Chemosphere 120 (2015) 165–170

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Chemosphere

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Natural humic substances effects on the life history traits of Latonopsisaustralis SARS (1888) (Cladocera – Crustacea)

http://dx.doi.org/10.1016/j.chemosphere.2014.06.0250045-6535/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +55 7199932211.E-mail addresses: carvalhopereira.tsa@hotmail.com (T.S.d.A.d. Carvalho-Pereira),

marenba@gmail.com (E.M. da Silva).1 NR, for Negro River.

Ticiana Soares de Andrade de Carvalho-Pereira ⇑, Thirza de Santana Santos, Edilene M.S. Pestana,Fábio Neves Souza, Vivian Marina Gomes Barbosa Lage, Bárbara Janaína Bezerra Nunesmaia,Palloma Thaís Souza Sena, Eduardo Mariano-Neto, Eduardo Mendes da SilvaBiology Institute – Federal University of Bahia (UFBA), Campus de Ondina, 40170-115 Salvador, Bahia, Brazil

h i g h l i g h t s

� NR treatments: trade-off between survival and reproduction to L. australis.� HA treatments: adverse results among L. australis life history traits analyzed.� NR20 (mgDOC L�1): recommended as supplement to L. australis artificial cultivation.

a r t i c l e i n f o

Article history:Received 11 December 2013Received in revised form 17 May 2014Accepted 10 June 2014

Handling Editor: A. Gies

Keywords:Dissolved organic carbonLatonopsis australisLife history traitsArtificial cultivationTrade-off

a b s t r a c t

Cultivation medium is one of the first aspects to be considered in zooplankton laboratory cultivation. Theuse of artificial media does not concern to reproduce natural conditions to the cultivations, which may beachieved by using natural organic compounds like humic substances (HS). This study aimed to evaluatethe effects of a concentrate of dissolved organic carbon (DOC) from the Negro River (NR1) and an extrac-tion of humic acids (HA) from humus produced by Eisenia andrei on the life history traits of laboratory-basedLatonopsis australis SARS (1888). A cohort life table approach was used to provide information about theeffectiveness of NR and HA as supplements for the artificial cultivation of L. australis. Additionally, we seekto observe a maximization of L. australis artificial cultivation fitness by expanding the range of HS concen-trations. The first experiment demonstrated that the females of L. australis reared under NR10 (mgDOC L�1)may have experienced an acceleration of the population life cycle, as the females have proportionally repro-duced more and lived shorter than controls. By contrast, the use of the HA did not improve life history traitsconsidered. The expansion of the concentration range (5, 10, 20 and 50 mgDOC L�1) corroborated thepatterns observed on the first assay. Results for the fitness estimates combined with shorter lifespans thancontrols demonstrated trade-offs between reproductive output and female longevity reared under NRconditions, with NR20 been suggested as the best L. australis cultivation medium. This response might beassociated with hormone-like effects.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Cultivation medium is one of the first aspects to be consideredin the laboratory standard zooplanktonic cultivation for populationand ecotoxicological studies (Klüttgen et al.,1994; Kilham et al.,1998; Loureiro et al., 2011). However the main interests in usingartificial media are the health or optimal ecotoxicologicalresponses of the cultivated organisms, regardless of reproducing

natural conditions in the cultivations. Approximating laboratorystudies to in situ conditions requires not only providing the naturalsalts and ions required for the organisms, but also the naturalorganic compounds.

Humification processes play the second roll, after photosynthe-sis, in ensuring the stability of the global cycling of energy andmaterials (Steinberg, 2003). The organic matter of soil and watercan be viewed as a mixture of plant and animal products in variousstages of decomposition, forming nonhumic and humic substances(HS) (Wetzel, 1983). In freshwater ecosystems, the dissolved HS isthe major carbon pool in the biosphere (Thurman, 1985). Typicallyranging from 50% to 75% in dissolved organic carbon (DOC), this

166 T.S.d.A.d. Carvalho-Pereira et al. / Chemosphere 120 (2015) 165–170

carbon source is available for both individuals and the simulta-neous development of populations (Steinberg, 2003). The dissolvedHS, which consists mainly of humic and fulvic acids, might beabsorbed by organisms cuticula, and can be adsorbed on bacterialcells or in algal surfaces (Steinberg et al., 2002; Koukal et al., 2003),before potentially being taken up by zooplankton, as an indirectsupplement source.

