Effects of chronic internal alpha irradiation on physiology, growth and reproductive success of Daphnia magna

Aquatic Toxicology 80 (2006) 228–236

Effects of chronic internal alpha irradiation on physiology, growth and reproductive success of Daphnia magna F. Alonzo ∗ , R. Gilbin, S. Bourrachot, M. Floriani, M. Morello, J. Garnier-Laplace Laboratory of Radioecology and Ecotoxicology, DEI/SECRE/LRE, Institute of Radioprotection and Nuclear Safety (IRSN), Cadarache Building 186, BP3, 13115 St-Paul-lez-Durance Cedex, France Received 24 March 2006; received in revised form 30 August 2006; accepted 1 September 2006

Abstract Daphnids were chronically exposed to waterborne Am-241, an alpha-emitting radionuclide, ranging in concentration from 0.4 to 40 Bq ml−1 . Am-241 amounts were monitored in the medium, daphnid tissues and cuticles. Corresponding average dose rates of 0.02, 0.11 and 0.99 mGy h−1 were calculated for whole organisms with internal ␣-radiation contributing 99% of total dose rates. Effects of internal alpha irradiation on respiration and ingestion rates, adult, egg and neonate individual dry masses, fecundity and larval resistance to starvation were examined in 23-day experiments. Daphnids showed increased respiratory demand after 23 days at the highest dose rate, suggesting increased metabolic cost of maintenance due to coping with alpha radiological stress. Although no effect was detected on ingestion rates between contaminated and control daphnids, exposure to dose rates of 0.11 mGy h−1 or higher, resulted in a significant 15% reduction in body mass. Fecundity remained unchanged over the 23-day period, but individual masses of eggs and neonates were significantly smaller compared to the control. This suggested that increased metabolic expenditure in chronically alpha-radiated daphnids came at the expense of their energy investment per offspring. As a consequence, neonates showed significantly reduced resistance to starvation at every dose rate compared to the control. Our observations are discussed in comparison with literature results reported for cadmium, a chemical toxicant which affects feeding activity and strongly reduces individual energy uptake. © 2006 Elsevier B.V. All rights reserved. Keywords: Daphnia; 241 Am; Internal alpha irradiation; Respiration; Growth; Reproduction

1. Introduction In the radioprotection field, the FASSET programme underlined that available data relating to effects of chronic exposure to ionising radiation in non-human species are scarce, or even absent for many wildlife groups and endpoints (morbidity, mortality, mutation and reproductive capacity) (Daniel et al., 2003). Laboratory experiments concern mainly exposure to acute and external ␥ radiation whereas ␣ and ␤ particles are known to be biologically more efficient, especially when internally deposited in organisms (i.e. contamination). Recently studies have started to focus on effects of ␤ radiation at low dose rates (<1 mGy h−1 ) in invertebrate species (Hagger et al., 2005; Jha et al., 2005) but alpha contamination was mainly studied in small mammals and cultured human lymphocytes at the cellular level (Barquinero et al., 2004; ERICA, 2006). Impact of chronic low-level exposure to ionising contaminants at higher levels of organisation

∗

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0166-445X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2006.09.001

(individual, population, and ecosystem) needs to be investigated (Gamier-Laplace et al., 2004). Fate and effects of long-lived, alpha-emitting radionuclides such as 241 Americium (Am-241) have been poorly studied until now. Trace concentrations of Am-241 are found worldwide in aquatic ecosystems (10−6 to 10−5 Bq l−1 ) as a consequence of former atmospheric nuclear weapon testing and accidental releases from nuclear reactors including Chernobyl fallouts, with regional higher levels of 10−5 to 10−2 Bq l−1 in freshwaters (Matsunaga et al., 1998; Choppin, 2006). Contaminated soils (10−2 to 101 Bq kg−1 on average, peaking locally up to 101 to 104 Bq kg−1 ) are a potential source of Am-241 for surface water and groundwater (Pourcelot et al., 2003; Agapkina et al., 1995), while the highest concentrations are found in the sediments of continental shelves (up to 105 Bq kg−1 ). Furthermore, in the future Am-241 will become one of the dominant pollutants in the Chernobyl affected areas due to ingrowth from 241 Pu (Muravitsky et al., 2005). Therefore, knowledge on biological effects of chronic exposure to alpha-emitting radionuclides is urgently needed both for post-accidental situations and for the long-term management of radioactive nuclear waste disposals.

