Population dynamics hide phenotypic changes driven by subtle chemical exposures: implications for risk assessments

Eco oxicology h ps://doi.o g/10.1007/s10646-023-02637-8 Popula ion dynamics hide pheno ypic changes d i en by sub le chemical exposu es: implica ions o isk assessmen s Ana del A co 1,2 ●Lu z Becks 1,2 ●Inmaculada de Vicen e 3 Accep ed: 15 Feb ua y 2023 © The Au ho (s) 2023 Abs ac Ecological isk assessmen o chemicals ocuses on he esponse o di e en axa in isola ion no aking ecological and e olu iona y in e play in communi ies in o accoun . I s conside a ion would, howe e , allow o an imp o ed assessmen by es ing o implica ions wi hin and ac oss ophic le els and changes in he pheno ypic and geno ypic di e si y wi hin popula ions. We p esen a simple expe imen al sys em ha can be used o e alua e he ecological and e olu iona y esponses o chemical exposu e a mic obial communi y le els. We exposed a mic obial model sys em o he cilia e Te ahymena he mophila (p eda o ) and he bac e ium Pseudomonas fluo escens (p ey) o i on eleased om Magne ic Pa icles (MP-Fedis), which a e Phospho us (P) adso ben s used in lake es o a ion. Ou esul s show ha while he esponses o p eda o single popula ion size di e ed ac oss concen a ions o MP-Fedis and he esponses o p ey om communi ies di e ed also ac oss concen a ion o MP-Fedis, he communi y esponses (species a io) we e simila o he di e en MP-Fedis concen a ions. Looking u he a an e olu iona y change in he bac e ial p eys’de ence, we ound ha MP-Fedis d o e di e en pa e ns and dynamics o de ence e olu ion. O e all, ou s udy shows how simila communi y dynamics mask changes a e olu iona y le els ha would be o e looked in he design o cu en isk assessmen p o ocols whe e e olu iona y app oaches a e no conside ed. Keywo ds Magne ic pa icles ●Eco-e olu iona y ●P eda ion ●Pseudomonas fluo escens ●Te ahymena he mophila ● Expe imen al e olu ion In oduc ion Popula ions a e con inuously exposed o bio ic and abio ic changes which al e popula ion densi ies as well as e olu- iona y esponses (Ma hews e al. 2011). Consume - esou ce in e ac ions a e iconic examples o con inuous bio ic changes wi h o en apid fluc ua ions in p eda o and p ey popula ion sizes o con inuous adap a ion. Ano he ele an example is coun e adap a ion o hos and pa asi e (Mu doch e al. 2003). Many p e ious s udies showed how abio ic s ess can p omo e apid e olu iona y esponses and how i p e en s popula ion ex inc ion h ough adap a ion in esponse o such abio ic s ess (Bell and Gonzalez 2011; Lindsey e al. 2013; Ramsaye e al. 2013; Bell 2017). The esponses o bo h abio ic and bio ic changes can howe e , be mo e complex when demog aphic and e olu iona y changes occu simul aneously and po en ially d i e each o he as eco-e olu iona y dynamics (Yoshida e al. 2003; Pos and Palko acs 2009; Palko acs and Hend y 2010). The significance o ecology and e olu ion in e play o unde - s and communi y changes aises he conce n abou i s ole in ecological isk assessmen s, as chemical exposu e can impac bo h ecology (e.g., popula ion ex inc ion and bo - lenecks, species so ing wi hin communi ies) and e olu ion (e.g., d i , fixa ion o alleles ela ed o adap a ion o he chemical, loss o gene ic di e si y, epigene ic modifica- ions), which ul ima ely may eed back in o each o he . Cu en isk assessmen s do no e alua e he po en ial o eco-e olu iona y changes despi e o he inc easing awa e- ness o i s ele ance o unde s anding he mechanisms *Ana del A co ana.del-a co@uni-kons anz.de 1Communi y Dynamics G oup, Depa men o E olu iona y Ecology, Max Planck Ins i u e o E olu iona y Biology, 24306 Plön, Ge many 2Limnological Ins i u e, Biology Depa men , Uni e si y o Kons anz, 78464 Kons anz/Egg, Ge many 3Depa amen o de Ecología, Facul ad de Ciencias, Uni e sidad de G anada, G anada 18071, Spain 1234567890();,: 1234567890();,: d i ing