Miquel Lürling
email: miquel.lurling@wur.nl

Elisabeth J Faassen

Justine Van

Justine S Van Eenennaam

β-N-methylamino-L-alanine (BMAA) on the survival, mobility and reproduction of Daphnia magna

Keywords: Bioconcentration, cyanotoxins, life history, LC-MSMS, bioaccumulation Bioconcentration, cyanotoxins, life-history, LC-MSMS

Effects of the cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA) on the survival, mobility and reproduction of Daphnia magna

INTRODUCTION

Pelagic cyanobacteria are ubiquitous inhabitants of freshwater systems and well-known for their ability to produce a variety of toxic secondary metabolites, cyanotoxins [START_REF] Sivonen | Cyanobacterial toxins and toxin production[END_REF][START_REF] Wiegand | Ecotoxicological effects of selected cyanobacterial secondary metabolites a short review[END_REF][START_REF] Funari | Human health risk assessment related to cyanotoxines exposure[END_REF]. Hundreds of these compounds, mostly peptides and alkaloids, have been structurally identified, but their physiological functions remain unknown [START_REF] Dietrich | Guidance values for microcystins in water and cyanobacterial supplement products (blue-green algal supplements): a reasonable or misguided approach?[END_REF].

Recently, a novel cyanotoxin, the non-protein amino acid β-N-methylamino-Lalanine (BMAA) was identified in cultures of the genus Nostoc, isolated from coralloid roots of the cycad Cycas micronesia [START_REF] Cox | Biomagnification of cyanobacterial neurotoxins and neurodegenerative disease among the Chamorro people of Guam[END_REF]. Subsequent analysis revealed that BMAA production is not restricted to symbiotic Nostoc, but occurred in about 95% of free-living cyanobacterial strains from different origins and all five taxonomic sections [START_REF] Cox | Diverse taxa of cyanobacteria produce ß-N-methylamino-l-alanine, a neurotoxic amino acid[END_REF][START_REF] Esterhuizen | ß-N-methylamino-L-alanine (BMAA) in novel South African cyanobacterial isolates[END_REF]. Although the results of these first studies could not be reproduced by some other groups and the analytical techniques used are under discussion [START_REF] Rosén | Determination of the neurotoxin BMAA (β-Nmethylamino-L-alanine) in cycad seed and cyanobacteria by LC-MS/MS (liquid chromatography tandem mass spectrometry)[END_REF]Hellenäs, 2008, Krüger et al., 2010), BMAA has recently been detected in cyanobacteria by highly specific analytical techniques, albeit in lower concentrations than reported by [START_REF] Cox | Diverse taxa of cyanobacteria produce ß-N-methylamino-l-alanine, a neurotoxic amino acid[END_REF] and [START_REF] Esterhuizen | ß-N-methylamino-L-alanine (BMAA) in novel South African cyanobacterial isolates[END_REF] [START_REF] Faassen | Determination of the neurotoxins BMAA (β-N-methylamino-L-alanine) and DAB (α-,γdiaminobutyric acid) by LC-MSMS in Dutch urban waters with cyanobacterial blooms[END_REF][START_REF] Jonasson | Transfer of a cyanobacterial neurotoxin within a temperate aquatic ecosystem suggests pathways for human exposure[END_REF][START_REF] Spáčil | Analytical protocol for identification of BMAA and DAB in biological samples[END_REF].

Since BMAA has been linked to the human neurodegenerative disease amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS/PDC), the role of BMAA in this disease has been studied over the last years (reviewed in [START_REF] Bradley | Beyond Guam: The cyanobacteria/BMAA hypothesis of the cause of ALS and other neurodegenerative diseases[END_REF]. However, the effect of BMAA on organisms that are in direct contact with cyanobacteria, such as the suspension-feeding zooplankton species Daphnia, has not yet been studied. Suspension-feeding zooplankton is often an important component of lake and pond ecosystems. These key organisms form a major link between limnetic primary production and higher trophic levels and therefore play an important role in the trophic energy flow [START_REF] Mcqueen | Trophic relationships in freshwater pelagic ecosystems[END_REF]. When cyanobacteria appear however, large-bodied Daphnia may decline and the cyanobacteria develop dense blooms [START_REF] Edmondson | Daphnia in Lake Washington[END_REF]).

An explanation for the decrease of Daphnia in the presence of cyanobacteria may be that cyanobacteria are in general poor food for Daphnia [START_REF] Wilson | Effects of cyanobacterial toxicity and morphology on the population growth of freshwater zooplankton: Meta-analyses of laboratory experiments[END_REF]Tillmanns et al., 2008). Several causal factors have been proposed, such as lack of essential compounds, a hard-to-handle morphology and the presence of toxic compounds. Traditionally, microcystins have received considerable interest, but these cyanotoxins seem not to be the sole cause of cyanobacteria being a poor food, since the growth of Daphnia is not necessarily significantly different with diets of nonmicrocystin producing cyanobacteria compared to microcystin producing ones [START_REF] Wilson | Effects of cyanobacterial toxicity and morphology on the population growth of freshwater zooplankton: Meta-analyses of laboratory experiments[END_REF]Tillmanns et al., 2008). Studies in which morphology and biochemical deficiencies could be eliminated support the hypothesis that yet unknown cyanotoxins might be present in the cyanobacterial matrix inhibiting Daphnia growth [START_REF] Jungmann | Toxic compounds isolated from Microcystis PCC7806 that are more active against Daphnia than two microcystins[END_REF][START_REF] Jungmann | Isolation, purification, and characterization of new Daphniatoxic compound from axenic Microcystis aeruginosa strain PCC7806[END_REF]Lürling, 2003a,b). These compounds might be peptides, but might also belong to the family of non-protein amino acids. In fact, the structural isomer of BMAA, 2,4-diaminobutyric acid (2,4-DAB) has been detected in a cyanobacterial matrix [START_REF] Rosén | Determination of the neurotoxin BMAA (β-Nmethylamino-L-alanine) in cycad seed and cyanobacteria by LC-MS/MS (liquid chromatography tandem mass spectrometry)[END_REF][START_REF] Faassen | Determination of the neurotoxins BMAA (β-N-methylamino-L-alanine) and DAB (α-,γdiaminobutyric acid) by LC-MSMS in Dutch urban waters with cyanobacterial blooms[END_REF]. Because of the many potential factors acting simultaneously on zooplankton grazers, we decided not to use a cyanobacterium as transport vehicle of BMAA at this stage. Moreover, upon bloom senescence, BMAA, like other cyanotoxins, may be released into the surrounding environment. Although no data are available at this point on dissolved BMAA concentrations in situ, we assumed that BMAA could also be present in dissolved form in surface waters, as has been found for microcystins that may be present in dissolved form for months at concentrations between 3 -100 µg L -1 [START_REF] Gurbuz | Analysis of dissolved microcystins in surface water samples from Kovada Lake, Turkey[END_REF]. Since Daphnia can take up dissolved organic carbon directly from the water [START_REF] Speas | Uptake of dissolved organic carbon (DOC) by Daphnia pulex[END_REF], we expected uptake of dissolved BMAA as well.

As non-protein amino acids are classical anti-metabolites inhibiting selected reactions in the primary metabolism of predators and herbivores [START_REF] Fowden | Toxic amino acids: Their action as antimetabolites[END_REF][START_REF] Bell | Nonprotein amino acids of plants: significance in medicine, nutrition, and agriculture[END_REF][START_REF] Zenk | Evolution and current status of the phytochemistry of nitrogenous compounds[END_REF], we hypothesize that BMAA inhibits the grazer Daphnia. Because one of the mechanisms of toxicity is the incorporation of nonprotein amino acids in the proteins of predators [START_REF] Crine | Incorporation of canavanine into rat pars intermedia proteins inhibits the maturation of proopiomelanocortin, the common precursor to adrenocorticotropin and beta-lipotropin[END_REF][START_REF] Rosenthal | l-Canavanine: a higher plant insecticidal allelochemical[END_REF], we also expect that Daphnia accumulates BMAA and thereby transports BMAA along the pelagic food chain. To test our hypotheses, we studied the effect of synthetic BMAA on survival, mobility, growth and reproduction of Daphnia in shortterm tests and life-history assays. In addition, we analyzed concentrations of BMAA in medium and animals, using a highly selective LC-MSMS technique [START_REF] Faassen | Determination of the neurotoxins BMAA (β-N-methylamino-L-alanine) and DAB (α-,γdiaminobutyric acid) by LC-MSMS in Dutch urban waters with cyanobacterial blooms[END_REF].