F. Alonzo et al. / Aquatic Toxicology 80 (2006) 228–236

During the last two decades, effects of toxic stress have been increasingly studied using a physiologically integrated approach, aimed at establishing individual energy budgets (Kooijman, 2000). For example in the cladoceran microcrustacean Daphnia magna, a species widely used for freshwater ecotoxicity testing, investigated endpoints have included metabolic rates of individuals, obtained from measuring oxygen consumption, energy uptake through food ingestion, reproduction and somatic growth (expressed as increase in carapace length or dry mass) (Knops et al., 2001; Baillieul et al., 2005). Energy available for production (SFG, for scope for growth) was evaluated from the difference between energy uptake and expenditure. Due to organismal and environmental constraints, any increase in energy demand induced by toxic stress was assumed to come at the expense of energy-dependent processes which have strong consequences for population dynamics (Calow and Sibly, 1990; Calow, 1991). Available literature on radiobiological effects in daphnids only refers to external gamma irradiation at dose rates ranging from 20 to 600 mGy h−1 (Marshall, 1962, 1966) and to acute response with alpha-emitting natural uranium which shows a relatively low radiotoxicity compared to its chemotoxicity (Poston et al., 1984; Barata et al., 1998). The present study examines for the first time effects of chronic internal alpha irradiation at dose rates below 1 mGy h−1 , using Am-241 contaminated D. magna. The objectives of the study were three-fold: first to quantify the bioaccumulation of Am-241 by daphnids in relation to concentration and the corresponding internal alpha dose rates; second to test how increasing internal alpha dose rate affects individual energy budgets through changes in respiratory demand and feeding activity; third to examine the consequences of such changes for somatic growth and reproductive success. 2. Materials and methods 2.1. Culture of D. magna Laboratory conditions aimed at maintaining continuous parthenogenetic reproduction following the OECD guideline 211 (1998). D. magna (clone obtained from INERIS, Verneuilen-Halatte, France) was reared at a density of one animal per 50 ml in artificial freshwater (Elendt M4 medium, hereafter ‘M4’) renewed every week. The green algae Chlamydomonas reinhardtii was used as food at a daily ration of 100 ␮g C daphnid−1 . Daphnid cultures were maintained at 20 ◦ C (±1 ◦ C) on a 16:8 light:dark photoperiod. 2.2. Test conditions Am-241 was purchased from CERCA-LEA (Framatome ANP, Pierrelatte, France). Controlled experiments were started with <24 h old neonates. For each condition, 24 daphnids were reared individually in 50 ml of M4 (pH 8.0) for 23 days. Four different exposure conditions were tested, including three concentrations of Am-241 (0.4, 4.0 and 40 Bq ml−1 ), plus a control without Am-241. Food (C. reinhardtii) was supplied everyday except weekends at a ration of 100 ␮g C daphnid−1 . Medium

229

was renewed three times a week. Each time, aliquots of 1 ml were collected to quantify initial and final concentrations of Am-241 in the medium. Vials were changed every week to limit Am241 adsorption on vial walls. Mortality was very low over the course of experiments, with only one daphnid dead for all conditions after 23 days. All daphnids met the OCDE requirement of 60 neonates produced per daphnid over the standard 21-day period. 2.3. Dry mass Changes in individual dry mass of daphnids, moults, neonates and eggs were examined over the course of the experiment in each exposure condition. For day 1, three replicate samples of 10 neonates (<24 h old) were collected from daphnid culture. Samples of daphnids (three adults per condition) were collected on days 7, 10, 16 and 23 among females that had deposited a new brood during the previous 24 h. This selection was made in order to reduce the individual variability associated with gonadal and embryonic developments. Daphnids were placed in ultrapure water. Eggs were carefully dissected out of the brood pouch under a binocular microscope and counted. Moults were collected and pooled. Samples of <24 h old neonates were collected on days 10, 13, 16, 19 and 23. Eggs and neonates were weighed as whole broods. All samples were rinsed with ultrapure water and transferred separately into pre-weighed aluminium pans, dried for 24 h at 60 ◦ C and weighed (precision of 0.1 ␮g, SE2 ultramicrobalance, Sartorius AG, G¨ottingen, Germany). 2.4. Calculation of dose rates Am-241 concentrations were measured in the medium, and in the dry samples of daphnids, moults, neonates and eggs after dry masses were measured. Dry samples were transferred from weighing pans to scintillation vials and mineralised on a sand bath at 105 ◦ C in 1 ml of HNO3 69% and 1 ml of H2 O2 30% until evaporated. Mineralised samples and aliquot samples of medium were taken up in 1 ml of HNO3 1 M and 19 ml of Insta-gel® Plus (Perkin-Elmer, Boston, USA). Alpha radioactivity was analysed by alpha liquid scintillation counting using a low background spectrometer (Quantulus 1220, Wallac Oy, Turku, Finland) with a detection limit of ∼0.03 Bq. Dose conversion coefficients (DCC) were calculated by a method used by Beaugelin-Seiller et al. (2006) based on Monte Carlo calculations (MCNP 4C software). Am-241 concentrations (Bq ml−1 ) measured in each compartment ‘c’ (medium, daphnid tissues and cuticles acting as sources of radiation) were converted to dose rates DR delivered to daphnids (mGy h−1 ), using  DCCc × [Am-241]c (1) DR = c