communi y changes (B ady e al. 2017; De Mees e e al. 2019). Assessing eco-e olu iona y esponses o che- mical exposu e is challenging because popula ions a e pa o communi ies whe e mul iple species in e ac . Gene ally, esponses o chemicals can lead o dec easing (e.g., che- mical oxici y) o inc easing popula ion densi ies (e.g. compe i ion elease, e ilise e ec ) depending on he concen a ion, axa, and species in e ac ion (Beke o and Liess 2006; B ooks e al. 2009; Lopez Pascua e al. 2012; del A co e al. 2015; Sen is e al. 2017), and whe he o no he e ec on he in e ac ing species is symme ical o asymme ical. Fo ins ance, Van den B ink e al. 2017 concluded ha he in e ac ion be ween bio ic (compe i ion and p eda ion) and abio ic (insec icide exposu e) s esso s on aqua ic in e eb a es is species- and con ex -specific. In addi ion o changes in popula ion densi ies, chemical exposu e can lead o al e ed selec ion on ai s in ol ed in species in e ac ions. Finally, he ecological and e olu- iona y esponses can influence each o he (Hend y e al. 2011), making p edic ions on how popula ions and com- muni ies espond o he p esence o abio ic s esso s di fi- cul . De eloping hese p edic ions a e, howe e , impo an when i comes o unde s anding how communi ies espond o an h opogenic changes (B ady e al. 2017; De Mees e e al. 2019; S aub e al. 2020). To e alua e he impo ance o conside ing e olu iona y changes o isk assessmen in communi ies, we in es iga e he e bo h single species popula ion (p ey o p eda o ) and communi y (p eda o -p ey) ecological esponses and popula ion (p ey) e olu iona y esponses o he p esence o a chemical. We do his o a specific chemical elease (dissol ed i on) om he used o no el abso ben (Magne ic Pa icles, MP) in es o a ion o eu ophica ed aqua ic eco- sys ems. Eu ophica ion is mainly an h opogenically d i en (Smi h and Schindle 2009) and is he esul o excessi e phospho us (P) loads (ex e nal and in e nal) causing wha is ul ima ely obse ed as lake wa e de e io a ion (Jeppen- sen e al. 1991; Sønde gaa d e al. 1993; Ca pen e 2008). Res o a ion p og ams aim o con ol P loads by using a wide ange o adso ben s o emo e excess P (i.e. Phoslock, Zeoli e o Me so b). Recen ly, Magne ic Pa icles (MP) ha e been p oposed as an al e na i e because hey ha e a high P adso p ion capaci y, as adso p ion kine ics, a e pH and edox condi ion independen (de Vicen e e al. 2010; Me ino-Ma os e al. 2011), and hey can be eused a e P-deso bing (Ál a ez- Manzaneda e al., pe sonal commu- nica ion). Beside he emo al o P, MP can elease low o unde ec able amoun s o dissol ed i on (MP-Fedis) (Funes e al. 2016). P e ious assessmen s obse ing he e ec o MP applica ion o 24 h and MP-Fedis on lake communi ies (Funes e al. 2016; Ál a ez-Manzaneda e al. 2019) demons a ed ha a single applica ion o MP educed P in he wa e column and had no impac on he plank on communi ies in e ms o o al abundance, species ichness and species di e si y (Ál a ez-Manzaneda e al. 2019; del A co e al. 2021). Howe e , in a labo a o y expe imen wi h glass ja s (1 L) MP exposu e e ec s (also 24 h con ac ime) on Daphnia magna esul ed in d as ic abundance dec ease independen ly o he deg ee o in a-specific compe i ion (i.e. lowe o highe densi ies wi h same ood a ailabili y and space) and/o communi y s uc u e (i.e. p esence o absence o ege a ion) (del A co e al. 2017). These eco- oxicological s udies did, howe e , no conside e olu- iona y esponses o he p esence o MP-Fedis and i is unclea i o ins ance gene ic so ing wi hin a species a ec ed he obse ed communi y dynamics. We es ed he ecological and e olu iona y esponses o MP-Fedis ( eleased om an MP concen a ion mimicking eal field applica ions) in a mic obial p eda o -p ey sys em wi h he bac e ium Pseudomonas fluo escens as he p ey and he cilia e Te ahymena he mophila as he p eda o . We ocused on mic obial communi