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Toxicon 55 (2010) 1244–1254 Contents lists available at ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon Chronic effects of cyanobacterial toxins on Daphnia magna and their offspring Thanh Son Dao a, b, Lan-Chi Do-Hong c, Claudia Wiegand b, d, * a Institute for Environment and Resources, 142 To Hien Thanh Street, District 10, Ho Chi Minh City, Vietnam Leibniz Institute of Freshwater Ecology and Inland Fisheries, Mueggelseedamm 301, 12587 Berlin, Germany c Vietnam National University- HoChiMinhCity, Linh Trung Ward, Thu Duc District, Ho Chi Minh City, Vietnam d University of Southern Denmark, Institute of Biology, Campusvej 55, 5230 Odense M, Denmark b a r t i c l e i n f o a b s t r a c t Article history: Received 10 July 2009 Received in revised form 14 December 2009 Accepted 26 January 2010 Available online February 2010 The zooplankton grazer Daphnia magna endures living in water bodies up to moderate densities of cyanobacteria, such as Microcystis spp., known for producing toxic secondary metabolites Although daphnids are affected via decreased food filtering, inhibition of digestive proteases and lethality, development of tolerance against cyanobacterial toxins has also been observed Aim of our study was to investigate in detail chronic effects of cyanobacterial toxins, with emphasis on microcystin, on D magna The animals were exposed chronically for two generations to either microcystin-LR in or 50 mg LÀ1, or to cyanobacterial crude extract containing the same amount of total microcystin, starting at neonate stadium Survival, growth, maturation and fecundity were observed for the first generation during two months In the offspring survival, maturation, and growth were followed for the first week Low concentration of microcystin-LR slightly affected the growth and reproduction of parent daphnids Survivorship decreased during chronic exposure with increasing microcystin concentration Age to maturity of the offspring increased and their survival decreased after parent generation was exposed to the toxin, even if the offspring were raised in control medium Besides, cessation of the eggs/embryos was observed and malformation of neonates caused by cyanobacterial toxins was firstly recorded Ó 2010 Elsevier Ltd All rights reserved Keywords: Chronic effects Daphnia magna Microcystin Cyanobacterial crude extract Life traits Malformation Introduction In nature, cyanobacteria are not only low nutrient food for zooplankton (Lampert, 1977; Muăller-Navarra et al., 2000; Von Elert et al., 2003) but also difficult to consume due to commonly big colonial or long filamentous formation and mucilage production (Rohrlack et al., 1999; Ebert, 2005) Furthermore, cyanobacteria are capable of producing toxic metabolites and bioactive compounds such as microcystin (MC), anatoxin-a, cylindrospermopsin, microviridin J or microcin SF608 (Sivonen and Jones, 1999; * Corresponding author at: University of Southern Denmark, Institute of Biology, Campusvej 55, 5230 Odense M, Denmark Tel.: ỵ45 6550 2785 E-mail address: wiegand@biology.sdu.dk (C Wiegand) 0041-0101/$ – see front matter Ó 2010 Elsevier Ltd All rights reserved doi:10.1016/j.toxicon.2010.01.014 Banker and Carmeli, 1999; Rohrlack et al., 2003) Most of those bioactive metabolites are contained inside the cells and released into the water during cell lysis Zooplankton directly suffer from toxic cyanobacteria as they are primary consumers feeding on suspended particles, and phytoplankton including small cyanobacteria Zooplankton accumulated MC at concentrations ranging from 0.3 to 16.4 mg gÀ1 dried weight (DW) which is much higher than present in natural water, from undetectable À5.8 ng gÀ1 DW (Ferra˜o-Filho et al., 2002a) The negative correlation between the density of cladoceran and the biomass of cyanobacteria producing MC was recorded in the field reflecting the nutritional and toxin effects on the animals (Ferra˜o-Filho et al., 2002b) Similarly, Hansson et al (2007) found a clear negative correlation between MC in water and T.S Dao et al / Toxicon 55 (2010) 1244–1254 