIsexposure leads to increased plasma serotonin levels that constrictsthe arterio-arterial branchial vasculature, leading to impaired gasexchange and hypoxia, ultimately leading to death (Richards et al.,2004). Studies of antidepressants in aquatic systems have provided,and will continue to develop, an advanced understanding of haz-ards and risks from pharmaceuticals and other environmentalcontaminants (Brooks, 2014).

3. Uptake, effects and ecotoxicological data

Below we review the SSRIs uptake, effects and ecotoxicologicaldata in the aquatic biota. Table 2 presents the SSRIs and their mainmetabolites mean concentrations found in different wild fish tis-sues collected from the real aquatic environment, whereas Table 3shows the toxicity data obtained from exposure studies for non-target organisms.

3.1. Citalopram

3.1.1. Uptake and bioaccumulationThe presence of citalopram in wastewater impacted ecosystems

is a reality that might pose health risks to the aquatic wildlife (Silvaet al., 2012). Despite this, few studies have investigated the uptakeand bioaccumulation of this SSRI in aquatic organisms (Table 2).Gelsleichter and Szabo (2013) studied the uptake of 9 pharma-ceuticals, including six antidepressants by bull sharks (Carcharhinusleucas) residing in pristine and wastewater impacted rivers. Cit-alopram was observed at mean levels of 0.4 ng mL�1 in plasma.Conversely, citalopram was undetectable in all plasma samples ofrainbow trout (Oncorhynchus mykiss), and of fry and adult maleguppies (Poecilia reticulata) exposed to waterborne citalopram,ranging from environmentally relevant to high concentrations (1,10, 100 mg L�1) for 3e7 days. An exception was observed in 2 out of5 samples from the 10 mg L�1 group (0.044 and 0.080 ng mL�1),corresponding to a bioconcentration factor (BCF) fromwater to fishblood plasma of 0.004e0.008 or less. It cannot be excluded that theexposure time applied in the present study (3e7 days) was tooshort for citalopram to elicit an effect (Holmberg et al., 2011). Infact, citalopram with a log Kow of 1.39 (Table 1), similar to that offluoxetine and sertraline, and with significantly higher aqueousconcentrations when compared to these SSRIs (about an order ofmagnitude higher) (Silva et al., 2012), ranged between not detectedand 0.07 ng g�1 in white sucker (Catostomus commersoni) brains,values an order of magnitude lower, as compared to fluoxetine andsertraline. This fact suggests that mechanisms, other than hydro-phobicity, also contribute to the distribution of antidepressants infish brains, and that, whereas aquatic organisms are exposed to thespectrum of antidepressants in surface water, specific

Table 2SSRIs and their main metabolites mean concentrations found in different wild fish tissues collected from the real aquatic environment.

SSRI Species (common name) Effluent-dominated collection point Matrix Meanconcentration(ng g�1 orng mL�1)

MQL orMDL(ng g�1 orng mL�1)

Methodofanalysis

Reference

Citalopram Catostomus commersoni (white suckers) Boulder Creek, Colorado, USAFourmile Creek, Iowa, USA

Brain 0.01e0.07 MQL:0.015

SLE-LC/MS/MS

(Schultzet al., 2010)

Citalopram Pimephales promelas (fathead minnows) Grand River, Ontario, Canada Whole fishhomogenate

2.9 MQL: 0.5 SPE-LC/MS/MS

(Metcalfeet al., 2010)

Desmethylcitalopram 2.84 MQL: 0.5Citalopram Carcharhinus leucas (bull sharks) Caloosahatchee River, Florida, USA Plasma 0.40 MQL: 0.25 SPE-LC/

MS/MS(Gelsleichterand Szabo,2013)

Fluoxetine Lepomis macrochirus (bluegill), Ictaluruspunctatus (channel catfish), Cyprinus carpio(carp),Pomoxis nigromaculatus (black crappie)

Pecan Creek, Texas, USA BrainLiverMuscle

1.581.340.11

MQL: 0.05 SPE-LC/MS/MS

(Brooks et al.,2005)

Norfluoxetine BrainLiverMuscle

8.8610.271.07

MQL: 0.05

Fluoxetine Ameiurus nebulosus (brown bullhead),Dorosoma cepedianum (gizzard shad),Morone americana (white perch)

Hamilton Harbour, Ontario, Canada Tissuehomogenate


(Chu andMetcalfe,2007)

Norfluoxetine Tissuehomogenate

0.27 MQL: 0.14

Fluoxetine Fish, not specified specie(s) Pecan Creek, Texas, USA Muscle ND MQL: 2.54 SLE-LC/MS/MS

(Ramirezet al., 2007)

Norfluoxetine 4.37 MQL: 1.08Fluoxetine Sonora sucker, Largemouth bass, Common

carp, Bowfin,White sucker, Smallmouth buffalo

Rivers in Chicago, Illinois; Dallas,Texas; Orlando, Florida; Phoenix,Arizona; and West Chester,Pennsylvania, USA

LiverMuscle

19e70ND

MDL: 4.09 SLE-LC/MS/MS


Norfluoxetine LiverMuscle

37e733.2e4.0

MDL: 4.36MDL: 2.9

Fluoxetine Catostomus commersoni (white suckers) Boulder Creek, Colorado, USAFourmile Creek, Iowa, USA

Brain 0.02e0.6 MQL:0.015

SLE-LC/MS/MS


Norfluoxetine 0.07e0.9 MQL:0.015

Fluoxetine Pimephales promelas (fathead minnows) Grand River, Ontario, Canada Whole fishhomogenate

<MQL MQL: 0.5 SPE-LC/MS/MS


Norfluoxetine 1.22 MQL: 0.5Fluoxetine Carcharhinus leucas (bull sharks) Caloosahatchee River, Florida, USA Plasma <MQL MQL: 0.25 SPE-LC/


Norfluoxetine 4.08 MQL: 2.50Fluvoxamine Catostomus commersoni (white suckers) Boulder Creek, Colorado, USA

Fourmile Creek, Iowa, USABrain ND MQL:

0.015SLE-LC/MS/MS


Fluvoxamine Carcharhinus leucas (bull sharks) Caloosahatchee River, Florida, USA Plasma 0.83 MQL: 0.25 SPE-LC/MS/MS

(Gelsleichterand Szabo,2013)

Paroxetine Ameiurus nebulosus (brown bullhead),Dorosoma cepedianum (gizzard shad),Morone americana (white perch)

Hamilton Harbour, Ontario, Canada Tissuehomogenate


(Chu andMetcalfe,2007)

Paroxetine Catostomus commersoni (white suckers) Boulder Creek, Colorado, USAFourmile Creek, Iowa, USA

Brain 0.01e0.02 MQL:0.015

SLE-LC/MS/MS


Paroxetine Pimephales promelas (fathead minnows) Grand River, Ontario, Canada Whole fishhomogenate

ND MQL: 0.5 SPE-LC/MS/MS


Paroxetine Carcharhinus leucas (bull sharks) Caloosahatchee River, Florida, USA Plasma 0.55 MQL: 1.25 SPE-LC/MS/MS

(Gelsleichterand Szabo,2013)

Sertraline Lepomis macrochirus (bluegill), Ictaluruspunctatus (channel catfish), Cyprinus carpio(carp), Pomoxis nigromaculatus (blackcrappie)

Pecan Creek, Texas, USA BrainLiverMuscle

4.273.590.34

MQL: 0.05 SPE-LC/MS/MS

(Brooks et al.,2005)

Desmethylsertraline BrainLiverMuscle

15.612.940.69

MQL: 0.05

Sertraline Not specified fish specie(s) Pecan Creek, Texas, USA Muscle ND MQL: 0.71 SLE-LC/MS/MS


Sertraline Sonora sucker, Largemouth bass, Commoncarp, BowfinWhite sucker, Smallmouth buffalo

Rivers in Chicago, Illinois; Dallas,Texas; Orlando, Florida; Phoenix,Arizona; and West Chester,Pennsylvania, USA

LiverMuscle

27e8411

MDL: 4.36MDL: 2.9

SLE-LC/MS/MS


Sertraline Catostomus commersoni (white suckers) Boulder Creek, Colorado, USAFourmile Creek, Iowa, USA

Brain 0.005e1.8 MQL:0.015

SLE-LC/MS/MS



Table 2 (continued )

SSRI Species (common name) Effluent-dominated collection point Matrix Meanconcentration(ng g�1 orng mL�1)

MQL orMDL(ng g�1 orng mL�1)

Methodofanalysis

Reference

Desmethylsertraline 0.01e3 MQL:0.015

Sertraline Pimephales promelas (fathead minnows) Grand River, Ontario, Canada Whole fishhomogenate

1.65e3.83 MQL: 0.5 SPE-LC/MS/MS


Desmethylsertraline <MQL e 1.50 MQL: 0.5Sertraline Carcharhinus leucas (bull sharks) Caloosahatchee River, Florida, USA Plasma 0.48 MQL: 0.25 SPE-LC/


ND eNot detected.MQL e Method quantification limit.MDL e Method detection limit.


antidepressants are selectively taken up by brain tissues (Schultzet al., 2010).

