- Email: [email protected]
Leaf litter leachates have the potential to increase lifespan, body size, and offspring numbers in a clone of Moina macrocopa
Chemosphere 86 (2012) 883–890
Contents lists available at SciVerse ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Leaf litter leachates have the potential to increase lifespan, body size, and offspring numbers in a clone of Moina macrocopa Sylva Hofmann a, Maxim A. Timofeyev b,c, Anke Putschew d, Nadine Saul a, Ralph Menzel a, Christian E.W. Steinberg a,⇑ a
Humboldt-Universität zu Berlin, Department of Biology, Laboratory of Freshwater and Stress Ecology, Arboretum, 12437 Berlin, Germany Irkutsk State University, Institute of Biology, Ul. Karl-Marx 1, 664003 Irkutsk, Russia Baikal Research Centre, Ul. Karl-Marx 1, 664003 Irkutsk, Russia d Technical University Berlin, Department of Water Quality Control, Sekr. KF4, Straße des 17, Juni 135, 10623 Berlin, Germany b c
a r t i c l e
i n f o
Article history: Received 1 June 2011 Received in revised form 19 October 2011 Accepted 24 October 2011 Available online 23 November 2011 Keywords: Coniferous trees litter Deciduous trees litter Mitohormesis Oxidative stress Moina macrocopa Lifespan extension
a b s t r a c t Leaf litter processing is one major pathway of the global organic carbon cycle. During this process, a variety of small reactive organic compounds are released and transported to the aquatic environment, and may directly impact aquatic organisms as natural xenobiotics. We hypothesize that different forest stockings produce different leachate qualities, which in turn, stress the aquatic communities and, eventually, separate sensitive from tolerant species. Particularly, leachates from coniferous trees are suspected to have strongly adverse impacts on sensitive species. We exposed individuals of a clone of the model organism, Moina macrocopa, to comparable concentrations (approximately 2 mM) of litter leachates of Norway spruce, Picea abies, Colorado blue spruce, Picea pungens, black poplar, Populus nigra, and sessile oak, Quercus petraea. The animals were fed ad libitum. The following life trait variables were recorded: growth, lifespan, and lifetime offspring. To identify, whether or not exposure to litter leachates provokes an internal oxidative stress in the exposed animals we measured the superoxide anion radical scavenging capacity via photoluminescence. Except of P. abies, exposure to the leachates reduced this antioxidant capacity by approximately 50%. Leachate exposures, except that of Quercus, increased body size and extended lifespan; furthermore, particularly the leachates of both Picea species significantly increased the offspring numbers. This unexpected behavior of exposed Moina may be based on food supplements (e.g., high carbohydrate contents) in the leachates or on yet to be identified regulatory pathways of energy allocation. Overall, our results suggest that the potentially adverse effects of litter leachates can be overruled by either bacterial-growth supporting fractions in the leachates or an internal compensation mechanism in the Moina individuals. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Although it is well known that, after photosynthesis, humification is the second largest complex of biogeochemical reactions on Earth and is mandatory to recycle energy and organic carbon and, thus, facilitate an almost closed cycling of organic matter (Schlesinger, 1997), its ecological impacts are much less studied and understood than that of photosynthesis. For instance, leaf litter fuels detritus food chains via microbial processing. Whereas leaf litter leachates with humic substances, HSs, as one of its major components were thought to be inert in terms of direct interactions with organisms, recent reports show that HSs can be taken up through epithelia or indirectly via food (Wang et al., 1999; Steinberg et al., 2003). Consequently, an array of stress symptoms ⇑ Corresponding author. Tel.: +49 30 6322 4715; fax: +49 30 6369 446. E-mail address: [email protected] (C.E.W. Steinberg). 0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2011.10.041
have been reported in organisms exposed to HSs. On the phenotypic level, adverse effects including lethality were found in HS-exposed organisms, both aquatic invertebrates (Rey et al., 2000; David et al., 2001; Steinberg, 2003; Canhoto and Laranjeira, 2007) and fishes (Tremolieres, 1988; McMaster and Bond, 2008; Morrongiello et al., 2011). Also, one of the European freshwater pearl mussels, Margaritifera margaritifera L., declined significantly after forestry in the catchment changed from deciduous to coniferous trees as reported from Sweden and Austria (Björk, 2004; Csar et al., 2004), and the authors assumed that this decline is more than a pure coincidence. Probably, these changes led to HSs with higher aromatic moieties that supported, due to the low nitrogen content, less microbial growth (He et al., 2007), and poor food for the mussels (Steinberg et al., 2006). On the histological, biochemical, and molecular biological level, the stress symptoms include degeneration of the midgut epithelium (Rey et al., 1999), energy consumption (Cazenave et al.,
884
S. Hofmann et al. / Chemosphere 86 (2012) 883–890
2006), reactive oxygen species accumulation and reduced antioxidant capacity, membrane lipid peroxidation, increased activity of biotransformation and antioxidant enzymes (Tilquin et al., 2004), increase of stress proteins, such as a-crystalline small heat shock proteins (sHSPs) and HSP70 (Timofeyev et al., 2004; Timofeyev et al., 2006; Bedulina et al., 2010; Steinberg et al., 2010a), and chemosenzitation of MXR exporters (Timofeyev et al., 2007). Rey et al. (2000) and David et al. (2001) attribute the adverse effects on arthropods to tannin-phenolic compounds. Later, Steinberg et al. (2003) coined the oxymoron ‘‘natural xenobiotics’’ for HSs in relation to their interactions with the biota – in analogy to secondary plant compounds (Li et al., 2007); yet, in contrast to the latter, HSs are not vectored against herbivores, but challenge any exposed organism. In the meantime, some direct effects are described; for instance, HSs and tannins can Act as xenohormones as shown in invertebrates, fishes, amphibians, and Arabidopsis (Höss et al., 2001; Meinelt et al., 2004; Lutz et al., 2005; Trevisan et al., 2010); Interact with the Ah receptor (Bittner et al., 2006; Matsuo et al., 2006; Janošek et al., 2007); Modulate biotransformation enzymes of phase I and phase II (Meems et al., 2004; Tilquin et al., 2004; Matsuo et al., 2006; Andersson et al., 2010); Oxidatively damage DNA (Hu et al., 2010). The effects of HSs in exposed organisms are by no means unequivocal, since specific building blocks (mainly phenolic and polyphenolic) ones, are suspected to be responsible for adverse effects on the freshwater mollusks and are, on the other hand, shown to have beneficial effects on the nematode Caenorhabditis elegans Maupas. This organism actively migrated into HS-rich environments, although these were clearly linked to chemical stress (Menzel et al., 2005), yet with the benefit of lifespan extension (Steinberg et al., 2007). These and subsequent papers have shown that mild chemical stress induced by HSs or their building blocks may ultimately not only extend lifespans, but also increase resistance to multiple stressors, including netting, high temperatures, or oxidative environments (Meinelt et al., 2004; Pietsch et al., 2009; Saul et al., 2010; Pietsch et al., 2011). Litter leachates do not contain only HSs, but also a variety of non-humic materials as recently reported by Kiikkilä et al. (2011) who showed that dissolved organic matter derived from fresh spruce needle litter had high concentrations of carbohydrates and phenolic compounds. The carbohydrate content was easily degradable and the remainder was characterized by a high C:N ratio as mentioned by He et al. (2007) and in a pedological textbook (Tan, 2009). From previous studies with C. elegans, we understand that carbohydrate-rich HSs tend to reduce the worm’s lifespan (Steinberg et al., 2007). Whether the leachate contains high share of non-humic materials depends on the sampling season and their environmental fate. Since C. elegans is an endogeic animal and the water percolation usually slow, it can be assumed that, in nature, it will be exposed to remainders of the litter rather than to carbohydrates. It is also likely that pelagic organisms are rather soon exposed to the leachates carried into the water by the run-off water after storm events and these organisms appear to be the more appropriate model organisms in this respect. This assumption receives support by the fact that leaf litter alone contributes up to 30% of the total dissolved organic carbon (DOC) in streams (Meyer et al., 1998). The aforementioned partly contradictory observations intrigued us to figure out, which effects prevail. We comparatively evaluated the impact of litter leachates of two deciduous and two coniferous tree species in terms of life trait variables in a Moina macrocopa
Straus clone. The deciduous species were black poplar, Populus nigra L., and sessile oak, Quercus petraea (Mattuschka) Liebl., and the coniferous ones were Norway spruce, Picea abies (L.) Karst., and Colorado blue spruce, Picea pungens Engelm. All trees are common in European forests, parks, or gardens. To characterize the various leachates, we applied a size-exclusion-chromatographic fingerprint technique with double detection (UV, dissolved organic carbon) which gives sufficient information about the humic and non-humic materials (Sachse et al., 2001). Moina macrocopa is a world-wide distributed cladoceran and belongs to a group of large-bodied Moina species; it is characteristic inhabitant of small, usually ephemeral water bodies in the temperate zone, often rich in DOC (Petrusek, 2002). The tested clone was isolated from a puddle in Rio de Janeiro, Brazil (Elmoor-Loureiro et al., 2010) and was successfully used in effect evaluations before (Pietsch et al., 2010; Steinberg et al., 2010b; Suhett et al., 2011); for the sake of comparison, this tropical clone was applied also in the present study. 2. Material and methods 2.1. Test design The stock culture was maintained in Berlin tap water with a mean Ca2+ concentration of 2.5 mM. This relatively hard water was chosen to avoid any risk of potential acidification by adding the leachates, which might be acidic. In order to evaluate only the impact of the biogeochemicals on the life performance of the cladocerans, the development of further stresses, such as crowding, with the subsequent development of males, was avoided. The densities of individuals, therefore, were evaluated in pre-tests which allow pure parthenogenetic reproductions. All exposure scenarios with the leachates were not stressful with respect to crowding, because males did not occur. Only 3rd generation neonates were used for the experiments. In each 500 mL Erlenmeyer vessel, 30 individuals were placed; the vessels were kept in a water bath at 30 ± 1 °C and illuminated by cool white light in a 14:10 h rhythm. Lifespan and reproduction assays were carried out in duplicates in two independent trials. Growth tests were carried out in separate series. Three trials with 30 animals each were conducted. The microscopic measurements of the entire body length of 30 animals each were taken at the end of 5-d cultivation after the animals were narcotized with ethanol. The presented data are means of the individual trials and subject of the statistical evaluation. Animals and offspring were counted and exposure was exchanged every second day. M. macrocopa was fed with the green algae Pseudokirchneriella subcapitata (Koršikov) Hindák. P. subcapitata strain NIVA-CHL 1 was obtained from the Culture Collection of Algae at the University of Göttingen, Germany. The algae were cultivated in freshwater medium (Nicklisch et al., 2008), and grown in batch cultures under day light conditions. The animals were fed daily ad libitum with 10 mL of a logarithmically growing culture which corresponds to 107 cells or approximately 2.5 mg L 1 C. 2.2. Leachate preparation and DOC fingerprint characterization Litter samples were collected in August/September 2009 in the Arboretum, the adjacent urban forest Königsheide, and a private garden in Berlin. The litter was collected during a long drought period. Immediately after collecting the material, the leaves were sorted; earthy remains and soil animals were removed and leaves were dried for 48 h at 56 °C. The samples were powdered in a household coffee grinder and 50 g litter were leached with 0.75 L de-ionized water for 48 h. During the leaching process, the suspen-
885
S. Hofmann et al. / Chemosphere 86 (2012) 883–890 Table 1 Exposure concentrations and shares of the finger print fractions of the various leachates. Leachate
Exposure concentration (mM)
Polysaccharides
Populus nigra Quercus petraea Picea abies Picea pungens
2.38 2.00 2.15 1.50
2.5 17.4 16.8 7.1
Humic substances
LMW organic acids
LMW substances
% of the dissolved organic carbon 64.6 73.7 65.1 78.8
28.0 5.7 5.7 6.3
4.9 3.2 12.4 7.8
LMW = low-molecular weight.
sion was stirred with a magnetic stirrer. Then the suspension was centrifuged with 4000 rpm at 4 °C and filtered through glass fiber filters (Whatman G6, Whatman, England). This means that, in compliance to the definition of DOC, the filtrate contained colloidal and even fine-particulate carbon. All stock solutions were diluted with Berlin tap water until they had an identical light extinction. The exposure concentrations lay between 1.5 mM (P. pungens) and 2.38 mM (P. nigra) (Table 1) due to different specific UV absorption of the leachates. Concentrations of non-fractionated dissolved organic carbon samples were determined by means of a TOC-505 A analyzer (Shimadzu, Germany). Automated size-exclusion-chromatography with UV- and organic carbon detection (IR) was used for characterization, as described in technical detail by Sachse et al. (2001). Four different groups of DOC could be fractionated: polysaccharides, humic substances (HSs), low molecular weight acids, and low molecular weight substances. The polysaccharide fraction is high molecular weight carbohydrates, which show no absorption in the UV range. HSs include humic and fulvic acids and their building blocks, which are UV active. Low molecular weight acids are defined as low molecular weight carboxylic acids such as several metabolites in biological and chemical processes. DOC in the low molecular weight range after the low molecular weight acids peak was described as other low molecular weight substances.