ies because hey o m he bo om o any ood web and changes in abundances and in e ac ions a his le el can ha e significan e ec s on whole sys ems and hei unc ion (Madan e al. 2005; Wo den and No 2008). P e ious wo k wi h bac e ia and p o is s showed he e ec s o Fedis whe e popula ion sizes dec eased (Dayeh e al. 2005; Wang e al. 2014). The e o e, Fedis eleased om he use o MP in field applica ions o eu ophic es o a ion migh impose u he p essu es on he communi y. In addi ion, mic obial communi ies ha e sho gene a ions imes, o en la ge popula ion sizes and hus a highe po en ial o e ol e a esponse o he exposu e o s esso s (Bell 2017), which migh inc ease he ela i e ole o e olu iona y change in mi iga ing ecological esponses o chemical exposu e. Simple expe imen al assays whe e one can compa e e olu iona y changes as changes in fi ness o indi iduals and/o popula ions ela i e o he ances o (Kassen 2014) will acili a e he possibili y o in eg a e e olu iona y esponse in o isk assessmen o unc ional changes o mic obial communi ies. In he Pseudomonas -Te ahymena sys em, p eda ion by he cilia es o en leads o he e olu ion o a de ence in he p ey popula ion agains consump ion by he p eda o such as biofilm o ma ion o g ow h in agg ega es which a ec s he p eda o -p ey in e ac ion and he ela i e oles o ecological and e olu iona y change depending on he en i onmen al condi ions (Ma z and Kjellebe g 2005; F iman e al. 2014; Hil unen e al. 2015). In his s udy, we mimic an en i on- men al MP applica ion in he field (Funes e al. 2016)by exposing bac e ia and cilia es o he MP-Fedis eleased om MP, we ha e ocused on dissol ed i on because MP would be e ie ed om he lake a e 24 h. Specifically, ea men s co e ed a ange o MP concen a ions mimicking di e en MP field applica ions: 0 g/L (con ol), 1 g/L and 2 g/L (he eina e 0 MP, 1 MP and 2 MP). A. del A co e al. Ma e ial and me hods Model sys em We used he cilia e Te ahymena he mophila 1630/1U (CCAP) as he p eda o and he bac e ium Pseudomonas fluo escens s ain SBW25 as he p ey (he eina e p eda o and p ey). P eda o s ocks we e cul u ed axenically in o ganic medium (20 g o p o eose pep one and 2.5 g o yeas ex ac in 1 L o deionized wa e ) p io o he expe imen (Hil unen e al. 2015). P ey was inocula ed om a single colony om a ozen s ock o minimise ini ial gene ic a iabili y in he popula ion. Thus, e olu iona y changes we e expec ed o esul om de-no o e olu ion as p e iously shown in his sys em (Hil unen e al. 2015). Magne ic pa icles The MP (97.5% i on, 0.9% ca bon, 0.5% oxygen, and 0.9% ni ogen) we e kindly supplied by BASF (Ge many). MP a e sphe ical and polydispe se wi h an a e age size ange o 805 nm ± 10 nm and a densi y o 7.5 g cm−3(de Vicen e e al. 2010). Maximum P adso p ion capaci y has been epo ed as 1 g MP:18.8 mg P (de Vicen e e al. 2010). Mic ocosm expe imen : single popula ions and communi y dynamics Ecological and e olu iona y dynamics o p eda o -p ey single popula ions and communi ies we e ollowed o e ime in semi-con inuous cul u es. Ou expe imen al design aimed o assess he in e play be ween ecology and e olu ion in he sho - e m (e.g. P. fluo encens could apidly e ol e (F iman e al. 2014)), which may ha e he po en ial o lead o eco-e olu iona y eedbacks in he long e m a commu- ni y le els (De Mees e e al. 2019). Single popula ions and p eda o -p ey communi ies we e exposed o an ini ial 24 h exposu e o MP concen a ions (see expe imen al de ails below). We se up ou eplica es wi h only (i) p eda o o (ii) p ey and (iii) p eda o and p ey oge he . Flasks we e inocula ed wi h 100 µL om an o e nigh cul u e o bac e ia s ock and 3,850 p eda o s (Hil unen e al. 2015) ei he alone (single popula ion ea - men s) o oge he (communi y ea men s). We used issue cul u e flasks (40 mL) supplied wi h 11 mL o M9 sal s and King´s B (KB) nu ien s [0.05x; 200 mL o Minimal Sal s (5x), 20 