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Transgenerational effects of two antidepressants (sertraline and venlafaxine) on Daphnia magna life history traits Laetitia Minguez, celine ballandonne, Christiane Rakotomalala, Christelle Dubreule, Valérie Kientz-Bouchart, and Marie-Pierre Halm-Lemeille Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es504808g • Publication Date (Web): 15 Dec 2014 Downloaded from http://pubs.acs.org on December 20, 2014
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Environmental Science & Technology
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Transgenerational effects of two antidepressants (sertraline and venlafaxine) on
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Daphnia magna life history traits
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Laëtitia Minguez1,2,*, Céline Ballandonne2, Christiane Rakotomalala1, Christelle Dubreule3,
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Valérie Kientz-Bouchart3, Marie-Pierre Halm-Lemeille2
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1
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Normandie, SFR ICORE, IBFA, Esplanade de la Paix, CS 14032, 14032 Caen cedex, France
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2
UMR BOREA, MNHN, UPMC, UCBN, CNRS-7208, IRD-207, Université de Caen Basse-
CERMN, UFR des Sciences Pharmaceutiques, UPRES EA4258 - FR CNRS INC3M – SF
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4206 ICORE, Université de Caen Basse-Normandie, Bd Becquerel, 14032 Caen cedex,
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France
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LABÉO Franck Duncombe, Saint-Contest, 14053 Caen, FRANCE
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* Corresponding author at: UMR BOREA, MNHN, UPMC, UCBN, CNRS-7208, IRD-207,
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Université de Caen Basse-Normandie, SFR ICORE, IBFA, Esplanade de la Paix, CS 14032,
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14032 Caen cedex, France.
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Tel.: +33 0231565294; fax: +33 0231565346.
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Email address:
[email protected] (L. Minguez).
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TOC/Abstract Art Day 1
Day 21 S: More offspring V: Few offspring
F0
F1
Brood 2 Exposure
Brood 5 Recovery
Exposure
14 days
21
S:
No significant effects
V:
No significant effects
Recovery 14 days
Few offspring
Related to the maternal exposure concentration
No significant effects
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ABSTRACT
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The low levels of antidepressants detected in surface waters currently raise concern about
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their potential long-term risks to non-target aquatic organisms. We investigated the
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transgenerational effects of sertraline, a selective serotonin reuptake inhibitor, and
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venlafaxine, a serotonin-norepinephrine reuptake inhibitor, on the life traits of Daphnia
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magna over two generations under environmentally realistic concentrations. We also studied
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the reversibility of the effect using recovery experiments. We assessed daphnid survival,
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growth and reproduction over 21 days and evidenced detectable effects of the antidepressants.
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Sertraline increased the F0-daphnid fecundity whereas it decreased the offspring number of
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F1-daphnids. Transfer to clean medium caused negative effects on the offspring of daphnids
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exposed to 0.3 µg L-1, but improved the fecundity of offspring of daphnids exposed to 100 µg
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L-1. Venlafaxine exposure decreased the offspring number of F0-daphnids and resulted in
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drug tolerance in the F1 generation. Sertraline, unlike venlafaxine, may turn out to be a true
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environmental threat due to its accumulation in algae and the physiological weakness
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observed over generations. These effects across generations point out to the need to perform
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multi-generation tests to assess the environmental risk of pharmaceuticals in non-target
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organisms.
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INTRODUCTION
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The improvement of analytical methods now makes it possible to detect the presence of
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pharmaceutical residues in the environment. These compounds are prescribed for medical and
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veterinary purposes. They are designed to strongly interact with biological systems and thus
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provide beneficial effects.1 Their presence in aquatic ecosystems may represent a threat to
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non-target aquatic organisms. More and more studies point to their toxicity to freshwater and
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marine organisms, but mainly by performing acute toxicity tests (reviewed by Fent et al.1 and
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Santos et al.2). Few studies report chronic toxicity data (reviewed by Santos et al.2).
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Nonetheless, given their continuous discharge into the environment, organisms are exposed
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throughout their entire lifetimes over several generations. Studies assessing transgenerational
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effects through multi-generational exposure are needed to have a more ecologically-relevant
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vision of the impact of pharmaceutical residues on ecosystems.3 Studies investigating the
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multi-generational effects of pharmaceuticals are still scarce3–8 and often provide
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contradictory results. For example, fecundity decreased and/or mortality increased in the
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second generation of Daphnia magna exposed to environmental estrogens, like 4-
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nonylphenol, diethylstilbestrol and 17β-estradiol.9 By contrast, second-generation D. magna
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were less sensitive to endocrine-active pharmaceuticals and developed resistance to these
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contaminants.5 As pointed out by Dietrich et al.6, these contradictory studies emphasize the
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need to perform multigenerational tests, as effects could be under- or overestimated if
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experiments target only one generation.