METHOD

Daphnia culture

The cladoceran Daphnia magna Straus was isolated from Lake Zwemlust (The Netherlands) in 1999 and has been cultured in the laboratory in 1 liter jars containing 800 mL artificial RT-medium with a pH of 7.6, a conductivity of 270 µS cm -1 and a total hardness of 88 mg CaCO 3 L -1 [START_REF] Tollrian | Neckteeth formation in Daphnia pulex as example of continuous phenotypic plasticity:morphological effects of Chaoborus kairomone concentration and their quantification[END_REF]. Jars with D. magna were kept Scenedesmus obliquus (about 4 mg C L -1 ), which was grown at 20 °C in a 1-L chemostat on a modified WC medium (Lürling and Beekman, 2006) in continuous light (~100 µmol photons m -2 s -1 ) and with a dilution rate of 1.0 d -1 . The S. obliquus population was dominated by single cells (linear dimensions 16 x 5 µm) with some two-and four-celled colonies.

Prior to the experiments, a new isofemale lineage was started. One juvenile D. magna was placed in a 125 mL glass-tube with 100 mL fresh RT-medium and ~5 mg C L -1 S. obliquus. Every two/three days medium and food were refreshed. The neonates of the first brood were discarded, the neonates of the second brood were collected and placed individually in 125 mL glass-tubes with 100 mL RT-medium and ~5 mg C L -1 S. obliquus. Every two/three days medium and food were refreshed.

From these second generation animals, first brood neonates were again discarded and ten neonates of the second brood were transferred to 1 L jars with 800 mL artificial RT-medium and ~5 mg C L -1 S. obliquus. Again every two/three days medium and food were refreshed. From these third generation animals, only the adults were transferred to fresh medium, all neonates born on the same day were transferred to separate jars to avoid crowding effects. Before each experiment, neonates (<24 h) or juveniles (>48 h and <72 h) born on the same day were collected from the jars and treated as described below.

Short-term toxicity experiment

Since no data on the effects of BMAA on D. magna were available, a short-term experiment was performed where neonate D. magna were exposed for 48 hours to BMAA (BMAA Hydrochloride, Sigma-Aldrich) was dissolved in RT-medium at concentrations of 0, 1, 10, 100, 1000 and 10000 µg L -1 . The experiment was performed in plastic 190 mL jars with 100 mL RT-medium. Each jar received 10 neonate (< 24 h) D. magna and for each treatment four jars were used. The jars were placed in a climate controlled chamber in constant light of 30 µmol photons m -2 s -1 and a constant temperature of 20 o C. Animals were not fed during the experiment.

After 48 h the number of living, dead and immobile (not swimming, but with heart beat) D. magna were recorded in each jar. The BMAA concentration at which 50% of the D. magna had died (LC 50 ) or were immobilized (IC 50 ) was determined by nonlinear regression (4 parameter logistic model) in the tool pack SigmaPlot 11.0.

BMAA concentration in the medium was checked by collecting 20 mL medium from each jar at the end of the experiment and transferring the medium to a glass-bottle.

Bottles were stored at -20°C until BMAA analysis (see section BMAA analysis). The stability of BMAA in solution was checked by sampling an additional jar without D.

magna, but with a BMAA concentration of 10000 µg L -1 at the start and end of the experiment.

Short term bioconcentration experiment

The short term toxicity experiment was repeated to determine the bioconcentration of BMAA in D. magna during short term exposure without food. This experiment was conducted in the same way as the short term toxicity experiment, only older animals (> 48 h and < 72 h) were used and exposure time was extended to 96 h. The endpoint of this experiment was BMAA concentration per amount of wet weight (WW) and dry

Life-history experiment

Long-term effects of sub lethal BMAA concentrations on D. magna were tested in an experiment where neonate D. magna were exposed for 15 days to different concentrations of dissolved BMAA. Somatic growth and population growth were chosen as endpoints, and levels of BMAA in the animals were analyzed.

Neonates (< 24 h) were selected and placed individually in 15 mL glass-tubes containing 10 mL of RT-medium with a S. obliquus concentration of ~5 mg C L -1 .

BMAA was dissolved in the RT-medium in the concentrations 0, 1, 10, 50 and 100 µg L -1 ; each treatment was replicated ten times. The glass-tubes were incubated in a temperature-controlled incubator at 22°C in darkness. The animals were transferred daily to new tubes with fresh medium containing ~5 mg C L -1 S. obliquus and the assigned BMAA concentration. Before the animals were transferred into these new tubes their body length was measured under a stereo-binocular microscope. Body length is defined as the distance from the most posterior point on the eye to the base of the junction of the tail spine with the carapace. The number of survivors, time of reproduction, and number of neonates were also recorded daily. The neonates were counted and then discarded, except for the last brood. The experiment ran until the control animals had reached their third broods. The mother animals as well as the neonates were then rinsed with RT-medium and placed separated from each other for 3-4 hours in clean tubes with fresh RT-medium. After wet weight determination, animals were transferred into pre-weighed aluminum boats in groups and were dried for 24 h at 55°C. After drying, dry weight was determined on a Sartorius MC5 microbalance (± 1 µg). After weighing, dried animals were pooled per treatment, but juveniles and adults were kept separate from each other. These groups of animals were stored at -20°C until BMAA-analysis (see section BMAA-analysis).

To assess the weight and length of the animals at the start of the experiment, twenty neonates that would not be used in the experiment were collected at the same time as the neonates that were used in the experiment. Body-length was measured for each of these twenty animals and animals were pooled in five groups of four animals for the dry weight determination.

At the end of the experiment, the increase in body-length over time (somatic growth) was statistically evaluated and population growth rates were calculated. Somatic S. obliquus was inoculated to a concentration of 5 mg C L -1 in 300 mL Erlenmeyer flasks that contained 150 mL sterile WC-medium. BMAA was added to the flasks at concentrations of 0, 1, 10, 100, 1000 and 10000 µg L -1 . The flasks were closed with a cellulose plug and the experiment was run in triplicate for 72 hours in a climatecontrolled incubator at 22°C, in continuous light of 150 µmol photons m -2 s -1 and continuous orbital shaking at 80 rpm. Every 24 hours cell densities (# mL -1 ) and biovolumes (µm 3 mL -1 ) were measured with a cell-counter system (Innovatis Casy® model TT), while chlorophyll-a concentrations (µg L -1 ) and Photosystem II efficiencies were determined using a PHYTO-PAM phytoplankton analyzer (Walz).

Growth rates (µ, d -1 ) were estimated using linear regression over natural logarithm transformed particle, biovolume and chlorophyll-a concentrations against time.

Growth rates were statistically compared in a one-way ANOVA (PASW Statistics 17).

BMAA analysis

BMAA concentrations were analyzed as described in

RESULTS

Short-term toxicity experiment

The short-term exposure (48 h) to BMAA had a significant effect on the activity of D.

magna. The number of swimming animals in the 0 and 1 µg BMAA L -1 treatments was significantly higher than in the treatments with 10 -10000 µg BMAA L -1 (Fig. 1; Tukey test following one-way ANOVA; F 5,18 = 23.1; p < 0.001).

The BMAA concentration at which the number of swimming animals had been reduced to 50% was estimated at 40 µg BMAA L -1 (r 2 adj = 0.798). In parallel, the number of immobile animals increased significantly (χ 2 = 17.4; p = 0.004) with increasing concentrations of BMAA (Fig. 1). Mortality also increased with higher concentrations of BMAA reaching a maximum of 45% (SD 17%) at the highest BMAA concentration (Fig. 1). Hence, the LC 50-48h is higher than 10000 µg BMAA L -1 .