3.1.2. Effects and toxicityAs for other SSRIs, most citalopram ecotoxicity data is available

from studies describing endpoints that do not include direct mea-surements of survival, development or reproduction but ratherdescribe behavioural effects (Holmberg et al., 2011; Ols�en et al.,2014), that are uncertain for an environmental risk assessment.

The acute and chronic toxicity of five SSRIs, including citalopramwere evaluated in the daphnid Ceriodaphnia dubia. The 48 hmedianlethal concentration (LC50) was determined in three static testswith neonates, and chronic (8 d) tests were conducted to determineno-observable-effect concentrations (NOEC) and lowest-observable-effect concentrations (LOEC) for reproduction end-points. Citalopram showed to be the SSRI with lower acute (LC50 of3.90mg L�1) and chronic (NOEC of 0.8mg L�1) toxicity (Henry et al.,2004). This was also observed by Christensen et al. (2007) thatreported a median effective concentration (EC50) ranging from 1.6to 20 mg L�1 in toxicity tests on biomass with Pseudokirchneriellasubcapitata and with Daphnia magna, respectively (Table 3).Recently, citalopram was reported to produce anxiolytic effects onguppy fish (Poecilia wingei) after 21 days exposure affectingecologically relevant behaviours important to their survival.Exposure to a concentration of 2.3 mg L�1 decreased the freezingbehaviour in females but no effects were observed in males. Inmales, a significant effect was found after exposure to 15 mg L�1.These results might suggest sex differences with females beingmore sensitive in connection to stressful situations. Male repro-ductive behaviour and connected swimming activity were notaffected by citalopram exposure (Ols�en et al., 2014). Accordingly,Holmberg et al. (2011) reported that citalopram, alone, does notaffect the aggressive or reproductive behaviour in fish at waterconcentrations regularly found in the environment. Nonetheless, inthe environment, fish may be exposed for longer times to severalSSRIs acting additively and in concert with other contaminants andother abiotic or biotic factors.

One should bear in mind that citalopram is the least toxic of theSSRIs however it is also the least tested of the SSRIs for ecotoxi-cological effects (Christensen et al., 2009).

3.2. Fluoxetine

3.2.1. Uptake and bioaccumulationFluoxetine, due to its environmental persistence, acute toxicity

to nontarget organisms, and unique pharmacokinetics associatedwith a readily ionizable compound (Oakes et al., 2010), nor-fluoxetine, is undoubtedly the SSRI most investigated both in the

aquatic compartments (Silva et al., 2012) and biota samples(Table 2). The first data that regarded fluoxetine and norfluoxetineaccumulation in brain, liver, and muscle tissues of different fishspecies of effluent-dominated ecosystems was reported by Brookset al. (2005). Among the three fish species examined, averagenorfluoxetine levels were higher in liver (10.27 ng g�1), brain(8.86 ng g�1), and muscle (1.07 ng g�1) tissues when compared tofluoxetine (1.34, 1.58, and 0.11 ng g�1, respectively). In fact, theseobservations were also made later on by other researchers(Lajeunesse et al., 2011; Ramirez et al., 2009, 2007; Schultz et al.,2010). Accordingly, fluoxetine was not possible to quantify in fishhomogenates, nor in plasma, but norfluoxetine was detected at1.22 ng g�1 and 4.08 ng mL�1, respectively (Gelsleichter and Szabo,2013; Metcalfe et al., 2010). In fact, ratios of norfluoxetine tofluoxetine of approximately 2:1 were reported for Japanese medakaexposed to fluoxetine for 7 days (Paterson and Metcalfe, 2008). Inany event, it should not be assumed that the profile of metabolitesthat accumulate in fish is the same as in man, as even amongmammalian species, the metabolism of antidepressants can differmarkedly (Metcalfe et al., 2010). A norfluoxetine pseudo-BCF ofapproximately 60 was calculated by Metcalfe et al. (2010), which isconsistent with the pseudo-BCFs for Japanese medaka calculated byNakamura et al. (2008) of 100 for exposures at pH 7, and of 170 forexposures at pH 8. This suggests the active metabolism of fluoxe-tine and may have implications for fish health because nor-fluoxetine, like some other SSRI metabolites, is less polar than itsparent compound and is therefore more prone to bioaccumulationthan fluoxetine, while still capable of eliciting the same or evenworse biological effects (Gelsleichter and Szabo, 2013).

In contrast, Chu and Metcalfe (2007) reported lower concen-trations of norfluoxetine (0.27 ng g�1) than of fluoxetine(0.37 ng g�1) in fish tissues homogenates. Nonetheless, one shouldnote that, since fluoxetine and most of the other target antide-pressants are weak bases which are present in water primarily inthe neutral form at high pH and in the ionic form at low pH,accumulation in fish is influenced by the pH of the water (Metcalfeet al., 2010) thusmodifying their expected and actual accumulation,partitioning behaviour, and even toxicity (Ramirez et al., 2009). Inthis context, accumulation assumptions for pharmaceuticals solelyon log Kow may lead to erroneous and inaccurate partitioning es-timates on a site-specific basis (Ramirez et al., 2009).

Recently, Franzellitti et al. (2014) reported BCF values forfluoxetine ranging from 200 to 800 in the marine mussel Mytilusgalloprovincialis, after a 7 day treatment with 30 and 300 ng L�1.

3.2.2. Effects and toxicityFrom a top 20 prioritization of pharmaceuticals and personal

care products and endocrine-disrupting chemicals, which was

Table 3Toxicity data from exposure studies of SSRIs and their main metabolites for non-target organisms.

Pharmaceutical Species e taxonomic group(common name)

Acute toxicological endpoint Acute ecotoxicitydata

Chronic toxicologicalendpoint

Chronicecotoxicitydata

Reference

CitalopramAlgaeCitalopram Pseudokirchneriella

subcapitata e chlorophyta(green microalgae)

EC50 (48 h) (biomass) 1.6 mg L�1 e e (Christensenet al., 2007)

InvertebrateCitalopram Ceriodaphnia dubia e

arthropod, crustacean(waterflea)

LC50 (48 h) (survival) 3.90 mg L�1 NOEC (8 d) (neonatesproduced)LOEC (8 d) (neonatesproduced)

0.80 mg L�1

4.00 mg L�1(Henry et al.,2004)

Citalopram Daphnia magna e


EC50 (48 h) (biomass) 20 mg L�1 e e (Christensenet al., 2007)

Citalopram Chlorostoma funebralis emollusk, gastropod

LOEC (4 h) (adhesion to substrate) 405 mg L�1 e e (Fong andMolnar, 2013)

Citalopram Lithopoma americanum e

mollusk, gastropodLOEC (4 h) (adhesion to substrate) 405 mg L�1 e e (Fong and

Molnar, 2013)Citalopram Tegula fasciatus e mollusk,

gastropodLOEC (4 h) (adhesion to substrate) 405 mg L�1 e e (Fong and

Molnar, 2013)Citalopram Nucella ostrina e mollusk,

gastropodLOEC (4 h) (adhesion to substrate) 4.05 mg L�1 e e (Fong and

Molnar, 2013)FluoxetineAlgaeFluoxetine Pseudokirchneriella


EC50 (120 h)(growth e turbidity)(growth e cell density)

24 mg L�1

39 mg L�1e e (Brooks et al.,

2003a; Brookset al., 2003b)

Fluoxetine Pseudokirchneriellasubcapitata e chlorophyta(green microalgae)

IC10 (96 h) (growth inhibition)IC50 (96 h) (growth inhibition)

31.34 mg L�1

44.99 mg L�1e e (Johnson et al.,

2007)

Fluoxetine Scendesmus acutus echlorophyta(freshwater greenmicroalgae)


55.60 mg L�1


2007)

Fluoxetine Scendesmus quadricauda e

chlorophyta(freshwater greenmicroalgae)


97.76 mg L�1


2007)

Fluoxetine Chlorella vulgaris echlorophyta(single-cell green algae)


2901.57 mg L�1


2007)

Fluoxetine Dunaliella tertiolecta e

chlorophytaEC50 (96 h) (population cell density) 169.81 mg L�1 e e (DeLorenzo and

Fleming, 2008)Fluoxetine Pseudokirchneriella


EC50 (24 h) (growth rate) 90 mg L�1 e e (Neuwoehneret al., 2009)

Norfluoxetine EC50 (24 h) (growth rate) 242 mg L�1 e e

Fluoxetine Scendesmus vacuolatus echlorophyta(freshwater greenmicroalgae)

EC50 (24 h) (cell volume growth) 93 mg L�1 e e (Neuwoehneret al., 2009)

Norfluoxetine EC50 (24 h) (cell volume growth) 189 mg L�1 e e

InvertebrateFluoxetine Ceriodaphnia dubia e


LC50 (48 h) (survival) 234 mg L�1 NOEC (7 d) (reproduction)LOEC (7 d) (reproduction)

56 mg L�1

112 mg L�1(Brooks et al.,2003a; Brookset al., 2003b)

Fluoxetine Daphnia magnaarthropod, crustacean(waterflea)

LC50 (48 h) (survival) 820 mg L�1 e e (Brooks et al.,2003a; Brookset al., 2003b)

Fluoxetine Hyalella azteca e arthropod,crustacean

e e EC50 (42 d) (survival)LOEC (42 d) (growth)