Estimation of DCC took account of changing body shape and volume in growing daphnids (Table 1). Daphnid volumes were calculated assuming that animals are ellipsoids growing from 0.8 to 6.0 mm (binocular observation). Cuticle volumes were calculated assuming a constant cuticle thickness of 1 ␮m

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Table 1 Dose conversion coefficients (DCC) estimated for Am-241 in internal tissues, cuticle and medium and used to estimate dose rates (mGy h−1 ) delivered to daphnids in relation to daphnid age and Am-241 concentrations (Bq ml−1 ) Age (days)

1 7 10 16 23

DCC Tissues (×10−3 mGy h−1 Bq−1 ml)

Cuticle (×10−6 mGy h−1 Bq−1 ml)

Medium (×10−4 mGy h−1 Bq−1 ml)

2.8 3.0 3.0 3.0 3.0

17.0 5.2 4.3 3.7 3.5

2.47 0.76 0.62 0.54 0.51

(measured by electronic transmission microscopy). Am-241 dissolved in the medium was taken into account (external irradiation), because ␣ particles (5.5 MeV) and ␤− particles (from 4.6 keV to 1.0 MeV) emitted by Am-241 propagate over distances from 2 to 400 ␮m. Am-241 adsorbed on vial walls contributed to the dose with a DCC of 9.0 × 10−5 mGy Bq−1 . The duration of all calculations was adjusted so that the statistical uncertainty of DCC estimates was <1%. 2.5. Reproduction and moulting Each experimental vial was monitored on a daily basis for egg deposition, neonate release and moulting. All neonates were collected within 24 h of their release and counted. Fecundity rates fi (eggs daphnid−1 day−1 ) and mass of eggs produced ri (␮g DW daphnid−1 day−1 ) at age i were calculated for each exposure condition using the equation: fi = pe (i) ne (i)

(2)

ri = we (i)fi

(3)

where at age i, pe (i) is the probability for depositing eggs, ne (i) the average number of eggs deposited per brood and we (i) is the dry weight of an egg. Moult-loss mi (␮g DW daphnid−1 day−1 ) was obtained from mi = wm (i)pm (i)

(4)

where at age i, pm (i) is the probability for moulting and wm (i) the dry weight of a moult. Daphnid sampling on days 7, 10 and 16 for dry mass was taken into account to correct values of pe (i) and pm (i). 2.6. Respiration Respiration was measured on days 10, 16 and 23 using three adult daphnids per condition. Daphnids were those collected for dry mass before eggs were dissected out. Females were placed individually into respiration chambers containing 1 ml of test medium at a constant temperature of 20 ◦ C. The decrease in oxygen partial pressure associated with respiration was recorded for l h using the Unisense microrespiration system ˚ (Unisense S/A, Arhus, Denmark). Signal of oxygen sensors was calibrated using vigorously bubbled M4 (100% of saturation) and a solution of sodium ascorbate 0.1 M in NaOH 0.1 M (0% of saturation). Percentage of saturation was converted into oxy-

gen concentration using an equilibrium concentration in water of 282.3 ␮mol O2 l−1 (20 ◦ C, 1 atm). Oxygen consumption rates Ri (␮mol O2 ␮g−1 DW min−1 ) were calculated as Ri =

[O2 ]0 × (exp−k×t − 1) × V t × wi

(5)

where [O2 ]0 is the oxygen concentration (␮mol ml−1 ) measured at t = 0, V the volume (ml) of M4 in the respiration chamber, wi the daphnid dry mass (␮g), t = 1 min, and k is the consumption coefficient (min−1 ) obtained by fitting exponential models to observed oxygen concentrations: [O2 ]t = [O2 ]0 exp−k×t

(6)