2.3. Superoxide anion radical scavenging capacity The antioxidative capacity of a cell or an organism indicates the potential to balance reactive oxygen species, ROS, namely superoxide anion radicals, hydroxyl radicals, and hydrogen peroxide. ROS are, e.g., generated in any heterotrophic reaction, in defense of pathogens, or after uptake of heavy metals or xenobiotic chemicals. Since superoxide anion radicals are the first products of triplet oxygen reduction, monitoring these ROS appears to be representative of all ROS produced by reduction. In M. macrocopa, this capacity was measured using 25 individuals exposed for 5 d against the leachates. The animals were deep frozen in liquid nitrogen in phosphate buffer, cleaved and homogenized in a Speedmill (AnalytikJena, Germany), and centrifuged at 13 000 rpm. The cooled supernatant of the homogenate was analyzed via photoluminescence for the superoxide anion radical scavenging capacities (SOSC in ascorbic acid equivalents) in a PhotoChem device (AnalytikJena, Germany) and the corresponding ACW-Kit (AnalytikJena, Germany) according to Popov and Lewin (1998). This assay measures the ability of an antioxidant to quench free, photochemically generated superoxide anion radicals by hydrogen donation. The sensitive luminometric detection uses luminol as detector substance. In biological samples, they may be quenched by antioxidant substances and enzymes. This quenching gives an inverted measure of SOSC and must be calculated in comparison to a standard substance, such as ascorbic acid. SOSC values in the animals were related to their protein content, which was measured according to Bradford (1976) using Coomassie Brilliant Blue G250 (Sigma Aldrich, USA). All measurements were done in triplicates.
2.4. Statistics Statistically significant differences between entire lifespans were tested by means of the log-rank test, which was developed for lifespan curves (Anonymous, without year); hence, statistical information for each single data point, such as standard deviation, is not necessary and not presented in the graph. To gauge reproduction, the offspring numbers were checked daily. Mean values of the reproductive output were taken to check the statistical significance. The significance of SOSC, body size after 5 d, and reproductive output was calculated by one-way ANOVA (Sigma Stat 3.5, SPSS Inc., USA). Cumulative curves of the reproductive output are additionally presented in the graphs in order to identify potential phases of modulated reproduction. 3. Results The fingerprint characterization revealed that all leachates contained considerable amounts of polysaccharides (UV-inactive, but DOC-active with retention times of approximately 45 min) with P. nigra having the lowest and Q. petraea the highest share; as negative control, the carbohydrate-free preparation HuminFeedÒ1 was also chromatographed (Fig. 1). The litter of both Picea species contained between 7 and almost 17% of carbohydrate carbon (Table 1). This indicates that the humification process was at its infancy when the samples were collected. The HS fractions comprised the highest carbon concentration and covered between 65% (P. nigra, P. abies) and 79% (P. pungens). Interestingly, the P. nigra leachate contained a high share of low-weight molecular acids, and the P. abies leachate of low-weight substances. Except of the P. abies leachate which did not significantly modulate SOSC, all leachates significantly reduced SOSC in the exposed M. macrocopa specimens (Fig. 2). Furthermore, the body size significantly increased upon exposure to all but one (Quercus) litter leachates by 0.1 to 0.15 mm (Fig. 3). The enlarged animals also lived significantly longer than the control ones or those ones exposed to the Quercus leachate (Fig. 4). The mean and median lifespan values are presented in Table 2. Both coniferous leachates highly significantly increased the reproductive output of the females (Fig. 5), whereby reproduction happened until the individuals pass away (Fig. 6). Both deciduous leachates only tended to increase reproduction. It is interesting to note that this increase in reproduction by the coniferous leachates was not only due to the longer lifespan, but took place also during the shorter lifespans which applied to the control individuals (Fig. 5). Relating the life trait variables to the fingerprint fractions of Table 1, there was no fraction identifiable which could explain why the Quercus leachate was not effective in terms of body growth, lifespan extension, or reproductive output. The missing reproductive output in Populus-exposed animals, however, could be due to the high share of unidentified low-molecular weight organic acids. 1 http://www.humintech.com/001/animalfeeds/products/huminfeed.html (accessed May, 2011). See this site for more information; the use of HuminFeedÒ is by no means an advertisement for this product.
886
S. Hofmann et al. / Chemosphere 86 (2012) 883–890 Humic substances LMW LMW & building blocks org acids substances
Polysaccharides
100
Picea pungens*
Survival , %
Dissolved organic carbon, arbitrary units
Populus nigra **
Picea pungens Quercus petraea
Populus nigra
Control
50
Picea abies
Quercus petraea Picea abies** Picea abies** HuminFeed®
0
1
5
10
15
Days Fig. 4. Lifespan modulation in Moina macrocopa females exposed against four different litter leachates. ⁄p < 0.05; ⁄⁄p < 0.01.
Retention time, min
5.0
Table 2 Mean and median lifespan values, d ± SD. Leachate
Mean lifespan
Median lifespan
Control Populus nigra Quercus petraea Picea abies Picea pungens
8.9 ± 0.3 10.7 ± 0.4 8.8 ± 0.2 10.6 ± 0.4 9.2 ± 0.2
8.9 ± 0.3 11.2 ± 0.2 8.7 ± 0.3 10.7 ± 0.5 9.1 ± 0.3
Leachate Fig. 2. Mean superoxide anion radical quenching capacity of 5 d old Moina macrocopa females exposed to four different litter leachates. ⁄p < 0.05; ⁄⁄p < 0.01.
** **
**
100
50
Picea pungens
0
Picea abies
Picea
150
Quercus
pungens
abies
Populus
Quercus
**
Populus
**
**
**
**
Control
2.5
Reproductive output per individual, N
200
Control
Superoxide anion radical scavenging capacity nM ascorbic acid per mg protein
Fig. 1. Qualitative DOC-fingerprint (size exclusion chromatography) analyses of the four leachates plus HuminFeedÒ which serves as polysaccharide-free standard.
0
Leachate
Picea pungens
Quercus
Populus
1.25
Picea abies
Fig. 5. Mean lifetime reproductive output of Moina macrocopa females exposed against four different litter leachates. ⁄⁄p < 0.01.
Control
Length, mm
1.50
1.00
Leachate Fig. 3. Mean body length of 5 d old Moina macrocopa females exposed against four different litter leachates. ⁄⁄p < 0.01.
4. Discussion We applied litter leachates in the concentration range of ca. 2 mM DOC and did not find throughout adverse effects in the exposed M. macrocopa individuals, rather particularly the litter
leachates from coniferous species increased lifespan, body size, and offspring numbers. These findings contrast the expectations based on empirical analyses with L. stagnalis and on the hypotheses for the decline of M. margaritifera (Björk, 2004; Csar et al., 2004) as well as reports on other stream invertebrates (Canhoto and Laranjeira, 2007) and diptera larvae (David et al., 2000; Rey et al., 2000). Furthermore, the Moina results also contrast those of Daphnia magna Straus who was exposed to HuminFeedÒ (Euent et al., 2008; Bouchnak and Steinberg, 2010; Steinberg et al., 2010a). D. magna responded to HS-exposure with lifespan reduction, if in higher population densities (Euent et al., 2008), or with slight lifespan extension, if in lower population densities (Steinberg et al., 2010a). In any case, exposed females reduced the life-time reproduction, mostly in a concentration-dependent manner. In these Daphnia studies, the exposed material was polysaccharide-free HuminFeedÒ (Fig. 1) and it is very likely that the polysaccharide
Cumulative offspring per female, N
S. Hofmann et al. / Chemosphere 86 (2012) 883–890
Picea abies**
150
Quercus petraea
Picea pungens**
100 Populus nigra
Control
50
0
1
5
10
15
Days Fig. 6. Lifetime reproductive output of Moina macrocopa females exposed against four different litter leachates. ⁄⁄p < 0.01.
fraction in the leachates of the present study is responsible, at least in parts, for the observed differences between Moina’s and Daphnia’s responses. This assumption gets support by the fact that HuminFeedÒ does not significantly modulate the life trait variables in Moina (Steinberg et al., 2010b). Both cladoceran species, D. magna and M. macrocopa, are widely used model animals in ecotoxicity testing, with M. macrocopa being the replacement for Daphnia in those regions where the latter does not occur naturally (Sarma et al., 2005; Sarma and Nandini, 2006). There exist only very few studies comparing sensitivities of D. magna and M. macrocopa. For instance, for cadmium the LC50 was 120 lg L 1 in 48 h for D. magna and 680 lg L 1 in 24 h for M. macrocopa (Sarma and Nandini, 2006); for Microcystis aeruginosafed D. magna the mean lifespan was between 5 and 6 d (Trubetskova and Haney, 2006) and for M. macrocopa 8.5 d (Sarma and Nandini, 2006). Very recently, Menzel et al. (2011) reported that exposure to humic substances resulted in DNA-methylation in either cladoceran species at comparable rates. The present study shows that Moina mostly benefited from being exposed to leachates, although oxidatively stressed in three out of four trials. Therefore, four aspects will be discussed in detail: leachates as potential food supplement; leachates as xenohormones; modulation of antioxidant capacity and mitohormesis hypothesis; and potential pathways of lifespan extension.