>43 mg kg�1

5.4 mg kg�1(Brooks et al.,2003a; Brookset al., 2003b)

Fluoxetine Chironomus tentans earthropod, insect (midge)

e e LC50 (10 d) (survival)LOEC (10 d) (growth)

15.2 mg kg�1

1.3 mg kg�1(Brooks et al.,2003a; Brookset al., 2003b)

Fluoxetine Ceriodaphnia dubia e


LC50 (48 h) 0.51 mg L�1 NOEC (8 d) (neonatesproduced)LOEC (8 d) (neonatesproduced)

0.089 mg L�1

0.447 mg L�1(Henry et al.,2004)

Fluoxetine Gammarus pulex e

arthropod, crustaceanLOEC (1.5 h) (activity) 100 ng L�1 e e (De Lange et al.,

2006)FluoxetineNorfluoxetine

Thamnocephalus platyuruse arthropod, crustacean

LC50 (24 h) 0.76 mg L�1

0.47 mg L�1e e (Nałecz-

Jawecki, 2007)







Reference

Fluoxetine Spirostomum ambiguum e

protistaEC50 (24 h) (deformity)LC50 (24 h)

0.41 mg L�1

0.55 mg L�1e e (Nałecz-

Jawecki, 2007)Norfluoxetine EC50 (24 h) (deformity)

LC50 (24 h)0.30 mg L�1

0.39 mg L�1e e

Fluoxetine Potamopyrgus antipodarume mollusk, gastropod

e e EC10 (56 d) (embryoswithout shell)NOEC (56 d) (embryoswithout shell)

0.81 mg L�1

0.47 mg L�1(Nentwig,2007)

Fluoxetine Daphnia magna e


e e LOEC (21 d) (reproduction)NOEC (21 d) (reproduction)

429 mg L�1

(R-Fluoxetine)430 mg L�1

(Racemic)444 mg L�1

(S-Fluoxetine)170 mg L�1


(Racemic)195 mg L�1

(S-Fluoxetine)

(Stanley et al.,2007

Fluoxetine Hyalella Azteca e

arthropod, crustaceane e LOEC (28 d) (growth)

NOEC (28 d) (growth)100 mg L�1

33 mg L�1(P�ery et al.,2008)

Fluoxetine Daphnia magna e


e e LOEC (21 d) (new borneslength)NOEC (21 d) (new borneslength)

31 mg L�1

8.9 mg L�1(P�ery et al.,2008)

Fluoxetine Potamopyrgus antipodarume mollusk, gastropod

e e LOEC (28 d) (reproduction)NOEC (28 d) (reproduction)

69 mg L�1

13 mg L�1(P�ery et al.,2008)

Fluoxetine Valvata piscinalis emollusk,gastropod(European valve snail)

e e NOEC (42 d) (cumulatenumber of eggs)

100 mg L�1 (Gust et al.,2009)

Fluoxetine Potamopyrgus antipodarume mollusk, gastropod (NewZealand mudsnail)

e e LOEC (42 d) (number ofneonates)NOEC (42 d) (number ofneonates)

100 mg L�1

33.3 mg L�1(Gust et al.,2009)

Fluoxetine Chlorostoma funebralis emollusk, gastropod


Fluoxetine Lithopoma americanum e

mollusk, gastropodLOEC (4 h) (adhesion to substrate) 3.45 mg L�1 e e (Fong and

Molnar, 2013)Fluoxetine Tegula fasciatus e mollusk,


Molnar, 2013)Fluoxetine Urosalpinx cin�erea e


Molnar, 2013)Fluoxetine Nucella ostrina e mollusk,


Molnar, 2013)Fluoxetine Lampsilis siliquoidea and

Ligumia recta e mollusk,freshwater musselsglochidiajuvenileadult female

EC50 (24 h) (valve closure)EC50 (48 h) (foot movement orheartbeat)EC50 (96 h) (foot movement orheartbeat)

239e624.8 mg L�1

179e265.7 mg L�1

62.0e96.9 mg L�1

LOEC (28 d) (footprotrusion, mantle luredisplay and glochidiaparturition)

29.3e300 mg L�1

(Hazelton et al.,2013)

Fish and other vertebratesFluoxetine Pimephales promelas e fish

(fathead minnow)LC50 (48 h) 705 mg L�1 e e (Brooks et al.,

2003a; Brookset al., 2003b)

Fluoxetine PLHC-1 (Poeciliopsis lucidahepatoma cell)RTG-2 (rainbow troutgonadal cell line)

EC50 (24 h) (MTT assay e The MTTassay is based on the uptake ofthiazolyl blue tetra-zolium bromide (MTT) and itsfollowing reduction in themitochondria of living cells to MTTformazan)

6.34 mg L�1

3.31 mg L�1e e (Caminada

et al., 2006)

Fluoxetine Xenopus laevis e amphibian EC10 (96 h) (deformity)EC50 (96 h) (deformity)LC10 (96 h)LC50 (96 h)NOEC (deformity)

3.0 mg L�1

4.9 mg L�1

7.1 mg L�1

7.5 mg L�1

2.0 mg L�1

e e (Richards andCole, 2006)

Fluoxetine LC50 (48 h)

(continued on next page)







Reference

Pimephales promelas e fish(fathead minnow)

212 mg L�1 (R-Fluoxetine)198 mg L�1

(Racemic)216 mg L�1 (S-Fluoxetine)

LOEC (7 d) (growth)NOEC (7 d) (growth)

170 mg L�1


(Racemic)51 mg L�1 (S-Fluoxetine)118 mg L�1


(Racemic)9 mg L�1 (S-Fluoxetine)

(Stanley et al.,2007)

Fluoxetine Cophixalus riparius eamphibian

e e LOEC (28 d) (emergence) 1.12 mg kg�1 (Nentwig,2007)

Fluoxetine Gambusia affinis e fish(mosquito fish)

LC50 (7 d) 546 mg L�1 e e (Henry andBlack, 2008)

Fluoxetine Oryzias latipes e fish(Japanese medaka)

LC50 (96 h)NOEC (96 h)

5.5, 1.3, and0.20 mg L�1 at pH 7,8, and 9,respectively3.8 mg L�1 at pH 7.1

e e (Nakamuraet al., 2008)

FluvoxamineAlgaeFluvoxamine Pseudokirchneriella


EC10 (96 h) (growth inhibition)EC50 (96 h) (growth inhibition)

3887.38 mg L�1


2007)

Fluvoxamine Scendesmus acutus echlorophyta(freshwater greenmicroalgae)


2503.65 mg L�1


2007)

Fluvoxamine Scendesmus quadricauda e



1662.91 mg L�1


2007)

Fluvoxamine Chlorella vulgaris echlorophyta(single-cell green algae)


6162.86 mg L�1

10,208.47 mg L�1e e (Johnson et al.,

2007)

Fluvoxamine Pseudokirchneriellasubcapitata e chlorophyta(green microalgae)


InvertebrateFluvoxamine Ceriodaphnia dubia e



0.366 mg L�1

1.466 mg L�1(Henry et al.,2004)

Fluvoxamine Daphnia magnaarthropod, crustacean(waterflea)

EC50 (48 h) (biomass) 13 mg L�1 e e (Christensenet al., 2007)

Fluvoxamine Chlorostoma funebralis emollusk, gastropod


Fluvoxamine Lithopoma americanum e


Molnar, 2013)Fluvoxamine Tegula fasciatus e mollusk,


Molnar, 2013)Fluvoxamine Urosalpinx cin�erea e

mollusk, gastropodLOEC (4 h) (adhesion to substrate) 434 mg L�1 e e (Fong and

Molnar, 2013)Fluvoxamine Nucella ostrina e mollusk,


Molnar, 2013)ParoxetineAlgaeParoxetine Pseudokirchneriella



InvertebrateMain metabolite Daphnia magna


EC 50 (48 h) (immobilization) 35.0 mg L�1 (Cunninghamet al., 2004)

Paroxetine Ceriodaphnia dubia e



0.22 mg L�1

0.44 mg L�1(Henry et al.,2004)







Reference

Paroxetine Daphnia magnaarthropod, crustacean(waterflea)


Fish and other vertebratesParoxetine Xenopus laevis e amphibian EC10 (96 h) (deformity)

EC50 (96 h) (deformity)LC10 (96 h)LC50 (96 h)NOEC (deformity)

3.6 mg L�1

4.1 mg L�1

4.4 mg L�1

5.12 mg L�1

2.0 mg L�1


SertralineBacteriaSertraline Vibrio fischeri e bacteria EC50 (30 min) (inhibition)

NOEC (30 min) (inhibition)LOEC (30 min) (inhibition)

10.72 mg L�1

2.25 mg L�1

4.5 mg L�1

e e (Minagh et al.,2009)

AlgaeSertraline Pseudokirchneriella



4.57 mg L�1


2007)

Sertraline Scendesmus acutus echlorophyta(freshwater greenmicroalgae)


54.59 mg L�1


2007)

Sertraline Scendesmus quadricauda e



48.19 mg L�1


2007)

Sertraline Chlorella vulgaris echlorophyta(single-cell green algae)


152.73 mg L�1


2007)