2.7. Ingestion Feeding activity was examined on days 8, 15 and 22 based on changes in algal density in the test vials between t = 0 h (when food is supplied), t = 2 and 6 h. Algal densities were measured by a particle analyser (Beckman-Coulter Counter Z2, Roissy, France) counting particles from 3.0 to 10 ␮m in diameter (C. reinhardtii ranged from 4.0 to 7.0 ␮m in the cultures). Exponential models were fitted to observed algal densities according to Frost (1972): Ct = C0 expk×t ,

Ct = C0 exp(k−f )×t

(7)

where C0 and Ct are algal densities at time 0 and time t, k and f (h−1 ) are respectively the algal growth rate measured in vials without daphnids and the daphnid grazing rate in the test vials. Mean algal density C (cell ml−1 ) and daphnid ingestion I (cell daphnid−1 day−1 ) were calculated using the following equations: C=

Ct − C0 (k − f ) × t

I =V ×f ×C×t

(8) (9)

where V is the experimental volume per individual (ml) and assuming a feeding time t = 10 h day−1 . Values were converted into carbon uptake using a TOC-content of 25 pg C cell−1 , measured in an aliquot of algal culture (Shimadzu TOC-5000A, Kyoto, Japan).

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Fig. 1. Concentration of Am-241 measured in medium, daphnid tissues and cuticle over the course of experiments at average concentrations of 0.4 Bq ml−1 (A), 4.0 Bq ml−1 (B) and 40 Bq ml−1 (C). Bold lines represent average concentrations in the medium. Error bars indicate ± standard deviations.

2.8. Larval resistance to starvation To examine potential consequences of changes in neonate mass on survival, <24 h old neonates (three groups of 10 neonates per condition) were collected on days 10, 16 and 23 and transferred into 50 ml of M4 at the same concentration of Am-241. Neonates were starved and survival was monitored on a daily basis for 10 days. Immobilized neonates were considered as dead and removed within 24 h. 2.9. Data analysis Differences between samples were tested by ANOVA (Sokal and Rohlf, 1981) using the SYSTAT 8.0 software (SPSS Inc., USA). Alpha levels of statistical significance were * p < 0.05, ** p < 0.01 and *** p < 0.001. The Hill equation (1910) was used to describe the relationship between larval survival and starvation time. For each exposure condition, the starvation time (days) that caused 50% survival in neonates was calculated using the ExcelTM macro REGTOX based on the Marquardt algorithm (Vindimian, 2001). Confidence intervals on calculated values were estimated by a bootstrap non parametric simulation. 3. Results 3.1. Dose rates Initial concentrations of Am-241 in the medium were 0.44, 4.3 and 42.5 Bq ml−1 in the different conditions. Am-241

declined in the medium by 4–8% per day, due to adsorption on the vials and uptake by algae and daphnids (Fig. 1). Concentrations reached ∼84–92% of nominal concentrations after 2 days (weekdays), and ∼75% of nominal concentrations after 3 days (weekends). Mean concentrations over the course of experiments were respectively 0.41, 4.0 and 38.6 Bq ml−1 in the different exposure conditions (Fig. 1). Absolute amounts of Am-241 taken up by daphnids increased progressively until day 16 and reached 0.4–0.5, 2.5–3.5 and 27.5–28.3 Bq daphnid−1 in the different exposure conditions. Carapace accounted for a significant fraction of Am-241 per daphnid, decreasing from 45–50 to 16% of total uptake. Between days 7 and 23, concentration of Am-241 in daphnid tissues decreased slightly at the low concentration whereas it increased significantly at the high concentration (Fig. 1). At the same time, concentration of Am241 in the cuticle showed opposite trends, increasing slightly at the low concentration whereas it decreased significantly at the high concentration. Daphnid were exposed to average dose rates of 0.02, 0.11 and 0.99 mGy h−1 , respectively, at the Am-241 concentrations of 0.4, 4.0 and 40 Bq ml−1 (Table 2). Alpha, ␤− and ␥ radiation contributed respectively >99, 1 and <0.1% to the total dose absorbed by daphnids independent of exposure condition, except on day 1 when ␤− and ␥ radiation accounted for 2% and 1% of total dose rates. Depending on daphnid age and exposure condition, Am-241 accumulated in tissues and Am-241 adsorbed on cuticles contributed from 52 to 89% and from 11 to 47% to total dose rates respectively (Table 2). Contribution of Am-241 in the medium ranged from 0.1 to 0.4% of total dose rate suggesting

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Table 2 Dose rates (mGy h−1 ) delivered to daphnids at the different Am-241 concentrations in relation to age and contribution of tissues, cuticle and medium to total dose rates Exposure condition

Age (days)

Dose rate (mGy h−1 )