4.1. Leachates as potential food supplement In comparative life-table experiments, the impact of three food algae on the performance of M. macrocopa was evaluated: the coccal green algae P. subcapitata, Monoraphidium minutum (Nägeli) Komárková-Legnerová, and Desmodesmus armatus (R. Chodat) E. Hegewald were tested, with P. subcapitata supporting all life trait variables best (Steinberg, 2011). Therefore, this food algal species was chosen for the present experiment. Nevertheless, we cannot exclude that the different leachates provided different, yet unidentified substances which could have served as food supplement. ‘‘Dissolved’’ leachates also include colloidal and fine-particulate organic carbon which pass given pore sizes of a filter, thus the nutritional utilization of this carbon source has to be considered. Indeed, there is increasing evidence that this carbon fraction is directly available to filter feeding zooplankton (Salonen and Hammar, 1986; Hessen et al., 1990). More recently, stable isotope analyses proved that allochthonous material of terrestrial origin can contribute significantly to zooplankton biomass (Pace et al.,
887
2004, 2007; Taipale et al., 2009). These studies support the idea of fine-particulate leachate material as an additional food source for exposed animals. As indicated in Table 1, all leachates contained considerable amounts of carbohydrates. M. macrocopa behaved contrary to C. elegans who responded to exposure against polyphenol-rich, but carbohydrate-poor humic substances with lifespan extension (Steinberg et al., 2007). In the Moina assay, carbohydrates could have supported bacterial growth which, in turn, could serve as good additional food source for the cladocerans. Furthermore, the leachate with the highest share of polysaccharides, Q. petraea, did not significantly affect any of the life trait variables, which can be due contrasting bioavailability of the polysaccharide fractions of the different leachates. 4.2. Leachates as xenohormones On the phenotypic level, Höss et al. (2001) and Meinelt et al. (2004) showed that HSs have a potential to act as xenohormones. Höss et al. (2001) reported that a variety of humic isolates increased the life-time offspring number in C. elegans, whereas in an ornamental fish (swordtail, Xiphophorus helleri Heckel), increasing exposure concentrations led to increased shares of females if fertilized eggs were exposed. Lutz et al. (2005) tested whether HSs can interfere with endocrine regulation in the amphibian Xenopus laevis Daudin. The in vivo results showed significant estrogenic effects on X. laevis during its larval development and resulted in a marked increase of the estrogenic biomarker estrogen receptor mRNA (ER-mRNA). Furthermore, the thyroid-stimulating hormone (TSHb-mRNA) is enhanced after exposure to HS1500, indicating a weak adverse effect on T3/T4 availability. Very recently, Trevisan et al. (2010) presented evidence that the positive effect of HSs on plant performance, which was presumed as phytohormone-like effect, is in fact transcriptionally controlled. HSs activated an auxin synthetic reporter in Arabidopsis thaliana (L.) Heynh. and enhanced transcription of the early auxin responsive gene. Taken together, these examples indicate that a hormone-like effect of the leachates on the life trait variables of M. macrocopa cannot be excluded and should be a concern of future evaluations. In particular, the juvenile hormone III and its derivatives which significantly impact the reproduction rate of daphnids (Tatarazako et al., 2002) strongly resembles terpenoids which are major constituents of some HSs (Leenheer and Rostad, 2004). Furthermore, also alkylphenols modulate the reproduction of daphnids, and alkylphenols, in turn, are major building blocks of HSs (SchmittKopplin et al., 1998). 4.3. Modulation of antioxidant capacity The SOSC value in P. abies-exposed individuals did not significantly differ from the control with the underlying mechanism remaining obscure. However, three exposures caused an oxidative stress in the water fleas, which could be facilitated by two mechanisms: external production of ROS from irradiated leachates (Zepp et al., 1977; Cooper and Zika, 1983) or, after internalization of the leachates via food or epithelia, internal activation of oxygen (=ROS production). These mechanisms are not mutually exclusive, but a comparison of both pathways in a cyanobacterium by Sun et al. (2005, 2006) emphasized the dominance of the internal over the external pathway. Most likely, this applies also to exposed Moina. With the macrophytes, coontail Ceratophyllum demersum L. and moss Vesicularia dubyana Brotherus, it has been shown that exposure to leachates of reed Phragmites australis (Cav.) Trin. ex Steud., English oak Quercus robur L., and European beech Fagus sylvatica L.
888
S. Hofmann et al. / Chemosphere 86 (2012) 883–890
led to increased hydrogen peroxide concentrations, oxidized glutathione (GSSG), increased peroxidase activity, and reduced photosynthetic oxygen production (Kamara and Pflugmacher, 2008; Nimptsch and Pflugmacher, 2008) – all symptoms of oxidative stress; the latter as oxidative stress avoidance reaction (Mittler, 2002). These findings indicate also that litter leachates contain natural xenobiotics which are responsible for the oxidative stress. From previous studies with C. elegans, it is well understood that mild oxidative stress by biogeochemicals can lead to multiple stress resistance and extension of the individual lifespans (Steinberg et al., 2007; Kampkötter et al., 2008; Saul et al., 2010). The mitohormesis theory provides a possible explanation for these observations and for the beneficial effects of the oxidatively acting leachates in this study. Mitohormesis, or more precisely mitochondrial hormesis, describes the assumption that an increase of mitochondrial activity and the subsequent increase of ROS production leads to a hormetic activation of the stress defense system (Tapia, 2006; Schulz et al., 2007; Ristow and Zarse, 2010; Ristow and Schmeisser, 2011). This might also apply as a potential background mechanism for the leaf litter effects in Moina and should be considered in future analyses. Another assumption is based on the observation that longevity is often combined with reduced offspring numbers and/or reduced body growth (Kirkwood, 1977). Hence, it is interesting to identify whether or not stressed Moina individuals showed either extended lifespans, larger bodies, or produced more offspring. 4.4. Pathways of lifespan extension In this work, we refer several times to studies with C. elegans, since this model animal is well understood and several recent studies and reviews indicate that there are many similarities between the nematode and arthropods in terms of stress response and lifespan extension (Minois, 2000; Gems and McElwee, 2003; Schrimpf et al., 2009; Ristow and Schmeisser, 2011). More general, Schrimpf et al. (2009) found that the abundances of ortholog proteins from worm and fly correlate well. Hence, it appears justified to refer to nematode studies in order to create hypotheses on potential regulatory pathways in arthropods, in general, and Moina macrocopa, in particular. For lifespan extension, several pathways are described. For instance, Murphy et al. (2003) identified a large set of genes whose activity is linked to lifespan and aging. A prime example of an interesting gerontogene is daf-16 which encodes the forkhead transcription factor DAF-16 in C. elegans. Murphy et al. (2003) identified two classes of genes that are influenced by DAF-16. Class 1 genes are switched on by DAF-16 and are associated with increased lifespan, whereas class 2 genes are repressed by DAF-16 and are associated with reduced lifespan and increased reproduction. This means that this regulatory pathway allocates energy either to body maintenance (growth, longevity) or reproduction. Since growth, longevity, and reproduction are stimulated simultaneously in exposed Moina individuals, it is questionable if this or an equivalent pathway applies. Almost simultaneously to the emerging hypothesis of DAF-16 being central in lifespan extension, reports showed that this is only one of several regulatory pathways. For instance, by using a daf16(mgDf50) mutant strain of C. elegans, Saul et al. (2008) showed that exposure to the polyphenol quercetin led to significantly increased mean lifespans. If components of the leachates trigger a DAF-16-independent pathway of longevity one may assume that the choice of body maintenance or reproduction is less strict than with DAF-16 pathway involved. This assumption is supported by Kenyon (2010) who reviewed the connection between lifespan, reproduction, and DAF-16. If a comparable regulatory pathway applies even to the Moina study is open to future studies.
5. Conclusion The present study shows that three of four litter leachates induced oxidative stress, thereby confirming the natural xenobiotic character of the leachates. However, three of four leachates increased body size and extended lifespan; furthermore, particularly the leachates of both studied Picea species significantly increased the offspring numbers. Increased body size, increased lifespan, and increased offspring question the assumption that energy is allocated either to body maintenance and repair (lifespan), growth, or offspring. This surprising behavior of exposed Moina may be based on food supplements (e.g., high carbohydrate contents) in the leachates or on a DAF-16-independent pathway of energy allocation. Overall, the results suggest that adverse stress effects can be overruled by either bacterial-growth supporting fractions in the leachates or an internal, yet to be discovered, compensation mechanism in the Moina individuals. These strategies are not mutually exclusive, rather they reinforce each other. Acknowledgment The scholarship by the Deutsche Forschungsgemeinschaft to R. M. (Ste 673/16-1) is gratefully acknowledged. We also thank the chemical laboratory of the Leibniz-Institute of Agricultural Engineering Potsdam-Bornim, Germany, for providing the DOC values. References Andersson, C., Abrahamson, A., Brunström, B., Örberg, J., 2010. Impact of humic substances on EROD activity in gill and liver of three-spined sticklebacks (Gasterosteus aculeatus). Chemosphere 81, 156–160. Anonymous, without year. Bioinformatics at the Walter and Eliza Hall Institute of Medical Research.
S. Hofmann et al. / Chemosphere 86 (2012) 883–890 Hessen, D.O., Andersen, T., Lyche, A., 1990. Carbon metabolism in a humic lake: pool sizes and cycling through zooplankton. Limnol. Oceanogr. 35, 84–99. Höss, S., Bergtold, M., Haitzer, M., Traunspurger, W., Steinberg, C.E.W., 2001. Refractory dissolved organic matter can influence the reproduction of Caenorhabditis elegans (Nematoda). Freshw. Biol. 46, 1–10. Hu, C.W., Yen, C.C., Huang, Y.L., Pan, C.H., Lu, F.J., Chao, M.R., 2010. Oxidatively damaged DNA induced by humic acid and arsenic in maternal and neonatal mice. Chemosphere 79, 93–99. Janošek, J., Bittner, M., Hilscherova, K., Blaha, L., Giesy, J.P., Holoubek, I., 2007. AhRmediated and antiestrogenic activity of humic substances. Chemosphere 67, 1096–1101. Kamara, S., Pflugmacher, S., 2008. Effects of leaf extracts on glutathione reductase expression, hydrogen peroxide and glutathione contents in the aquatic macrophyte Ceratophyllum demersum. Aquat. Sci. 70, 204–211. Kampkötter, A., Timpel, C., Zurawski, R.F., Ruhl, S., Chovolou, Y., Proksch, P., Wätjen, W., 2008. Increase of stress resistance and lifespan of Caenorhabditis elegans by quercetin. Comput. Biochem. Physiol. B 149, 314–323. Kenyon, C., 2010. A pathway that links reproductive status to lifespan in Caenorhabditis elegans. Ann. N.Y. Acad. Sci. 1204, 156–162. Kiikkilä, O., Kitunen, V., Smolander, A., 2011. Properties of dissolved organic matter derived from silver birch and Norway spruce stands: degradability combined with chemical characteristics. Soil Biol. Biochem. 43, 421–430. Kirkwood, T.B.L., 1977. Evolution of ageing. Nature 270, 301–304. Leenheer, J.A., Rostad, C., 2004. Tannins and terpenoids as major precursors of Suwannee River fulvic acid. Scientific Investigations Report 2004-5276. US Geological Survey, pp. 1–16. Li, X., Schuler, M.A., Berenbaum, M.R., 2007. Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics. Annu. Rev. Entomol. 52, 231– 253. Lutz, I., Jie, Z., Opitz, R., Kloas, W., Ying, X., Menzel, R., Steinberg, C.E., 2005. Environmental signals: synthetic humic substances act as xeno-estrogen and affect the thyroid system of Xenopus laevis. Chemosphere 61, 1183– 1188. Matsuo, A.Y.O., Woodin, B.R., Reddy, C.M., Val, A.L., Stegeman, J.J., 2006. Humic substances and crude oil induce cytochrome P450 1A expression in the Amazonian fish species Colossoma macropomum (Tambaqui). Environ. Sci. Technol. 40, 2851–2858. McMaster, D., Bond, N., 2008. A field and experimental study on the tolerances of fish to Eucalyptus camaldulensis leachate and low dissolved oxygen concentrations. Mar. Freshw. Res. 59, 177–185. Meems, N., Steinberg, C., Wiegand, C., 2004. Direct and interacting toxicological effects on the waterflea (Daphnia magna) by natural organic matter, synthetic humic substances and cypermethrin. Sci. Total Environ. 319, 123–136. Meinelt, T., Schreckenbach, K., Knopf, K., Wienke, A., Stuber, A., Steinberg, C.E.W., 2004. Humic substances affect physiological condition and sex ratio of swordtail (Xiphophorus helleri Heckel). Aquat. Sci. 66, 239–245. Menzel, R., Stürzenbaum, S., Bärenwaldt, A., Kulas, J., Steinberg, C.E.W., 2005. Humic material induces behavioral and global transcriptional responses in the nematode Caenorhabditis elegans. Environ. Sci. Technol. 39, 8324–8332. Menzel, S., Bouchnak, R., Menzel, R., Steinberg, C.E.W., 2011. Dissolved humic substances initiate DNA-methylation in cladocerans. Aquat. Toxicol. 105, 640– 642. Meyer, J.L., Wallace, J.B., Eggert, S.L., 1998. Leaf litter as a source of dissolved organic carbon in streams. Ecosystems 1, 240–249. Minois, N., 2000. Longevity and aging: beneficial effects of exposure to mild stress. Biogerontology 1, 15–29. Mittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7, 405–410. Morrongiello, J.R., Bond, N.R., Crook, D.A., Wong, B.B.M., 2011. Eucalyptus leachate inhibits reproduction in a freshwater fish. Freshw. Biol. 56, 1736–1745. Murphy, C.T., McCarroll, S.A., Bargmann, C.I., Fraser, A., Kamath, R.S., Ahringer, J., Li, H., Kenyon, C., 2003. Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature 424, 277–284. Nicklisch, A., Shatwell, T., Köhler, J., 2008. Analysis and modelling of the interactive effects of temperature and light on phytoplankton growth and relevance for the spring bloom. J. Plankton Res. 30, 75–91. Nimptsch, J., Pflugmacher, S., 2008. Decomposing leaf litter: the effect of allochthonous degradation products on the antioxidant fitness and photosynthesis of Vesicularia dubyana. Ecotoxicol. Environ. Saf. 69, 541–545. Pace, M.L., Carpenter, S.R., Cole, J.J., Coloso, J.J., Kitchell, J.F., Hodgson, J.R., Middelburg, J.J., Preston, N.D., Solomon, C.T., Weidel, B.C., 2007. Does terrestrial organic carbon subsidize the planktonic food web in a clear-water lake? Limnol. Oceanogr. 52, 2177–2189. Pace, M.L., Cole, J.J., Carpenter, S.R., Kitchell, J.F., Hodgson, J.R., Van De Bogert, M.C., Bade, D.L., Kritzberg, E.S., Bastviken, D., 2004. Whole-lake carbon-13 additions reveal terrestrial support of aquatic food webs. Nature 427, 240–243. Petrusek, A., 2002. Moina (Crustacea: Anomopoda, Moinidae) in the Czech Republic (a review). Acta Soc. Zool. Bohem. 66, 213–220. Pietsch, K., Hofmann, S., Henkel, R., Saul, N., Menzel, R., Steinberg, C.E.W., 2010. The plant polyphenol caffeic acid affects life traits differently in the nematode Caenorhabditis elegans and the cladoceran Moina macrocopa. Fresenius Environ. Bull. 19, 1238–1244. Pietsch, K., Saul, N., Chakrabarti, S., Stürzenbaum, S.R., Menzel, R., Steinberg, C.E., 2011. Hormetins, antioxidants and prooxidants: defining quercetin-, caffeic acid- and rosmarinic acid-mediated life extension in C. elegans. Biogerontology 12, 329–347.