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The cladoceran Daphnia magna is a suitable biological model for multi-generational studies
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because of its short life span, high fecundity, parthenogenetic reproduction and easy
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laboratory handling.10 It is therefore widely used in the assessment of pollutant effects and
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ecological risk assessment procedures.11–12 However, the chronic tests in the OECD 211
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guideline13 last over only one generation. The “maternal effects” (i.e. all the information
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about environmental disturbances transmitted from mothers to their offspring14) and the health
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status of the offspring are not taken into account. Nonetheless, Daphnia parental exposure can
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lead to higher sensitivity or acclimation/adaptation of the following generation.14–16 Since
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Daphnia spp. play a critical role in aquatic food webs by serving as an intermediate between
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primary producers and fishes, changes in their life history over several generations could
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trigger responses at the community- or ecosystem-levels.17
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Antidepressants used for the treatment of clinical depression and other psychiatric disorders,
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i.e. selective serotonin reuptake inhibitors (SSRIs) and serotonin and norepinephrine reuptake
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inhibitors (SNRIs), represent a potential threat to aquatic ecosystems. These drugs act by
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blocking the reuptake of serotonin and/or norepinephrine at the level of the nerve synapses; as
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a result, the effective concentration of these neuromediators increases in the intrasynaptic
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space and stimulates serotoninergic and noradrenergic neurons.18 For example, in crustaceans
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neuromediators control a wide variety of biological functions including reproduction, growth,
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immune function, and metabolism. They also stimulate the release of other hormones like
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hyperglycaemic and gonad-stimulating hormones. Thus, any chemicals in the environment
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with the ability to modulate these neuromediators can disrupt the normal endocrine and
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biological function in exposed organisms (reviewed by Fong and Ford19).
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The Organization for Economic Co-operation and Development20 reports a significantly
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increasing consumption of antidepressants in most OECD countries since 2000. In 2012,
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antidepressants ranked third in the list of the most frequently prescribed drugs.20 Due to the
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continuously increasing use of antidepressants, greater concentrations of these compounds are
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likely to be present in the environment. For example, sertraline, an SSRI, and venlafaxine, an
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SNRI, were the second and the tenth most prescribed psychiatric drugs in the United States in
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2013, respectively.21 Concentrations of sertraline in wastewater effluents ranged from 1 ng L-1
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to 0.6 µg L-1 22–23, but reached 17 µg L-1 in untreated sewage.24 Venlafaxine is often measured
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at concentrations up to 10 times higher than sertraline. Schultz and Furlong22 measured
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venlafaxine up to 1,000 ng L-1 downstream from treatment plants in Texas but as much as
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2.19 µg L-1 in wastewater effluents. In addition to their presence in waters, sertraline and
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venlafaxine have been shown to accumulate in tissues of several aquatic organisms like fishes
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(reviewed by Daughton and Brook25), mussels26, crabs27, insect larva and leeches28, and
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algae29.
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Although sertraline and venlafaxine are detected in aquatic ecosystems, to our knowledge
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only one study has assessed the effects of sertraline over generations3 and no such studies
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exist for venlafaxine. Nonetheless, studies assessing the effects of these two compounds on
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non-target organisms show significant effects on metabolic responses30, survival3,7,31–34,
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reproduction3,31–32,35, and behaviors (e.g. the escape response33; predation36; moving
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velocity37) at concentrations similar to environmental levels. These effects on life history
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traits can have impacts on the following generations and on the whole population.
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We investigated the potential transgenerational effects of two antidepressants highly
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prescribed, i.e. sertraline and venlafaxine, on D. magna. In a previous study, we found that
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these two antidepressants differ by their acute lethal concentrations for 50% of Daphnia
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magna (LC50). Indeed, sertraline displayed a LC50 of 1.15 mg L-1 (i.e. the most toxic
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antidepressant tested) whereas venlafaxine had a LC50 of 141.28 mg L-1.38 But have they also
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different long-term toxicities? To answer this question, we studied their effects on life traits
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(survival, reproduction and growth) of the parental generation (F0) and of the offspring (F1)
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born under exposure conditions. For this offspring F1, two broods were studied and
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compared: the second brood (F1-B2), i.e. neonates of mothers exposed for about 10 days, and
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the fifth brood, i.e. neonates from mothers exposed during 21 days. Thus, by studying two
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successive generations, we can assess (1) whether antidepressant toxicity might differ
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between parental generation and offspring; and (2) whether offspring transferred to a clean
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medium after parental exposure might recover from maternal effects.