Contribution to dose rate (%) Tissues

Cuticle

Medium

0.4 Bq ml−1

7 10 16 23

0.025 0.021 0.024 0.013

87.1 89.0 86.5 76.3

12.8 10.8 13.4 23.5

0.1 0.1 0.1 0.2

4.0 Bq ml−1

7 10 16 23

0.09 0.08 0.15 0.09

58.1 64.9 86.3 77.9

41.5 34.8 13.6 21.8

0.3 0.3 0.1 0.2

40 Bq ml−1

7 10 16 23

0.90 0.62 1.25 0.99

53.7 52.1 85.4 85.9

46.0 47.5 14.4 13.9

0.3 0.4 0.2 0.2

that any potential toxicity of Am-241 was mainly attributable to internal alpha radiation from Am-241 bioaccumulation in daphnid tissues and adsorption on cuticles. 3.2. Individual dry mass Daphnids exposed to alpha radiation showed a reduced body dry mass at the dose rate of 0.99 mGy h−1 (Fig. 2). Dry mass (without brood) was significantly smaller than in the control after 16 days of exposure (p = 0.004) and 23 days of exposure (p = 0.02). Similarly, average daphnid dry mass was smaller at 0.11 mGy h−1 than in the control but the difference was not significant, due to high individual variability at this dose rate. Dry mass observed at 0.02 mGy h−1 did not differ from the control (except on day 16). Changes in dry mass w(i) with age i were described using an exponential model for the juvenile stage (age i ≤ 7 days): w(i) = w(1) eg(i−1) with w(1) = 9.8 ␮g daphnid−1 and g = 0.43 day−1 independent of exposure condition. As soon as daphnids started producing eggs (age i ≥ 7 days, adult stage), dry mass w(i) followed a Von

Fig. 2. Changes in body dry mass per daphnid in relation to age and dose rate: (dotted line) juvenile growth; (continuous line) adult growth in control and at 0.02 mGy h−1 ; (dashed line) adult growth at 0.11 and 0.99 mGy h−1 . Error bars indicate ± standard deviations.

Bertalanffy model: w(i) = w(max) − [w(max) − w(7)] × expk×(i−7)

(10)

with w(7) = 132.3 ␮g daphnid−1 and with w(max) and k differing between exposure conditions: w(max) = 468.6 ␮g daphnid−1 and k = −0.12 day−1 (in control and 0.02 mGy h−1 , n = 24, r2 = 0.94*** ); w(max) = 377.1 ␮g daphnid−1 and k = −0.19 day−1 (in 4.0 and 0.99 mGy h−1 , n = 24, r2 = 0.84*** ). Mass-specific somatic growth rate decreased strongly with age, from 43% day−1 during the juvenile stage, independent of exposure condition, to an average value in adults of 7.3% day−1 in control and 0.02 mGy h−1 conditions and of 6.4% day−1 in 0.11 and 0.99 mGy h−1 . Mass of moults increased with age of daphnids, following a linear relationship with the body dry mass (y = 0.12x + 3.41, n = 12, r2 = 0.95*** ). 3.3. Maintenance Duration of instars progressively increased with age of daphnids. During juvenile stage, new carapaces were produced every day or every second day, in association with intensive somatic growth. After daphnids had started producing eggs, carapaces were produced every 3–4 days, in close relation with breeding cycle. Exposure to alpha radiation did not affect moulting frequency with the same timing observed in exposed daphnids as in the control. Moulting represented an average mass-specific loss rate of 10.9% day−1 in juveniles and 3.0% day−1 (i.e. 9.0% every 3 days) in adults. Oxygen consumption rate per daphnid increased with age, from 13.4 ␮mol O2 h−1 on day 10 to 17.8 ␮mol O2 h−1 on day 23 in the control, as a result of increasing mass of daphnids. Oxygen consumption rates were higher at 0.99 mGy h−1 than in the control, with values ranging from 15.4 ␮mol O2 h−1 on day 10 to 20.0 ␮mol O2 h−1 on day 23, but differences were significant only on day 23. Mass-specific consumption rates decreased with age (Fig. 3). This decrease was significant in the control (n = 9, p = 0.04* ) and at the dose rate of 0.02 mGy h−1

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Fig. 3. Changes in daphnid mass-specific respiration rates in relation to age and dose rate. ANOVA: * p < 0.05. Values are means ± standard deviations.