889
Pietsch, K., Saul, N., Menzel, R., Stürzenbaum, S.R., Steinberg, C.E., 2009. Quercetin mediated lifespan extension in Caenorhabditis elegans is modulated by age-1, daf-2, sek-1 and unc-43. Biogerontology 10, 565–578. Popov, I., Lewin, G., 1998. Antioxidative homeostasis: characterization by means of chemiluminescent technique. Methods Enzymol. 300, 437–456. Rey, D., David, J.P., Martins, D., Pautou, M.P., Long, A., Marigo, G., Meyran, J.C., 2000. Role of vegetable tannins in habitat selection among mosquito communities from the Alpine hydrosystems. Comptes Rendus de l’Academie des Sciences Serie III 323, 391–398. Rey, D., Pautou, M.P., Meyran, J.C., 1999. Histopathological effects of tannic acid on the midgut epithelium of some aquatic diptera larvae. J. Invertebr. Pathol. 73, 173–181. Ristow, M., Schmeisser, S., 2011. Extending life span by increasing oxidative stress. Free Rad. Biol. Medic. 51, 327–336. Ristow, M., Zarse, K., 2010. How increased oxidative stress promotes longevity and metabolic health: the concept of mitochondrial hormesis (mitohormesis). Exp. Gerontol. 45, 410–418. Sachse, A., Babenzien, D., Ginzel, G., Gelbrecht, J., Steinberg, C.E.W., 2001. Characterization of dissolved organic carbon (DOC) in a dystrophic lake and an adjacent fen. Biogeochemistry 54, 279–296. Salonen, K., Hammar, T., 1986. On the importance of dissolved organic matter in the nutrition of zooplankton in some lake waters. Oecologia 68, 246–253. Sarma, S.S.S., Nandini, S., 2006. Review of recent ecotoxicological studies on cladocerans. J. Environ. Sci. Health B 41, 1417–1430. Sarma, S.S.S., Nandini, S., Gulati, R.D., 2005. Life history strategies of cladocerans: comparisons of tropical and temperate taxa. Hydrobiologia 542, 315–333. Saul, N., Pietsch, K., Menzel, R., Steinberg, C.E., 2008. Quercetin-mediated longevity in Caenorhabditis elegans: is DAF-16 involved? Mech. Ageing Dev. 129, 611–613. Saul, N., Pietsch, K., Menzel, R., Stürzenbaum, S.R., Steinberg, C.E., 2010. The longevity effect of tannic acid in Caenorhabditis elegans: Disposable Soma meets hormesis. J. Gerontol. A 65, 626–635. Schlesinger, W.H., 1997. Biochemistry. An Analysis of Global Change. Academic Press, San Diego. Schmitt-Kopplin, P., Hertkorn, N., Schulten, H.R., Kettrup, A., 1998. Structural changes in a dissolved soil humic acid during photochemical degradation processes under O2 and N2 atmosphere. Environ. Sci. Technol. 32, 2531–2541. Schrimpf, S.P., Weiss, M., Reiter, L., Ahrens, C.H., Jovanovic, M., Malmström, J., Brunner, E., Mohanty, S., Lercher, M.J., Hunziker, P.E., Aebersold, R., von Mering, C., Hengartner, M.O., 2009. Comparative functional analysis of the Caenorhabditis elegans and Drosophila melanogaster proteomes. PLoS Biol. 7, e1000048. Schulz, T.J., Zarse, K., Voigt, A., Urban, N., Birringer, M., Ristow, M., 2007. Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress. Cell Metab. 6, 280–293. Steinberg, C.E.W., 2003. Ecology of Humic Substances in Freshwaters. Determinants from Geochemistry to Ecological Niches. Springer, Berlin. Steinberg, C.E.W., 2011. Stress Ecology – Environmental Stress as Ecological Driving Force and Key Player in Evolution. Springer, Dordrecht. Steinberg, C.E.W., Kamara, S., Prokhotskaya, V.Y., Manusadzianas, L., Karasyova, T.A., Timofeyev, M.A., Jie, Z., Paul, A., Meinelt, T., Farjalla, V.F., Matsuo, A.Y.O., Kent Burnison, B., Menzel, R., 2006. Dissolved humic substances – ecological driving forces from the individual to the ecosystem level? Freshw. Biol. 51, 1189–1210. Steinberg, C.E.W., Ouerghemmi, N., Herrmann, S., Bouchnak, R., Timofeyev, M.A., Menzel, R., 2010a. Stress by poor food quality and exposure to humic substances: Daphnia magna responds with oxidative stress, lifespan extension, but reduced offspring numbers. Hydrobiologia 652, 223–236. Steinberg, C.E.W., Paul, A., Pflugmacher, S., Meinelt, T., Klöcking, R., Wiegand, C., 2003. Pure humic substances have the potential to act as xenobiotic chemicals – a review. Fresenius Environ. Bull. 12, 391–401. Steinberg, C.E.W., Saul, N., Pietsch, K., Meinelt, T., Rienau, S., Menzel, R., 2007. Dissolved humic substances facilitate fish life in extreme aquatic environments and have the potential to extend lifespan of Caenorhabditis elegans. Ann. Environ. Sci. 1, 81–90. Steinberg, C.E.W., Vic´entic´, L., Rauch, R., Bouchnak, R., Suhett, A.L., Menzel, R., 2010b. Exposure to humic material modulates life history traits of the cladocerans Moina macrocopa and Moina micrura. Chem. Ecol. 26, 135–143. Suhett, A.L., Steinberg, C.E.W., Santangelo, J.M., Bozelli, R.L., Farjalla, V.F., 2011. Natural dissolved humic substances increase the lifespan and promote transgenerational resistance to salt stress in the cladoceran Moina macrocopa. Environ. Sci. Pollut. Res. 18, 1004–1014. Sun, B.K., Tanji, Y., Unno, H., 2005. Influences of iron and humic acid on the growth of the cyanobacterium Anabaena circinalis. Biochem. Eng. J. 24, 195–201. Sun, B.K., Tanji, Y., Unno, H., 2006. Extinction of cells of cyanobacterium Anabaena circinalis in the presence of humic acid under illumination. Appl. Microbiol. Biotechnol. 72, 823–828. Taipale, S., Kankaala, P., Hämäläinen, H., Jones, R.I., 2009. Seasonal shifts in the diet of lake zooplankton revealed by phospholipid fatty acid analysis. Freshw. Biol. 54, 90–104. Tan, K.H., 2009. Environmental Soil Science. CRC Press, Boca Raton. Tapia, P.C., 2006. Sublethal mitochondrial stress with an attendant stoichiometric augmentation of reactive oxygen species may precipitate many of the beneficial alterations in cellular physiology produced by caloric restriction, intermittent fasting, exercise and dietary phytonutrients: ’’Mitohormesis’’ for health and vitality. Med. Hypotheses 66, 832–843.
890
S. Hofmann et al. / Chemosphere 86 (2012) 883–890
Tatarazako, N., Takao, Y., Kishi, K., Onikura, N., Arizono, K., Iguchi, T., 2002. Styrene dimers and trimers affect reproduction of daphnid (Ceriodaphnia dubia). Chemosphere 48, 597–601. Tilquin, M., Meyran, J.C., Marigo, G., 2004. Comparative capability to detoxify vegetable allelochemicals by larval mosquitoes. J. Chem. Ecol. 30, 1381–1391. Timofeyev, M.A., Shatilina, Z.M., Bedulina, D.S., Menzel, R., Steinberg, C.E., 2007. Natural organic matter (NOM) has the potential to modify the multixenobiotic resistance (MXR) activity in freshwater amphipods Eulimnogammarus cyaneus and E. verrucosus. Comput. Biochem. Physiol. B 146, 496–503. Timofeyev, M.A., Shatilina, Z.M., Kolesnichenko, A.V., Kolesnichenko, V.V., Steinberg, C.E., 2006. Specific antioxidant reactions to oxidative stress promoted by natural organic matter in two amphipod species from Lake Baikal. Environ. Toxicol. 21, 104–110. Timofeyev, M.A., Wiegand, C., Burnison, B.K., Shatilina, Z.M., Pflugmacher, S., Steinberg, C.E.W., 2004. Impact of natural organic matter (NOM) on freshwater amphipods. Sci. Total Environ. 319, 115–121.
Tremolieres, M., 1988. Deoxygenating effect and toxicity of ground-up dried coniferous needles and deciduous leaves of Canadian trees in water: A preliminary study in comparison with litter of European trees. Water Res. 22, 21–28. Trevisan, S., Pizzeghello, D., Ruperti, B., Francioso, O., Sassi, A., Palme, K., Quaggiotti, S., Nardi, S., 2010. Humic substances induce lateral root formation and expression of the early auxin-responsive IAA19 gene and DR5 synthetic element in Arabidopsis. Plant Biol. 12, 604–614. Trubetskova, I.L., Haney, J.F., 2006. Effects of differing concentrations of microcystin-producing Microcystis aeruginosa on growth, reproduction, survivorship and offspring of Daphnia magna. Arch. Hydrobiol. 167, 533–546. Wang, W.H., Bray, C.M., Jones, M.N., 1999. The fate of 14C-labelled humic substances in rice cells in culture. J. Plant Physiol. 154, 203–211. Zepp, R.G., Wolfe, N.L., Baughman, G.L., Hollis, R.C., 1977. Singlet oxygen in natural waters. Nature 267, 421–423.
