Effects of experimental long-term CO 2 exposure on Daphnia magna (Straus 1820): From physiological effects to ecological consequences

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Daphnia magna was used in a CO 2 injection experiment, simulating a CCS leak scenario. Survival, individual growth, RNA:DNA ratio, and neonates production were analysed. Secondary production effects were detected, highlighting the ecological
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  Effects of experimental long-term CO 2  exposure on  Daphnia magna (Straus 1820): From physiological effects to ecological consequences Gema Parra  a ,  * , Andr  ea Galotti  a , Raquel Jim  enez-Melero  a , Francisco Guerrero  a ,Emilio S  anchez-Moyano  b , Francisco Jim  enez-G  omez  a , Mercedes Conradi  b a Department of Animal Biology, Plant Biology and Ecology, Centre of Advanced Studies in Earth Sciences, University of Ja  en, Spain b Department of Zoology, University of Sevilla, Spain h i g h l i g h t s   Daphnia magna  was used in a CO 2  injection experiment, simulating a CCS leak scenario.   Survival, individual growth, RNA:DNA ratio, and neonates production were analysed.   Secondary production effects were detected, highlighting the ecological implications. a r t i c l e i n f o  Article history: Received 29 January 2016Received in revised form17 March 2016Accepted 18 April 2016Handling Editor: Jim Lazorchak Keywords: Carbon capture and storage (CCS)technologiesZooplanktonBiomarkersSecondary production a b s t r a c t The carbon capture and storage (CCS) technologies that were proposed to mitigate environmentalproblems arising from anthropogenic CO 2  emissions, also have potential environmental risks. Aneventual CCS leak might induce very low pH values in the aquatic system. Due to the lack of knowledgeof long-term CO 2  exposures with very low pH values, this study aims to know the effects and conse-quences of such a situation for zooplankton, using the  Daphnia magna  experimental model. A CO 2  in- jection system was used to provide the experimental condition. A twenty-one days experiment withcontrol and low pH treatment (pH  ¼  7) replicates was carried out under light and temperature-controlled conditions. Survival, individual growth, RNA:DNA ratio, and neonates production were ana-lysed during the aforementioned period. No differences on survival (except last day), individual growthand RNA:DNA ratio were observed between both control and low pH treatments. However, clear dif-ferences were detected in neonates production and, consequently, in population growth rates and sec-ondary production. The observed differences could be related with an energy allocation strategy toensure individual survival but would have ecological consequences affecting higher trophic levels. ©  2016 Elsevier Ltd. All rights reserved. 1. Introduction New technologies that capture CO 2  have been proposed tomitigate environmental problems arising from man-made CO 2 emissions (Halsband and Kurihara, 2013a,b). The IPCC (2005) esti- mates that carbon capture and storage (CCS) technologies couldhave an economic potential between 10% and 55% of the totalcarbon mitigation strategies by the end of the century. Thesetechnologies pose their own risks for instance, an eventual CCSleak, where very low pH values might be reached. So a pre-requisite, more knowledge is required to complete riskassessments on CCS. The recent and vast bibliography concerningpotential effects of ocean acidi 󿬁 cation (OA) is based on a futurescenario inwhich the average surface seawater pH ranges from 8.2to 7.6 by year 2100 (Caldera and Wickett, 2003; IPCC, 2007), and isalso based on short-term CO 2  experimental exposures. Neverthe-less,someauthorssuggestpHvalueslowerthan6,neartothepointof leakages (Herzog et al., 1996). The environmental risk assess-ment must be based primarily on laboratory and small-scale ex-periments, because such large amounts of relatively pure CO 2  havenever been introduced into the deep ocean in a controlled experi-ment (IPCC, 2005). However, since most of the current informationcame from short-term CO 2  experimental exposures, it is necessaryto invest more effort in decreasing the gap between these pre-dominant short-term experiments and the long-term exposure *  Corresponding author. E-mail address:  gparra@ujaen.es (G. Parra). Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere http://dx.doi.org/10.1016/j.chemosphere.2016.04.0660045-6535/ ©  2016 Elsevier Ltd. All rights reserved. Chemosphere 156 (2016) 272 e 279  that might take place near to a CCS leak. This applies especially tothose organisms, such as zooplankton, that have a limited ability tomove across water masses (Herzog et al., 1996) even though arecent study has reported higher zooplankton capacity to respondunder moderate levels of turbulence (Michalec et al., 2015).The present research is based on laboratory experiments, butwith the intent of understanding the effects of CO 2  leakages innatural populations, as was recommended by G  omez et al. (2001),who argued that laboratory investigations with model zooplank-ters are needed in order to improve  󿬁 eld measurements. The mainshortcoming of those experimental tests is the lack of ecologicalrelevance, because the ecological interactions are ignored andsolely standard species are used, instead of local species. However,theyarenecessaryasa 󿬁 rststeptoachievingtheinitialinformationabout long-term effects. In this sense, Raimondo and Mckenney(2006) state the importance of reproduction in population-levelrisk assessment and the need for complete life-cycle test data tomakeanexplicitlinkbetweentheorganismandhigherhierarchicallevels. For instance, secondary production research has beenclaimed, because it allows to know how material and energy aretransferred,andcouldbeused to detecttheeffectsofperturbationson the ecosystem ( Jim  enez-Melero et al., 2013). Ecologicallyimportant endpoints, such as those related to reproduction, havebeen previously used (Calow et al., 1997; Van Straalen andKammenga, 1998). Some of them have been proposed to be usedas references to calculate the safety factors needed in the Envi-ronmental Risk Assessment (ERA) procedures (Roex et al., 2000).The intrinsic rate of population increase  r   is regarded as being oneof the most ecologically relevant, and as a standardised parametercould be used to compare toxic effects (Calow et al., 1997; Forbesand Calow, 2002), and is a more relevant measurement of toxi-cant effects than traditional measures of mortality or reproduction(Barata et al., 2012). The reduction of population growth rate of akeyspecies could induce signi 󿬁 cant ecological effects on the restof a lake community (Hanazato and Dodson,1995). Although realisticeffects at population level are dif  󿬁 cult to determine and requirelong-term observation, the step in seeking the effect on  r   might betaken. Multi-generation incubations to detect selection pressure of acidi 󿬁 cation on life cycle traits and adaptation would be desirable(Halsband and Kurihara, 2013a,b) similarly to those described bySouissi et al. (2014) where a generational selection occurred after just  󿬁 ve generations under a climate change scenario. From a hi-erarchical point of view, a link between physiological effects andthe population and community consequences is needed to under-stand the mode of action of toxicants and/or changeable environ-mental factors. For this reason, biomarkers are being used,increasingly, to bridge the aforementioned gap (De Coen et al.,2000). A biochemical endpoint, such as the RNA:DNA ratio, hasalso been proposed to achieve the effects of, for instance, foodquality or toxic substances on somatic growth and secondary pro-duction (Gusm ~ ao and McKinnon, 2009; Vrede et