BMAA remained stable in RT-medium during the experiment; the levels in the control jar did not decrease. BMAA levels in the exposure media with 0 and 10 µg BMAA L -1 were below level of quantification (~ 15 µg L -1 ), while the mean concentration in the 100 µg BMAA L -1 treatments was 77 (SD 2) µg L -1 , 804 (SD 6) µg L -1 in the 1000 µg BMAA L -1 treatments and 9932 (SD 85) µg L -1 in the 10000 µg BMAA L -1 . During the short-term bioconcentration experiment dose related immobilization and death were observed (data not shown). BMAA was not detected in control animals. In contrast, BMAA was detected in D. magna that had been exposed to BMAA in exposures of 1 to 1000 µg L -1 , albeit below the level of quantification (sample concentration 4.3 -7.1 µg L -1 ; LOQ = 15 µg L -1 ). BMAA could be quantified in animals exposed to 10000 µg BMAA L -1 (Table 1). Based on their dry-weights, animals exposed to 10000 µg L -1 had an averaged 125 times higher concentration of BMAA in their tissue than in the surrounding medium (Table 1). When wet-weights were used the BCFs was 44 for animals from the 10000 µg L -1 treatment (Table 1).

Short-term bioconcentration experiment

Life-history experiment

Survival in the life-history experiment varied between 70 and 90% (Table 2), the majority of deaths were caused by handling the animals. All living animals were swimming, no immobility was observed.

D. magna showed somatic growth in all treatments. Body-length increased from 0.91 (± 0.02) mm at the beginning of the experiment to on average 2.75 (± 0.03) mm at the end (Fig. 2). Despite the fact that differences in size were minor and between -1% to 12% in treatments compared to control animals, BMAA had a significant effect on body-size (repeated-measure ANOVA F 4,35 = 3.83; p = 0.011), where animals exposed to 10 and 50 µg BMAA L -1 were significantly (Tukey test; p < 0.05) larger than the controls (Fig. 2). At the end of the experiment the DW of animals from these treatments was also higher (Table 2). Animals in the 50 µg BMAA L -1 were significantly larger than those in the controls (t-test; t = 5.71; p = 0.029), and the 1 µg BMAA L -1 treatments (t = 7.33; p = 0.018), but they were not different from the 10 and 100 µg BMAA L -1 treatments (p ≥ 0.251). In line with the somatic growth over time, animals exposed to BMAA were significantly larger at first reproduction than control animals (one-way ANOVA F 4,36 = 3.41; p = 0.018; Table 2), but only control animals and those at 10 µg BMAA L -1

were significantly different from each other (Tukey test). Neonates that were produced at the end of the experiment were equally heavy and had an average dryweight of 6.7 (SD 0.5) µg per individual

The mean number of offspring produced per female decreased slightly from 11.3 to 8.8 with higher concentrations of BMAA, but was not significantly different among treatments (F 4,36 = 1.08; p = 0.382; Table 2). Age at first reproduction also showed a tendency to increase with higher BMAA concentration, but this was not a significant increase (one-way ANOVA on natural-log transformed data F 4,36 = 1.39; p = 0.259; Table 2). Nonetheless, population growth rates decreased gradually and linearly from 0.174 d -1 to 0.159 d -1 with increasing BMAA concentrations (Table 2): r = 0.173 -1.36 10 -4 × [BMAA] (r 2 adj = 0.964, F = 109.6; p = 0.002).

BMAA was detected in adults exposed to 50 and 100 µg BMAA L -1 and their neonates, no BMAA was detected in adults and their neonates exposed to 0, 1 and 10 µg BMAA L -1 (Table 3). On a wet weight basis, for adults the BCF was 31 and 51, while it was 1574 and 3327 for their neonates (Table 3). On a dry-weight basis BCFs were higher: 196 and 275 for adults, and 1808 and 3821 for their neonates. The neonates, that had been in the medium for less than 24 hours, had a 31 to 108 times higher BMAA concentration than their parents based on wet weight; based on dry weight their concentration was 7 to 20 times higher (Table 3). The green alga S. obliquus showed good growth in controls and the different BMAA treatments. Chlorophyll-a based growth rates were significantly different among treatments (F 5,12 = 4.75; p = 0.013). Mean growth rates (± 1SD) at 10000 µg BMAA L -1 were 0.92 (0.01) d -1 and significantly lower than those in controls, the 100 and 1000 µg BMAA L -1 treatments (0.98 ± 0.03 d -1 ). However, differences were small and only significant because of low within group variability. Particle-based growth rates (0.86 ± 0.02 d -1 ) and volume-based growth rates (0.97 ± 0.03 d -1 ) were similar among controls and treatments (F 5,12 = 2.55; p = 0.086 and F 5,12 = 1.52; p = 0.256, respectively). Photosystem II efficiencies were also similar between treatments (0.70 ± 0.01).

Growth experiment with S. obliquus

DISCUSSION

Our results show that the neurotoxic non-protein amino acid BMAA is not acutely lethal to D. magna when dissolved in medium. The acute LC 50 found in this study for neonate D. magna after 48 hours of exposure is higher than 10000 µg BMAA L -1 . D.

magna therefore seems to be less sensitive to BMAA than some other aquatic organisms: fish larvae Danio rerio (LC 50 10 µg BMAA L -1 ), brine shrimps Artemia salina (LC 50 1500 -5000 µg BMAA L -1 ) and the ciliate Nassula sorex (LC 50 5000 µg BMAA L -1 ) all showed mortality at lower BMAA concentrations [START_REF] Purdie | Toxicity of the cyanobacterial neurotoxin β-N-methylamino-l-alanine to three aquatic animal species[END_REF].

The fifteen day exposure to lower concentrations in the medium (maximum 100 µg BMAA L -1 ) also did not cause death in D. magna. Although D. magna with a history of regular exposure to cyanobacteria could be more tolerant to cyanobacterial toxins than naïve specimens (Gustafson & Hansson, 2004;[START_REF] Sarnelle | Effects of cyanobacteria on fitness components of the herbivore Daphnia[END_REF], our clone has been in the laboratory for more than 10 years without any exposure to cyanobacteria making increased resistance unlikely. Nevertheless, it would be worthwhile quantifying the between and within-species variation in tolerance of Daphnia to BMAA.

While BMAA was not acutely lethal to D. magna, it reduced mobility when the animals were exposed without food. Behavioral changes have also been reported

for the fruit-fly Drosophila where exposure to BMAA reduced the climbing performance [START_REF] Zhou | BMAA neurotoxicity in Drosophila[END_REF] and for the brine shrimp A. salina where concentrations ≥ 50 µg BMAA L -1 caused a loss of the group swarm response and loss of phototaxis [START_REF] Purdie | Toxicity of the cyanobacterial neurotoxin β-N-methylamino-l-alanine to three aquatic animal species[END_REF]. However, in the life-history experiment, in the presence of a good quality green algal food, no immobility of the Daphnia was observed. The presence of good quality food therefore seemed to reduce the toxic effects of BMAA, which could be through uptake and inactivation of BMAA by the algae and/or a stronger health of fed animals. Interestingly, [START_REF] Zhou | BMAA neurotoxicity in Drosophila[END_REF] found a similar pattern in fruit-flies: females that were given a diet enriched with BMAA and leucine did not show a reduced climbing performance, whereas females that were only given a BMAA enriched diet did. The authors suggested that leucine blocked the same amino acid transporter system used by BMAA, thereby reducing toxicity.

However, in our study it remains unknown whether the presence of algae decreased the toxicity of BMAA as has been found for pesticides [START_REF] Barry | Effect of algal food concentration on toxicity of two agricultural pesticides to Daphnia carinata[END_REF], or that Daphnia are less capable of dealing with the adverse effects of toxicants in the absence of food.

BMAA did not cause mortality or immobilization during the 15 day exposure period in the life-history experiment, but it had an effect on reproduction. Exposed animals showed a tendency to a delayed age at first reproduction and produced a lower number of offspring. Consequently, population growth rates declined with increasing concentrations of BMAA in the medium. Interestingly, the animals that showed delayed reproduction and smaller clutch sizes were larger than the nonexposed controls. In general, at a high resource level Daphnia has a larger size at maturity, and these larger Daphnia have larger eggs and carry more eggs than smaller individuals (Lampert, 1993). However, in our study, the tax Daphnia seemed to pay for increased body-size was a delay in maturation, thus at the expense of early reproduction [START_REF] Cole | The population consequences of life history phenomena[END_REF]. The reaction of D. magna in the life-history experiment shows some resemblance with the inducible defense reactions of certain Daphnia species to the chemical cues from their invertebrate natural enemy, the midge larva

Chaoborus [START_REF] Tollrian | Inducible defenses in cladocera: Constraints, costs and multi-predator environments[END_REF]. When these Daphnia sense the presence of Chaoborus larvae, they allocate a higher portion of energy to maternal growth (larger size at first reproduction, later reproduction, lower relative reproductive investment)

to avoid size-selective predation on the smaller juvenile instars [START_REF] Tollrian | Inducible defenses in cladocera: Constraints, costs and multi-predator environments[END_REF].