Contents lists available at SciVerse ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Leaf litter leachates have the potential to increase lifespan, body size, and offspring numbers in a clone of Moina macrocopa Sylva Hofmann a, Maxim A. Timofeyev b,c, Anke Putschew d, Nadine Saul a, Ralph Menzel a, Christian E.W. Steinberg a,⇑ a
Humboldt-Universität zu Berlin, Department of Biology, Laboratory of Freshwater and Stress Ecology, Arboretum, 12437 Berlin, Germany Irkutsk State University, Institute of Biology, Ul. Karl-Marx 1, 664003 Irkutsk, Russia Baikal Research Centre, Ul. Karl-Marx 1, 664003 Irkutsk, Russia d Technical University Berlin, Department of Water Quality Control, Sekr. KF4, Straße des 17, Juni 135, 10623 Berlin, Germany b c
a r t i c l e
i n f o
Article history: Received 1 June 2011 Received in revised form 19 October 2011 Accepted 24 October 2011 Available online 23 November 2011 Keywords: Coniferous trees litter Deciduous trees litter Mitohormesis Oxidative stress Moina macrocopa Lifespan extension
a b s t r a c t Leaf litter processing is one major pathway of the global organic carbon cycle. During this process, a variety of small reactive organic compounds are released and transported to the aquatic environment, and may directly impact aquatic organisms as natural xenobiotics. We hypothesize that different forest stockings produce different leachate qualities, which in turn, stress the aquatic communities and, eventually, separate sensitive from tolerant species. Particularly, leachates from coniferous trees are suspected to have strongly adverse impacts on sensitive species. We exposed individuals of a clone of the model organism, Moina macrocopa, to comparable concentrations (approximately 2 mM) of litter leachates of Norway spruce, Picea abies, Colorado blue spruce, Picea pungens, black poplar, Populus nigra, and sessile oak, Quercus petraea. The animals were fed ad libitum. The following life trait variables were recorded: growth, lifespan, and lifetime offspring. To identify, whether or not exposure to litter leachates provokes an internal oxidative stress in the exposed animals we measured the superoxide anion radical scavenging capacity via photoluminescence. Except of P. abies, exposure to the leachates reduced this antioxidant capacity by approximately 50%. Leachate exposures, except that of Quercus, increased body size and extended lifespan; furthermore, particularly the leachates of both Picea species significantly increased the offspring numbers. This unexpected behavior of exposed Moina may be based on food supplements (e.g., high carbohydrate contents) in the leachates or on yet to be identified regulatory pathways of energy allocation. Overall, our results suggest that the potentially adverse effects of litter leachates can be overruled by either bacterial-growth supporting fractions in the leachates or an internal compensation mechanism in the Moina individuals. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Although it is well known that, after photosynthesis, humification is the second largest complex of biogeochemical reactions on Earth and is mandatory to recycle energy and organic carbon and, thus, facilitate an almost closed cycling of organic matter (Schlesinger, 1997), its ecological impacts are much less studied and understood than that of photosynthesis. For instance, leaf litter fuels detritus food chains via microbial processing. Whereas leaf litter leachates with humic substances, HSs, as one of its major components were thought to be inert in terms of direct interactions with organisms, recent reports show that HSs can be taken up through epithelia or indirectly via food (Wang et al., 1999; Steinberg et al., 2003). Consequently, an array of stress symptoms ⇑ Corresponding author. Tel.: +49 30 6322 4715; fax: +49 30 6369 446. E-mail address: [email protected] (C.E.W. Steinberg). 0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2011.10.041
have been reported in organisms exposed to HSs. On the phenotypic level, adverse effects including lethality were found in HS-exposed organisms, both aquatic invertebrates (Rey et al., 2000; David et al., 2001; Steinberg, 2003; Canhoto and Laranjeira, 2007) and fishes (Tremolieres, 1988; McMaster and Bond, 2008; Morrongiello et al., 2011). Also, one of the European freshwater pearl mussels, Margaritifera margaritifera L., declined significantly after forestry in the catchment changed from deciduous to coniferous trees as reported from Sweden and Austria (Björk, 2004; Csar et al., 2004), and the authors assumed that this decline is more than a pure coincidence. Probably, these changes led to HSs with higher aromatic moieties that supported, due to the low nitrogen content, less microbial growth (He et al., 2007), and poor food for the mussels (Steinberg et al., 2006). On the histological, biochemical, and molecular biological level, the stress symptoms include degeneration of the midgut epithelium (Rey et al., 1999), energy consumption (Cazenave et al.,
884
S. Hofmann et al. / Chemosphere 86 (2012) 883–890
2006), reactive oxygen species accumulation and reduced antioxidant capacity, membrane lipid peroxidation, increased activity of biotransformation and antioxidant enzymes (Tilquin et al., 2004), increase of stress proteins, such as a-crystalline small heat shock proteins (sHSPs) and HSP70 (Timofeyev et al., 2004; Timofeyev et al., 2006; Bedulina et al., 2010; Steinberg et al., 2010a), and chemosenzitation of MXR exporters (Timofeyev et al., 2007). Rey et al. (2000) and David et al. (2001) attribute the adverse effects on arthropods to tannin-phenolic compounds. Later, Steinberg et al. (2003) coined the oxymoron ‘‘natural xenobiotics’’ for HSs in relation to their interactions with the biota – in analogy to secondary plant compounds (Li et al., 2007); yet, in contrast to the latter, HSs are not vectored against herbivores, but challenge any exposed organism. In the meantime, some direct effects are described; for instance, HSs and tannins can Act as xenohormones as shown in invertebrates, fishes, amphibians, and Arabidopsis (Höss et al., 2001; Meinelt et al., 2004; Lutz et al., 2005; Trevisan et al., 2010); Interact with the Ah receptor (Bittner et al., 2006; Matsuo et al., 2006; Janošek et al., 2007); Modulate biotransformation enzymes of phase I and phase II (Meems et al., 2004; Tilquin et al., 2004; Matsuo et al., 2006; Andersson et al., 2010); Oxidatively damage DNA (Hu et al., 2010). The effects of HSs in exposed organisms are by no means unequivocal, since specific building blocks (mainly phenolic and polyphenolic) ones, are suspected to be responsible for adverse effects on the freshwater mollusks and are, on the other hand, shown to have beneficial effects on the nematode Caenorhabditis elegans Maupas. This organism actively migrated into HS-rich environments, although these were clearly linked to chemical stress (Menzel et al., 2005), yet with the benefit of lifespan extension (Steinberg et al., 2007). These and subsequent papers have shown that mild chemical stress induced by HSs or their building blocks may ultimately not only extend lifespans, but also increase resistance to multiple stressors, including netting, high temperatures, or oxidative environments (Meinelt et al., 2004; Pietsch et al., 2009; Saul et al., 2010; Pietsch et al., 2011). Litter leachates do not contain only HSs, but also a variety of non-humic materials as recently reported by Kiikkilä et al. (2011) who showed that dissolved organic matter derived from fresh spruce needle litter had high concentrations of carbohydrates and phenolic compounds. The carbohydrate content was easily degradable and the remainder was characterized by a high C:N ratio as mentioned by He et al. (2007) and in a pedological textbook (Tan, 2009). From previous studies with C. elegans, we understand that carbohydrate-rich HSs tend to reduce the worm’s lifespan (Steinberg et al., 2007). Whether the leachate contains high share of non-humic materials depends on the sampling season and their environmental fate. Since C. elegans is an endogeic animal and the water percolation usually slow, it can be assumed that, in nature, it will be exposed to remainders of the litter rather than to carbohydrates. It is also likely that pelagic organisms are rather soon exposed to the leachates carried into the water by the run-off water after storm events and these organisms appear to be the more appropriate model organisms in this respect. This assumption receives support by the fact that leaf litter alone contributes up to 30% of the total dissolved organic carbon (DOC) in streams (Meyer et al., 1998). The aforementioned partly contradictory observations intrigued us to figure out, which effects prevail. We comparatively evaluated the impact of litter leachates of two deciduous and two coniferous tree species in terms of life trait variables in a Moina macrocopa
Straus clone. The deciduous species were black poplar, Populus nigra L., and sessile oak, Quercus petraea (Mattuschka) Liebl., and the coniferous ones were Norway spruce, Picea abies (L.) Karst., and Colorado blue spruce, Picea pungens Engelm. All trees are common in European forests, parks, or gardens. To characterize the various leachates, we applied a size-exclusion-chromatographic fingerprint technique with double detection (UV, dissolved organic carbon) which gives sufficient information about the humic and non-humic materials (Sachse et al., 2001). Moina macrocopa is a world-wide distributed cladoceran and belongs to a group of large-bodied Moina species; it is characteristic inhabitant of small, usually ephemeral water bodies in the temperate zone, often rich in DOC (Petrusek, 2002). The tested clone was isolated from a puddle in Rio de Janeiro, Brazil (Elmoor-Loureiro et al., 2010) and was successfully used in effect evaluations before (Pietsch et al., 2010; Steinberg et al., 2010b; Suhett et al., 2011); for the sake of comparison, this tropical clone was applied also in the present study. 2. Material and methods 2.1. Test design The stock culture was maintained in Berlin tap water with a mean Ca2+ concentration of 2.5 mM. This relatively hard water was chosen to avoid any risk of potential acidification by adding the leachates, which might be acidic. In order to evaluate only the impact of the biogeochemicals on the life performance of the cladocerans, the development of further stresses, such as crowding, with the subsequent development of males, was avoided. The densities of individuals, therefore, were evaluated in pre-tests which allow pure parthenogenetic reproductions. All exposure scenarios with the leachates were not stressful with respect to crowding, because males did not occur. Only 3rd generation neonates were used for the experiments. In each 500 mL Erlenmeyer vessel, 30 individuals were placed; the vessels were kept in a water bath at 30 ± 1 °C and illuminated by cool white light in a 14:10 h rhythm. Lifespan and reproduction assays were carried out in duplicates in two independent trials. Growth tests were carried out in separate series. Three trials with 30 animals each were conducted. The microscopic measurements of the entire body length of 30 animals each were taken at the end of 5-d cultivation after the animals were narcotized with ethanol. The presented data are means of the individual trials and subject of the statistical evaluation. Animals and offspring were counted and exposure was exchanged every second day. M. macrocopa was fed with the green algae Pseudokirchneriella subcapitata (Koršikov) Hindák. P. subcapitata strain NIVA-CHL 1 was obtained from the Culture Collection of Algae at the University of Göttingen, Germany. The algae were cultivated in freshwater medium (Nicklisch et al., 2008), and grown in batch cultures under day light conditions. The animals were fed daily ad libitum with 10 mL of a logarithmically growing culture which corresponds to 107 cells or approximately 2.5 mg L 1 C. 2.2. Leachate preparation and DOC fingerprint characterization Litter samples were collected in August/September 2009 in the Arboretum, the adjacent urban forest Königsheide, and a private garden in Berlin. The litter was collected during a long drought period. Immediately after collecting the material, the leaves were sorted; earthy remains and soil animals were removed and leaves were dried for 48 h at 56 °C. The samples were powdered in a household coffee grinder and 50 g litter were leached with 0.75 L de-ionized water for 48 h. During the leaching process, the suspen-
885
S. Hofmann et al. / Chemosphere 86 (2012) 883–890 Table 1 Exposure concentrations and shares of the finger print fractions of the various leachates. Leachate
Exposure concentration (mM)
Polysaccharides
Populus nigra Quercus petraea Picea abies Picea pungens
2.38 2.00 2.15 1.50
2.5 17.4 16.8 7.1
Humic substances
LMW organic acids
LMW substances
% of the dissolved organic carbon 64.6 73.7 65.1 78.8
28.0 5.7 5.7 6.3
4.9 3.2 12.4 7.8
LMW = low-molecular weight.
sion was stirred with a magnetic stirrer. Then the suspension was centrifuged with 4000 rpm at 4 °C and filtered through glass fiber filters (Whatman G6, Whatman, England). This means that, in compliance to the definition of DOC, the filtrate contained colloidal and even fine-particulate carbon. All stock solutions were diluted with Berlin tap water until they had an identical light extinction. The exposure concentrations lay between 1.5 mM (P. pungens) and 2.38 mM (P. nigra) (Table 1) due to different specific UV absorption of the leachates. Concentrations of non-fractionated dissolved organic carbon samples were determined by means of a TOC-505 A analyzer (Shimadzu, Germany). Automated size-exclusion-chromatography with UV- and organic carbon detection (IR) was used for characterization, as described in technical detail by Sachse et al. (2001). Four different groups of DOC could be fractionated: polysaccharides, humic substances (HSs), low molecular weight acids, and low molecular weight substances. The polysaccharide fraction is high molecular weight carbohydrates, which show no absorption in the UV range. HSs include humic and fulvic acids and their building blocks, which are UV active. Low molecular weight acids are defined as low molecular weight carboxylic acids such as several metabolites in biological and chemical processes. DOC in the low molecular weight range after the low molecular weight acids peak was described as other low molecular weight substances.