(n = 9, p = 0.001*** ) but not at the two highest dose rates. On day 23, mass-specific respiration rate was significantly higher at 0.99 mGy h−1 than in the control, as a result of combined high oxygen consumption and small body mass. 3.4. Reproductive investment Daphnids started producing eggs on day 6 and released five successive broods over the 23-day period (peaks of egg deposition on days 7, 10, 12, 16 and 19). Each time, brood deposition in the pouch occurred within 24 h of moulting. Deposition of a sixth brood was in progress when experiments were ended. A slight delay in brood 5 was observed at 0.99 mGy h−1 , with ∼30% of daphnids depositing eggs on day 21 (against 13% in the control). However, fecundity did not differ significantly between exposure conditions, with 75.8 (±5.8) to 80.2 (±4.8) eggs produced per daphnid over the 23-day period (Fig. 4A). Egg dry mass (Fig. 5A) increased significantly with age of daphnids, from 4.4–5.0 ␮g egg−1 on day 7 to 10.5–

Fig. 5. Changes in (A) individual dry mass of egg (at time of deposition in the brood pouch) and (B) individual dry mass of neonate (at time of release from the brood pouch) in relation to daphnid age and dose rate. ANOVA: * p < 0.05; ** p < 0.01. Values are means ± standard deviations.

13.5 ␮g egg−1 on day 23. Exposure to alpha radiation affected egg dry mass, with a significant decrease observed with increasing dose rate. Due to reduced egg dry mass, alpha radiation caused a slight but significant decrease in total mass of eggs produced per daphnid over the 23-day period at every dose rate (Fig. 4B). Average values ranged from 722 ␮g of eggs in the control to 618–654 ␮g of eggs in exposed daphnids. Concomitant reductions in daphnid individual mass and total mass of eggs produced resulted in unchanged mass-specific fecundity rates between exposure conditions, with an average value of 11.3% day−1 during the adult stage (starting at first egg deposition on day 6). 3.5. Neonate mass and resistance to starvation

Fig. 4. (A) Cumulative number of eggs produced per daphnid in relation to age and dose rate and (B) cumulative dry mass of eggs produced per daphnid in relation to age and dose rate. Values are means ± standard deviations.

Changes in individual dry mass of <24 h old neonates closely followed those observed in eggs (Fig. 5B), with average value increasing with age of daphnids from 8.1 ␮g neonate−1 in brood 1 to 14.0 ␮g neonate−1 in brood 5. On day 23, neonate dry mass differed between exposure conditions. A significantly smaller value was observed at the dose rate of 0.99 mGy h−1 (12.1 ␮g neonate−1 ) compared to the control (15.7 ␮g neonate−1 ). Differences in dry mass between exposure conditions might reflect reduced amounts of energy reserves invested per egg. As a possible consequence for larval capacity of survival, neonates from exposed daphnids showed a smaller resistance to starvation than those from the control. In broods 1 and 5, mortality

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Fig. 6. Changes in ingestion rates of daphnid in relation to age and dose rate. Values are means ± standard deviations.

occurred earlier in neonates from exposed daphnids (starting after 3 days of starvation) than in those from the control (starting after 4 days of starvation). In neonates from exposed daphnids, 50%-mortality was observed within 3.0–4.6 days (broods 1) and 4.6–4.8 days (brood 5) of starvation, whereas this occurred later in control neonates, after 6.3–6.4 days of starvation. With brood 3, 50% mortality occurred after 3.3–4.0 days of starvation in every condition, but survival after 10 days of starvation was higher in the control (∼20%) than in exposed daphnids (0–3%). 3.6. Ingestion At the tested dose rates, alpha radiation did not show any effect on filtering rates of daphnids (Fig. 6). Although ingestion was higher on day 8 than on day 15 (respectively 35.4 and 26.2 ␮g C daphnid−1 day−1 ), highest values were observed on day 22 in every exposure condition (average of 54.0 ␮g C daphnid−1 day−1 ), suggesting that ingestion increased with age. Although differences in filtration rates between exposure conditions were significant, no consistent variation with dose rate was observed.