al., 2002). In adultzooplankters, RNA:DNA ratios have been, generally, positivelycorrelated with growth rates and egg production (EP) (Wagneret al., 1998; Gorokhova, 2003; Speekmann et al., 2007). Thisbiomarker could be used to assess the effect of water acidi 󿬁 cationfrom a physiological point of view.Consideringzooplanktonasthelinkbetweenprimaryproducersandhighertrophic levels,theaim of this paperis to understandtheeffects and consequences of a long-term CO 2  exposure that simu-lates eventual CCS technology leakages. In order to achieve thisgoal,  Daphnia magna  (Cladocera: Crustacea  e  Straus, 1820), wasusedastheexperimentalmodelsystem.Owingtoitssmallsize,butwhich is big among the zooplankters, its short life cycle, ubiquityand capacity for parthenogenetic reproduction,  D. magna  has beenwidely-used as an experimental animal in aquatic environmentaltoxicity testing, and has been adopted by different environmentalagencies(US-EPA,2002;ASTM,1988;OECD,1984,1998).Theeffectsat individual levels and ecological consequences were analysedusing survival, individual length, RNA:DNA ratio and egg produc-tion as endpoints. 2. Materials and methods  2.1. Experimental conditions Theexperimental model chosen,  D. magna , has ashort life cycle,the lasting of the experiment (21 days) has been considered aslong-term experimental period. A monoclonal population of  D. magna wasraisedfromasecond-generation neonate (Neonaten.2) in a natural wetland Laguna Grande (Baeza, Ja  en, Spain). Theindividuals were reared in mineral water plus a mix diet of   Scene-desmus obliquus  (Turpin) Kützing 1833 (Chemical EngineeringLaboratory, University of Ja  en) and  Cryptomonas pyrenoidifera  Gei-tler 1922 (Water Institute, University of Granada) as food in arelation of at least, 1.5    10 6 algae cell/individual. The aforemen-tioned algae were routinely maintained in 3N-BBM  þ  V culturemedium pH 8.3 e 8.5 (modi 󿬁 ed from CCAP, Scotland) under condi-tions of 20   C and supplied with a cycle of 12 h light:12 h dark.The experiments were designed to test the effects on the or-ganismsofCO 2 injectionintothewater, drawingonthesubsequentacidi 󿬁 cation using the system-simulator, as described in detail byBasallote et al. (2012), De Orte et al. (2014), and Rodríguez-Romeroet al. (2014). Aqua Medic AT Control System (Europe) was used tocontrolandmaintainthepHineachvesselwherethepHelectrodeswere placed. Before use, pH electrodes of the CO 2 -injection systemwere calibrated and the values obtained throughout the tests wereregularly veri 󿬁 ed by a portable pH-meter (Crison GLP 22). A sole-noid valve allowed the adjusting of the pH values when it wasdetectedthatthepHhadincreasedby0.01unitsormore;then,CO 2 gas bubbles were injected into each vessel until the required pHvalue was reached. A computer connected to the ATcontrol systemallowed modi 󿬁 cation of the pH values, as required. In this experi-ment, all exposure tests were carried out in a temperature- andlight-controlled chamber (20  ±  1   C and a photoperiod of 12:12 hlight:dark). Twenty neonates, with no more than 24 h, were placedin each 2 L vessel. Four replicates for control and for low-pHtreatments (pH  ¼  7) were used during 21 days of experimentalperiod. Both  S. obliquus  and  C. pyrenoidifera  (mixed) were used asfood during the experimental period. The microalgae density, toensure no food limitation conditions (at least 1.5    10 6 cell/daphnia/day;Díaz-Baez et al., 2004) on each vessel, was monitoreddaily during the experimental period. Cell density was calculatedusingthechlorophyll 󿬂 uorescencemeasurement(Aqua 󿬂 uorTurnerDesigns) in each vessel. The following correlations between  󿬂 uo-rescence (  X  ) and abundance ( Y  ) were calculated and used for C. pyrenoidifera  and  S. obliquus , respectively  y  ¼  14 : 912  X    1283 : 2  R 2 ¼  0 : 99  and  y  ¼  61 : 543  X   þ 55494  R 2 ¼  0 : 94  Water samples, each