2.3. Superoxide anion radical scavenging capacity The antioxidative capacity of a cell or an organism indicates the potential to balance reactive oxygen species, ROS, namely superoxide anion radicals, hydroxyl radicals, and hydrogen peroxide. ROS are, e.g., generated in any heterotrophic reaction, in defense of pathogens, or after uptake of heavy metals or xenobiotic chemicals. Since superoxide anion radicals are the first products of triplet oxygen reduction, monitoring these ROS appears to be representative of all ROS produced by reduction. In M. macrocopa, this capacity was measured using 25 individuals exposed for 5 d against the leachates. The animals were deep frozen in liquid nitrogen in phosphate buffer, cleaved and homogenized in a Speedmill (AnalytikJena, Germany), and centrifuged at 13 000 rpm. The cooled supernatant of the homogenate was analyzed via photoluminescence for the superoxide anion radical scavenging capacities (SOSC in ascorbic acid equivalents) in a PhotoChem device (AnalytikJena, Germany) and the corresponding ACW-Kit (AnalytikJena, Germany) according to Popov and Lewin (1998). This assay measures the ability of an antioxidant to quench free, photochemically generated superoxide anion radicals by hydrogen donation. The sensitive luminometric detection uses luminol as detector substance. In biological samples, they may be quenched by antioxidant substances and enzymes. This quenching gives an inverted measure of SOSC and must be calculated in comparison to a standard substance, such as ascorbic acid. SOSC values in the animals were related to their protein content, which was measured according to Bradford (1976) using Coomassie Brilliant Blue G250 (Sigma Aldrich, USA). All measurements were done in triplicates.
2.4. Statistics Statistically significant differences between entire lifespans were tested by means of the log-rank test, which was developed for lifespan curves (Anonymous, without year); hence, statistical information for each single data point, such as standard deviation, is not necessary and not presented in the graph. To gauge reproduction, the offspring numbers were checked daily. Mean values of the reproductive output were taken to check the statistical significance. The significance of SOSC, body size after 5 d, and reproductive output was calculated by one-way ANOVA (Sigma Stat 3.5, SPSS Inc., USA). Cumulative curves of the reproductive output are additionally presented in the graphs in order to identify potential phases of modulated reproduction. 3. Results The fingerprint characterization revealed that all leachates contained considerable amounts of polysaccharides (UV-inactive, but DOC-active with retention times of approximately 45 min) with P. nigra having the lowest and Q. petraea the highest share; as negative control, the carbohydrate-free preparation HuminFeedÒ1 was also chromatographed (Fig. 1). The litter of both Picea species contained between 7 and almost 17% of carbohydrate carbon (Table 1). This indicates that the humification process was at its infancy when the samples were collected. The HS fractions comprised the highest carbon concentration and covered between 65% (P. nigra, P. abies) and 79% (P. pungens). Interestingly, the P. nigra leachate contained a high share of low-weight molecular acids, and the P. abies leachate of low-weight substances. Except of the P. abies leachate which did not significantly modulate SOSC, all leachates significantly reduced SOSC in the exposed M. macrocopa specimens (Fig. 2). Furthermore, the body size significantly increased upon exposure to all but one (Quercus) litter leachates by 0.1 to 0.15 mm (Fig. 3). The enlarged animals also lived significantly longer than the control ones or those ones exposed to the Quercus leachate (Fig. 4). The mean and median lifespan values are presented in Table 2. Both coniferous leachates highly significantly increased the reproductive output of the females (Fig. 5), whereby reproduction happened until the individuals pass away (Fig. 6). Both deciduous leachates only tended to increase reproduction. It is interesting to note that this increase in reproduction by the coniferous leachates was not only due to the longer lifespan, but took place also during the shorter lifespans which applied to the control individuals (Fig. 5). Relating the life trait variables to the fingerprint fractions of Table 1, there was no fraction identifiable which could explain why the Quercus leachate was not effective in terms of body growth, lifespan extension, or reproductive output. The missing reproductive output in Populus-exposed animals, however, could be due to the high share of unidentified low-molecular weight organic acids. 1 http://www.humintech.com/001/animalfeeds/products/huminfeed.html (accessed May, 2011). See this site for more information; the use of HuminFeedÒ is by no means an advertisement for this product.
886
S. Hofmann et al. / Chemosphere 86 (2012) 883–890 Humic substances LMW LMW & building blocks org acids substances
Polysaccharides
100
Picea pungens*
Survival , %
Dissolved organic carbon, arbitrary units
Populus nigra **
Picea pungens Quercus petraea
Populus nigra
Control
50
Picea abies
Quercus petraea Picea abies** Picea abies** HuminFeed®
0
1
5
10
15
Days Fig. 4. Lifespan modulation in Moina macrocopa females exposed against four different litter leachates. ⁄p < 0.05; ⁄⁄p < 0.01.
Retention time, min
5.0
Table 2 Mean and median lifespan values, d ± SD. Leachate
Mean lifespan
Median lifespan
Control Populus nigra Quercus petraea Picea abies Picea pungens
8.9 ± 0.3 10.7 ± 0.4 8.8 ± 0.2 10.6 ± 0.4 9.2 ± 0.2
8.9 ± 0.3 11.2 ± 0.2 8.7 ± 0.3 10.7 ± 0.5 9.1 ± 0.3
Leachate Fig. 2. Mean superoxide anion radical quenching capacity of 5 d old Moina macrocopa females exposed to four different litter leachates. ⁄p < 0.05; ⁄⁄p < 0.01.
** **
**
100
50
Picea pungens
0
Picea abies
Picea
150
Quercus
pungens
abies
Populus
Quercus
**
Populus
**
**
**
**
Control
2.5
Reproductive output per individual, N
200
Control
Superoxide anion radical scavenging capacity nM ascorbic acid per mg protein
Fig. 1. Qualitative DOC-fingerprint (size exclusion chromatography) analyses of the four leachates plus HuminFeedÒ which serves as polysaccharide-free standard.
0
Leachate
Picea pungens
Quercus
Populus
1.25
Picea abies
Fig. 5. Mean lifetime reproductive output of Moina macrocopa females exposed against four different litter leachates. ⁄⁄p < 0.01.
Control
Length, mm
1.50
1.00
Leachate Fig. 3. Mean body length of 5 d old Moina macrocopa females exposed against four different litter leachates. ⁄⁄p < 0.01.
4. Discussion We applied litter leachates in the concentration range of ca. 2 mM DOC and did not find throughout adverse effects in the exposed M. macrocopa individuals, rather particularly the litter
leachates from coniferous species increased lifespan, body size, and offspring numbers. These findings contrast the expectations based on empirical analyses with L. stagnalis and on the hypotheses for the decline of M. margaritifera (Björk, 2004; Csar et al., 2004) as well as reports on other stream invertebrates (Canhoto and Laranjeira, 2007) and diptera larvae (David et al., 2000; Rey et al., 2000). Furthermore, the Moina results also contrast those of Daphnia magna Straus who was exposed to HuminFeedÒ (Euent et al., 2008; Bouchnak and Steinberg, 2010; Steinberg et al., 2010a). D. magna responded to HS-exposure with lifespan reduction, if in higher population densities (Euent et al., 2008), or with slight lifespan extension, if in lower population densities (Steinberg et al., 2010a). In any case, exposed females reduced the life-time reproduction, mostly in a concentration-dependent manner. In these Daphnia studies, the exposed material was polysaccharide-free HuminFeedÒ (Fig. 1) and it is very likely that the polysaccharide
Cumulative offspring per female, N
S. Hofmann et al. / Chemosphere 86 (2012) 883–890
Picea abies**
150
Quercus petraea
Picea pungens**
100 Populus nigra
Control
50
0
1
5
10
15
Days Fig. 6. Lifetime reproductive output of Moina macrocopa females exposed against four different litter leachates. ⁄⁄p < 0.01.
fraction in the leachates of the present study is responsible, at least in parts, for the observed differences between Moina’s and Daphnia’s responses. This assumption gets support by the fact that HuminFeedÒ does not significantly modulate the life trait variables in Moina (Steinberg et al., 2010b). Both cladoceran species, D. magna and M. macrocopa, are widely used model animals in ecotoxicity testing, with M. macrocopa being the replacement for Daphnia in those regions where the latter does not occur naturally (Sarma et al., 2005; Sarma and Nandini, 2006). There exist only very few studies comparing sensitivities of D. magna and M. macrocopa. For instance, for cadmium the LC50 was 120 lg L 1 in 48 h for D. magna and 680 lg L 1 in 24 h for M. macrocopa (Sarma and Nandini, 2006); for Microcystis aeruginosafed D. magna the mean lifespan was between 5 and 6 d (Trubetskova and Haney, 2006) and for M. macrocopa 8.5 d (Sarma and Nandini, 2006). Very recently, Menzel et al. (2011) reported that exposure to humic substances resulted in DNA-methylation in either cladoceran species at comparable rates. The present study shows that Moina mostly benefited from being exposed to leachates, although oxidatively stressed in three out of four trials. Therefore, four aspects will be discussed in detail: leachates as potential food supplement; leachates as xenohormones; modulation of antioxidant capacity and mitohormesis hypothesis; and potential pathways of lifespan extension.