g o p o eose pep one and 10 g o glyce ol o 1 L o medium]. We used h ee di e en s ocks o he medium wi h di e en concen a ions o MP; 0 g/L (con ol), 1 g/L and 2 g/L (he eina e 0 MP, 1 MP and 2 MP) which simula ed a ealis ic en i onmen al MP applica ion (Funes e al. 2016). Fo his, we p epa ed a s ock solu ion o MP by dilu ing 1 o 2 g o MP in 20 mL o 0.05x KB medium. A e 24 h, MP we e emo ed om he s ock solu ion using a magne ic g adien (Block magne 40 × 40 × 20 mm, Webc a GMbH, Ge many) and he medium wi h he dissol ed i on o e 24 h (MP-Fedis) was au ocla ed (121 °C o 20 min) o es ablish s e ile es ing condi ions. Fo he single and he communi y expe imen , we ans e ed 10% o he olume om expe imen al flasks e e y 24 h o e-seed new flasks wi h esh medium wi hou any mo e MP exposu e. The single popula ion communi y, ans e s we e done du ing 2 (48 h) days. And, o he communi y expe imen o 7 days (168 h), co e ing app oxima ely 21 p eda o and 42 p ey gene a ions. Flasks we e kep a 28 °C ± 2 °C unde s a ic condi ions. A each ans e , a 2 mL subsample om each flask was ozen o c yop ese e he p ey a e adding 0.5 mL o 80% glyce ol o he sample and s o ed a −80 °C un il u he use. Ano he 300 µL we e aken o measu e p ey densi y using Op ical Densi y (OD) a 600 nm (Tecan abso bance mic opla e eade ). OD measu emen s we e ans o med o p ey Colony Fo ming Uni s (CFU). A sample o ances al p ey and expe imen al samples (n=3) g own o e nigh , la e dilu ed, pla ed on solid media and we de e mined CFUs o es ablish he con e sion be ween OD and CFU/ mL. In addi ion, ano he 1 mL subsample was fixa ed wi h o maldehyde (4% final concen a ion) o la e es ima ion o p eda o densi y using flow cy ome y (FACS Calibu ). We used he au o sample (3 × 10 μL samples) in he flow cy ome e using well pla es. P io o flow cy ome e , sam- ples we e s ained wi h 1 μL o 100x concen a ion o SYBER G een I (Sigma-Ald ich), and incuba ed o 1 h a 24 °C in he da k (Ayan 2018). P ey and p eda o densi ies we e used o calcula e g ow h a es, p eda o -p ey a ios, and ollow popula ion dynamics. Fe measu emen s We p epa ed addi ional cul u e flasks o measu e MP-Fedis om each MP ea men wi hou he addi ion o p ey o p eda o s. F om hese suspensions, samples we e aken a di e en imes: 0 h ( igh a e he addi ion o he MP) and a e 24 h (a e emo ing he MP). To al MP-Fedis was measu ed using he spec opho ome ic e ozine me hod (Gibbs 1979). MP-Fedis was measu ed wi hin 24 h o mimic en i onmen al MP applica ion in he field whe e MP would be emo ed a e 24 h. In addi ion, in he mic ocosm expe imen s, 90% o medium was enewed wi h uncondi- ioned medium e e y 24 h a e he ini ial 24-h exposu e o he popula ions and communi y. E olu iona y changes in p ey popula ions The e olu ion o a p ey de ence agains p eda o g azing was es ima ed by using a simple, ecologically ele an Popula ion dynamics hide pheno ypic changes d i en by sub le chemical exposu es: implica ions o isk. . . bioassay desc ibed in de ail in Hil unen and Becks (2014). A e hawing c yop ese ed p ey popula ions aken om he s a and a e e y ans e and g owing popula ions o ~10 gene a ions (24 h) in liquid cul u e (0.05% KB), 100 μL o he cul u e was added o 2 mL o esh cul u e medium. 3850 p eda o s we e added om he p eda o s ock cul u e (i.e., naï e p eda o s as a s anda d o con- sume eeding on po en ially gene ically di e en ia ed p ey) and p eda o g ow h a e was es ima ed o e 48 h. Di e ences in p eda o densi ies compa ed o p eda o s’ g ow h o naï e p ey we e aken as an es ima e o he p ey de ence le el (D). P ey de ence ai alues we e calcula ed as ela i e fi ness by D =1−(p eye o/p eyanc) whe e p eye o is he p eda o densi y a e eeding on e ol ed p ey, and p eyanc is p eda o densi y a e eeding on ances al p ey (coming om ozen cul u es, same s ain used o s a bo h popula ions and communi y ea men s). S a is ical analysis