tration) suggesting that chemotoxicity could be neglected. Any potential toxicity of Am-241 was attributed to internal alpha radiation in this study since 99% of total absorbed doses were due to alpha particles from tissues and cuticles. Dose conversion coefficients were strongly dependent on the geometry of daphnids. Approximations were required in order to simplify calculations. Despite these approximations, dose rates reported in this study are the best estimation available to date and can be used for a robust comparison between exposure conditions. However, on an absolute basis, calculated values are only representative of the order of magnitude of alpha dose rates delivered to daphnids. The effect of chronic external gamma irradiation was studied in Daphnia pulex (Marshall, 1962, 1966). Dose rates tested ranged approximately from 0 to 200 mGy h−1 and from 230 to 700 mGy h−1 . The author reported declining birth rate above a dose rate of 300 mGy h−1 and mortality rising abruptly above 420 mGy h−1 . In experimental populations of daphnids, extinction was observed above a dose rate of 170 mGy h−1 . These values were far greater than experimental dose rates reported here (0.02–0.99 mGy h−1 ). This may explain why effects observed in the present study were not as strong as those reported earlier for gamma radiation. Our results also contrasted with those reported recently where tritium as internal ␤-emitter was shown to induce mortality and developmental defects in larval stages and genetic damage in adults of marine mussels at dose rates as low as 2 ␮Gy h−1 (Hagger et al., 2005; Jha et al., 2005). A difference in radiosensitivity might be involved between the various species, organisms and/or life stages tested. Finally, although ␣ particles are assumed to be biologically more damaging than ␤ and ␥ rays, the concept of relative biological effectiveness and associated weighting factors for alpha dose rates is still under debate for non-human species (Chambers et al., 2006). 4.2. Individual energy balance

4. Discussion 4.1. Bioaccumulation and dose rates The present study aimed at quantifying the Am-241 biokinetics in D. magna on a range of exposure concentrations, and evaluating the equivalent internal alpha dose rates delivered to contaminated organisms. Our results showed that accumulation of Am-241 in daphnids depended on the concentration in the medium. Ten-fold increase in Am-241 concentration in the medium led approximately to 10-fold increase in the amount of Am-241 deposited in organisms. Transfer factors of Am-241 (defined as the activity accumulated in daphnids in Bq g−1 wet weight, relative to the activity in the water) ranged from 213 to 388, decreasing with increasing concentration in the medium. These values were in good agreement with those reported earlier for marine crustaceans (Coughtrey et al., 1985) but much lower than those obtained with freshwater amphipod and annelid species (Sibley and Stohr, 1990). Considering the high-specific activity of Am-241 (1.268 × 1011 Bq g−1 ), bioaccumulation in daphnids was equivalent to a very small amount of Am-241 (0.98 × 10−12 mol or 0.24 ng daphnid−1 at the highest concen-

This study aimed at examining how exposure to chronic lowlevel internal alpha radiation might affect the daphnids energy balance (energy acquisition through nutrition, and energy consumption through respiration), and how any reduction in the amount of energy available (‘SFG’ for scope for growth) might alter growth, reproduction and survival ability under low food resource. In our experiments, despite a reduced body mass observed at the highest dose rates, absolute oxygen consumption (expressed daphnid−1 ) was greater after 23 days at 0.99 mGy h−1 than in uncontaminated daphnids. This strongly supported the assumption that exposure to internal alpha radiation increases overall metabolic costs of organisms. Decreasing mass-specific rates of oxygen consumption with age in control daphnids were in agreement with previous studies (Kooijman, 2000; Baillieul et al., 2005). With no dose-dependent trend in ingestion rates, our observations suggest that the decreased SFG in daphnids exposed to Am-241 was entirely associated with an increase in metabolic expenditure while energy acquisition remained constant. These results, however, have to be interpreted with caution as energy

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intake also depends on how efficiently ingested food is assimilated. Internal alpha radiation might affect assimilation rates but this parameter was not examined here. This contrasted with the results obtained when daphnids were chronically exposed to heavy metals (copper and cadmium) or a cationic surfactant (CTAB). With these toxic chemicals, decreasing SFG was mainly attributable to a reduction in energy intake while respiration rates showed only little or no alteration (Bodar et al., 1988b; Baird et al., 1990; Barber et al., 1990; Knops et al., 2001; Baillieul et al., 2005). 4.3. Growth and reproduction Consequences of internal alpha radiation for growth and reproduction were examined in this study. Daphnids exposed to the two highest dose rates (≥0.11 mGy h−1 ) showed a reduction of 15% in dry somatic mass compared to the control. As a consequence, the observed increase of 29% in mass-specific respiratory demand at 0.99 mGy h−1 yielded only 12% increase in absolute oxygen consumption. These results were consistent with earlier observations that daphnids can adjust their somatic growth and reproduction depending on how much food is available (Taylor, 1985; Trubetskova and Lampert, 1995). Reducing adult growth when energy supplies are low is a mechanism for controlling body maintenance costs and increasing the likelihood of survival. As maximum fecundity rate depends on body size, this implies tradeoffs between reproduction and survival (Stearns, 1992). In a similar manner, reduced feeding activity under cadmium stress was accompanied with decreased growth and reproduction (Bodar et al., 1988a; Baird et al., 1990; Knops et al., 2001). Alpha radiological stress offered a different perspective where decreasing SFG resulted only from increasing metabolic costs while food ingestion was unchanged. Over the tested range of dose rates, no significant change was observed in the number of eggs produced during the 23 days of experiments despite reduced adult body mass, suggesting that fecundity might be determined by food intake independent of actual SFG. 4.4. Offspring fitness One major trade-off resides in how energy invested in reproduction is divided between individual offspring (Williams, 1966; Ebert, 1993). Although fecundity was unchanged over the range of dose rates, reduced SFG and/or body mass of daphnids resulted in significantly smaller individual egg mass at the dose rate of 0.99 mGy h−1 than in the control. In parallel, neonates showed reduced resistance to starvation at every dose rate compared to the control. These observations suggested that under increasing alpha radiological stress, daphnids maintained a constant egg production at the expense of energy reserves deposited in each egg, with strong consequences for the ability of offspring to cope with food deprivation. This interpretation is in good agreement with earlier studies. Daphnids were shown to produce small broods of large eggs, with increased resistance to starvation when food availability is low (Tessier and Consolatti, 1991; Glazier, 1992; Gliwicz and Guisande, 1992). Authors supposed