4.1. Leachates as potential food supplement In comparative life-table experiments, the impact of three food algae on the performance of M. macrocopa was evaluated: the coccal green algae P. subcapitata, Monoraphidium minutum (Nägeli) Komárková-Legnerová, and Desmodesmus armatus (R. Chodat) E. Hegewald were tested, with P. subcapitata supporting all life trait variables best (Steinberg, 2011). Therefore, this food algal species was chosen for the present experiment. Nevertheless, we cannot exclude that the different leachates provided different, yet unidentified substances which could have served as food supplement. ‘‘Dissolved’’ leachates also include colloidal and fine-particulate organic carbon which pass given pore sizes of a filter, thus the nutritional utilization of this carbon source has to be considered. Indeed, there is increasing evidence that this carbon fraction is directly available to filter feeding zooplankton (Salonen and Hammar, 1986; Hessen et al., 1990). More recently, stable isotope analyses proved that allochthonous material of terrestrial origin can contribute significantly to zooplankton biomass (Pace et al.,
887
2004, 2007; Taipale et al., 2009). These studies support the idea of fine-particulate leachate material as an additional food source for exposed animals. As indicated in Table 1, all leachates contained considerable amounts of carbohydrates. M. macrocopa behaved contrary to C. elegans who responded to exposure against polyphenol-rich, but carbohydrate-poor humic substances with lifespan extension (Steinberg et al., 2007). In the Moina assay, carbohydrates could have supported bacterial growth which, in turn, could serve as good additional food source for the cladocerans. Furthermore, the leachate with the highest share of polysaccharides, Q. petraea, did not significantly affect any of the life trait variables, which can be due contrasting bioavailability of the polysaccharide fractions of the different leachates. 4.2. Leachates as xenohormones On the phenotypic level, Höss et al. (2001) and Meinelt et al. (2004) showed that HSs have a potential to act as xenohormones. Höss et al. (2001) reported that a variety of humic isolates increased the life-time offspring number in C. elegans, whereas in an ornamental fish (swordtail, Xiphophorus helleri Heckel), increasing exposure concentrations led to increased shares of females if fertilized eggs were exposed. Lutz et al. (2005) tested whether HSs can interfere with endocrine regulation in the amphibian Xenopus laevis Daudin. The in vivo results showed significant estrogenic effects on X. laevis during its larval development and resulted in a marked increase of the estrogenic biomarker estrogen receptor mRNA (ER-mRNA). Furthermore, the thyroid-stimulating hormone (TSHb-mRNA) is enhanced after exposure to HS1500, indicating a weak adverse effect on T3/T4 availability. Very recently, Trevisan et al. (2010) presented evidence that the positive effect of HSs on plant performance, which was presumed as phytohormone-like effect, is in fact transcriptionally controlled. HSs activated an auxin synthetic reporter in Arabidopsis thaliana (L.) Heynh. and enhanced transcription of the early auxin responsive gene. Taken together, these examples indicate that a hormone-like effect of the leachates on the life trait variables of M. macrocopa cannot be excluded and should be a concern of future evaluations. In particular, the juvenile hormone III and its derivatives which significantly impact the reproduction rate of daphnids (Tatarazako et al., 2002) strongly resembles terpenoids which are major constituents of some HSs (Leenheer and Rostad, 2004). Furthermore, also alkylphenols modulate the reproduction of daphnids, and alkylphenols, in turn, are major building blocks of HSs (SchmittKopplin et al., 1998). 4.3. Modulation of antioxidant capacity The SOSC value in P. abies-exposed individuals did not significantly differ from the control with the underlying mechanism remaining obscure. However, three exposures caused an oxidative stress in the water fleas, which could be facilitated by two mechanisms: external production of ROS from irradiated leachates (Zepp et al., 1977; Cooper and Zika, 1983) or, after internalization of the leachates via food or epithelia, internal activation of oxygen (=ROS production). These mechanisms are not mutually exclusive, but a comparison of both pathways in a cyanobacterium by Sun et al. (2005, 2006) emphasized the dominance of the internal over the external pathway. Most likely, this applies also to exposed Moina. With the macrophytes, coontail Ceratophyllum demersum L. and moss Vesicularia dubyana Brotherus, it has been shown that exposure to leachates of reed Phragmites australis (Cav.) Trin. ex Steud., English oak Quercus robur L., and European beech Fagus sylvatica L.
888
S. Hofmann et al. / Chemosphere 86 (2012) 883–890
led to increased hydrogen peroxide concentrations, oxidized glutathione (GSSG), increased peroxidase activity, and reduced photosynthetic oxygen production (Kamara and Pflugmacher, 2008; Nimptsch and Pflugmacher, 2008) – all symptoms of oxidative stress; the latter as oxidative stress avoidance reaction (Mittler, 2002). These findings indicate also that litter leachates contain natural xenobiotics which are responsible for the oxidative stress. From previous studies with C. elegans, it is well understood that mild oxidative stress by biogeochemicals can lead to multiple stress resistance and extension of the individual lifespans (Steinberg et al., 2007; Kampkötter et al., 2008; Saul et al., 2010). The mitohormesis theory provides a possible explanation for these observations and for the beneficial effects of the oxidatively acting leachates in this study. Mitohormesis, or more precisely mitochondrial hormesis, describes the assumption that an increase of mitochondrial activity and the subsequent increase of ROS production leads to a hormetic activation of the stress defense system (Tapia, 2006; Schulz et al., 2007; Ristow and Zarse, 2010; Ristow and Schmeisser, 2011). This might also apply as a potential background mechanism for the leaf litter effects in Moina and should be considered in future analyses. Another assumption is based on the observation that longevity is often combined with reduced offspring numbers and/or reduced body growth (Kirkwood, 1977). Hence, it is interesting to identify whether or not stressed Moina individuals showed either extended lifespans, larger bodies, or produced more offspring. 4.4. Pathways of lifespan extension In this work, we refer several times to studies with C. elegans, since this model animal is well understood and several recent studies and reviews indicate that there are many similarities between the nematode and arthropods in terms of stress response and lifespan extension (Minois, 2000; Gems and McElwee, 2003; Schrimpf et al., 2009; Ristow and Schmeisser, 2011). More general, Schrimpf et al. (2009) found that the abundances of ortholog proteins from worm and fly correlate well. Hence, it appears justified to refer to nematode studies in order to create hypotheses on potential regulatory pathways in arthropods, in general, and Moina macrocopa, in particular. For lifespan extension, several pathways are described. For instance, Murphy et al. (2003) identified a large set of genes whose activity is linked to lifespan and aging. A prime example of an interesting gerontogene is daf-16 which encodes the forkhead transcription factor DAF-16 in C. elegans. Murphy et al. (2003) identified two classes of genes that are influenced by DAF-16. Class 1 genes are switched on by DAF-16 and are associated with increased lifespan, whereas class 2 genes are repressed by DAF-16 and are associated with reduced lifespan and increased reproduction. This means that this regulatory pathway allocates energy either to body maintenance (growth, longevity) or reproduction. Since growth, longevity, and reproduction are stimulated simultaneously in exposed Moina individuals, it is questionable if this or an equivalent pathway applies. Almost simultaneously to the emerging hypothesis of DAF-16 being central in lifespan extension, reports showed that this is only one of several regulatory pathways. For instance, by using a daf16(mgDf50) mutant strain of C. elegans, Saul et al. (2008) showed that exposure to the polyphenol quercetin led to significantly increased mean lifespans. If components of the leachates trigger a DAF-16-independent pathway of longevity one may assume that the choice of body maintenance or reproduction is less strict than with DAF-16 pathway involved. This assumption is supported by Kenyon (2010) who reviewed the connection between lifespan, reproduction, and DAF-16. If a comparable regulatory pathway applies even to the Moina study is open to future studies.
5. Conclusion The present study shows that three of four litter leachates induced oxidative stress, thereby confirming the natural xenobiotic character of the leachates. However, three of four leachates increased body size and extended lifespan; furthermore, particularly the leachates of both studied Picea species significantly increased the offspring numbers. Increased body size, increased lifespan, and increased offspring question the assumption that energy is allocated either to body maintenance and repair (lifespan), growth, or offspring. This surprising behavior of exposed Moina may be based on food supplements (e.g., high carbohydrate contents) in the leachates or on a DAF-16-independent pathway of energy allocation. Overall, the results suggest that adverse stress effects can be overruled by either bacterial-growth supporting fractions in the leachates or an internal, yet to be discovered, compensation mechanism in the Moina individuals. These strategies are not mutually exclusive, rather they reinforce each other. Acknowledgment The scholarship by the Deutsche Forschungsgemeinschaft to R. M. (Ste 673/16-1) is gratefully acknowledged. We also thank the chemical laboratory of the Leibniz-Institute of Agricultural Engineering Potsdam-Bornim, Germany, for providing the DOC values. References Andersson, C., Abrahamson, A., Brunström, B., Örberg, J., 2010. Impact of humic substances on EROD activity in gill and liver of three-spined sticklebacks (Gasterosteus aculeatus). Chemosphere 81, 156–160. Anonymous, without year. Bioinformatics at the Walter and Eliza Hall Institute of Medical Research.
S. Hofmann et al. / Chemosphere 86 (2012) 883–890 Hessen, D.O., Andersen, T., Lyche, A., 1990. Carbon metabolism in a humic lake: pool sizes and cycling through zooplankton. Limnol. Oceanogr. 35, 84–99. Höss, S., Bergtold, M., Haitzer, M., Traunspurger, W., Steinberg, C.E.W., 2001. Refractory dissolved organic matter can influence the reproduction of Caenorhabditis elegans (Nematoda). Freshw. Biol. 46, 1–10. Hu, C.W., Yen, C.C., Huang, Y.L., Pan, C.H., Lu, F.J., Chao, M.R., 2010. Oxidatively damaged DNA induced by humic acid and arsenic in maternal and neonatal mice. Chemosphere 79, 93–99. Janošek, J., Bittner, M., Hilscherova, K., Blaha, L., Giesy, J.P., Holoubek, I., 2007. AhRmediated and antiestrogenic activity of humic substances. Chemosphere 67, 1096–1101. Kamara, S., Pflugmacher, S., 2008. Effects of leaf extracts on glutathione reductase expression, hydrogen peroxide and glutathione contents in the aquatic macrophyte Ceratophyllum demersum. Aquat. Sci. 70, 204–211. Kampkötter, A., Timpel, C., Zurawski, R.F., Ruhl, S., Chovolou, Y., Proksch, P., Wätjen, W., 2008. Increase of stress resistance and lifespan of Caenorhabditis elegans by quercetin. Comput. Biochem. Physiol. B 149, 314–323. Kenyon, C., 2010. A pathway that links reproductive status to lifespan in Caenorhabditis elegans. Ann. N.Y. Acad. Sci. 1204, 156–162. Kiikkilä, O., Kitunen, V., Smolander, A., 2011. Properties of dissolved organic matter derived from silver birch and Norway spruce stands: degradability combined with chemical characteristics. Soil Biol. Biochem. 43, 421–430. Kirkwood, T.B.L., 1977. Evolution of ageing. Nature 270, 301–304. Leenheer, J.A., Rostad, C., 2004. Tannins and terpenoids as major precursors of Suwannee River fulvic acid. Scientific Investigations Report 2004-5276. US Geological Survey, pp. 1–16. Li, X., Schuler, M.A., Berenbaum, M.R., 2007. Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics. Annu. Rev. Entomol. 52, 231– 253. Lutz, I., Jie, Z., Opitz, R., Kloas, W., Ying, X., Menzel, R., Steinberg, C.E., 2005. Environmental signals: synthetic humic substances act as xeno-estrogen and affect the thyroid system of Xenopus laevis. Chemosphere 61, 1183– 1188. Matsuo, A.Y.O., Woodin, B.R., Reddy, C.M., Val, A.L., Stegeman, J.J., 2006. Humic substances and crude oil induce cytochrome P450 1A expression in the Amazonian fish species Colossoma macropomum (Tambaqui). Environ. Sci. Technol. 40, 2851–2858. McMaster, D., Bond, N., 2008. A field and experimental study on the tolerances of fish to Eucalyptus camaldulensis leachate and low dissolved oxygen concentrations. Mar. Freshw. Res. 59, 177–185. Meems, N., Steinberg, C., Wiegand, C., 2004. Direct and interacting toxicological effects on the waterflea (Daphnia magna) by natural organic matter, synthetic humic substances and cypermethrin. Sci. Total Environ. 319, 123–136. Meinelt, T., Schreckenbach, K., Knopf, K., Wienke, A., Stuber, A., Steinberg, C.E.W., 2004. Humic substances affect physiological condition and sex ratio of swordtail (Xiphophorus helleri Heckel). Aquat. Sci. 66, 239–245. Menzel, R., Stürzenbaum, S., Bärenwaldt, A., Kulas, J., Steinberg, C.E.W., 2005. Humic material induces behavioral and global transcriptional responses in the nematode Caenorhabditis elegans. Environ. Sci. Technol. 39, 8324–8332. Menzel, S., Bouchnak, R., Menzel, R., Steinberg, C.E.W., 2011. Dissolved humic substances initiate DNA-methylation in cladocerans. Aquat. Toxicol. 105, 640– 642. Meyer, J.L., Wallace, J.B., Eggert, S.L., 1998. Leaf litter as a source of dissolved organic carbon in streams. Ecosystems 1, 240–249. Minois, N., 2000. Longevity and aging: beneficial effects of exposure to mild stress. Biogerontology 1, 15–29. Mittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7, 405–410. Morrongiello, J.R., Bond, N.R., Crook, D.A., Wong, B.B.M., 2011. Eucalyptus leachate inhibits reproduction in a freshwater fish. Freshw. Biol. 56, 1736–1745. Murphy, C.T., McCarroll, S.A., Bargmann, C.I., Fraser, A., Kamath, R.S., Ahringer, J., Li, H., Kenyon, C., 2003. Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature 424, 277–284. Nicklisch, A., Shatwell, T., Köhler, J., 2008. Analysis and modelling of the interactive effects of temperature and light on phytoplankton growth and relevance for the spring bloom. J. Plankton Res. 30, 75–91. Nimptsch, J., Pflugmacher, S., 2008. Decomposing leaf litter: the effect of allochthonous degradation products on the antioxidant fitness and photosynthesis of Vesicularia dubyana. Ecotoxicol. Environ. Saf. 69, 541–545. Pace, M.L., Carpenter, S.R., Cole, J.J., Coloso, J.J., Kitchell, J.F., Hodgson, J.R., Middelburg, J.J., Preston, N.D., Solomon, C.T., Weidel, B.C., 2007. Does terrestrial organic carbon subsidize the planktonic food web in a clear-water lake? Limnol. Oceanogr. 52, 2177–2189. Pace, M.L., Cole, J.J., Carpenter, S.R., Kitchell, J.F., Hodgson, J.R., Van De Bogert, M.C., Bade, D.L., Kritzberg, E.S., Bastviken, D., 2004. Whole-lake carbon-13 additions reveal terrestrial support of aquatic food webs. Nature 427, 240–243. Petrusek, A., 2002. Moina (Crustacea: Anomopoda, Moinidae) in the Czech Republic (a review). Acta Soc. Zool. Bohem. 66, 213–220. Pietsch, K., Hofmann, S., Henkel, R., Saul, N., Menzel, R., Steinberg, C.E.W., 2010. The plant polyphenol caffeic acid affects life traits differently in the nematode Caenorhabditis elegans and the cladoceran Moina macrocopa. Fresenius Environ. Bull. 19, 1238–1244. Pietsch, K., Saul, N., Chakrabarti, S., Stürzenbaum, S.R., Menzel, R., Steinberg, C.E., 2011. Hormetins, antioxidants and prooxidants: defining quercetin-, caffeic acid- and rosmarinic acid-mediated life extension in C. elegans. Biogerontology 12, 329–347.