total zooplankton biomass In addition, big cladoceran had an apparently negative response while smaller cladoceran seemed to have a weakly positive response to MC This was assumed as the effects of toxic cyanobacteria which induced a shift in zooplankton size and community composition in nature (Hansson et al., 2007) Considerable numbers of laboratorial studies investigated acute effects of cyanobacterial toxins on daphnids concerning survival, food filtering, molting or enzyme activities of the animals Survival of Daphnia spp was rapidly reduced by toxic Microcystis aeruginosa (Nizan et al., 1986; Ferra˜o-Filho et al., 2000) The 48-h lethal concentration (LC50) of purified microcystin-LR (MC-LR) for three Daphnia species ranged from 9.6 to 21.4 mg MC-LR mLÀ1(DeMott et al., 1991) Similarly, cyanobacterial crude extract containing MC varied in concentrations of 48-h LD50 or 48-h effective concentration (EC50) ranging from 36 to 162.45 mg mLÀ1 or 34.2 to 1380 mg MC gÀ1 DW in Daphnia pulicaria and Daphnia similis respectively (Jungmann and Benndorf, 1994; Sotero-Santos et al., 2006; Okumura et al., 2007) Hietala et al (1997a) showed a discrepancy among the 48-h EC50 concentration of toxic M aeruginosa to ten clones of Daphnia pulex, varying from 0.022 to >2.61 mg C LÀ1 Hence, susceptibility to cyanobacterial toxins differs not only between zooplankton species but also within clones of Daphnia The food filtering rate of Daphnia spp was inhibited when the organisms were exposed to toxic cyanobacteria and purified MC-LR (Lampert, 1982; Nizan et al., 1986; DeMott, 1999; Ferra˜o-Filho et al., 2000; Ghadouani et al., 2004) Toxic M aeruginosa caused sudden stops in swimming and filtering activity in daphnids (Rohrlack et al., 2001) Deformation of carapace and interruption of molting process of daphnids were induced by the cyanobacterial metabolite microviridin J (Kaebernick et al., 2001; Rohrlack et al., 2004) Physiological responses included elevated activities of the biotransformation enzyme glutathione S-transferase in D magna after exposure to MC-LR or Cylindrospermopsis raciborskii but inhibition after exposure to microcin SF608 or Aphanizomenon issatschenkoi (Wiegand et al., 2002; Nogueira et al., 2004a,b) In addition, the digestive enzyme trypsin of daphnids was inhibited by cyanobacterial metabolites obtained from a widely distributed cyanobacterial genus Planktothrix (Rohrlack et al., 2005) In chronic and semi-chronic exposures, toxin accumulation and detrimental influence on the animals such as survival, growth, maturation and fecundity were observed Exposed to toxic M aeruginosa, daphnids accumulated MC at very high concentrations, up to 0.0712 mg MC per Daphnia and 24.5 mg MC gÀ1 DW of daphnids respectively (Thostrup and Christoffersen, 1999; Mohamed, 2001) Similar to acute exposures, survival of Daphnia was decreased by toxic M aeruginosa, both at species and clone levels (DeMott et al., 1991; Hietala et al., 1995, 1997b; Rohrlack et al., 2001) Body length of daphnids was significantly reduced when they were fed with M aeruginosa (Luărling, 2003; Luărling and Van der Grinten, 2003; Trubetskova and Haney, 2006) Consequently, maturation and time to first reproduction of daphnids were delayed if they fed on toxic M aeruginosa (Hietala et al., 1995; Lauren-Maăaăttaă et al., 1245 1997) Toxic M aeruginosa also caused aborted eggs (Gustafsson et al., 2005) and reduced the fecundity of D magna (Thostrup and Christoffersen, 1999; Luărling and Van der Grinten, 2003; Trubetskova and Haney, 2006) or even completely inhibited the reproduction of D pulex (Hietala et al., 1997b) In contrast, dissolved MC-LR at the concentration of 3.5 mg LÀ1 had no effect on growth and reproduction of D magna (Luărling and Van der Grinten, 2003) By continuous exposure to low densities of M aeruginosa over generations, D magna increased their tolerance to the toxic cyanobacterium, had higher fitness and earlier reached the maturity age and first clutch of reproduction These adaptations were assumed as the result of maternal effects (Gustafsson and Hansson, 2004; Gustafsson et al., 