The population growth rate r has often been used as a measure of Daphnia fitness (e.g. [START_REF] Mccauley | Growth, reproduction, and mortality of Daphnia pulex Leydig: life at low food[END_REF]Lürling, 2003a,b) and represents a relevant measure of ecological consequences of stressors [START_REF] Forbes | Is the per capita rate of increase a good measure of population-level effects in ecotoxicology?[END_REF]. Although a good estimate of r requires life-table experiments with at least the first three broods included [START_REF] Stearns | The evolution of life-histories[END_REF], a close proxy for r can also be obtained by assays including only the period until release of the first clutch [START_REF] Guilhermino | A Daphnia magna first-brood chronic test: an alternative to the conventional 21-day chronic bioassay? Ecotox[END_REF][START_REF] Trubetskova | The juvenile growth rate of Daphnia: a shortterm alternative to measuring the per capita rate of increase in ecotoxicology?[END_REF]. Hence, the chronic assay employed in this study might be sufficient to predict population level consequences of BMAA to Daphnia; r was about 9% lower when animals were exposed chronically to 100 µg L -1 BMAA.

Leakage from damaged cyanobacterial cells could result in a rather "chronic exposure" as, for example, has been revealed recently for microcystins that were present in the surface water in dissolved form in concentration ranging from 3 -100 18 µg l -1 during the entire study period May -October [START_REF] Gurbuz | Analysis of dissolved microcystins in surface water samples from Kovada Lake, Turkey[END_REF]). However, even though no data on dissolved BMAA concentrations in situ are available, the concentrations required to observe negative effects were exceedingly high (10-100 µg L -1 ). In a recent study, a BMAA concentration of 1.9 µg L -1 was found in a cyanobacteria bloom from the Baltic Sea [START_REF] Spáčil | Analytical protocol for identification of BMAA and DAB in biological samples[END_REF], but more data on particulate and dissolved BMAA concentrations are required.

In this study, when BMAA was added to the medium without food, BMAA accumulation in the animals was observed, which is in line with the findings that Daphnia can take up dissolved organic carbon directly from the water [START_REF] Speas | Uptake of dissolved organic carbon (DOC) by Daphnia pulex[END_REF]. The presence of protease activity in the gut of D. magna [START_REF] Elert | Protease activity in gut of Daphnia magna: evidence for trypsin and chymotrypsin enzymes[END_REF] supports the ability of the animals to take up amino acids actively from ingested water. Daphnia can, however, be viewed as a highly efficient filter-feeder that concentrates food items from the water before ingestion [START_REF] Lampert | Feeding and nutrition in Daphnia[END_REF]. Hence, intake of a toxin dissolved in water is presumably less than the uptake of toxin containing particles collected from the water, which suggests that the mode of toxin delivery (dissolved or in food particles) might be very important as a determinant for the actual dose. For example, intact cells of the cyanobacterium Microcystis aeruginosa containing the toxin microcystin were lethal at a concentration of four orders of magnitude lower than dissolved microcystin [START_REF] Demott | Effects of toxic cyanobacteria and purified toxins on the survival and feeding of a copepod and three species of Daphnia[END_REF]. Although presence of other toxic compounds in the cyanobacterial matrix can not be excluded in that study, the huge difference suggests a strong influence of the mode of delivery.

In analogy, when BMAA is offered as particles, such as cyanobacterial cells, higher BMAA uptake might be possible. We decided not to use a cyanobacterium as transport vehicle of BMAA, since the presence of other metabolites, a deficiency in essential compounds, grazing resistance and/or feeding deterrents could all influence (Lürling, 2003a,b). We did not detect any effect of BMAA on S. obliquus, our algal food source, and since BMAA might be incorporated into proteins [START_REF] Murch | A mechanism for slow release of biomagnified cyanobacterial neurotoxins and neurodegenerative disease in Guam[END_REF][START_REF] Rodgers | Misincorporation of amino acid analogues into proteins by biosynthesis[END_REF]) further studies will focus on the green alga S. obliquus as possible transport vehicle for BMAA and selected toxins to zooplankton grazers. Moreover, synergistic effects of BMAA with other cyanobacterial neurotoxins on zooplankton grazers should also be studied, since low concentrations of BMAA can enhance the effects of other neurotoxins at the cellular level [START_REF] Lobner | ß-Nmethylamino-L-alanine enhances neurotoxicity through multiple mechanisms[END_REF] and BMAA co-occurrence with other cyanotoxins has already been observed [START_REF] Metcalf | Co-occurrence of β-N-methylamino-l-alanine, a neurotoxic amino acid with other cyanobacterial toxins in British waterbodies, 1990-2004[END_REF][START_REF] Faassen | Determination of the neurotoxins BMAA (β-N-methylamino-L-alanine) and DAB (α-,γdiaminobutyric acid) by LC-MSMS in Dutch urban waters with cyanobacterial blooms[END_REF].

Although we measured a direct effect of BMAA on population growth rates, we did not include possible transgenerational effects in our experimental design.

Because toxicants can significantly reduce neonate fitness, transgenerational effects cannot be excluded a priori [START_REF] Hammers-Wirtz | Offspring fitness in Daphnia: Is the Daphnia reproduction test appropriate for extrapolating effects on the population level?[END_REF]. The high BMAA concentrations in neonates (up to 3821 times as high as the medium and up to 108 times as high as their mothers) that were exposed for a maximum of 24 hours to BMAA containing medium indicate that the effect of BMAA exposure might also be transgenerational, cumulative and/or delayed. Significantly higher bioconcentration in D. magna neonates than in adults has also been observed for other compounds, such as nonylphenol [START_REF] Preuss | Life stage-dependent bioconcentration of a nonylphenol isomer in Daphnia magna[END_REF]. A higher concentration in neonates could be the result of the dependency of bioconcentration kinetics on the surface-to-volume ratio as bioconcentration seems to be inversely related to body-size [START_REF] Preuss | Life stage-dependent bioconcentration of a nonylphenol isomer in Daphnia magna[END_REF].

Another possible mechanism is an active maternal transfer to neonates, as observed for mercury [START_REF] Tsui | Uptake and elimination routes of inorganic mercury and methylmercury in Daphnia magna[END_REF]. [START_REF] Murch | A mechanism for slow release of biomagnified cyanobacterial neurotoxins and neurodegenerative disease in Guam[END_REF].

BMAA concentrations in zooplankton from the Baltic Sea were 6-fold higher than in cyanobacteria and up to 200-fold higher in certain fish species [START_REF] Jonasson | Transfer of a cyanobacterial neurotoxin within a temperate aquatic ecosystem suggests pathways for human exposure[END_REF],

indicating biomagnification scenarios of BMAA may also be present in temperate regions [START_REF] Jonasson | A novel cyanobacterial toxin (BMAA) with potential neurodegenerative effects[END_REF]. Our study provides independent evidence that BMAA may be biomagnified in aquatic systems as BMAA was clearly bioconcentrated in Daphnia. µg L -1 ); n.q. means detected, but not quantified (LOQ = 15 µg L -1 ).

F

  20° C in low light of about 15 µmol photons m -2 s -1 with a 14:10 hours light-dark cycle. The animals were fed three times a week with the green alga

  dissolved BMAA. Immobilization and death were chosen as endpoints.