Among the freshwater zooplankton, the order cladocera com-prises the most efficient filter feeders in the lake pelagic commu-nity (Mourelatos and Lacroix, 1990). The effects of HS oncladocerans remain adverse. Extemporaneous sexual reproduction,reduction in longevity and production of reactive oxygen specieshave been observed in the Daphnia magna STRAUS (1820) life cycle(Euent et al., 2008; Steinberg et al., 2010). However, Moina macro-copa STRAUS (1820), in the presence of a natural HS (5, 10 and20 mgDOC L�1), increased the body size and longevity (Suhettet al., 2011). Therefore, it becomes relevant to ask if natural humicsubstances should be applied as a supplement to the artificial cul-tivation of cladocerans in the laboratory.

Two sources of natural humic substances were considered: (1) aconcentrate of Negro River DOC – which is mostly concentrated inHS, as fulvic and humic acids (Rocha et al., 1999) – and (2) anextraction of humic acids from humus produced by the oligochaeteEisenia andrei BOUCHÉ (1972) on a standard prepared substrate.While it is recognized that the Negro River is the most representa-tive in the humic and fulvic acids flux (70%) to the Amazonas River(Ertel et al., 1986), humic acids extracted from humus produced byoligochaetes from the genus Eisenia have shown to be a good sup-plement for the root development and increase in height and leafarea of plants, probably through hormone-like effects (Atiyehet al., 2002; Canellas et al., 2002).

The main objective of this study was to evaluate the effects ofthese natural humic substances on life history traits of a laboratorypopulation of Latonopsis australis SARS (1888), in order to find aDOC concentration which could be used as a supplement for theartificial cultivation of this species. The first specific objective wasto test both sources of natural HS using a similar DOC concentrationfound in the Negro River on the life history traits of L. australis. Thesecond, was to expand the range of DOC concentrations for both HSused (remaining in a range naturally found in freshwaters), and testfor a maximization of L. australis population fitness compared tocultivation in artificial medium alone. Population fitness was mea-sured as the intrinsic rate of increase (rm) and the net reproductiverate (Ro). This would be carried out using a single-female life table,in a cohort approach (Townsend et al., 2006).

2. Material and methods

2.1. Organisms and laboratory cultivation

The benthic L. australis has been found mostly in the littoralzone of freshwater tropical ponds (Korovchinsky, 1992; Crispimand Watanabe, 2001; Elmoor-Loureiro, 2007; Korovchinsky andSanoamuang, 2008), in waters ranging from 2.5 to 25 mgDOC L�1

(Mladenov et al., 2005; Lindholm et al., 2009; Amaral et al.,2011; Ghidini, 2011). The animals were collected in a small reser-voir in Cruz das Almas (BA, Brazil), and a single female (clone) waschosen to start the cultivation. The cultivations consisted of vesselscarrying around 40 individuals in aged and filtered natural waterfrom a humic acid rich wetland (20 mgDOC L�1) (�12.819606,�38.220255, Jauá Lake, Camaçari-BA, Brazil). This clone has provedto be an useful species in ecotoxicological assessment (Araújoet al., 2008), as well as an effective species in identifying interspe-cific differences in sensitivity to acidity and in the ability tocompete, with potential to future recolonize an acidified environ-ment (Saro et al., 2011).

Twenty-four hours old females from third generation were cho-sen randomly from the natural medium laboratory cultivation toinitiate the cultivations in an artificial medium made of reconsti-tuted hard water from ASTM (1980). This medium carries no chelat-ing agent, leaving metal ions available (OECD, 2008). The artificialcultivations have been carried out in the laboratory for five yearsin glass vessels carrying 470 mL of medium, with 30 individualsin each vessel. They have been kept under a 12 h:12 h (light:dark)photoperiod, with food available (ca. 104 cells mL�1 of standardizedcultivated green algae Pseudokirchneriella subcapitata/day) and at25 ± 2 �C.

2.2. Humic acids

Humic acids (HA) were extracted once from two differenthumus produced by the oligochaete E. andrei – cultivated in thelaboratory according to standard ISO (1998) under 25 �C – generat-ing two different solutions used in each experiment. The extrac-tions followed the methods proposed by Rosa et al. (2000), withsome modifications on the centrifugation frequency (3000 rpm),and final procedures to obtain the HA in liquid phase.

2.3. Negro River DOC

Reverse osmosis (RO) procedures were carried out to obtain theDOC concentrated solutions from the Negro River (Serkiz andPerdue, 1990; Sun et al., 1995), called hereafter ‘‘NR’’. All the pro-cedures were repeated, from sampling to concentration efforts, togenerate another concentrated of NR DOC for the second test.