Sertraline Pseudokirchneriellasubcapitata e chlorophyta(green microalgae)

EC50 (72 h) (inhibition)NOEC (72 h) (inhibition)LOEC (72 h) (inhibition)

0.14 mg L�1

0.05 mg L�1

0.075 mg L�1


Sertraline Pseudokirchneriellasubcapitata e chlorophyta(green microalgae)


InvertebrateSertraline Daphnia magna



Sertraline Ceriodaphnia dubia e



0.009 mg L�1

0.045 mg L�1(Henry et al.,2004)

Sertraline Thamnocephalus platyuruse arthropod, crustacean

LC50 (24 h)NOEC (24 h) (lethality)LOEC (24 h) (lethality)

0.6 mg L�1

0.4 mg L�1

0.6 mg L�1


Sertraline Daphnia magnaarthropod, crustacean(waterflea)

EC50 (48 h) (immobilization)NOEC (24 h) (immobilization)LOEC (24 h) (immobilization)

1.3 mg L�1

0.10 mg L�1

0.18 mg L�1

EC50 (21 d) (reproduction)NOEC (21 d) (reproduction)LOEC (21 d) (reproduction)EC50 (21 d) (lethality)NOEC (21 d) (lethality)LOEC (21 d) (lethality)

0.066 mg L�1

0.032 mg L�1

0.1 mg L�1

0.12 mg L�1

0.032 mg L�1

0.1 mg L�1

(Minagh et al.,2009)

Sertraline Ceriodaphnia dubia e


EC50 (48 h) (offspring) 126 mg L�1 LOEC (1st and 2ndgenerations) (fecundity andgrowth)LOEC (3rd generation)(fecundity and growth)EC50 (mean) (offspring)

53.4 mg L�1

4.8 mg L�1

17.2 mg L�1

(Lamichhaneet al., 2014)

Fish and other vertebratesSertraline Oncorhynchus mykiss e fish LC50 (96 h)

NOEC (96 h) (lethality)LOEC (96 h) (lethality)

0.38 mg L�1

0.1 mg L�1

0.32 mg L�1


Sertraline Pimephales promelas e fish(fathead minnow)

LC50 (48 h) pH 6.5LC50 (48 h) pH 7.5LC50 (48 h) pH 8.5

647 mg L�1

205 mg L�1

72 mg L�1

EC50 (7 d) (growth,survival) pH 6.5EC50 (7 d) (feeding rate) pH6.5EC50 (7 d) (growth,survival) pH 7.5EC50 (7 d) (feeding rate) pH7.5EC50 (7 d) (growth,survival) pH 8.5

544.4 mg L�1

199.7 mg L�1

131.4 mg L�1

149.5 mg L�1

50 mg L�1

80.3 mg L�1

(Valenti et al.,2009)

(continued on next page)







Reference

EC50 (7 d) (feeding rate) pH8.5

Sertraline Xenopus laevis e amphibian EC10 (96 h) (deformity)EC50 (96 h) (deformity)LC10 (96 h)LC50 (96 h)NOEC (deformity)

3.0 mg L�1

3.3 mg L�1

3.6 mg L�1

3.9 mg L�1

1.0 mg L�1


SSRIs in mixturesFluoxetine,

Fluvoxamine,and Sertraline

Cyanobacteria EC10 (abundance) (7 d)EC50 (abundance) (7 d)EC10 (abundance) (35 d)EC50 (abundance) (35 d)

53.3 nM1040.1 nM198.8 nM957.9 nM

(Johnson et al.,2007)

Fluoxetine,Fluvoxamine,and Sertraline

Chlorophyta EC10 (abundance) (7 d)EC50 (abundance) (7 d)EC10 (abundance) (35 d)EC50 (abundance) (35 d)

49.4 nM536.6 nM274.9 nM1169.4 nM



Heterokonts EC10 (abundance) (7 d)EC50 (abundance) (7 d)EC10 (abundance) (35 d)EC50 (abundance) (35 d)

51.4 nM3614.18 nM316.6 nM1346.3 nM



Cryptophtya/Dinophtya EC10 (abundance) (7 d)EC50 (abundance) (7 d)EC10 (abundance) (35 d)EC50 (abundance) (35 d)

15.2 nM99.6 nM152.2 nM664.4 nM


Sertraline, andFluoxetine

Ceriodaphnia dubia e


LC50 (48 h) 1.09 mM e e (Henry andBlack, 2007)

Sertraline, andParoxetine




Sertraline, andCitalopram




Fluoxetine, andParoxetine




Fluoxetine, andCitalopram




Paroxetine, andCitalopram




Fluoxetine,Citalopram,Sertraline andParoxetine




Fluoxetine andTriclosan

Dunaliella tertiolecta e

algae chlorophytaEC50 (96 h) (population cell density) 135.28 mg L�1 e e (DeLorenzo and

Fleming, 2008)Sertraline and Diphenhydramine Ceriodaphnia dubia e arthropod,

crustacean(waterflea)

LC50 (48 h) 0.433 mg L�1 EC50 (7 d) (reproduction)

0.184 mg L�1 (Goolsby et al., 2013)

EC e Effective concentration.IC e Inhibitory concentration.LC e Lethal concentration.LOEC e Lowest observed effect concentration.NOEC e No observed effect concentration.


based on the overall score and the three criteria (occurrence,ecological effects and health effects), fluoxetine was selected as acompound of concern in USA (Zenker et al., 2014). In fact, amongSSRIs, fluoxetine is the most studied and was reported as being themost acute toxic (Brooks et al., 2003a), in general, at levels of atleast one order of magnitude lower when compared with the otherSSRIs (Table 3). There is great variability in the sensitivity ofdifferent aquatic species to this SSRI, nonetheless, it affects a widerange of aquatic organisms, both vertebrate and invertebrate(Sumpter et al., 2014). Fluoxetine seems to have a stronger effect onphytoplankton than on other aquatic organisms (Fent et al., 2006),

and whereas it may affect pelagic organisms, this compound mayalso bind to sediments and affect benthic organisms (Brooks et al.,2003a). According to the available scientific data (Table 3), fluoxe-tine acute toxicity ranges from a LOEC (1.5 h activity, Gammaruspulex amphipod) of 100 ng L�1 (De Lange et al., 2006) to a LC50(96 h, amphibian Xenopus laevis) of 7.5 mg L�1 (Richards and Cole,2006). Lesser chronic toxicity data are available, nonetheless, itranges from a NOEC (56 d embryos without shell, Potamopyrgusantipodarum mollusk) of 0.47 mg L�1 (Nentwig, 2007) to a LOEC(21 d reproduction, arthropod D. magna) of 447 mg L�1 (Henry et al.,2004).


It has been recently found that significant enantioselectivity intoxicity occurs in the aquatic environment and that the differentenantiomers of fluoxetine exert different levels of toxicity onaquatic organisms at different trophic levels. Therefore, it is highlydesirable to further consider the enantiomeric differences whenevaluating the ecotoxicological effects of chiral pharmaceuticalssuch as fluoxetine (Barclay et al., 2011). Stanley et al. (2007) eval-uated endpoints such as D. magna immobilization, reproduction,and grazing rate and Pimephales promelas survival, growth, andfeeding rate and conclude that S-Fluoxetine was found to be moretoxic to sublethal standardized and behavioural endpoints in P.promelas, potentially because its primary active metabolite, S-nor-fluoxetine, is more potent than the same metabolite of R-fluoxetinein mammals. Up to a 9.4 fold difference in toxicity between enan-tiomers was observed; P. promelas growth EC10 for R- and S-fluoxetine were 132.9 and 14.1 mg L�1, respectively. Nonetheless,this was not observed for D. magna responses (Stanley et al., 2007).

Regarding norfluoxetine, its reported acute toxicity ranges froman EC50 (24 h cell volume growth, Scendesmus vacuolatus greenmicroalgae) of 189 mg L�1 (Neuwoehner et al., 2009) to a LC50 (24 h,Thamnocephalus platyurus protist) of 470 mg L�1 (Nałecz-Jawecki,2007). In Spirotox and Thamnotoxkit F tests, it was found thatnorfluoxetine is 50% more toxic than the parent compound (Celizet al., 2009).

3.2.2.1. Algae and plants. In an environmental risk assessment,using the European guideline (EMEA 2006), green algae wereidentified as the most sensitive species to fluoxetine, with a NOECof <0.6 mg L�1. From this value, a predicted no effect concentrationfor surface waters (PNECSW) of 0.012 mg L�1 was derived. The PEC/PNEC ratio was above the trigger value of 1 in worst case exposurescenarios indicating a potential risk to the aquatic compartment.Similarly, risks of fluoxetine for sediment dwelling organisms couldnot be excluded (Oakes et al., 2010). Regarding algae and plants, thelowest toxicity values (EC50 growth) were reported for P. sub-capitata at 24 and 39 mg L�1 (Brooks et al., 2003b). Nonetheless,fluoxetine, caused no visible or statistically significant phytotoxiceffects or endpoint increases over the treatment levels analysed(0e1000 mg L�1) in a study with Lemna gibba (Brain et al., 2004b).Indeed, an IC50 (96 h growth inhibition) of 4339.25 mg L�1 wasdetermined by Johnson et al. (2007) for Chlorella vulgaris, a single-cell green algae. Also, according to Neuwoehner et al. (2009),fluoxetine is an example of a compound of high therapeutic po-tency in humans that has baseline toxic effects in aquatic in-vertebrates like daphnia and in fish, but does pose an unexpectedspecific hazard to algae. Nonetheless, given the nearlyconcentration-additive effect of fluoxetine and its human mainmetabolite, norfluoxetine, not to mention potential mixture effectswith other pharmaceuticals and environmental pollutants, it isimportant not to neglect the contribution of this metabolite to therisk assessment that is as abundant and toxic as the parent com-pound. Regarding norfluoxetine, an EC50 (24 h cell volume growth)of 242 and 189 mg L�1 was reported for P. subcapitata andS. vacuolatus (green microalgae) (Neuwoehner et al., 2009).