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EXPERIMENTAL SECTION Chemical preparation. Sertraline hydrochloride (CAS No. 79559-97-0) and
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venlafaxine hydrochloride (CAS No. 99300-78-4) were purchased from Interchim
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(Montluçon, France) at analytical grade (purity ≥ 99%). At each medium change, solutions of
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antidepressants were made by sequential dilutions from a freshly prepared solution at 100 mg
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L-1. No carrier solvent was used.
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Chemical analyses. The stability of the compounds throughout the test was studied
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using ultra performance liquid chromatography coupled to tandem mass spectrometry. The
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concentrations of test substances were determined in freshly prepared solution (t0) and at the
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time of renewals (i.e. after exposure, t48 and t72h), in triplicates. These determinations were
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repeated at weekly intervals. Analytical methods are detailed in SI (SI Methods).
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Experimental design. Daphnia magna reproduction tests were conducted with respect
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to OECD guideline 21113. The stock culture was maintained in continuous parthenogenetic
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reproduction under laboratory conditions (breeding in Elendt medium M4 at pH 7, chamber
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set at 20 ± 1 °C with a 16:8h light-dark photoperiod, fed daily with a mixture of three algal
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suspensions composed of Pseudokirchneriella subcapitata, Chlorella vulgaris and
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Desmodesmus subspicatus). Daphnids less than 24h old were randomly selected from the
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third brood of the age-synchronized stock culture. Ten neonates per antidepressant
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concentration and control were individually transferred to 100-mL glass beakers containing
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50 mL of the test medium (pH 7.2 ± 0.1; Dissolved oxygen: 5.56 ± 0.23 mg/L). Daphnids
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were exposed to four nominal concentrations of antidepressants (0, 0.3, 30 and 100 µg L-1).
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They were fed daily with a mixture of three algal suspensions composed of P. subcapitata, C.
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vulgaris and D. subspicatus at a density of 3 x 105 cells mL-1 each (50 µL of each algal
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solution, 0.16 mg C/daphnid/day, C:N ratio = 6.6). Test solutions were renewed three times
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per week. The exposure period for the parental generation (F0) was 21 days. The F1
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generation was started with neonates, less than 24h old, of the second and fifth broods of the
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parental generation (named F1-B2 and F1-B5, respectively). Offspring were either exposed to
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the same concentration as their parents (10 replicates per concentration) or returned to a clean
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medium to assess their capacity to recover from parental exposure (10 replicates per
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condition) for 14 days. During this time, both the control and exposed groups produced three
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broods. We counted the new offspring and removed them daily from the beakers in the
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parental and F1-exposure experiments. The experimental design is presented in SI Figure S1.
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Response variables. For each daphnid, we monitored survival, body length and the
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number of offspring per brood. Survival and the total number of neonates per female were
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recorded daily. The body length of neonates and adults at the end of the exposure period,
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defined as the distance from the upper edge of the compound eye to the base of the spine, was
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measured by image analysis (NIS-Elements D3.0 software) using a Nikon Digital camera
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DXM 1200C connected to a Nikon Eclipse 80i microscope. To extrapolate toxic effects from
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the organism level to the population level, we calculated the asymptotic population growth
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rate (λ) for each population, considered as the dominant eigenvalue of Leslie matrix. The
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projection matrix contained fecundity rates in the first row and survival rates in the
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subdiagonal. Experimental results from the different generations, i.e. F0, F1-B2 and F1-B5,
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were considered either separately (i.e. singly-F-based population) or jointly (i.e. multi-F-
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based population). The asymptotic population growth rate relates to the intrinsic rate of
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population increase r with r = ln(λ).
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Data analysis. All statistical analyses were performed with R 2.15.1 (R Core Team39).
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We compared body length of F0, F1-B2 and F1-B5 and the offspring number of control and
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exposed daphnids using t-tests, after validation of normality and variance homoscedasticity of
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observations. Differences between groups were considered significant when p