2005) Overall, toxic cyanobacteria and their toxins severely impacted daphnids in both acute and chronic exposures The organisms were not only suppressed in their survival but impaired in development as well As mentioned above, investigations of the chronically detrimental influence on daphnids have been conducted with cyanobacterial cultures or pure cyanobacterial toxin In most of the mentioned chronic studies, exposures were performed with one generation of daphnids and lasted for around three weeks Gustafsson et al (2005) were conducting transgenerational experiments in which the F1 daphnids produced up to seven clutches; hence for the F1 generation the exposure lasted for around one month In this study, chronic effects of dissolved MC-LR and a cell free cyanobacterial crude extract containing MC on life history of D magna were investigated over their entire life duration of two months Their offspring were raised in either toxic or control medium until maturation to follow consequences of maternal exposure The observed life traits of the daphnids were survival, growth, maturation, time to first reproduction and fecundity The investigation on chronic effects was carried out with two concentrations of MC (5 and 50 mg LÀ1) which would fall within the range of dissolved MC in natural waters in the world, from trace concentration to 200 mg LÀ1 (Sivonen and Jones, 1999) and have already impacted enzyme activities in D magna (Wiegand et al., 2002) In all exposure, Scenedesmus spp were provided as food source to exclude effects due to malnutrition, hence to be able to attribute effects to cyanobacterial toxin or toxic compounds Materials and methods 2.1 Microcystin-LR and cyanobacterial crude extract Microcystin-LR and a cyanobacterial crude extract containing MC were used for the exposures The MC-LR was purchased from Axxora (Germany), and the cyanobacterial crude extract was produced from our batch cultures of toxic M aeruginosa The cyanobacterium, strain AB 2005/47, was isolated from Chaohu Lake in China in 2005 and has been cultivated as single strain in the laboratory of the Leibniz Institute of Freshwater Ecology and Inland Fisheries, Berlin, Germany since then The crude extract was prepared according to Fastner et al (1998) with modification Briefly, the biomass of cultures on GF/C filters (Whatman) were homogenized and firstly extracted over night in 70% MeOH 1246 T.S Dao et al / Toxicon 55 (2010) 1244–1254 (Carl Roth, Germany) containing 5% acetic acid (Merck, Germany) and 0.1% triflouracetic acid (TFA; Merck, Germany) followed by  60 in MeOH 90% containing 5% acetic acid and 0.1% TFA with 30 s sonication during the last extraction Each extraction step was followed by centrifugation (4500 rpm, 10 min, C) The supernatants of all extractions were pooled, dried at 35 C, re-dissolved in MeOH (100%) and centrifuged at 14 000 rpm, C for 10 The supernatant was kept at À20 C prior to highperformance liquid chromatography (HPLC) analysis Microcystin content was analyzed according to Pflugmacher et al (2001) by high-performance liquid chromatography (HPLC, Waters Alliance, Eschborn, Germany) system equipped with a reverse phase column (RP 18; mM LIChroSpher 100) and photodiode array detection Injection volume of the samples was 80 mL Separation was achieved at 40 C by a gradient of Milli-Q water and acetonitrile (ACN, Rathburn, Walkerburn, UK), both enriched with 0.1% (v/v) TFA The flow rate was mL minÀ1, starting at 35% ACN, linearly increasing to 55% ACN within 15 min, followed by a cleaning step of 100% ACN and 10 equilibration to start conditions MC-LR was used as the reference toxin The HPLC analysis shown that there were several different variants of MC and their total concentration in the crude extract was 6.92 mg mLÀ1 MC-LR equivalent Mass spectrometer measurements for the cyanobacterial crude extract were performed using an API 165 mass spectrometer (Applied Biosystems, Foster City, CA, USA) with an atmospheric pressure ionization source operating in turbo ion-spray mode Quantitative determination of the toxins was carried out by comparing peak areas in