3.2.2.2. Invertebrate. According to several studies, fluoxetine maydisrupt invertebrate endocrine systems and increase fecundity byincreasing the bioavailability of serotonin. Serotonin, which stim-ulates ecdysteroids, ecdysone, and juvenile hormones, is respon-sible for regulating oogenesis and molting in invertebrates(Flaherty and Dodson, 2005). In fact, fluoxetine was shown to be aneffective spawning inducer in zebra mussels (Dreissena poly-morpha), causing 100% of males to spawn at 1.55 mg L�1. Theconcentration required to induce a significant percentage ofspawning was as low as 15.5 mg L�1 for males and 1.55 mg L�1 for

females. More than 60% of the males spawned within the first hourof exposure (Fong, 1998). Fluoxetine also induced spawning in darkfalse mussels (Mytilopsis leucophaeata) at concentrations as low as0.31 mg L�1 (Fong andMolnar, 2008). The effects of norfluoxetine onspawning and parturition in bivalves were also investigated, and asignificant induction of spawning in zebra mussels and dark falsemussels at concentrations as low as 1.5 mg L�1, was observed.Norfluoxetine also induced significant parturition in fingernailclams (Sphaerium striatinum) at 3 mg L�1 (Fong and Molnar, 2008).Brooks et al. (2003b) found that C. dubia reproduction wasincreased when exposed to 56 mg L�1 of fluoxetine for 7 days.Flaherty and Dodson (2005) exposed D. magna to 36 mg L�1

fluox-etine, a concentration that induced zebra mussel spawning andobserved that chronic exposure (30 d) elicited a significant increasein the total number of Daphnia offspring produced.

In 96 h lab tests with freshwater mussels, fluoxetine signifi-cantly induced parturition of nonviable larvae from female Elliptiocomplanata exposed to 300 mg L�1 and 3000 mg L�1. Moreover,exposure at 300 mg L�1 and 3000 mg L�1 resulted in stimulation oflure display behaviour in female Lampsilis fasciola and Lampsiliscardium, respectively. In male E. complanata, 3000 mg L�1 signifi-cantly induced release of spermatozeugmata during a 48 h expo-sure. These results suggest that fluoxetine accumulates in musseltissues and has the potential to disrupt several aspects of repro-duction of these organisms, a faunal group recognized as one of themost imperiled in the world (Bringolf et al., 2010). The study ofS�anchez-Argüello et al. (2009) also showed that fluoxetine canaffect reproduction of freshwater molluscs; a stimulation of Physaacuta (44 d) reproduction was observed at lower concentrations(31.25 and 62.5 mg L�1), while the opposite effect was observed atthe highest treatment (250 mg L�1). According to Hazelton et al.(2013) chronic fluoxetine exposures (28 d) affected behaviour ofadult L. fasciola (lure display and foot protrusion) at concentrations3e10 times less than previously published by Bringolf et al. (2010),and much closer to concentrations commonly measured in theenvironment. Exposure (24 h) of glochidia to fluoxetine did notaffect viability duration, but likelihood of metamorphosis to thejuvenile stage significantly increased with 1 and 100 mg L�1 treat-ments. Later on Hazelton et al. (2014) conducted a 67 d exposure ofadult L. fasciola to fluoxetine up to 22.3 mg L�1 and assessed impactson behaviour and metabolism. Mussels treated with 2.5 and22.3 mg L�1 displayed mantle lures and those treated with22.3 mg L�1 were statistically more likely to have shorter time-to-movement, greater total movement, and initiate burrowing sooner.

Henry et al. (2004) reported a NOEC for fluoxetine for the meannumber of neonates produced of 89 mg L�1, whichwas similar to theNOEC reported by Brooks et al. (2003b), however, this exposurereduced the mean number of neonates produced by C. dubia. P�eryet al. (2008) also found a reproduction decrease for the mollusk P.antipodarum, observing a 28 d NOEC of 13 mg L�1. These findingswere also observed by Nentwig (2007) in a 56 d exposure test withthe same species. Therefore, fluoxetine appears to have differenteffects on fecundity and reproduction depending on the species(P�ery et al., 2008). For instance, two gastropod species, P. anti-podarum and Valvata piscinalis, presented different sensitivity tofluoxetine (42 d exposure). Perhaps this could be explained by themetabolic breakdown of fluoxetine that differs between species.Depending on the species, cytochrome P-450 dependent mono-oxygenase biotransforms, at different rates, fluoxetine into nor-fluoxetine, that has shown to be 50% more toxic than the parentcompound in 24 h lethality tests. Moreover, the lipid content ofdifferent species might also explain the bioaccumulation andconsequently, the effects of the lipophilic fluoxetine (Gust et al.,2009). Accordingly, Franzellitti et al. (2014) observed adverseoutcome pathways of fluoxetine on M. galloprovincialis mussel


physiology, after a 7 day administration encompassing a range ofenvironmentally relevant values (0.03e300 ng L�1), not solelyrelated to its mammalian therapeutic mode of action, and alsoprovided some significant advances about transduction pathwayscoupled to 5-HT receptors in mussel tissues. Fluoxetine affectedcAMP/PKA signalling, which is at the basis of energymetabolism forgonad development, heart beating, and cilia movements in mus-sels. Moreover, it lowered mRNA levels of an ABCB gene encodingtheMXR-related transporter P-glycoprotein, thus possibly reducingmussel potential to cope with further environmental stressors.Finally, it induced significant lysosome alterations, previouslyrelated to decrease of scope for growth and viability in mussels.

In a study that evaluated the multibiomarker response (assessingantioxidant enzymes activities as superoxide dismutase (SOD),catalase (CAT) and glutathione-S-transferase (GST); lipid peroxida-tion (LPO), acetylcholinesterase (AChE) neurotoxic response andendocrine disruption through alkali-labile phosphates (ALP) indirectmeasurement of vitellogenin-like proteins) on mussels, M. gallo-provincialis, during two weeks exposure to 75 ng L�1 of fluoxetine,results showed clearly ALP levels inhibition throughout time, in bothsex-differentiated gonads, what gives evidence to its reinforced ac-tion as an endocrine disruptor rather than an oxidative or neurotoxicinducer (Gonzalez-Rey and Bebianno, 2013).

Regarding survival, and growth, Chironomus tentans (42 d) wasmore sensitive to fluoxetine than Hyalella azteca (10 d). A plausibleexplanation for greater C. tentans sensitivity to fluoxetine isincreased exposure via ingestion of sediments. Because H. azteca isepibenthic, it is likely that dietary exposure to fluoxetine is lessthan that C. tentans would experience during sediment burrowing(Brooks et al., 2003b).

Recently, the study of Luna et al. (2013) also demonstrated thatfluoxetine affected the life history traits andpopulationgrowth ratesof the freshwater pulmonate snail Physa pomilia at environmentallyrelevant concentrations and, for the first time, the effects of lowdoses (1e100 ng L�1) of fluoxetine on defensive behaviours andbrain chemistry in young cuttlefish (Sepia officinalis) chronicallyexposed (2 weeks) at early life stages were described by Di Poi et al.(2014). At low dose, fluoxetine significantly affected cryptic perfor-mances of cuttlefish in a transitorymanner at hatching,whereas theanimals seemed to recover when the exposure was pursuing post-hatching. However, the deviation in the behaviours of young ani-mals isworrying because hatching is themost critical time in the lifeof S. officinalis.Whenexposed to10 and100ng L�1offluoxetine, timespent on activity by G. pulex was reduced by <20%. These concen-trations are much lower than previously established effect concen-trations for other organisms, indicating that behaviour is a muchmore sensitive endpoint than growth or survival. Environmentalconcentrations found in surfacewaters are high enough to reduceG.pulex activity. Whereas exposure to higher concentrations did notaffect the percentage of time spent at activity (De Lange et al., 2006).Accordingly, fluoxetine also significantly altered phototaxis andgeotaxis activity of the marine amphipod, Echinogammarus marinuswith the greatest behavioural changes observed at 100 ng L�1. Themain patterns of these behavioural responses were consistent be-tween twotrials and the3weeks exposurewith specimens spendingmore timewithin the light andoccurringhigher in thewater column(Guler and Ford, 2010). Fluoxetine also had a significant impact onthe behaviour and neurophysiology of this amphipod at environ-mentally relevant concentrations as low as 1 ng L�1, with effectsobserved after relatively short periods of time, after 1 day exposure(Bossus et al., 2014).