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that daphnids were able to assess their food environment to adjust their per-offspring energy investment. Under such assumption, our results imply that at constant food level, daphnids are unable to adjust their per-offspring investment according to SFG. One might hypothesize that, as suggested for adults, reducing individual mass of offspring acts as a mechanism for reducing their metabolic costs of living under stress, since eggs and neonates were exposed to alpha radiation, as their parents were. In conclusion, although no effect was detected on fecundity and mortality in the laboratory for the investigated range of dose rates, internal alpha irradiation due to Am-241 bioaccumulation appeared to reduce the capacity of offspring to cope with the variability in food resource, one of the most important environmental factors for controlling populations in the natural conditions. Consequences of a decrease in neonate body mass might not be restricted to a reduction in the resistance to starvation. Offspring might delay first egg deposition and/or reduce number of eggs if additional energy is allocated to catching up with somatic growth (Glazier, 1992). This needs further examination in a multigenerational study of the effects of Am-241 in daphnids. Acknowledgements The authors would like to thank A. Arbez for editing this manuscript. This work is part of the EC’s ERICA program (Environmental Risk from Ionising Contaminants: Assessment and Management, contract FI6R-CT-2003-508847). The support of EC is gratefully acknowledged. This research received additional funding by IRSN, as part of the ENVIRHOM program, and by EDF “GGP Environment”. References Agapkina, G.I., Tikhomirov, F.A., Shcheglov, A., Kracke, W., Bunzl, K., 1995. Association of Chernobyl-derived 239+240 Pu, 241 Am, 90 Sr and 137 Cs with organic matter in the soil solution. J. Environ. Radioactiv. 29, 257–269. Baillieul, M., Smolders, R., Blust, R., 2005. The effect of environmental stress on absolute and mass-specific scope for growth in Daphnia magna Strauss. Comp. Biochem. Physiol. 140C, 364–373. Baird, D.J., Barber, I., Calow, P., 1990. Clonal variation in general responses of Daphnia magna Straus to toxic stress. I. Chronic life-history effects. Funct. Ecol. 4, 399–407. Barata, C., Baird, D., Markich, S.J., 1998. Influence of genetic and environmental factors on the tolerance of Daphnia magna Straus to essential and nonessential metals. Aquat. Toxicol. 42, 115–137. Barber, I., Baird, D.J., Calow, P., 1990. Clonal variation in general responses of Daphnia magna Straus to toxic stress. 2. Physiological-effects. Funct. Ecol. 4, 409–414. Barquinero, J.F., Stephan, G., Schmid, E., 2004. Effect of americium-241 alphaparticles on the dose–response of chromosome aberrations in human lymphocytes analysed by fluorescence in situ hybridization. Int. J. Radiat. Biol. 80, 155–164. Beaugelin-Seiller, K., Jasserand, F., Garnier-Laplace, J., Gariel, J.C., 2006. Modelling the radiological dose in non-human species: principles, computerization and application. Health Phys. 90, 485–493. Bodar, C.W.M., Van der Sluis, I., Voogt, P.A., Zandee, D.I., 1988a. Effects of cadmium on consumption, assimilation and biochemical parameters of Daphnia magna: possible implications for reproduction. Comp. Biochem. Physiol. 90C, 341–346.

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