of 50 mL, were used for the alkalinityanalysis (automatic titrator, 848 Titrino Plus devise). Dissolvedoxygen and conductivity data were obtained using a multi-parametric probe (YSI-556 MPS).Measured responses included RNA:DNA ratio, survival, adultand embryo size, and reproduction parameters. G. Parra et al. / Chemosphere 156 (2016) 272 e  279  273   2.2. RNA:DNA ratio estimation Theorganismswerecollectedforbiochemicalanalysisafter7,14and 21 days. Each  Daphnia  individual was placed alive in anuclease-free Eppendorf tube and immediately frozen at   80   C.Methods for quanti 󿬁 cation of nucleic acids largely followed themethod described in Vrede et al. (2002) with some modi 󿬁 cations,as described below. RNA and DNA were extracted and analysedusing the  󿬂 uorochrome RiboGreen in combination with RNasetreatment (Gorokhova and Kyle, 2002). A RiboGreen ™ RNA Quan-titation Kit (Molecular Probes, cat. # R-11490) was used. Further,Ribonuclease A from bovine pancreas (RNase), Triton X-100, Pro-tease from  Bacillus licheniformis  (Type VIII, lyophilised powder,7 e 15 units/mg solid) were supplied by Sigma. Nuclease-free waterwas also supplied by Sigma. DNA standard (SIGMA) sets wereprepared from frozen (  20   C) aliquoted stock. 1  TE buffer pre-pared by diluting the 20xTE buffer with nuclease-free water.RiboGreen reagent solution was prepared by diluting the stocksolution 200-fold with 1  TE buffer. The reagent solutionwas keptdark and cold and was used within a few hours.RNA and DNA were extracted from individual zooplankters in asolution of 1  TE buffer containing Triton X-100 (0.1%  󿬁 nal con-centration) and protease (0.1 mg mL   1 󿬁 nal concentration).Extraction buffer (50 mL) was added to each Eppendorf vial con-taining one zooplankton individual. The animal was thoroughlycrushed with a plastic pellet micropestle (Eppendorf). Another250 mL of the extraction buffer was added afterwards. Sampleswere shaken at room temperature on a multiple vial head for 1 hand then spun in a centrifuge for 1 min, plus another 1 min, at2000 rpm.Fluorescence measurements were performed using a  󿬂 uorom-eterBioTekSynergyHT( 󿬁 lters:485/20forexcitationand528/20foremission) and black solid  󿬂 at-bottom microplates (Greiner bio-one). In each plate, two replicates with 100  m L well  1 of theextracted samples, controls and RNA and DNA standards wereincluded. Samples, DNA standards and RNA standards received100  m Lwell  1 of RiboGreen, followed by 5 min incubation in dark-ness. Then, the plate was read using the  󿬂 uorometer with a sensi-tivity of 55 (RNA and DNA together,  󿬁 rst reading). After the  󿬁 rstscan, 25  m Lwell  1 of RNase working solution was added and theplatewasincubatedfor20minindarkness.Aftertheincubationtheplate was scanned again to quantify solely the DNA (secondreading). RNase solution was found to increase the background 󿬂 uorescence, somewhat, and this increase was factored into thecalculations (Gorokhova and Kyle, 2002).  2.3. Stage structure Every four days, the age-speci 󿬁 c survival (l x ), the proportion of the females surviving to age x (days) and reproduction (m x ) rate,and the number of juveniles produced per surviving female be-tween the ages x and x  þ  1, were computed, from which life-cycletables were constructed. The net reproductive rate (R  0 ) is the meannumber of offspring per individual and it was calculated using thefollowing equation: R 0  ¼ X m x ¼ 1 l  x  m  x The generation length (G; days) was calculated using thefollowing equation: G  ¼ P m x ¼ 1 l  x  x  x m  x R 0 The life-table studies were  󿬁 nished at day 21, after which theintrinsic rates of population increase  r   (days  1 ) were calculated(Lotka,1913). Then  r   was approximated, according to: r    ln ð R  0 Þ G  2.4. Secondary production Secondary production was estimated by considering biomassincreaseineachvessel.Bothindividualgrowth(fromthebeginningto