  ). Dry weight and BMAA concentration were determined as described for the life-history experiment (see section Life-history experiment), animals were pooled per treatment for the BMAA analysis (see section BMAA analysis).

  growth was evaluated statistically by a repeated-measures ANOVA, which was followed by a Tukey's post-hoc comparison in the program PASW Statistics 17 (SPSS Inc.). Population growth rates, or the instantaneous rates of population increase (r) were estimated from the abbreviated life-tables including the first two broods using the equationr = rate of population increase (d -1 ), x =age class (0…N), l x = probability of surviving to age x, m x = fecundity at age x, and T the generation time (d). The life-history characteristics age at first reproduction (AFR, d), size at first reproduction (SFR, d) and total number of offspring per female, as well as the dry-weight (DW, µg) of the experimental animals at the end of the experiment were analyzed by one-way ANOVAs (PASW Statistics 17). Means that were significantly different were distinguished by a Tukey post-hoc comparison test (p < 0.05).

  served as food in the life history experiment and was therefore also exposed to different concentrations of BMAA. Because it was unknown whether BMAA has a negative effect on the survival and growth of S. obliquus and therefore on the food availability of D. magna, we exposed S. obliquus to different concentrations of BMAA and measured different growth parameters.

  [START_REF] Faassen | Determination of the neurotoxins BMAA (β-N-methylamino-L-alanine) and DAB (α-,γdiaminobutyric acid) by LC-MSMS in Dutch urban waters with cyanobacterial blooms[END_REF] with slight adaptations. BMAA was analysed by LC-MSMS using an Agilent 1200 LC and an Agilent G6410A QQQ. Chromatography was performed on a 2.1 × 150 mm, 5 µm diameter ZIC ® -HILIC column (SeQuant, Sweden) with a Direct-Connect™ Filter ). The mobile phase consisted of acetonitrile with 0.1 % formic acid (v:v, eluent A) and nanopure water with 0.1% formic acid (v/v, eluent B). The elution program was 0 to 2 min: 95% A, 4 min: 65% A, 8 to 17 min 55% A, 17 to 23 min 95% A. HPLC flow was 0.4 mL min -1 , injection volume 5 µL and column temperature 40 °C. The LC-MSMS was operated in positive mode with an ESI source. Nitrogen was used as the drying and the collision gas. Both quadrupoles were operated in unit mode, fragmentor voltage was 50 V. In MRM mode, quadrupole 1 selected ions with a mass-to-charge ratio (m/z) of 119 and quadrupole 2 recorded the abundance of the following product ions: m/z 102 at 4 V collision energy and m/z 76 and 88 at 8 V.Calibration standards of BMAA were prepared on the day of analysis.Samples of RT-medium were analyzed without pretreatment. For these measurements, calibration standards of BMAA were dissolved in RT-medium.D. magna samples were hydrolysed before analysis in an EldexHydrolysis/Derivatization WorkStation. Hydrolysis was performed in 6 N HCl vapour at 105 °C for 20 h in the absence of oxygen. After hydrolysis, samples were dried down and reconstituted in 300 µl acetonitrile / nanopure water / formic acid (v/v 65:35:0.1) and were measured on the LC-MSMS. Calibration standards of BMAA for these measurements were also dissolved in acetonitrile / nanopure water / formic acid in the same ratio. The limit of BMAA detection in hydrolyzed Daphnia samples is 5 µg L -1 ; the limit of quantification (LOQ) is 15 µg L -1 . Recovery of this method, determined by spiking unexposed D. magna prior to hydrolysis, was 40%.The bioconcentration factor (BCF) of BMAA in the animals was calculated according to[START_REF] Walker | Principles of Ecotoxicology, 2 nd Edition[END_REF]: higher than 1 indicate that the compound is bioconcentrated in the organism. BCF was expressed per unit WW and DW (µg g -1 ).

  Guam ecosystem, cyanobacterially produced BMAA is biomagnified in cycads, pigs, deer and flying foxes, until it reaches the top trophic level in the Guam ecosystem: the indigenous Chamorro people

(Fig. 1 .

 1 FIGURE LEGENDS

TABLE LEGENDS Table 1 .

 LEGENDS1 BMAA concentrations (corrected for sample recovery) and the calculated bioconcentration factors (BCF) in juvenile Daphnia magna based on wet-and dryweight (µg g -1 ) after 96 h exposure to different concentrations of BMAA in the medium (µg L -1 ) in the absence of food. n.d. means not detected in sample (LOD = 5

Table 2 .

 2 Survival (%) until the end of the experiment (15 d), age at first reproduction

http://mc.manuscriptcentral.com/jplankt Journal of Plankton Research

Acknowledgements

We would like to thank Wendy Beekman for assistance in sample preparation. EF was supported by a grant (No. 817.02.019) from the Netherlands Organization for Scientific Research (NWO).


Anastasios Dimitriou

Samuel Cohen
email: scohen@uoi.gr

Andreas Lyras

Energy dependence of photoelectron angular distributions from two-and four-photon ionization of Mg in the vicinity of the 3p 2 1 S 0 doubly excited state

Keywords: 32.80.Rm, 33.60.-q, 32.80.Dz

Energy dependence of photoelectron angular distributions from two-and four-photon ionization of Mg in the vicinity of the 3p21S0 doubly excited state

Introduction

The Magnesium atom is a typical two-active-electron system for which the study of multiphoton ionization with visible or UV laser pulses is susceptible to thorough theoretical treatment. The latter has been achieved due to its relative simplicity with respect to heavier Alkaline Earth atoms (such as Ca, Sr and Ba), making a fairly accurate description of its structure tractable and thus allowing for a detailed examination of the laser-atom interaction aspect. As a consequence, the majority of experimental studies performed during the last fifteen years or so were quite efficiently supported theoretically, at various levels of complexity and sophistication. In both experiment and theory, particular attention was paid to multiphoton and stepwise ionization processes involving the highly correlated 3p 2 1 S 0 doubly excited state, located just above the first (3s 1/2 ) ionization threshold of Mg. For example, the spectral profile of this state was experimentally recorded (by collecting the produced Mg + ions after ionization by two photons from the 3s 2 1 S 0 ground state [1][2][3]) and theoretically calculated by several authors [3][4][5][6][7]. Additionally, the asymmetry parameters and related features of photoelectron angular distributions (PADs) for such processes (involving also the possibility of above-threshold-absorption) were computed over an extended excitation energy range [4,[7][8][9][10]. These data reveal a remarkable energy variation when the laser radiation is resonant with either intermediate or final atomic states (the latter embedded in the continuum). However, experimentally there is only one PAD measurement performed on the 3p 2 1 S 0 resonance line maximum after two-photon excitation [2]. On the other hand, it is well known by now that the quality of computed wavefunctions cannot be conclusively assessed solely on the basis of the spectral profile reproduction. In fact, wavefunctions and computational procedures as a whole, may be much more sensitively and stringently tested via the angular distributions of photoelectrons (as was also clearly demonstrated in a number of stepwise excitation studies on Mg [3pns] J=1 and [3pnd] J=1,3 autoionizing Rydberg series [11,12]). Moreover, the complexity of the already demanding task of multiphoton PADs computation is increasing considerably with the number of absorbed photons. This may explain the fact that there are no results for photon numbers higher than two, apart from a single theoretical work devoted to a fourphoton ionization PAD [13] that was experimentally recorded at the fixed wavelength of 532 nm [14]. Nevertheless, in particular cases multiphoton ionization with a large number of photons may reveal subtle structural and laser-atom interaction features. For example, in an older [15] as well as in a fairly recent work [16], where the 3p 2 1 S 0 state was excited with four photons, a number of unexpected features were observed. In [16] particularly, it was shown that this state is strongly onephoton coupled by the laser field with a bound (lower lying) as well as an autoionizing (higher lying) state. This strong coupling results in a significant red shift (~6 nm) of the four-photon resonance with respect to its unperturbed location. Furthermore, the three states involved, form a ladder system making feasible above-threshold-absorption, Mg + production into the excited 3p j states and double ionization with laser pulses of moderate intensity ( 10 12 W/cm 2 ) and ns duration.