All s a is ical analysis we e done wi h R 4.1.1 (De el- opmen Co e Team). Di e ences in MP-Fedis concen a- ions be ween con ol and ea men s we e assessed by using a non-pa ame ic Wilcoxon Rank Sum es , using he PMCMR package (Pohle 2014). We used Gene al- ised linea model (GLM), using he geepack package o assessing di e ences be ween p eda o and p ey densi ies in bo h p ey and p eda o coming om single popula ions o communi ies. Fo a single popula ion, we compa ed densi iesbe ween heMP ea men s(0MP,1MP,2MP) using ime as a ac o . Fo popula ions coming om communi ies, we assessed densi y di e ences be ween he MP ea men s and p eda ion ea men s (p ey s. p ey unde p eda ion).Also, we used Gene alised Es ima ing Equa ion models (GEE), using he geepack package o assessing di e ences be ween de ence le els, p eda o - p ey a ios o e ime wi h MP ea men s and ime as fixed e ec s. The GEE models co ec o au oco ela ion be ween ime poin s wi hin one eplica e and we used independence co ec ion (Halekoh e al. 2006). Fo da a analysis, di e en models we e used depending on he kind o da a; amily gamma o p eda o densi ies, amily gamma o p ey densi ies, and amily Poisson o p eda o -p ey dynamics, amily gamma o a ios and amily gaussian o de ence le els. We used species- specific models (p ey o p eda o ) o es he e ec s o MP-Fedis in he single species ea men s and models using only da a om he p eda o -p ey ea men s o es o he e ec o MP-Fedis on p eda o -p ey a ios and de ence e olu ion (Hil unen and Becks 2014; Hil unen e al. 2015). Resul s and discussion E ec s o MP-Fedis on p ey and p eda o g ow h The e we e significan ly di e en amoun s o MP-Fedis eleased in he 2 MP ea men s wi h espec o he 1 MP and 0 MP con ol (K uskal-Wallis: chi-squa ed =10; p- alue < 0.01; igh a e emo ing o he MP, K uskal- Wallis: chi-squa ed =10; p- alue < 0.01, Table 1). E en hese low amoun s o i on du ing he MP applica ion ha e he po en ial o impac mic obes o e mul iple gene a ions (i.e. ~8 o 16 gene a ions wi hin 24 h o bac e ium axa as Esche ichia,Pseudomonas,Salmonella,S aphylococcus o Vib io (Gibson e al. 2018)). De imen al e ec s o me als on mic obes ha e been p e iously epo ed (Madoni and Romeo 2006; Wang e al. 2014). The le hal concen a ion (LC50) o Fedis is ~7 mg/L - 4 mg/L (in alama Blue and CFDA-AM media) o he p eda o T. he mophila (Dayeh e al. 2005). The minimum inhibi o y concen a ion (MIC) o he p ey Pseudomonas has been epo ed as ≤0.349 mg/L (Wo ken ine e al. 2008; Chiadò e al. 2013). The MP-Fedis in ou s udy was lowe han he epo ed LC50 and in he ange o MIC. These low concen a ions migh no impose a di ec isk o oxic e ec o highe biological le els, bu migh impac he mic obial loop and i s ole o nu ien cycling as well as highe ophic le els. Fo ins ance, changes in he bac e ia and cilia es communi ies migh influence ood quali y o highe ophic le els. Such changes in ood quali y o g aze s as Cladoce a do no only impac g ow h and g azing e ficiency bu can igge changes in ep oduc ion cycles (sexual s. asexual) and es ing eggs p oduc ion (Koch e al. 2009). We used single popula ions wi h only p ey o p eda o o es o he e ec s o he MP-Fedis om he MP ea men s on he wo species sepa a ely o 48 h exposu e. A e age p eda o densi ies significan ly di e ed wi h inc easing MP concen a ions bu did no change o e ime (Gene alised linea model (GLM), MP: F =5.5133, d =2, p=0.0136; Table 1 Tempo al changes o dissol ed i on (MP-Fedis) concen a ions (mean ± s.d., n=4) measu ed igh a e applica ion o MP (H0) and a e 24 h (H24) Nominal MP concen a ions (g/L) I on dissol ed (MP-Fedis, in mg Fe/L) in he ea men s H0 H24 0MP 0.016 ± 0.032 0.023 ± 0.016 1MP 1.246 ± 0.204 1.182 ± 0.023 2 MP 2.913 ± 0.984 2.990 ± 0.517 Numbe s in bold indica e significan di e ences (p< 0.05) wi h espec o he i on eleased (Fedis) om magne ic pa icle concen a ions compa ed o