DOC concentrations of both HS (HA and NR) were estimatedaccording to Dabin (1976).

2.4. Cohort life table

2.4.1. Experiment one: NR and HA at 10 mgDOC L�1, similarconcentration naturally found in Negro River (Rocha et al., 1999)

Neonates from the third generation were randomly selected toinitiate the tests. Ebert (1993) has shown that before the 3rd broodthe distribution of Daphnia neonate length present larger varianceswithin clutch than later broods. This allowed for the exclusion ofthe two first broods to set cultivations and experiments to avoidconfounding variables (Hurlbert, 1984).

Twenty-four hours old females from reconstituted hard watercultivation were individually distributed to jars containing 80 mLof reconstituted hard water, with HS added whenever needed.There were 10 experimental units to each treatment as follows:Control (reconstituted hard water), HA (reconstituted hardwater + HA in 10 mgDOC L�1), NR (reconstituted hard water + NRDOC in 10 mgDOC L�1).

Treatments followed similar laboratory cultivation. Organismswere counted and fed daily, and then neonates were counted andremoved. Every three days, the media were changed, after thephysical and chemical variables of each treatment were measuredand controlled. The experiment terminated with the death of thelast female (Suhett et al., 2011). Each treatment provided data ofage at primipara, mean and maximum lifespan, mean and total off-spring produced per day, and total offspring per female at the endof the experiment. Additionally, age specific survival and broodsize were used to estimate measures of fitness as the net reproduc-tive rate (Ro) and the intrinsic rate of increase (rm).

2.4.2. Experiment two: NR and HA at 5, 10, 20 and 50 mgDOC L�1,range naturally found in freshwater ecosystems (Steinberg, 2003)

The selection of neonates from laboratory cultivations occurredas previously described. Twenty-four hours old females fromreconstituted hard water cultivation were individually distributed

T.S.d.A.d. Carvalho-Pereira et al. / Chemosphere 120 (2015) 165–170 167

to jars containing 80 mL of reconstituted hard water, with HSadded whenever needed. There were 10 experimental units to eachtreatment concentration as follows: Control (reconstituted hardwater, 0 mgDOC L�1), HA (reconstituted hard water + HA in fourDOC concentrations: 5, 10, 20 or 50 mgDOC L�1), NR (reconstitutedhard water + Negro River DOC in four concentrations: 5, 10, 20 or50 mgDOC L�1).

Treatments followed similar laboratory cultivation and experi-mental conditions as previously described. The life history traitsand measures of fitness for each concentration of each HS weresimilar to that in experiment one.

2.4.3. Statistical analysesMaximum lifespan, age at primipara, total number of offspring

per female per treatment and total number of offspring producedin broods 3–6 were compared in test one using one-way ANOVAor by permutation one-way ANOVA, whenever the ANOVAassumptions were not met (Anderson and Robinson, 2001). Inexperiment two, these traits were compared using a permutationtwo-way ANOVA in a nested design, wherein the factor concentra-tion (0, 5, 10, 20 and 50 mgDOC L�1) would be nested in the vari-able ‘substance’. Post-hoc comparisons were carried out usingFisher’s LSD test. Ro and rm were estimated according to Meyeret al. (1986). Age specific survival and brood size were used to esti-mate rm by 500 permutations, generating means and confidenceintervals (2.5% and 97.5%) for each treatment.

The statistical analyses were carried out using the R package2.14.2 (R Development Core Team, 2011), with a = 0.05.

3. Results

In experiment one, both HS treatments had shorter lifespansthan the Control (p < 0.05; Figs. 1A and 2A). On the other hand,females of NR treatment had proportionally better reproductiveresponses than the Control. The NR females reached maturity earlier(p < 0.05; Fig. 1B), presented with no significant difference in totalnumber of offspring produced per female (Fig. 1C) and providedthe highest rm (rm = 0.33, rm = 0.37, rm = 0.39, to Control, HA andNR, respectively).

Fig. 1. Experiment one – (A) lifespan; (B) age at primipara; (C) total number of offspringmedians are represented by the black bars crossing the boxes latitudinally. Different let

Females of HA treatment, except for reaching maturity, livedshorter and produced less offspring than the Control (p < 0.05;Fig. 1C). Also, the HA treatment presented with a significantlysmaller number of offspring produced in broods 3–6, than thoseproduced in the NR treatment (p < 0.05; Fig. 1D). The mean netcontribution of neonates for the next generation represented byRo of 41.5, 57.5 and 62.0 (HA, NR and Control, respectively) corrob-orates the worst reproductive response of HA in experiment one(Fig. 3A–C).