The above processes were experimentally probed by recording the Mg + and Mg +2 yields as well as the fluorescence yield from the ionic 3p j 3s 1/2 radiative decay. A theoretical model was developed in [16] in order to clarify the underlying dynamics of the laser-driven Mg atom. Atomic parameters were obtained by a nearly ab-initio configuration interaction method on two-electron B-splines basis sets [17] and the time evolution of the Mg atom and Mg + ion was described with a density matrix and rate equations formulation, correspondingly. The accurate modelling of the electronic structure and the detailed description of the laser-induced dynamics led to a nearly quantitative agreement between experiment and theory and facilitated a consistent interpretation of the aforementioned unanticipated results. Additional evidence on the interplay between the various competing processes can be obtained by photoelectron energy spectroscopy and wavelengthdependent PAD measurements. In particular, photoelectron energy analysis could provide wavelength-dependent information on the decay channels of the n-photon excited Mg atom (particularly for n 4) by resolving the corresponding groups of emitted photoelectrons, while PADs will identify the wavelength-dependent interplay between the various final-state channels as defined by the electronic structure (bare continuum vs autoionizing resonances embedded in it) and modified by the pulsed laser excitation. It is the purpose of the present experimental work to supply part of that missing piece of information by presenting the energy variation of photoelectron angular distributions across the 3p 2 1 S 0 resonance under both two-and four-photon excitation.

The rest of the paper is divided in four sections: In the following section, the experimental set-up and procedure are described. In the third section, our experimental results on photoelectron angular distributions are presented and discussed. Finally, in the last section our conclusions and outlook are given.

Experimental setup and procedure

The experimental set up, consisting of an atomic beam apparatus capable of electron and ion detection, is shown schematically in figure 1. Mg vapor is produced in an electrically heated stainless steel oven mounted at the top of a laser-atom interaction chamber and connected to it through a water-cooled baffle. The vapor passes through a 0.5 mm diameter hole where it is collimated via a ~15 cm long stainless steel tube of ~5 mm inner diameter. The laser-atom interaction point is located ~3 cm downstream the tube exit. The chamber is evacuated by a rotary pump and a turbo molecular pump (Leybold Turbovac TW 250S) system and is equipped with two liquid nitrogen traps. The achieved background pressure is ~1×10 -7 mbar.

The atomic beam interacts at right angle with the laser beam. The latter is provided by a Nd: AG pumped dye laser (Lamda Physik Scanmate 2EC-400 pumped by Brilliant BW) operating in the 580-596 nm wavelength range (using the dyes Rhodamine B or Rhodamine 6G or their appropriate mixture) and offering wavelength calibration within ~0.01Å. The laser linewidth is ~0.1 cm -1 and the amount of amplified spontaneous emission (ASE) is kept to a power level lower than 1.5% by appropriately adjusting the dye concentration of the laser oscillator and amplifier. For the two-photon ionization studies the fundamental frequency is doubled by a BBO type-III crystal. The system operates at a repetition rate of 10 Hz and delivers linearly polarized pulses of ~5 ns duration.

The laser pulse energy is controlled by a variable attenuator (Newport 735-5) without any spatial misalignment and it is measured in front of the chamber entrance by a digital joule meter (Ophir PD300). The pulse energy is kept constant within ~15% over the whole wavelength range examined here and amounts to ~10 mJ for the fundamental and a fraction of a mJ for the second harmonic radiation. When focused into the interaction chamber by an f ≈10 cm focal length lens, these values correspond to power densities of ~2×10 11 W/cm 2 and ~1×10 10 W/cm 2 respectively (as calculated under the assumption of a diffraction limited Gaussian beam profile). Before entering the chamber the laser linear polarization is first purified (from any imperfections caused by the right angle prisms guiding the beam from the laser to the chamber) and then the polarization plane is gradually rotated by means of a /2 device in order for the PADs to be recorded. For the fundamental frequency a quartz Glan-Laser polarizer is used in conjunction with an achromatic Quartz-MgF 2 zero-order /2 wave plate (Newport 10RP52-1). The corresponding system for the UV radiation is an alpha-BBO Rochon prism polarizer (EKSPLA 455-2110) and a double Fresnel rhomb.

The ions and electrons created by the laser-atom interaction are detected perpendicularly to both the atomic and laser beams. The photoelectrons are energy analyzed by an electrostatic 166 o spherical sector electron energy analyzer (Comstock AC-902B), operated in the constant transmission energy mode [18] and equipped with an electrostatic lens (Comstock EL-321) for the purpose of maximizing the collected electron signal. The latter is detected by a pair of microchannel plates (MCPs) mounted at the exit of the analyzer. The whole system is placed within a µ-metal box for protection against parasitic magnetic fields. The achieved energy resolution FWHM /E o is ~0.8% for transmission energies E o in the range 10-30 eV. The angular resolution is found to be ~3o with the electrostatic lens turned-off and ~4o when the lens is turned-on. It was verified that, apart from this small angular resolution degradation, the lens does not influence the recorded PADs in any other way (as was already mentioned previously [19]). Concerning the Mg + ions, they are discriminated against parasitic ionic signals by means of a pulsed time-of-flight (TOF) mass spectrometer. A positive ~1 kV pulse is applied on a hollow repeller electrode ~500 ns after the laser pulse. This delay is long enough so that the electron signal has completely decayed. The electric pulse pushes the ions towards a grounded plate with a ~3 mm diameter hole in its center which is followed by a ~18 cm long grounded tube. At the end of the tube ions are detected by another pair of MCPs. The TOF system easily resolves all three 24,25,26 Mg + ions.

All the voltages required for the operation of the analyzer are provided by a dedicated power supply (Comstock Model ES-101). The latter is driven by a 0-10 V voltage ramp provided by a programmable module (Stanford Research Systems SR245). The module itself is driven by a personal computer through a home-made LabView ® software. The electron and ion signals (either themselves or after they are processed by boxcar integrators (Stanford Research Systems SR 250)) are monitored by a digital oscilloscope (LeCroy 9310). The latter is connected to the personal computer via a GPIB ® port for the purpose of storing those signals.

The oven operates within a temperature range of 580-640 o C. The temperature is chosen so as to avoid any apparent space charge effects. For that purpose, for each wavelength it is reduced to a value resulting in quasi-symmetric electron line profiles, exhibiting the instrumental line widths (see figure 2). The estimated vapor density under these conditions is ~10 7 -10 8 atoms/cm 3 . The temperature is continuously monitored by a thermocouple and it is kept constant to within 1 o C. Furthermore, the stability of the atomic density during the PAD recording is verified by periodically monitoring the Mg + signal.

Results and discussion

Two-photon ionization

It is by now well established [20] that Mg atom can be described fairly well within the pure LS coupling scheme (at least for low excitation/ionization cases). Therefore, two-photon absorption of linearly polarized light from its 3s 2 1 S 0 ground state leads to even parity final states of 1 S 0 and 1 D 2 symmetry. The relevant continua are the 3s s (s-wave) and 3s d (d-wave) ones, respectively. Within the examined wavelength range (290.5-295 nm) there are no J=2 autoionizing levels and the spectrum is dominated by the broad 3p 2 1 S 0 doubly excited state. Its line profile however is distorted by the red tail of the one-photon resonant, two-photon ionization process via the 3s3p 1 P 1 bound intermediate state [1,2] (see inset of figure 2(a)). The recorded electron spectrum consists of a single peak (at ~0.81 eV for ≈293 nm -figure 2(a)). No electron peaks corresponding to abovethreshold-absorption [10] are observed for the laser intensity employed in the present experiment.

At any given wavelength, the transmission energy of the electron energy analyzer is set at the maximum of this peak and the signal S is recorded as a function of the angle between the linear laser polarization and the direction of electron detection. The recorded two-photon PADs vs wavelength are shown in figure 3. The data are fitted to the form [21] ( )

( ) ( ) ( ) ( ) θ ω θ ω β θ ω k N k k k N k k a P S 2 0 2 2 0 2 cos cos , = = ∝ ∝ (1)
where N=2 for two-photon ionization. In the absence of intermediate resonant levels the parameter 0 of equation ( 1) is given by [7],

( ) ( ) ( ) π ω σ ω β 4 0 N tot = (2)
with ( ) ( ) ω σ N tot the total integrated, generalized N-photon ionization cross section. Below saturation,

( ) ( ) ω σ N tot
is proportional to the total ionization (Mg + ) yield. We found more convenient to fit the recorded PADs using the second form of equation (1). The constant term is set equal to unity, i.e. a 0 =1. Therefore, the parameters a 2k , k=1,2, assembled in table 1, refer to the ratios a 2 /a 0 and a 4 /a 0 .