he con ols (0 MP) A. del A co e al. Time : F =3.3997, d =,p=0.0817; Time*MP: F=0.304, d =1, p=0.7413; Fig. 1a). Fo he p ey popula ions, we obse ed ha he a e age p ey densi ies we e significan ly di e en when compa ing he beginning and he end o he expe imen (GLM, Time: F=17.2743, d =1, p=0.00059) bu only ma ginally ac oss MP concen a ions (MP: F =3.1124, d =2, p=0.069, MP*Time: F =2.2286, d =1, p=0.137; Fig. 1b). O e all, he MP-Fedis eleased om MP concen a ions (in ended o be used in field applica ions) had an asym- me ic e ec on he species. MP-Fedis migh ha e al e ed he p eda o popula ions by ac ing as a mic onu ien igh a e exposu e, acili a ing he g ow h o p eda o (Fig. 1a). On he con a y, MP-Fedis nega i ely a ec ed he bac e ia esul ing in smalle popula ion size compa ed o he con ols igh a e exposu e (Fig. 1b), howe e , bac e ia eached simila densi ies a e 48 h in all ea men s wha migh sugges adap ion o he exposu e (e.g. geno ype so ing, de-no o mu a ions). E ec s o MP-Fedis on p eda o -p ey in e ac ion When p ey and p eda o g ew oge he , p eda o s sig- nifican ly educed p ey densi ies depending on he MP concen a ion o e ime (GLM, MP: F =227463702, d = 2, p<2×10 −16; P eda ion: F =18121171, d =1, p<2×10 −16; MP x P eda ion: F =0, d =2, p<2×10 −16; Fig. 2“All expe imen al days”), wi h inc easing p ey den- si ies wi h inc easing MP concen a ions unde p eda ion condi ions, specially by he end o he expe imen (GLM, MP: F =1330690697, d =2, p<2×10 −16; P eda ion: F=603799903, d =1, p<2×10 −16; MP x P eda ion: F=388377847, d =2, p<2×10 −16; Fig. 2, Las expe i- men al day). To u he compa e he e ec s o MP-Fedis on he com- muni y le el, we es ed o di e ences in p eda o -p ey a ios as an es ima e o species in e ac ion s eng h (Fig. 3); highe a ios indica e s onge p eda ion p essu e on he p ey. MP ea men s we e a sou ce o a ia ion be ween he commu- ni ies esul ing in empo al peaks o highe p eda o -p ey a ions in communi ies ea ed wi h he highe MP ea men bu esul ed on simila communi ies o e ime (GEE, MP: W=1.69 × 1019,d =2, p<2×10 −16,MPxTime: W=−4,41 × 1018,d =2, p=1; Time: W =−1.8 × 1017, d =2, p=1; Fig. 3). This esul o inc easing p eda ion p essu e a ea ly s ages o MP exposu e, is in line wi h he obse a ions in he single species du ing a pe iod o 48 h. In single species expe imen s, we ound a end owa ds highe p eda o densi ies in he MP ea men compa ed o 0 MP ea men s (Fig. 1a). Howe e , o e ime we obse ed simila a ios o he di e en MP ea men s, and hus simila communi y esponses a e a ew days om he exposu e e en . None heless, di e ences in p eda ion p essu e among MP ea men s esul ed in a delay in he highe p eda ion p essu e peak in 1 MP and 2 MP ea men s compa ed o he 0 MP ea men . This empo al a ia ion in p eda ion p es- su es migh influence species in e ac ions d i ing di e se popula ion dynamics and e olu iona y esponses. Specifi- cally, we obse ed ha o e ime he p eda o /p ey dynamics we e simila , howe e , hey migh be d i en by di e se e olu iona y esponses o he p ey. Fo ins ance, in 0 MP he peak o p eda ion p essu e occu ed a e 24 h o exposu e, howe e in 1 MP and 2 MP ea men s i occu ed a e 48 and 72h espec i ely. The MP exposu e migh impose a de imen al e ec on p eda o densi y delaying he peak o p eda ion o a longe ime a e exposu e. In his ega d, heo y and empi ical da a show how he pe u ba ion co- occu ence, pe u ba ion iming and s eng h influence coa- dap a ion and selec o di e ence p ey de ences (Hil unen e al. 2018; Raa z e al. 2019). The e o e, he obse ed empo al a ia ion in p eda ion p essu es migh selec o di e en p ey de ences in he mic obial communi y. As e olu ion o p ey de ences can al e p eda o -p ey in e ac ions (Yoshida e al. 2003; Hil unen e al. 2015) and Fig. 1 E ec s o MP-Fedis on p eda o and p ey popula ion size. P eda o (a) and p ey (b) popula ion size (109) changes in