the end of the experimental period) and egg production (fromthe day 7) were taken into account. As dry weight was notmeasured, juvenile and adult weights were obtained from thelength-weight regressions, as established by Kawabata and Urabe(1998): LnW   ¼  3 : 05 þ 2 : 16 LnL ; where W is  m g of dry weight, and L is the length in mm.For length measurements, each individual was photographedunder a stereomicroscope (Leica MZ 125, Spain) in order to analyseindividual size, using Image-J software (U. S. National Institutes of Health, Bethesda). Total carapace length is de 󿬁 ned as the lengthfrom the top of the daphnid head to the base of its spine. Thebiomass of a given stage was obtained by multiplying the abun-dance of this stage by the weight that was estimated from therespective length-weight regression. The most commonly-usedmethod for measuring secondary production (PR) is the weightincrement method that consists of multiplying the weight-speci 󿬁 cgrowth rate of each developmental stage (  g  i ) by its biomass ( B i )(Hirst et al., 2005): PR  ¼ X ð  g  i B i Þ þ   g   f  B  f   The  󿬁 rst term of the equation corresponds to the somaticgrowthrateof thejuvenilepopulation(inthepresentexperimentalperiod from day 1 to day 7), and adult individuals (from the day 7),and the second term corresponds to the speci 󿬁 c egg productionrateof the females  g   f   (from day 8 to day 21), and the biomass of theadult females  B  f  .The growth rate (  g  i ) was estimated as follows (modi 󿬁 ed from:Hirst et al. (2005):  g  i  ¼  ln  W  exit  W  entry  MR where  W  entry  is mean weight of individuals in the day  i ,  W  exit   ismeanweightofindividualsintheday i  þ  1 ,and MR istheinverse of the experimental period selected (modi 󿬁 ed from: Jim  enez-Meleroet al., 2013). In this particular case, since the measurements wereperformedatregularintervalsof7days, MR isequalto0.143days  1 .The speci 󿬁 c egg production rates were calculated by using thefollowing equation:  g   f   ¼  W  eggs W   females MR ; where W  eggs iscalculatedbyconsideringtheweightofanindividualegg multiplied by the average clutch size, and  MR  is the inverse of the experimental period selected (see above). Egg weight wascalculated using the length-weight regressions, as established byGlazier (1992).When comparing two groups, an unpaired  t  -test was applied. G. Parra et al. / Chemosphere 156 (2016) 272 e  279 274  Comparisonsofthreeandmoregroupswereperformedwithaone-wayANOVAor Repeated MeasuresANOVA, when suitable. When itwas necessary, the data were log (x  þ  1) transformed to improvenormality and variance homogeneities (Zar, 1996).  P   values  <  0.05were considered signi 󿬁 cant. 3. Results The pH was monitored during the experimental period by theAT Control computer (366 measurements). The pH values weredifferent in control and low-pH vessels, throughout the experi-mental period, being their mean values 8.77 ( ± 0.17) and 7.00( ± 0.03), respectively ( t   ¼  209.8;  p  <  0.05). Dissolved oxygen andconductivitywerehigherinlow-pHvesselsatday7,althoughtherewere no statistically signi 󿬁 cant differences between control andlow-pH treatment vessels in dissolved oxygen, conductivity andalkalinity at the end of the experimental period (Table 1). At thistime, however, differences were found in the amount of foodavailable (phytoplankton cell abundance), that was higher in low-pH treatment vessels than in control vessels.AfactorialANOVAshowedthatacidi 󿬁 cationdidnotaffect totheRNA:DNA ratios (F (1, 79)  ¼  0.022,  p  ¼  0.88), which decreasedsigni 󿬁 cantly(F (2, 79)  ¼ 16.489,  p < 0.01)throughoutthetime(Fig.1).However, the fall patternwas not the same in both treatments (F (2,79)  ¼ 4.675,  p  ¼ 0.01);intheacidi 󿬁 edtreatmentthisratiodecreasesconsiderably over the time, in the control it seems that stabilizesfrom the day 14.Therewereno signi 󿬁 cant differences in the survival of   