In experiment two, all HS treatments presented with shorterlifespans than the Control (p < 0.05; Figs. 4A and 2B and C). TheNR treatments and HA05 presented with similar lifespans, whileHA10, HA20 and HA50 have shown a clear pattern wherebyincreased DOC concentration led to an important reduction in life-span with an associated decrease in the daily (Fig. 3E–H) and totalnumber of offspring produced per female (Fig. 4C). This resulted intheir exclusion from the analysis for the total number of offspringproduced in broods 3–6 (Fig. 4D), in which NR50 presented with asignificantly higher response compared to the Control. It is note-worthy that HA10 in experiment two has shown both shorter life-span (p < 0.05) and lower number of offspring produced per female(p < 0.05) compared to the HA10 of experiment one.

There was no significant difference among ages at primipara ofeach treatment tested, typically occurring in the 7th day of life(Fig. 4B), even under the concentration of 10 mgDOC L�1. Thevarious NR treatments and the Control did not differ in the totalnumber of offspring produced per female (Fig. 4C). However,NR05 showed the smallest production among them, which wassignificantly different than the NR20 production. In this context,NR20 presented with the highest Ro in the experiment: 70.5.Also, while the concentration of NR increased, concomitant rm

increase of these treatments occurred, achieving its highest valuein NR20: rm = 0.42 (rm = 0.38, rm = 0.34, rm = 0.39, rm = 0.26,rm = 0.17, rm = 0.32, rm = 0.35, rm = 0.42, rm = 0.38, to Control,HA05, HA10, HA20, HA50, NR05, NR10, NR20 and NR50,respectively). These combined results for NR treatment have char-acterized a trade-off pattern between reproductive output and lon-gevity for NR females only observed with the HS concentrationsexpansion (Fig. 3I–L).

produced per female and (D) total number of offspring produced in broods 3–6. Theters represent statistical differences among treatments.

Fig. 2. Survival Curves – (A) experiment one; (B) experiment two: Control + HA treatments and (C) experiment two: Control + NR treatments.

Fig. 3. Reproduction patterns represented by the daily offspring production per female. (A)–(C) experiment one; (D)–(L) experiment two.

168 T.S.d.A.d. Carvalho-Pereira et al. / Chemosphere 120 (2015) 165–170

4. Discussion

Among the HS chosen to supplement to L. australis cultivation,only NR treatments presented with a compensatory response onreproduction traits. Generally, the females produced offspring inalternate days, which has been less variable in the NR treatment.Daily offspring production contributes to the definition of popula-tion broods, which facilitates decision making for the best period ofcollection for neonates in population studies. The NR treatmentsincreasing in total number of offspring produced in broods 3–6indicates not only a greater offspring production/female/broodbut also a high synchronization in the production. This is a positive

feature, as a higher offspring production in a brood means highernumber of organisms available to be used on population and eco-toxicological studies (Xi et al., 2005; Araújo et al., 2008).

The cost of reproduction suggests that an individual mayincrease its current allocation to reproduction, will likely decreaseits survival and/or its rate of growth, and therefore decrease itspotential for reproduction in the future (Sarma et al., 2002). Thecombination of trait responses and highest rm found for NR treat-ment in experiment one showed that this population might haveaccelerated its life cycle, despite no observed increased total off-spring production. The acceleration on organisms life cycles mightrepresent a positive strategy, as it ensures the maintenance of the

Fig. 4. Experiment two – (A) lifespan; (B) age at primipara; (C) total number of offspring produced per female and (D) total number of offspring produced in broods 3–6. Themedians are represented by the black bars crossing the boxes latitudinally. Different letters represent statistical differences among treatments.

T.S.d.A.d. Carvalho-Pereira et al. / Chemosphere 120 (2015) 165–170 169

population in the natural environments, where they are subject topredation (García and Guido-Pereira, 2000) and to environmentalvariations (Crispim and Watanabe, 2001; Suhett et al., 2011).

Humic and fulvic acids can originate from polyphenols of plantor microbial origin (Stevenson, 1994). The portion of proteins andphenols in the HA increases under the conditions of the oligochaetedigestive tract, where the alkaline conditions increase the solubil-ity of phenols and amino acids (Frouz et al., 2011). It has beenshown that phenols produce toxic effects on D. magna, howeverthere are mechanisms to reduce these effects, as phenols can formcomplexes with metals and become unavailable for biota (Kimet al., 2006). The artificial medium used carried no chelatingagents, rather the HA produced by E. andrei may have carried ahigh portion of phenols, turning L. australis sensitive to theconcentration of HA 10 mgDOC L�1, an opposite expectation tothe hormone-like effects in plants presented in literature.