889
Pietsch, K., Saul, N., Menzel, R., Stürzenbaum, S.R., Steinberg, C.E., 2009. Quercetin mediated lifespan extension in Caenorhabditis elegans is modulated by age-1, daf-2, sek-1 and unc-43. Biogerontology 10, 565–578. Popov, I., Lewin, G., 1998. Antioxidative homeostasis: characterization by means of chemiluminescent technique. Methods Enzymol. 300, 437–456. Rey, D., David, J.P., Martins, D., Pautou, M.P., Long, A., Marigo, G., Meyran, J.C., 2000. Role of vegetable tannins in habitat selection among mosquito communities from the Alpine hydrosystems. Comptes Rendus de l’Academie des Sciences Serie III 323, 391–398. Rey, D., Pautou, M.P., Meyran, J.C., 1999. Histopathological effects of tannic acid on the midgut epithelium of some aquatic diptera larvae. J. Invertebr. Pathol. 73, 173–181. Ristow, M., Schmeisser, S., 2011. Extending life span by increasing oxidative stress. Free Rad. Biol. Medic. 51, 327–336. Ristow, M., Zarse, K., 2010. How increased oxidative stress promotes longevity and metabolic health: the concept of mitochondrial hormesis (mitohormesis). Exp. Gerontol. 45, 410–418. Sachse, A., Babenzien, D., Ginzel, G., Gelbrecht, J., Steinberg, C.E.W., 2001. Characterization of dissolved organic carbon (DOC) in a dystrophic lake and an adjacent fen. Biogeochemistry 54, 279–296. Salonen, K., Hammar, T., 1986. On the importance of dissolved organic matter in the nutrition of zooplankton in some lake waters. Oecologia 68, 246–253. Sarma, S.S.S., Nandini, S., 2006. Review of recent ecotoxicological studies on cladocerans. J. Environ. Sci. Health B 41, 1417–1430. Sarma, S.S.S., Nandini, S., Gulati, R.D., 2005. Life history strategies of cladocerans: comparisons of tropical and temperate taxa. Hydrobiologia 542, 315–333. Saul, N., Pietsch, K., Menzel, R., Steinberg, C.E., 2008. Quercetin-mediated longevity in Caenorhabditis elegans: is DAF-16 involved? Mech. Ageing Dev. 129, 611–613. Saul, N., Pietsch, K., Menzel, R., Stürzenbaum, S.R., Steinberg, C.E., 2010. The longevity effect of tannic acid in Caenorhabditis elegans: Disposable Soma meets hormesis. J. Gerontol. A 65, 626–635. Schlesinger, W.H., 1997. Biochemistry. An Analysis of Global Change. Academic Press, San Diego. Schmitt-Kopplin, P., Hertkorn, N., Schulten, H.R., Kettrup, A., 1998. Structural changes in a dissolved soil humic acid during photochemical degradation processes under O2 and N2 atmosphere. Environ. Sci. Technol. 32, 2531–2541. Schrimpf, S.P., Weiss, M., Reiter, L., Ahrens, C.H., Jovanovic, M., Malmström, J., Brunner, E., Mohanty, S., Lercher, M.J., Hunziker, P.E., Aebersold, R., von Mering, C., Hengartner, M.O., 2009. Comparative functional analysis of the Caenorhabditis elegans and Drosophila melanogaster proteomes. PLoS Biol. 7, e1000048. Schulz, T.J., Zarse, K., Voigt, A., Urban, N., Birringer, M., Ristow, M., 2007. Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress. Cell Metab. 6, 280–293. Steinberg, C.E.W., 2003. Ecology of Humic Substances in Freshwaters. Determinants from Geochemistry to Ecological Niches. Springer, Berlin. Steinberg, C.E.W., 2011. Stress Ecology – Environmental Stress as Ecological Driving Force and Key Player in Evolution. Springer, Dordrecht. Steinberg, C.E.W., Kamara, S., Prokhotskaya, V.Y., Manusadzianas, L., Karasyova, T.A., Timofeyev, M.A., Jie, Z., Paul, A., Meinelt, T., Farjalla, V.F., Matsuo, A.Y.O., Kent Burnison, B., Menzel, R., 2006. Dissolved humic substances – ecological driving forces from the individual to the ecosystem level? Freshw. Biol. 51, 1189–1210. Steinberg, C.E.W., Ouerghemmi, N., Herrmann, S., Bouchnak, R., Timofeyev, M.A., Menzel, R., 2010a. Stress by poor food quality and exposure to humic substances: Daphnia magna responds with oxidative stress, lifespan extension, but reduced offspring numbers. Hydrobiologia 652, 223–236. Steinberg, C.E.W., Paul, A., Pflugmacher, S., Meinelt, T., Klöcking, R., Wiegand, C., 2003. Pure humic substances have the potential to act as xenobiotic chemicals – a review. Fresenius Environ. Bull. 12, 391–401. Steinberg, C.E.W., Saul, N., Pietsch, K., Meinelt, T., Rienau, S., Menzel, R., 2007. Dissolved humic substances facilitate fish life in extreme aquatic environments and have the potential to extend lifespan of Caenorhabditis elegans. Ann. Environ. Sci. 1, 81–90. Steinberg, C.E.W., Vic´entic´, L., Rauch, R., Bouchnak, R., Suhett, A.L., Menzel, R., 2010b. Exposure to humic material modulates life history traits of the cladocerans Moina macrocopa and Moina micrura. Chem. Ecol. 26, 135–143. Suhett, A.L., Steinberg, C.E.W., Santangelo, J.M., Bozelli, R.L., Farjalla, V.F., 2011. Natural dissolved humic substances increase the lifespan and promote transgenerational resistance to salt stress in the cladoceran Moina macrocopa. Environ. Sci. Pollut. Res. 18, 1004–1014. Sun, B.K., Tanji, Y., Unno, H., 2005. Influences of iron and humic acid on the growth of the cyanobacterium Anabaena circinalis. Biochem. Eng. J. 24, 195–201. Sun, B.K., Tanji, Y., Unno, H., 2006. Extinction of cells of cyanobacterium Anabaena circinalis in the presence of humic acid under illumination. Appl. Microbiol. Biotechnol. 72, 823–828. Taipale, S., Kankaala, P., Hämäläinen, H., Jones, R.I., 2009. Seasonal shifts in the diet of lake zooplankton revealed by phospholipid fatty acid analysis. Freshw. Biol. 54, 90–104. Tan, K.H., 2009. Environmental Soil Science. CRC Press, Boca Raton. Tapia, P.C., 2006. Sublethal mitochondrial stress with an attendant stoichiometric augmentation of reactive oxygen species may precipitate many of the beneficial alterations in cellular physiology produced by caloric restriction, intermittent fasting, exercise and dietary phytonutrients: ’’Mitohormesis’’ for health and vitality. Med. Hypotheses 66, 832–843.
890
S. Hofmann et al. / Chemosphere 86 (2012) 883–890
Tatarazako, N., Takao, Y., Kishi, K., Onikura, N., Arizono, K., Iguchi, T., 2002. Styrene dimers and trimers affect reproduction of daphnid (Ceriodaphnia dubia). Chemosphere 48, 597–601. Tilquin, M., Meyran, J.C., Marigo, G., 2004. Comparative capability to detoxify vegetable allelochemicals by larval mosquitoes. J. Chem. Ecol. 30, 1381–1391. Timofeyev, M.A., Shatilina, Z.M., Bedulina, D.S., Menzel, R., Steinberg, C.E., 2007. Natural organic matter (NOM) has the potential to modify the multixenobiotic resistance (MXR) activity in freshwater amphipods Eulimnogammarus cyaneus and E. verrucosus. Comput. Biochem. Physiol. B 146, 496–503. Timofeyev, M.A., Shatilina, Z.M., Kolesnichenko, A.V., Kolesnichenko, V.V., Steinberg, C.E., 2006. Specific antioxidant reactions to oxidative stress promoted by natural organic matter in two amphipod species from Lake Baikal. Environ. Toxicol. 21, 104–110. Timofeyev, M.A., Wiegand, C., Burnison, B.K., Shatilina, Z.M., Pflugmacher, S., Steinberg, C.E.W., 2004. Impact of natural organic matter (NOM) on freshwater amphipods. Sci. Total Environ. 319, 115–121.
Tremolieres, M., 1988. Deoxygenating effect and toxicity of ground-up dried coniferous needles and deciduous leaves of Canadian trees in water: A preliminary study in comparison with litter of European trees. Water Res. 22, 21–28. Trevisan, S., Pizzeghello, D., Ruperti, B., Francioso, O., Sassi, A., Palme, K., Quaggiotti, S., Nardi, S., 2010. Humic substances induce lateral root formation and expression of the early auxin-responsive IAA19 gene and DR5 synthetic element in Arabidopsis. Plant Biol. 12, 604–614. Trubetskova, I.L., Haney, J.F., 2006. Effects of differing concentrations of microcystin-producing Microcystis aeruginosa on growth, reproduction, survivorship and offspring of Daphnia magna. Arch. Hydrobiol. 167, 533–546. Wang, W.H., Bray, C.M., Jones, M.N., 1999. The fate of 14C-labelled humic substances in rice cells in culture. J. Plant Physiol. 154, 203–211. Zepp, R.G., Wolfe, N.L., Baughman, G.L., Hollis, R.C., 1977. Singlet oxygen in natural waters. Nature 267, 421–423.