They are also shown graphically in figures 4(a) and (b) for visualizing their variation across the 3s 2 1 S 0 + 2 UV 3p 2 1 S 0 transition. Thus, a 2 changes sign twice, becoming negative on both sides of the resonance (whose maximum is located at ~292.9 nm). Note that negative a 2 values can be associated with the appearance in the PADs of a lobe at =90 o (see figure 4(d)). In turn, the emergence of this lobe can, roughly speaking, be attributed to the increasing importance of the dwave component, relative to the s-wave one (see figure 8 of [4]). As for a 4 , it exhibits a pronounced on-resonance dip. For the purpose of comparison, figure 4 also includes the single measurement reported in [2] on the resonance maximum. It is obvious that these data complement nicely the present measurements. From the a 2k values the corresponding 2k / 0 , k=1,2 ratios can be subsequently computed (not shown here). The first ratio is found to be 2 / 0 ≈1.5 and nearly energyindependent, in good agreement with the calculation of [8] (figure 1 therein), where it is found to exhibit a small variation, which however lies within our error bars. The second ratio 4 / 0 , shows a remarkable on-resonance dip, following closely the theoretical data of references [8] (figure 1 therein) and [4] (figure 6 therein, showing the ratio

( ) ( ) ( ) ( ) ω σ ω σ 2 pol. lin. 2 pol. circ. / (
which is equal to (12/7) 4 / 0 [8]) being in close agreement with each other. The only discrepancy lies in the value of 4 / 0 at the dip minimum which is experimentally found to be lower by a factor of ~2 (~0.13 instead of their values of ~0.23 -see table II of [8]). Furthermore, multiplication of these ratios by the Mg + spectrum (∝ 0 ), recorded under the same UV radiation intensity, provides the wavelength dependence of 2k k=1,2 themselves. They are shown in figure 4(c) with the value of 0 set arbitrarily equal to unity at the longest wavelength of this set of measurements, i.e. at UV ≈295 nm.

The data of figure 4(c) may be compared to the theoretical calculations of [7] (figure 2(b) therein).

Apart from the resonance location, which is not well reproduced by that theoretical work, there is quite satisfactory overall agreement between the experimental and the theoretical energy variation for all 2k , k=0-2 parameters. Particularly for 2 , whose wavelength behaviour follows closely that of 0 , the agreement is quantitative within our reported error-bars. As for 4 (which is proportional to the 1 D 2 partial cross section [7]), a smooth monotonic increase with increasing energy was predicted. This increase is related to the one-photon resonant two-photon ionization through the 3s3p 1 P 1 state, corresponding to a significantly increased 1 D 2 partial cross section. Experimentally though, the increase of 4 is not monotonic, since a shallow dip (associated to the discrepancy mentioned above for the 4 / 0 ratio) appears around the center of the 3p 2 1 S 0 resonance (see figure 4(c)). Since our measurements are performed with a laser power density higher than the calculated saturation intensity [4,7,8], the existence of the dip could possibly be attributed to saturation effects (which are expected to affect the PAD shapes [22]). A theoretical study that takes saturation into account would be of great interest for clarifying this point. Nevertheless, in the event that saturation phenomena play indeed a role, the fact that they are evident solely on the energy dependence of 4 implies that the s-wave contribution is not particularly sensitive to them.

Four-photon ionization

We present next the results of the interaction of Mg atoms with the fundamental laser radiation within the examined wavelength range. Under such conditions the 3p 2 1 S 0 resonance is red-shifted by ~6 nm with respect to its unperturbed location due to its strong one-photon coupling with the bound 3s4p 1 P 1 state (third photon energy range) and the 3p3d 1 P 1 autoionizing level in the vicinity of the fifth photon (see inset of figure 2(b)). These two levels fall well outside the wavelength range scanned. Despite that, their interaction with the 3p 2 1 S 0 state is strong enough to facilitate the absorption of six photons (from the Mg ground state), leading to excitation of Mg atoms above the 3p j thresholds. As a consequence, ionization at the sixth photon level creates Mg + ions excited to the 3p j ionic states. The latter serve as initial levels for sequential double ionization [16]. The corresponding electron energy spectrum, shown in figure 2(b), provides conclusive evidence on the validity of the above picture and additional support to the thorough analysis offered in [16]. A similar electron energy spectrum was also presented in [15]. In the present work, we limit our presentation to the PADs of the main electron peak (numbered (1) in figure 2(b)) corresponding to four-photon ionization out of the Mg ground state. Furthermore, the laser intensity employed here is somewhat below the onset of saturation for four-photon ionization (as deduced from the theoretical cross sections given in [8] and the measurements of [16]). Our purpose here is to convincingly establish the potential of photoelectron energy analysis and PADs in shedding more light on the details of the underlying dynamics. Therefore, a direct comparison with the experimental and theoretical results reported in [16] should be possible. Consequently, we have limited our presentation to a specific laser intensity most appropriate for this comparison and deferred a detailed intensity-dependent study of the PADs to a later publication.

Apart of the 3s 2 1 S 0 + 4 VIS 3p 2 1 S 0 transition there are no other even parity autoionizing states reached by four photons (in particular, the [3pnf] J=4 states are found to be quasi-hydrogenic [23] and far from the examined energy range). For linear laser polarization there are three relevant continua, i.e. the s ( 1 S), d ( 1 D) and g ( 1 G) ones. As it was shown in [16], for a reliable interpretation of the experimental data both the J=0 and J=2 continua should be taken into account. On the contrary, the J=4 pathway was omitted, in accordance to theoretical calculations [8] showing that the J=4 channel four-photon partial cross section is significantly lower than the corresponding values for the J=0 channel (by about one order of magnitude) and for the J=2 channel (by about a factor of 3).

Equation ( 1) is now applied with N=4. The recorded and fitted PADs are shown in figure 5 and in polar form in figure 6(f). The a 2k , k=1-4 parameters (a 0 =1) are given in table 2 and graphically in figures 6(a)-(d). Finally, figure 6(e) shows the corresponding Mg + spectrum (proportional to 0 ) recorded under identical conditions. From the above data the 2k , k=1-4 parameters could be also computed. Nevertheless, as with the two-photon ionization case, the PADs resonant behavior is already evident in the energy dependence of the parameters a 2k . As it may be seen from figure 6, within the range of 590-594 nm, a 2 and a 4 exhibit a maximum and a minimum respectively, while a 6 and a 8 change sign. The two latter parameters are approximately mirror images of each other. All PADs exhibit a lobe at =90 o , explaining the negative a 2 values over the whole wavelength range. This fact implies that the d-wave contribution is always present while, as expected [16,8], the PADs are not suggestive of any important g-wave contribution. On the other hand, as it may be seen from the polar plots of figure 6(f) the ratio S(0 o )/S(90 o ) increases significantly for 591 and 592 nm. Stated in another way, the relative importance of the 90 o lobe shows an on-resonance decrease, presumably in favor of the s-wave continuum into which the 3p 2 1 S 0 state autoionizes. Furthermore, all the above resonance manifestations show an energy dependence which is broader than the apparent width of the 3p 2 1 S 0 resonance itself (figure 6(e)).

Additionally, the locations of the extrema and zero-crossing points of a 2k are found shifted with respect to the apparent maximum of the Mg + spectrum. This is expected however, because (i) there is a considerable ionization signal of 1 D character modifying the line profile and maximum of the resonance and (ii) the asymmetry parameters are indeed expected to display a shifted as well as a broader resonant behavior with respect to the spectral characteristics of the resonance [24,25].

While it would be tempting to apply the isolated autoionizing state formalism of [24] to the four-photon PADs, it was not attempted here, since the autoionizing resonance can be hardly considered as isolated, due to the strong light-induced couplings mentioned above. It turns out that a more rigorous theoretical treatment is required.

Not surprising perhaps, we also record a rise of the dominance of the s-wave contribution around 586 nm, a wavelength corresponding approximately to the field-free energy position of the autoionizing resonance. While the relative contribution of the two partial waves is less prominent in this energy range, it is nevertheless clearly discernible from the polar plots of the PADs. However, the spectral range, over which these features are present, is considerably narrower than the corresponding one around the field-shifted position of the resonance (~591 nm). Thus, the PADs reveal important details of the underlying electron correlation effects that shape the atom's response to the field excitation, details that cannot be revealed by any other observable quantity.