single popula ions (p eda o o p ey g owing alone, median, n=4) in he absence and p esence o MP-Fedis eleased om he h ee magne ic pa icles concen a ion es ed (0 MP, 1 MP and 2 MP) Popula ion dynamics hide pheno ypic changes d i en by sub le chemical exposu es: implica ions o isk. . . hus popula ion dynamics, we ollowed he e olu iona y esponse o he p ey o MP ea men (i.e. he i able phe- no ypic de ence ai changes) in he p esence and absence o he p eda o . Fo his we measu ed g ow h a es o he ances al p eda o (naï e p eda o ) when g own on same densi ies o ei he ances al p ey (naï e p ey) o isola ed p ey (po en ially e ol ed) aken om each ans e o he expe imen . F om his, we calcula ed he de ence le el D wi h ze o meaning ha he e ol ed p ey had a simila le el o de ence as he ances o ; alues close o 1 indica e a high le el o de ence compa ed o he ances o . As all pheno ypic measu emen s we e pe o med using isola es om he expe imen , which g ew a leas 10 gene a ions in he absence o he MP-Fedis and he cilia es unde s anda - dise condi ions be o e he pheno ypes we e measu ed, all obse ed de ence changes a e likely gene ically de e mined and no en i onmen ally induced. De ence e ol ed bu di e en ly in all MP ea men s. We ound di e ences o he in e ac ion o ime-MP ea - men (GEE, MP x Time: W =−4.63 × 1027,d =1, p<2×10 −16, Fig. 4), o p ey de ence e olu ion le els o he di e en MP ea men s (GEE, MP: W =1.07 × 1029, d =2, p<2×10 −16, Fig. 4) and o e ime (GEE, Time: W=1.6 × 10129,d =1, p<2×10 −16, Fig. 4). Specifically, de ence e ol ed o highe le el o de ence compa ed o ances al p ey in 0 MP ea men and kep a simila le els o e ime. Howe e , de ence in 1 MP and 2 MP fluc ua ed Fig. 2 E ec s o MP-Fedis on p ey popula ion dynamics. P ey popula- ion size (109) dynamics o e ime, p ey a e age densi y o all expe i- men al days by MP ea men and p ey densi y in he las expe imen al day densi y by MP ea men in single popula ion and in communi ies in he absence and p esence o MP-Fedis eleased om he h ee magne ic pa icles concen a ion es ed (0 MP, 1 MP and 2 MP) Fig. 3 E ec s o MP-Fedis on p eda o /p ey a ios. P eda o /p ey a ios (10−5) om he p eda o -p ey communi y expe imen o e ime a di e en MP-Fedis eleased om he h ee magne ic pa icles con- cen a ion es ed (0 MP, 1 MP and 2 MP). E o ba s ep esen he s anda d de ia ions o he mean (mean ± s.d., n=4) A. del A co e al. o e he cou se o he expe imen being: a) always lowe compa ed o de ence le els o p ey om 0 MP, and b) some imes simila o e en lowe han ances al p ey. In addi ion, o he p ey ai s (e.g. mo ili y) and nu i ional quali y (e.g. pala abili y) migh influence p eda ion s eng h, so hen, de ence le els. In ac , Cai ns e al. (2017) s udied genomic e olu ion o he bac e ium Pseudomonas fluo - escens unde an ibio ics and phage selec ion p essu es like he me al and p eda ion selec ion p essu e o he mic o- cosmos expe imen s p esen ed he e. They ound, gene ic e olu ion ela ed o mo ili y, nu ien anspo and me a- bolic pa hways. (Cai ns e al. 2017) We ha e assessed he e ec s o a chemical exposu e o MP-Fedis (coming om MP concen a ions in ended o be used in lake es o a ion) on ecological and e olu iona y esponses in a p ey-p eda o sys em. In single popula ions, we obse ed ha MP-Fedis a ou ed an inc ease o he p eda o ; while, p ey, a e an ini ial de imen al e ec , g ew o equal popula ion sizes independen ly o he MP-Fedis exposu e. In he p eda o -p ey communi y, we obse ed simila communi y esponses despi e o he di e en MP concen a ions. Howe e , he unde lying p ocesses ha d o e he popula ion size changes and a ios o e ime migh di e wi h and wi hou MP due o p ey de ence ai e olu ion. P ey de ence e ol ed di e en ly in he absence s. p esence o MP and had a significan e ec on he p eda o ’s popula ion g ow h a e. P ey de ence e olu ion fluc ua ed in he p esence o MP esul ing in simila