3.2.2.3. Fish and other vertebrates. Mennigen et al. (2011) havedescribed the evolutionary conservation of the 5-HT transporter,the therapeutic target of SSRIs, and reviewed the disruptive effects

of fluoxetine on several physiological endpoints of fish, includinginvolvement of neuroendocrine mechanisms. As above mentioned,in fish, fluoxetine has the potential of affecting processes such asbranchial nitrogen excretion and intestinal osmoregulation(Morando et al., 2009), and also constricting the arterio-arterialbranchial vasculature, leading to impaired gas exchange and hyp-oxia, ultimately leading to death (Richards et al., 2004). As shownbelow, several investigations, some of those very recently, havestudied the impact of this SSRI in fish, evaluating different end-points such as reproduction, physiology, and behaviour of fishspecies.

To assess the effects of fluoxetine on reproductive anatomy,physiology, and behaviour, adult male fathead minnows (P. prom-elas) were exposed for 21 d to different SSRIs including fluoxetine.Anatomical alterations were noted and, additionally, at 28 ng L�1

fluoxetine induced vitellogenin inmalefish, a common endpoint foroestrogenic endocrine disruption. Moreover, significant alterationsinmale secondary sex characteristics were also noted (Schultz et al.,2011). Conversely, another study also determined its potential todisrupt teleost reproductive function of Japanese medaka (Oryziaslatipes). Fish were exposed to fluoxetine at 0.1, 0.5,1 and 5 mg/L for 4weeks, nonetheless, no significant changes were observed in eggproduction, rate of fertilization and spawning, or hatching successof fertilized eggs. Adult gonadal somatic index, hepatic vitellogenin,and ex vivo gonadal steroidogenesis were also unaffected. A lowincidence (1.97e2.53%; 4.02e5.16 fold greater than controls) ofdevelopmental abnormalities was observed in offspring from allfluoxetine treatments (Foran et al., 2004). Accordingly, the resultsprovided by Lister et al. (2009) suggest that fluoxetine may disruptreproductive functioning in zebrafish (Danio rerio) at concentra-tions greater than those found in receiving environments.Compared with the control groups, fluoxetine (32 mg L�1) and 50%effluent treatment significantly reduced the average eggs spawnedby approximately 4.5 and 2 fold, respectively, and also decreasedovarian levels of 17 b-oestradiol without affecting the gonadoso-matic indices of the fish. Another study also demonstrated thatchronic exposure (91 d) of western mosquito fish (Gambusia affinis)to fluoxetine can affect sexual development; however, it does soonly at concentrations 3 to 4 orders of magnitude higher than thosepreviously found in the environment. Chronic exposure did notaffect survival, growth, or sex ratio; however, increased lethargy infish exposed to �0.5 mg L�1 of fluoxetine was observed. In fishexposed from age 59e159 days (juvenile to adult life stages),delayed development of external adult sexual morphology wasobserved at 71 mg L�1

fluoxetine (Henry and Black, 2008).The in vitro interference of fluoxetine with key enzymatic ac-

tivities, C17,20-lyase and CYP11b, involved in the synthesis of activeandrogens in gonads of male carp have also been investigated.Fluoxetine was the strongest inhibitors of C17,20-lyase and CYP11benzymes, with IC50s in the range of 244e550 mM. As oxy-androgens are known to influence spermatogenesis and stimulatereproductive behaviour and secondary sexual characteristics inmale fish, this work highlights the need for further investigatingthese endpoints when designing specific in vivo studies to assessthe endocrine disruptive effect in fish (Fernandes et al., 2011).Caminada et al. (2006) determined the cytotoxicity of fluoxetine inPLHC-1 (Poeciliopsis lucida hepatoma cell) and RTG-2 (rainbowtrout gonadal cell line) and observed that RTG-2 cells seemed to bemore sensitive. This SSRI was also found to inhibit CYP1A (carp andtrout) and CYP3A-like (carp) catalysed activities in vitro studies ofcarp liver microsomal fractions or primary rainbow trout hepato-cytes in a dose-dependent manner after 6 h of incubation (Thibautand Porte, 2008). The early molecular effects of SSRIs, includingfluoxetine, on the Na/K-dependent ATPase pump activity in brainsynatosomes were investigated in vitro and in fish exposed to the


municipal effluents. The Na/K-ATPase activity was significantly andnegatively correlatedwith brain tissue concentrations of fluoxetine,demonstrating its potential biological effects and corroboratingprevious findings on the serotonergic properties of municipal ef-fluents to aquatic organisms (Lajeunesse et al., 2011).

Regarding behaviour, a research characterized the impact ofsublethal fluoxetine exposures on the ability of hybrid striped bass(Morone saxatilis � Morone chrysops), exposed to fluoxetine for 6 d,followed by a 6 d recovery period in cleanwater, to capture fatheadminnows (P. promelas). Exposed fish exhibited a concentration andduration dependent decrease in ability to capture prey. Increasedtime to capture prey also correlated with decreases in brain sero-tonin activity; moreover, serotonin levels in exposed fish did notrecover to control levels during the 6 d recovery period. These re-sults suggest that sublethal exposure to fluoxetine decreases theability of hybrid striped bass to capture prey and that serotonin canbe used as a biomarker of exposure and effect (Gaworecki andKlaine, 2008). Exposure (28 d) of goldfish (Carassius auratus) tofluoxetine at 54 mg L�1, led to a significant decrease in food intakeand weight gain. Furthermore, a significant decrease occurred incirculating glucose levels in the group exposed to 540 ng L�1

fluoxetine (Mennigen et al., 2010).Recently, in 2014, was demonstrated that 4 week exposure of P.

promelas to environmentally relevant concentrations of fluoxetinehave an impact on specific behaviours important to reproductionand predator avoidance that are dose dependent and more pro-nounced in males. Moreover, they may also be brain region specificas not all behaviours are impacted at the same exposure dose andmales and females do not demonstrate the same effects. Forinstance, concentrations as low as 1 mg L�1, were found to signifi-cantly impact mating behaviour, specifically nest building anddefending in male fish. In addition, predator avoidance behavioursin males and females were also impacted at the same exposurelevel (Weinberger and Klaper, 2014).

In amphibians a decrease in food intake and consequent delay indevelopment was also observed at ecologically relevant levels(Foster et al., 2010).

3.3. Fluvoxamine

3.3.1. Uptake and bioaccumulationThe first published detection of fluvoxamine in wild fish was

reported by Gelsleichter and Szabo (2013), although it has beensurveyed in past investigations by Schultz et al. (2010). According toGelsleichter and Szabo (2013) fluvoxamine was observed at meanlevels of 0.83 ngmL�1 in plasma of bull sharks (C. leucas) residing inpristine and wastewater impacted rivers.

3.3.2. Effects and toxicityAs above mentioned, Fong (1998) studied the effect of SSRIs on

zebra mussel spawning. Fluvoxamine inducted spawning ofboth sexes at concentration as low as 10�5 M with the rankorder of toxicity (lowest to highest) beingparoxetine < fluoxetine < fluvoxamine. Fluvoxamine inducedspawning in 100% of both sexes of zebra mussels (D. polymorpha) at3.2mg L�1 and 0.32mg L�1, for females andmales, respectively. Theconcentration that induced a significant percentage of animals tospawnwas as lowas 0.32 mg L�1 formales and 32 mg L�1 for females.The lowest concentration of fluvoxamine to induce spawning was3.2 mg L�1 for females (40%) and 0.032 mg L�1 for males (20%).Gametes spawned in fluvoxamine (3.2 mg L�1 and lower) wereviable, and swimming trochophores were formed within 20 h.More than 60% of the males spawned within the first hour ofexposure. When compared to fluoxetine and to paroxetine, flu-voxamine was the most powerful spawning inducer in any bivalve

(Fong, 1998).Conversely, Henry et al. (2004) found a difference in the relative

rank order potency for SSRIs with the order of toxicity (lowest tohighest) beingcitalopram < fluvoxamine < paroxetine < fluoxetine < sertralinewith the cladoceran C. dubia. These authors performed both acuteand chronic toxicity studies and reported an LC50 (48 h) of0.84 mg L�1 and a NOEC (8 d) on the neonates produced of0.366 mg L�1. This order of toxicity was also observed for algae inthe study of Johnson et al. (2007). These authors performed acutetoxicity (96 h) tests on different species of greenmicroalgae and theEC50 (growth inhibition) ranged between 3563.34 and10,208.47 mg L�1. Accordingly, in the study of Christensen et al.(2007) with P. subcapitata and with D. magna, EC50s were of0.062 and 13 mg L�1, respectively. According with the previousreferred authors, fluvoxamine was ranked in the third place onwhat regards SSRIs toxicity. This compoundwasmore toxic to algaethan to daphnids.

A small but significant decrease in the viability of fish hepatomacell lines PLHC was observed in cells incubated with 20 mM flu-voxamine for 6 h (9e21%). The maximal induction response forfluvoxamine was observed at a concentration of 1 mM (13%).However, cell viability returned to control levels with longerexposure times (Thibaut and Porte, 2008). Conversely, fluvoxaminewas reported as one of the strongest inhibitors of C17,20-lyase andCYP11b enzymes, involved in the synthesis of active androgens ingonads of male carp, with IC50s in the range of 321e335 mM(Fernandes et al., 2011).