D. magna during the experimental period, except at day 21, where the low-pHvesselsshowedalowermeansurvival( t   ¼ 2.93,  p  ¼ 0.03;Fig.2).Differences in the population parameters R  0  and  r   were foundbetween control and low-pH treatments (vessels) (see Table 2).Although both experimental populations, controls and thosegrowing at pH  ¼  7, have the  󿬁 rst reproduction at the same time (9days), the net reproductive rate was signi 󿬁 cantly reduced by acid-i 󿬁 cation. Almost double the intrinsic rate of population increase ( r  )was observed in the control vessels (Table 2), whereas no differ-ences in the generation length (G), were observed between controland low-pH treatments.In fact, acidi 󿬁 cation had a signi 󿬁 cant effect on abundance of neonates from day 13 (ANOVA: F  ¼  3.05,  p  ¼  0.02). More than 70%of reduction in the accumulated neonates production occurred inlow-pH treatment vessels at the end of the experimental period(Fig. 3).In relation to adult total length, there were no differences be-tween control (3.49  ±  0.04 mm) and low-pH treatment vessels(3.35 ± 0.24 mm) at the end of the experimental period ( t   ¼  2.560;  p  ¼  0.12). But, differences were found in embryo size betweencontrol and low-pH treatments (0.34  ±  0.04 mm and0.29  ±  0.05 mm in control and low-pH treatment vessels, respec-tively;  t   ¼  4.697;  p  ¼  0.04) at the end of the experimental period.A repeated measures ANOVA showed a signi 󿬁 cant effect onsecondaryproduction of acidi 󿬁 cation( F   ¼  24.574;  p < 0.01), time of exposureofindividuals( F  ¼ 103.341;  p < 0.01)tothatconditionandtheir interaction ( F   ¼  22.527;  p  <  0.01) (Fig. 4). Indeed, secondaryproduction increased gradually throughout the time being signi 󿬁 -cantly higher at the end of the experiment, both in pH  ¼  7 (Bon-ferroni:  p  <  0.01) and the control (Bonferroni:  p  <  0.01).  Table 1 Mean values ( ± SD) of physical-chemical and biological parameters measured during the experimental period. * Denotes statistically signi 󿬁 cant differences between controland low-pH treatment (  p  <  0.05).Day 7 Day 14 Day 21Control pH 7 Control pH 7 Control pH 7Conductivity(mS cm  1 )0.84 (0.02)  0.88 * (0.01)  e e  0.85 (0.05) 0.91 (0.03)D.O.(mg O 2  l  1 )12.45 (0.94)  14.82 * (1.07)  e e  11.01 (1.48) 12.80 (1.18)Alkalinity(meq ml  1 )3.1 (0.7) 4.7 (1.1) 4.8 (0.8) 4.8 (0.3) 5.2 (0.9) 4.4 (0.5)Food(cells ml  1 )65,600 (4032) 68,287 (2583) 62,822 (3623) 77,485 (12,117) 69,762 (7562)  156,702 * (32,535)Note: Conductivity and Dissolved Oxygen data on day 14 were missed. 0510152025300 7 14 21    R   N   A  :   D   N   A  r  a   t   i  o Days controlpH 7 Fig.1.  RNA:DNA ratios mean value ( ± SD) obtained in  Daphnia magna  individuals fromcontrol and low pH vessels during the experimental period. Fig. 2.  Survival (%) mean value ( ± SD) obtained in  Daphnia magna  individuals fromcontrol and low pH vessels during the experimental period. * Denotes statisticallysigni 󿬁 cant differences between control and low pH vessels (  p  <  0.05). G. Parra et al. / Chemosphere 156 (2016) 272 e  279  275  Acidi 󿬁 cation caused a decline in production, such as can beobserved in Fig. 4.The contribution of every stage analysis (i.e. neonates-juvenileson one hand, and reproductive females on the other hand) on thesecondary productionwas preformed using differences on biomassseparately throughout the time. In the acidi 󿬁 ed vessels there wasnotdifferencesbetweenbiomassofneonates-juvenilesandfemales(Fig. 5a). In both stages biomass increased between T1 and T2 andthistrendcontinuedinthecaseofneonates-juvenilesbutnotinthefemales. Despite of this different trend in T3, no differences weredetected between biomass of both stages (Fig. 5a). The samepattern was observed in the control vessels, but in this case theincrement of neonates-juveniles 0 biomass between T2 and T3 wasmuch higherand signi 󿬁 cantly different (p < 0.05) to the biomass of females (Fig. 5b). Therefore, although both in control and pH  ¼  7the biomass of neonates increased over the time, this incrementwas considerably lower in the acidi 󿬁 ed vessels (Fig. 5c). In the caseof the females no differences were detected between treatmentsandtheincrementonlywassigni 󿬁 cantbetweenT1andT2(Fig.5d). 