o lowe de ence le els ha ances al p ey sugges ing ha p eda o ’s popula ion g ow h was mos ly d i en by p ey abundances in he communi y. Thus, al hough we obse ed simila p eda o -p ey a ios and dynamics o e ime in all ea men s, ou esul s sugges ha he ela i e oles o ecology and e olu ion di e ed significan ly depending on he p esence o absence o MP. In ag eemen wi h ou findings, o he s udies on di e se axa ( o i e -algae, phage-bac e ia, p o ozoan-mosqui o, plan -insec ) epo popula ion dynamics d i en by changes whe e ecology and e olu iona y oles a y in esponse o bo h abio ic and bio ic changes (Yoshida e al. 2007; Becks e al. 2010; Te Ho s e al. 2010; Ag awal e al. 2013). The ac ha he in e play o ecological and e olu iona y changes a e challenging o iden i y a communi y le els aises conce n o cu en ecological isk assessmen s. Unde s anding how and when e olu iona y changes ha e he po en ial o eedback on o ecological changes, and ice e sa, will en ich he in e p e a ion o isk assessmen s by iden i ying he mechanisms d i ing communi y changes (e.g. species so ing s. gene ic so ing). We ad oca e o mo e expe imen s conside ing he ele ance o ecological and e olu iona y changes a same ime scales unde en i - onmen ally ealis ic scena ios. I will p o ide c ucial in o ma ion o unde s anding how human-d i en selec ion shapes communi ies and imp o e ecological isk assess- men s. (Van den B ink e al. 2017) Acknowledgemen s We hank Goekce Ayan o sha ing aluable p o ocols and expe ience wi h he expe imen al sys em. This wo k was suppo ed by Jun a de Andalucía p ojec s P10-RNM-6630 and P11-FQM-7074 (P oyec os de Excelencia, Spain), MINECO CTM 2013-46951-R, MAT 2013-44429-R and PCIN 2015-051 p ojec s (Spain) and by he Eu opean Regional De elopmen Fund (ERDF). Au ho con ibu ions AdA concei ed he s udy, AdA, LB, IdV designed he s udy; AdA ca ied ou he expe imen , AdA, LB ana- lysed he da a; AdA, LB w o e he manusc ip , and all au ho s edi ed he manusc ip . Funding Open Access unding enabled and o ganized by P ojek DEAL. Compliance wi h e hical s anda ds Conflic o in e es The au ho s decla e no compe ing in e es . Publishe ’s no e Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a filia ions. Open Access This a icle is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License, which pe mi s use, sha ing, adap a ion, dis ibu ion and ep oduc ion in any medium o o ma , as long as you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The images o o he hi d pa y ma e ial in his a icle a e included in he a icle’s C ea i e Commons license, unless indica ed o he wise in a c edi line o he ma e ial. I ma e ial is no included in he a icle’s C ea i e Commons license and you in ended use is no pe mi ed by s a u o y egula ion o exceeds he pe mi ed use, you will need o ob ain pe mission di ec ly om he copy igh holde . To iew a copy o his license, isi h p://c ea i ecommons. o g/licenses/by/4.0/. Fig. 4 E ec s o MP-Fedis on p ey de ence dynamics. P ey de ence dynamics on he p eda o and p ey communi ies a di e en MP-Fedis eleased om he h ee magne ic pa icles concen a ion es ed (0 MP, 1 MP and 2 MP). Dashed ho izon al line a he alue o 0 is a e e ence line ep esen ing no e olu ion o de ence compa ed wi h he ances o s. E o ba s ep esen he s anda d de ia ions o he mean (mean ± s.d., n=4) Popula ion dynamics hide pheno ypic changes d i en by sub le chemical exposu es: implica ions o isk. . . Re e ences Ag awal AA, Johnson MTJ, Has ings AP, Ma on JL (2013) A field expe imen demons a ing plan li e-his o y e olu ion and i s eco- e olu iona y eedback o seed p eda o popula ions. Am Na 181:S35–S45. h ps://doi.o g/10.1086/666727 Ál a ez-Manzaneda I, Gue e o F, del A co AI e al. (2019) Do magne ic phospho us adso ben s used o lake es o a ion impac on zooplank on communi y. 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