Conclusions and Outlook

We have measured the photoelectron angular distributions resulting from two-and fourphoton ionization of ground state Mg in the energy range where the highly correlated 3p 2 1 S 0 doubly excited state is located. The study focused on the energy dependence of the asymmetry parameters a 2k and/or 2k , k=1-N, where N is the order of the ionization process. For the two-photon case, the agreement between experimentally derived 2,4 parameters and theoretically calculated ones by various authors were found to be in good agreement. However, a small remaining discrepancy observed for 4 implied that a perturbative treatment may not be sufficient and that an improved calculation (taking perhaps saturation phenomena into account) may be required. As for the fourphoton asymmetry parameters, for which theoretical predictions are not available, they also show a resonant behavior. The latter is manifested by the appearance of on-resonance extrema (a 2,4 ) and zero-crossing points (a 6,8 ) implying a wavelength-dependent interchange between the relative contribution of s-wave (on-resonance) and d-wave (off-resonance) character to the measured distributions. The resonant character was red-shifted and broader with respect to the apparent maximum and spectral width of the Mg + yield. The latter is, in turn, significantly red-shifted with respect to the unperturbed location of the 3p 2 1 S 0 state, due to a strong field-mediated interaction, dominating the multiphoton ionization process.

Evidently, for such a complex process, involving above-threshold-absorption and sequential double ionization as well, calculations within the lowest-non-vanishing-order of perturbation theory would not be sufficient. On the contrary, an elaborate time-dependent approach, along the lines of our earlier work [16], but appropriately extended to include PAD calculations, would be of great interest. Such a task, along with experimental measurements for high laser intensity and for an extended wavelength range, constitutes an ongoing project of our group and will be presented in a forthcoming publication. Moreover, having established the potential of photoelectron energy analysis and PAD measurements in clarifying the complex multiphoton dynamics of Mg, when autoionizing resonances and multichannel continua are involved, we plan to apply these techniques to more complex Alkaline Earth atoms such as Sr, for which similar excitation schemes, as the one reported herein, have provided equally unanticipated results [26]. While certain features observed in Sr are similar to the corresponding ones for Mg, thus establishing a set of propensity rules for the high-order multiphoton dynamics of alkaline-earths, there are also some striking differences. We hope that the combined experimental techniques presented herein, when applied to Sr, will provide additional clues as to the origin of these differences in the multiphoton-induced response of similar two-active electron atoms. Mg + (3p j ) + 6 VIS Mg 2+ (ground state) + e -(~2 eV). For a detailed analysis of these pathways see [16]. The vertical dashed line separates electron energy ranges recorded with different transmission energy due to the low signal level corresponding to peaks (2) and (4). For E<1 eV, E o =10 eV and for E>1 eV, E o =30 eV. The electron energy scales were calibrated using known two-photon resonant three-photon ionization processes on Xe atom [19] recorded under unsaturated and spacecharge-free conditions. 1)) determined from the fits of PADs for two-photon ionization of ground state Mg in the vicinity of the 3p 2 1 S 0 resonance. The tabulated wavelengths refer to the fundamental laser radiation (i.e. before doubling) for an easier comparison with the data corresponding to four-photon ionization. They are higher than those corresponding to twice the UV radiation wavelength by ~0.01 nm. By definition a 0 =1. Two-photon ionization VIS (nm) a 2 a 4

Table 2. Parameters a 2k , k=1-4 (see equation ( 1)) determined from the fits of PADs for four-photon ionization of ground state Mg in the vicinity of the (shifted) 3p

Figure 1 .

 1 Figure 1. Schematic drawing of the experimental set-up. (a) Optical arrangement. DL: Dye laser system, BBO: second harmonic generation crystal, F: UV-transmission filter (for suppressing the fundamental laser radiation), LP: Linear polarizer, HWP: half wave plate, L: lens. (b) Interaction chamber and electron and ion detection arrangement. i-TOF: positive ion time-of-flight mass spectrometer, V TOF : positive HV pulse. EL: Electrostatic Lens, SEA: spherical sector electron energy analyzer, MCP: microchannel plates.

Figure 2 .

 2 Figure 2. (a) Electron energy spectrum resulting from two-photon ionization of ground state Mg at UV ~293 nm exhibiting a single peak (P denotes a parasitic nearly zero kinetic energy electron peak stemming from the ionization of the parasitic background). The electron analyzer transmission energy is E o =10 eV and the experimental resolution is E FWHM 0.08E o . The inset shows a simplified energy level diagram of the process. (b) Electron energy spectrum resulting from the interaction of ground state Mg with the fundamental laser radiation ( VIS ~590 nm). Apart from the parasitic peak, the spectrum consists of four peaks originating from the pathways shown in the inset's simplified energy level diagram, namely: (1): Mg + 4 VIS Mg + (3s 1/2 ) + e -(~0.76 eV), (2): Mg + 5 VIS Mg + (3s 1/2 ) + e -(~2.9 eV): (3): Mg + 6 VIS Mg + (3p j ) + e -(~0.52 eV) and (4):

Figure 3 .

 3 Figure 3. Experimental and fitted PADs from two-photon ionization of Mg around the 3p 2 1 S 0 resonance. The quoted error bars are the standard deviation of the experimental data accumulated over a few thousand laser shots. The wavelengths shown refer to the fundamental laser radiation (i.e. before doubling) for an easier comparison with the corresponding four-photon PADs.These values are higher than those corresponding to twice the UV radiation wavelength by ~0.01 nm. All graphs have the same vertical scale. The parameters determined from the fits are assembled in table 1.

Figure 4 .

 4 Figure 4. Parameters (a) a 2 and (b) a 4 (equation (1)) determined from the fits of the two-photon ionization PADs (shown in figure 3 and listed in table 1), as a function of the fundamental laser radiation wavelength (i.e. before doubling). Note that a 0 =1. (c) The corresponding parameters 0 , 2 and 4 , obtained from the Mg + spectrum recorded in the vicinity of the 3p 2 1 S 0 resonance under the same laser intensity (dots) and the parameters a 2 and a 4 . In all (a), (b) and (c) graphs the points marked with (×) refer to the single measurement reported in reference [2] and the dashed lines are smooth interpolations through the data for guiding the eye. (d) Wavelength dependence of PADs polar plots in the same radial scale. The angle is measured with respect to the direction of the arrow.

Figure 5 .

 5 Figure 5. Experimental and fitted PADs from four-photon ionization of Mg around the (shifted) 3p 2 1 S 0 resonance for the laser wavelengths reported on each graph. The quoted error bars are the standard deviation of the experimental data accumulated over a few thousand laser shots. The vertical scale is the same for all graphs. The parameters determined from the fits are assembled in table 2.

Table Captions .Table 1 .

 Captions1 Parameters a 2 and a 4 (see equation (

  2 1 S 0 resonance. By definition a 0 =1.

		Four-photon ionization
	VIS (nm)	a 2	a 4	a 6	a 8
	585	6.4±0.3 19.9±0.5 -24.9±0.4 13.3±0.6
	586	8.3±0.9	33±2	-27±2	10.2±0.9
	587	8.2±0.3 31.5±0.6 -43.7±0.7 24.5±0.7
	588	7.8±0.4 28.0±0.7 -29.0±0.5 13.2±0.5
	589	7.5±0.2 26.2±0.6 -26.0±0.5 13.5±0.8
	590	6.7±0.7	26±2	-12.4±1.0 2.1±0.6
	591	5.6±0.8	18±2	-5±3	5±2
	592	6.0±0.9	14±3	13±3	-8±2
	593	5.5±0.6	12±1	9.3±0.9 -9.3±0.6
	594	4.8±0.8	9±2	2±5	-3±1
	595	6.5±0.2 15.7±0.5 -4.5±0.4 -0.6±0.4
	596	7.1±0.2 17.0±0.5	-7±3	1.7±0.4

Acknowledgments

The experiment was performed at the Central Laser Facility of the University of Ioannina. The research Project is co-funded by the European Union -European Social Fund (ESF) & National Sources, in the framework of the program "HRAKLEITOS II" of the "Operational Program Education and Life Long Learning" of the Hellenic Ministry of Education, Life Long Learning and Religious Affairs.