The expansion of the concentration range of both HS usedallowed for the identification of two different patterns, presentedby the females under each HS tested. However, the adverse resultsfor age at primipara among experiments leads us to question thescale in which this trait is measured, as ‘‘hours’’ may provide moreaccurate results, than ‘‘days’’, for this demographic variable. Eachpattern corroborated the effects observed on the first assay,whereby increased DOC concentration led to an important reduc-tion in lifespan and reproduction in HA treatments, while femalesof NR treatment have invested more in reproduction in spite ofreduced lifespan. NR20 has showed the best fitness of the experi-ment, presenting a higher rm and similar lifespan and offspringproduction per female compared to the results found by Saroet al. (2011). These authors have cultivated the same L. australisclone we used, however, under natural medium (Jauá Lake,Camaçari-BA, Brazil), which also presents around 20 mgDOC L�1.This finding corroborates the use of 20 mgDOC L�1 concentrationof NR in L. australis artificial cultivation.

Our results of increasing DOC concentrations in NR treatmentsoppose those found for increasing green algal carbon availabilityby Chaparro-Herrera et al. (2010). The reproduction numbertended to increase with concentration in NR treatments, while

reproduction number tended to decrease with the amount of foodconcentration. Therefore, NR might not be considered a foodsource, and another function might have influenced the increasein reproduction. Rocha et al. (1999) have shown that the NegroRiver HS contains high aromaticity, however, with no phenol con-tents, contrary to the HA extracted from E. andrei in humus.Steinberg et al. (2002) and Meinelt et al. (2004) have shown thatthe nematode Caenorhabditis elegans and the fish Xiphophorus hel-leri, respectively, presented hormone-like reactions in the presenceof alkylaromatics (alkylphenols) from HS contents. The potential toreproduce the amount of females cultivated in NR treatmentsmight, therefore, be associated with hormone-like effects assignedto L. australis females, which is characterized by the presence ofaromatic compounds other than phenols.

This is hypothesized as phenols may have been toxic to femalesunder HA from E. andrei as previously described. By contrast,Campos et al. (2012) showed that phenols can enhance the repro-duction of D. magna when presented in small concentrations whilealso decreasing reproduction in high concentrations. However theresponsible mechanisms remain unknown, and may explains thebetter reproductive responses of HA05 compared to the other HAtreatments. The adverse results found for HA10 extracted of differ-ent humus produced by E. andrei, in experiments one and two, onlycorroborate the avoidance to use this HS as a supplement toL. australis laboratory artificial cultivation.

5. Conclusions

This work demonstrates that the increasing concentrations ofDOC found in Negro River in L. australis artificial laboratory cultiva-tion led to trade-offs between survival and reproduction. The useof HA extracted from humus produced by E. andrei, in contrast,led to adverse results among the life history traits analyzed. It issuggested the use of NR concentrations, like 20 mgDOC L�1, as asupplement in L. australis artificial laboratory cultivation, due tothe benefits found in the reproductive responses and fitness forthis population. It is also recommended to conduct sensitivitytests, once L. australis females are reared in the concentration

170 T.S.d.A.d. Carvalho-Pereira et al. / Chemosphere 120 (2015) 165–170

suggested, to evaluate the supplemented cultivation viability inecotoxicological approaches. This would help to simulate naturalenvironmental responses in a laboratory context as HS are foundin all natural ecosystems, even in so-called clear-water lakes(Steinberg, 2003).

Acknowledgements

The authors are grateful to the Brazilian Coordination for theImprovement of Higher Level -or Education-Personnel (CAPES),which granted T.S.A. Carvalho-Pereira scholarship, for making thiswork possible. E.M. da Silva acknowledges receiving a scholarshipfrom the Brazilian Research Council (CNPq). The authors are alsograteful to the Laboratory of Ecophysiology and Molecular Evolu-tion of the National Institute for Amazonian Research (LEEM-INPA)for providing the concentrate of Negro River DOC used in the pres-ent study. The authors also acknowledge Gabriel Ghizzi and Aloy-sio Ferrão-Filho for providing statistical help and Antoine Leduc,Kathryn Hacker and Joshua Taylor for providing language help.

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