3.4. Paroxetine

3.4.1. Uptake and bioaccumulationGelsleichter and Szabo (2013) found paroxetine at mean levels

of 0.55 ng mL�1 in plasma of bull sharks (C. leucas) residing in awastewater impacted river, Caloosahatchee River from South Flor-ida, USA. Chu and Metcalfe (2007) reported detectable levels ofparoxetine ranging from ND to 0.58 ng/g in whole body homoge-nates of fish from Hamilton Harbor, Lake Ontario, Ontario, Canada,whereas Schultz et al. (2010) reported similar levels (i.e., rangingfrom ND-0.113 ng g�1) of this compound in brains of C. commersonifrom two creeks in Colorado, USA. Lajeunesse et al. (2011) analysedbrook trout exposed to 20% v/v of effluent. The highest concen-tration of paroxetine was retrieved in liver tissue at 0.35 ng g�1,with a BCF of 365, whereas in brain levels of 0.19 ng g�1 were found,with a BCF of 198. As seen, the highest BCFs were retrieved for livertissue extracts.

3.4.2. Effects and toxicityThe toxicity of paroxetine toward environmental organisms has

also been tested in a limited number of studies. Paroxetine was aweak spawning inducer of zebra mussels (D. polymorpha). At con-centrations of 3.29mg L�1 and 329 mg L�1, it induced significant, butlow (50% and 40%, respectively) percentages of males to spawn.Paroxetine did not induce significant spawning in females (Fong,1998). Henry et al. (2004) tested the acute toxicity of paroxetineon freshwater crustacean C. dubia and found LC50s of 0.58 mg L�1.Chronic toxicity studies (8 d test) were also performed and a NOECon the neonates produced of 0.22 mg L�1 was reported.Cunningham et al. (2004) tested the toxicity of paroxetine mainmetabolite to D. magna and found higher toxicity values, EC50(immobilization) of 35 mg L�1, than observed for paroxetine in thestudy of Christensen et al. (2007), with an EC50 (biomass) of6.3 mg L�1.

Paroxetine was also tested with eggs produced by X. laevis. Theinitial malformation in embryos exposed to paroxetine regarded


tail flexures beginning at 4.0 mg L�1. The EC10 was calculated to be3.6 mg L�1 and the EC50 was 4.1 mg L�1. The LC10 and LC50 valueswere determined to be 4.4 and 5.12 mg L�1, respectively. One-hundred percent embryo lethality occurred at 7.0 mg L�1. Theconcentrations shown to be toxic in the present study are orders ofmagnitude above that which are currently detected surface waters(Richards and Cole, 2006).

This SSRI also exerted cytotoxic effects on fish hepatoma celllines, and the cell viability decreased from 70% to 6%, after 24 h ofexposure to 20 mM paroxetine. The increased cytotoxicity of par-oxetine over time suggests either increased bioaccumulation orincreased metabolism that leads to the production of toxic entities(Thibaut and Porte, 2008).

3.5. Sertraline

3.5.1. Uptake and bioaccumulationRecently sertraline was found to accumulate in crabs, Carcinus

maenas, collected in Portugal from a moderately contaminated(Lima) and a low-impacted (Minho) estuary, after a 7 day exposure.Accumulation in crabs' soft tissues was found in Lima (5 mg L�1:15.3 ng g�1; 500 mg L�1: 1010 ng g�1) and Minho (500 mg L�1:605 ng g�1) animals. The exposure to waterborne elicited tissueaccumulation from both study sites, but clearly higher (almostdouble) in those from the moderately polluted Lima site. Lowdesmethylsertraline/sertraline ratios were found in crabs from bothsites (0.19 in Minho and 0.11 in Lima crabs), supporting the hy-pothesis of low metabolization and/or elimination by these in-vertebrates (Rodrigues et al., 2015).

In fish, sertraline was found at mean levels of 0.48 ng mL�1 in 10out of 10 plasma samples of bull sharks (C. leucas) residing in awastewater impacted river, Caloosahatchee River from South Flor-ida, USA (Gelsleichter and Szabo, 2013). Previously, sertraline hasbeen measured at mean levels of 0.34 ng g�1. in muscle of bluegill(Lepomis macrochirus), channel catfish (Ictalurus punctatus), andblack crappie (Pomoxis nigromaculatus) from the effluent domi-nated Pecan Creek in Denton County, Texas, USA (Brooks et al.,2005), and at levels ranging from not detected to 19 ng g�1 inresident fish species from rivers in 5 large metropolitan cities in theU.S. (Ramirez et al., 2009). Sertraline concentrations were shown tobe higher in liver from fish examined by Brooks et al. (2005) (meanof 3.59 ng g�1) and by Ramirez et al. (2009) (maximum of545 ng g�1), observations consistent with the role of this organ asthe primary site of xenobiotic metabolism. Brain concentrations ofsertraline found by Brooks et al. (2005) (mean of 4.27 ng g�1) andby Schultz et al. (2010), in the white sucker of C. commersoni, fromBoulder Creek, Colorado, USA and Fourmile Creek, Iowa, USA(427 ng g�1), have also been shown to exceed those observed inmuscle. This suggests selective uptake of this compound into braintissue and raises concern about the potential serotonergic effectsthat it may have in wild fish. According to Ramirez et al. (2009),sertraline consistently displayed the greatest maximum concen-tration among the other pharmaceuticals studied, including nor-fluoxetine, in both fillet and liver tissues.

Analysis of brain fish tissues from adult male fathead minnows(P. promelas) suggested increased uptake of sertraline whencompared to water concentrations (Schultz et al., 2011). Accord-ingly, the study of Lajeunesse et al. (2011), that examined the tis-sues distribution SSRIs in brook trout, exposed for 3 months tocontinuous flow-through primary-treated effluent before and afterozone treatment, reported that sertraline and its metabolite des-methylsertraline were the predominant substances observed inmost tissues (0.04e10.3 ng g�1), in decreasing order:liver > brain > muscle. Bioaccumulation studies also revealed thatthe N-desmethyl metabolite was mostly measured at higher

concentrations in liver tissues than the parent compound. Theconcentration of desmethylsertraline in liver tissue found byLajeunesse et al. (2011) (10.3 ± 0.8 ng g�1) was consistent with theconcentration previously reported by Brooks et al. (2005)(12.9 ± 10.5 ng g�1). Schultz et al. (2010) have reported lowerconcentrations of desmethylsertraline (1.8e3.0 ng g�1) in brainhomogenates from native white suckers species exposed down-stream to outfalls discharging of two effluent-impacted streamslocated in Colorado and Iowa, USA. The origin of metabolitesdetected in fish tissues could be assigned to direct uptake or byin vivometabolism (Lajeunesse et al., 2011). Lajeunesse et al. (2011),estimated a BCF for 20% v/v effluent mesocosm, retrieved for livertissue extracts, for sertraline and desmethylsertraline, of 264 and12,250, respectively. For brain, lower BCFs were retrieved, 2476 and191, respectively. Nevertheless, according to Lajeunesse et al.(2011), the remarkable efficiency observed with ozone treatmentcertainly decreases the bioavailability of antidepressants to fishexposed to wastewater effluents.

3.5.2. Effects and toxicityAs above mentioned, Henry et al. (2004) investigated the acute

and chronic toxicity of five SSRIs, including sertraline, in C. dubia.48 h LC50 and chronic (8 d) tests were conducted to determineNOECs and LOECs for reproduction endpoints. The 48 h LC50 forsertraline was 0.12 mg L�1, and mortality data for the 8 d chronictests were similar to the 48 h acute data. Nonetheless, for sertraline,the most toxic SSRI, the LOEC for the number of neonates per fe-male was 0.045 mg L�1 and the NOEC was 0.009 mg L�1. Theseresults indicate that sertraline, as the other SSRIs, can impact sur-vival and reproduction of C. dubia; however, only at concentrationsthat are considerably higher than those expected in theenvironment.

On the contrary, others argue that adverse effects may resultfrom lower concentration exposures, and that further research intochronic toxicity is advocated (Minagh et al., 2009). Johnson et al.(2007) reported green algae 96 h growth inhibitory concentra-tions (IC50) ranging between 12.10 and 763.66 mg L�1. Accordingly,48 h biomass EC50 were reported by Christensen et al. (2007),0.043 mg L�1. In a 7 d study with Lemna gibba (an aquatic higherplant), sertraline caused no statistically significant phytotoxicresponse over the concentration range utilized (0.33 and452.51 mg L�1). Nonetheless, caused a slight increase in wet mass,which was found to be significant (p ¼ 0.0032) at 300 and1000 mg L�1 treatment levels (Brain et al., 2004a). During theevaluation of sertraline using a battery of freshwater species, thespecies most sensitive were D. magna 21 d reproduction test (NOEC0.032 mg L�1), P. subcapitata 72 h growth inhibition (NOEC0.05 mg L�1), and O. mykiss 96 h lethality (NOEC 0.1 mg L�1)(Minagh et al., 2009). Chronic exposure of C. dubia to sertraline ledto fecundity and growth effects that occur at concentrations (LOEC1st and 2nd generations 53.4 mg L�1) only an order of magnitudehigher than predicted environmental concentrations. Moreover,standard toxicity tests do not account for increases in sensitivity insuccessive generations to toxicants, and multigenerational effects(LOEC 3rd generation 4.8 mg L�1) should be considered(Lamichhane et al., 2014).