4. Discussion Theresultspresentedinthisstudyhaveshownthatacidi 󿬁 cationduring 21 days affects negatively  D. magna  population growth rateand neonate production. However, no differences were detected inadult size, nor in RNA:DNA ratio. Previous studies have shown thatunder limited food conditions,  Daphnia  prioritisation of resourceallocation will affect  󿬁 rstly reproduction, then growth and ulti-matelymaintenanceandhence survival(Glazier,1992),butno foodlimitation occurred during the present experiment. Urabe et al.(2003) reported lower individual growth in  D. magna  exposed toincreased CO 2  concentration, and found a relationship with adecreased food nutritional quality rather than a food limitation.This issue is especially important when  D. magna  is fed just withone microalgae species (Sterner et al., 1993), in the present exper-iment the mixed diet was used trying to avoid nutritional de- 󿬁 ciencies and an algae culture medium with two phosphorous (P)sources was used raising the P content (Urabe et al., 2003).Nevertheless, no direct nutritional status indexes were measuredsuch as Fatty Acids (FA) composition (Rossoll et al., 2012). So, otherindirect effects associated to the algae nutritional status might beinterfering in the results. However, it is necessary to point out thatadult growth has not seemed to be affected, as does occur withother invertebrates that are exposed to acidi 󿬁 cation (Carter et al.,2012; Keppel et al., 2012; Stumpp et al., 2011), while a lower em-bryos size was obtained. Then, an energy deviation strategy couldbe part of the response. The energy provided by the food probablymighthavebeendeviatedinorderto 󿬁 ghtagainstthephysiologicalconsequences of a low-pH environmental condition, instead of beingdeviatedtothereproductionenergycompartment,leadingtoreduced reproduction rates and smaller embryos.Although the RNA:DNA ratio changed during the growth pro-cess, surprisingly, there were no differences between treatments,suggesting that the pH experimental conditions might not inducechange in RNA:DNA ratios or even this biomarker is not effective/  Table 2 Life table parameters in each experimental population (vessels). R  0  is the net reproductive output per female at the end of the 21-day period (numbers of offspring). G is thegeneration length (days) and  r   is the intrinsic rate of population increase (day  1 ). * Denotes statistically signi 󿬁 cant differences (  p  <  0.05).Vessel R  0  R  0  mean(SD)G G mean(SD) r r   mean(SD)Control C1 12.90 12.78(1.50)18.50 18.38(0.38)0.14 0.14(0.01)C2 11.30 18.80 0.13C3 14.80 18.30 0.15C4 12.10 17.90 0.14Low pH L1 4.80 4.28*(0.95)18.40 17.68(0.51)0.09 0.08*(0.01)L2 5.30 17.50 0.10L3 3.80 17.20 0.08L4 3.20 17.60 0.07 Fig. 3.  Numbers of accumulated neonates during the experimental period. * Denotesstatistically signi 󿬁 cant differences between control and low pH vessels (  p  <  0.05). Fig. 4.  Variation in the secondary production over time at different pH (pH 7 andcontrol). T1 is the period between the beginning of the experiment and the day 7, T2 isthe period between the day 7 and the day 14, and T3 is the period between day 14 andday 21. Means predicted by a repeated-measures ANOVA (see text). Bars show con 󿬁 -dence levels of 0.95. Signi 󿬁 cant differences throughout the time are indicated withasterisks in grey. Signi 󿬁 cant differences at a given time are indicated in black.*p  <  0.05; **p  <  0.01; ***p  <  0.001. G. Parra et al. / Chemosphere 156 (2016) 272 e  279 276
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