sample chromatograms with the corresponding peak areas obtained from the pure toxins Proportion of the main cyanobacterial toxins in the crude extract was as follows, MC-RR, 71.09%; MC-LR, 24%; MC-LA, 3.09%; MC-YR, 1.79% and MC-LF, 0.03% Kruăger et al., (submitted for publication) 2.2 Experimental organisms The test organism was D magna Straus, obtained from MicroBioTests Inc Belgium and has been maintained in the laboratory of the Leibniz Institute of Freshwater Ecology and Inland Fisheries, Berlin, for several generations The Daphnia medium provided by MicroBioTests Inc Belgium consisted of CaCl2, KCl, NaHCO3 and MgSO2 The living green algae Scenedesmus spp were used as the sole food for D magna The algae were cultivated in Z8 medium (Kotai, 1972) with continuous aeration Both culturing of Scenedesmus and Daphnia as well as the exposures were conducted at a temperature of 20 Ỉ C and a photoperiod of 14 h light and 10 h dark Prior start of the experiments, fifteen adolescent female D magna were incubated in a 500 mL beaker and fed with Scenedesmus spp for 2–3 weeks Offspring from the second to fifth clutch of these D magna were used for experiments and hereafter referred to as mother daphnids Thirty neonates less than 24 h old were randomly selected for each chronic exposure (Adema, 1978) and individually incubated in 50 mL beakers containing 20 mL of medium The animals were fed  106 Scenedesmus spp cells per daphnid per day equaling a cell density of  105 cells mLÀ1 (approximately mg C LÀ1) Density of Scenedesmus cells was adjusted daily The food density and availability were chosen according to Lampert (1977) and Gustafsson et al (2005) respectively, in which individual animal was fed with  104 Scenedesmus cells mLÀ1 in 60 mL beakers, equaling 3.6  106 cells per daphnid per day 2.3 Experimental setup For exposure, MC-LR or cell free cyanobacterial crude extract containing MC was added into Daphnia medium In total, there were four different exposures: and 50 mg LÀ1 of purified MC-LR, and and 50 mg LÀ1 of total MC in crude extract, hereafter referred as M5, M50, E5 and E50 respectively The control was implemented twice (C1 and C2) because the food was changed between exposure M5 and the others Daphnids of C1 and M5 treatments were fed with green alga Scenedesmus vacuolatus Due to unavailability of S vacuolatus, daphnids were fed with another Scenedesmus species, Scenedesmus armatus for the other exposures (M50, E5 and E50 respectively) Consequently a new control (C2) was conducted in which daphnids were fed with S armatus in order to get the same supply of nutrient quality Results of daphnids incubated in the same food regime were compared only (C1–M5; C2–M50, E5 and E50) Twice a week, half of toxic and non-toxic Daphnia medium was renewed whereas food density was kept constant on a daily basis Each exposure and control experiment lasted for two months 2.4 Life history traits For life history study, the survival, growth, maturation, time to first reproduction and fecundity of mother Daphnia were observed daily for two months Simultaneously, the survival, growth and maturation of their offspring were also recorded for one week Death of the animal was defined as the stop of heartbeat confirmed by microscopic observation (Olympus TH3) Maturation of Daphnia was defined as time point of first egg occurrence in the brood chamber The time to first reproduction was at first offspring release from brood chamber during molting Fecundity of animals was recorded as the number of clutches and number of offspring per clutch produced by every mother Daphnia during exposure time In case of release of decomposed eggs, embryos or neonates, the offspring number of that clutch was assumed as zero Twice a week, the growth of test organisms was monitored by measuring the distance from top of the head to the base of tail spine (body length) by photographing the animals under a microscope (Olympus SZX7) equipped with a digital camera (Olympus XC50) The body length of Daphnia was measured exactly to mm based on the software DT5 analysis (Soft Imaging System – Olympus) Neonates (