Rodrigues et al. (2015) investigated sertraline accumulation andeffects in crabs C. maenas collected from a moderately Portuguesecontaminated (Lima) and a low-impacted (Minho) estuary andexposed them to environmental and high levels of sertraline (0.05,5, 500 mg L�1). A battery of biomarkers related to sertraline mode ofaction was employed to assess neurotransmission, energy meta-bolism, biotransformation and oxidative stress pathways. After aseven-day exposure, it was observed that Lima crabs were alsomore sensitive to sertraline than those from Minho, exhibiting


decreased acetylcholinesterase activity, indicative of ventilatoryand locomotory dysfunction, inhibition of anti-oxidant enzymesand increased oxidative damage at concentrations �0.05 mg L�1,depicting an influence of the exposure history on differentialsensitivity and the need to perform evaluations with site-specificecological receptors to increase relevance of risk estimationswhen extrapolating from laboratory to field conditions.

To assess the effects of sertraline on fish reproductive anatomy,physiology, and behaviour, adult male fathead minnows (P. prom-elas) were exposed for 21 d to environmental relevant concentra-tions. The data demonstrated that this SSRI induced anatomicalalterations, significant alterations in male secondary sex charac-teristics, and that at 5.2 ng L�1 resulted in mortality. This studydemonstrated that anatomy and physiology, but not reproductivebehaviour, can be disrupted by exposure to environmental con-centrations of sertraline (Schultz et al., 2011). Hedgespeth et al.(2014) performed 7 day feeding trials using juvenile Eurasianperch (Perca fluviatilis) exposed to sertraline and observed that thisSSRI at concentrations of 89e300 mg L�1 alters foraging ecology ofthe fish in terms of their functional response, decreasing feedingwith increasing sertraline concentrations, at both low and highprey densities, indicating effects on both attack rate and handlingtime, respectively, that may alter the stability of predatorepreysystems and consequently, community structure.

Reduced feeding rates were observed in SSRIs exposed tadpoles(X. laevis), and nutritional status can influence growth and devel-opment in amphibians via effects on the neuroendocrine system.But, on the contrary to fluoxetine, sertraline was capable of causingdevelopmental toxicity in tadpoles at environmentally relevantconcentrations of 0.1 and 1 mg L�1 (Conners et al., 2009). Carfagnoand Fong (2014) also tested the effects of sertraline on developmentof wood frog (Lithobates sylvaticus) larvae when wild-collected eggmasses were exposed to concentrations ranging from 0 to10.0 mg L�1. Time to metamorphosis did not differ between treat-ments, but sertraline-exposed tadpoles grew less when raisedamong conspecifics. However, this effect was not detected whenthe study was repeated with tadpoles raised individually. This in-dicates that the effects of sertraline and other SSRIs may beenhanced when tadpoles are raised under more stressfulconditions.

Moreover, 48 h and 7 d toxicity tests were undertaken with P.promelas exposed to sertraline over a gradient of environmentallyrelevant surface water concentrations and pHs. For the 48 h ex-periments the LC50 values were 647, 205, and 72 mg L�1 at pH 6.5,7.5, and 8.5, respectively. Survivorship, growth, and feeding rate(non-traditional endpoints linked by others to sertraline's specificmode of action) were monitored during the 7 d experiment, andadverse effects were more pronounced when individuals wereexposed to sertraline at pH 8.5 (growth and survival EC50 of50 mg L�1, and feeding rate EC50 of 80.3 mg L�1) compared to pH 7.5(growth and survival EC50 of 131.4 mg L�1, and feeding rate EC50 of149.5 mg L�1) and 6.5 (growth and survival EC50 of 544.4 mg L�1, andfeeding rate EC50 of 199.7 mg L�1) (Valenti et al., 2009).

Sertraline was also shown to have a significant impact afterrelatively short periods of time on the behaviour and neurophysi-ology of Echinogammarus marinus, an amphipod, exposed at envi-ronmentally relevant concentrations from 0.001 to 1 mg L�1 duringshort-term (1 h and 1 day) andmedium-term (8 days) experiments.The behavioural analysis revealed a significant effect on movementvelocity which was observed after 1 h exposure to sertraline at0.01 mg L�1 (Bossus et al., 2014).

Finally, the early molecular effects of sertraline on the Na/K-dependent ATPase pump activity in brain synatosomes were alsoinvestigated in vitro and in fish exposed to the municipal effluents.The Na/K-ATPase activity was significantly and negatively

correlated with brain tissue concentrations of desmethylsertraline,and sertraline, demonstrating their potential biological effects andcorroborating previous findings on the serotonergic properties ofmunicipal effluents to aquatic organisms (Lajeunesse et al., 2011).

3.6. SSRIs in mixtures

Most of the bioassays and testing in the literature have focusedon the toxicological properties of individual SSRIs, but the need fordata relating to mixtures of pharmaceuticals has been identifiedsome years ago (Johnson et al., 2007) (Table 3). Therefore, in riskassessment based on chemical analysis of environmental samples,it is important to include the effect of all SSRIs that are present atlow concentrations, and the model of concentration addition maybe used to predict the combined effect of the mixture of SSRIs(Christensen et al., 2007).

Since SSRIs share the same pharmacological mode of action, aconcentration addition model may be expected (Christensen et al.,2007; Henry and Black, 2007; Johnson et al., 2007) and wasdemonstrated for fluvoxamine, fluoxetine and sertraline (Johnsonet al., 2007). According to Henry and Black (2007), concentrationaddition also adequately modelled the toxicity of mixtures of SSRIs(fluoxetine, citalopram, sertraline and paroxetine), and indicatedthat individual SSRI components may act through a similar mech-anism of toxic action. Conversely, Schultz et al. (2011), found thateffects of single compound exposures neither carried over, norbecame additive when adult male fathead minnows (P. promelas)were exposed for 21 days to an antidepressant mixture of fluoxe-tine and sertraline.

Because pharmaceuticals are continuously released at lowconcentrations into the environment and likely are present ascomplex mixtures, it is relevant to perform toxicity tests withmixtures of compounds belonging to different therapeutic groups(Richards et al., 2004). For instance, although fluoxetine, caused novisible or statistically significant phytotoxic effects or endpointincreases over the treatment levels analysed (0e1000 mg L�1) in astudy with Lemna gibba (Brain et al., 2004b), Richards et al. (2004)observed that, when exposed, for 35 days, at low (6e10 mg L�1),medium (60e100 mg L�1) and high (600e1000 mg L�1) concentra-tion treatments with ibuprofen (a nonsteroidal anti-inflammatorydrug), fluoxetine and ciprofloxacin (antibiomicrobial), Lemnagibba and Myriophyllum spp. showed mortality in the high con-centration treatment and that the growth of L. gibba was alsoreduced in the medium concentration treatment. Moreover, the96 h EC50 value (135.28 mg L�1) when Dunaliella tertiolecta, achlorophyta algae, was exposed to the mixture of triclosan andfluoxetine demonstrated additive toxicity (DeLorenzo and Fleming,2008). Finally, Goolsby et al. (2013), found additive effects ofdiphenhydramine and sertraline both when C. dubia was exposedfor 48 h (LC50 of 0.433 mg L�1) and for 7 days (EC50 on repro-duction of 0.184 mg L�1).

4. Final remarks and future needs

As mentioned SSRIs modulate the neurotransmitter serotonin,which, even at trace levels, regulates a wide range of physiologicalsystems in the aquatic biota. The reviewed literature data showedthat these antidepressants have remarkable effects on differenttrophic levels of non-target organisms, such as algae, plants,invertebrate and vertebrate, and that their uptake and bio-accumulation in fish is a reality.

The actual exposure scenario posing the most uncertaintyregarding SSRIs ecotoxicology is long-term and multi-generationalexposure at environmental relevant concentrations. Moreover,many of the toxicology studies regard only the parent compound,


with very little attention given to the potential contributions thatmetabolites may have. The importance of considering the enan-tiospecifictoxicity of chiral SSRIs also needs to be emphasized inecotoxicity studies. Knowledge on the consequences of a lifelongexposure to mixtures of low levels is also mandatory since SSRIs donot occur individually but in mixtures with other pharmacologi-cally active compounds that share similar modes of action, result-ing in possible additive or synergistic effects. Finally, more studiesare needed regarding specific molecular targets, such as serotonintransporter and receptors, and interactions with other hormonaland neurotransmitter systems.

Acknowledgements

The authors thank Fundaç~ao para a Ciencia e a Tecnologia (FCT)and FEDER/POCTI (project and fellowship PTDC/AAC-AMB/120889/2010) for the financial support. The authors also gratefully recog-nize the FCT for a post-PhD fellowship granted to L.J.G. Silva (SFRH/BPD/62877/2009).

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reviewing the serotonin reuptake inhibitors (ssris) footprint in the aquatic biota: uptake,...

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