Enrichment of Uranium in Particulate Matter during Litter

Environ. Sci. Technol. 2008, 42, 8721–8726

Enrichment of Uranium in Particulate Matter during Litter Decomposition Affected by Gammarus pulex L. ¨ R G S C H A L L E R , * ,† A R N D T W E I S K E , † JO M A R T I N M K A N D A W I R E , †,‡ A N D E. GERT DUDEL† Institute of General Ecology and Environmental Protection, Dresden University of Technology, D-01737 Tharandt, Germany, and Institute of Material Sciences, Dresden University of Technology, D-01062 Dresden, Germany,

Received May 26, 2008. Revised manuscript received September 22, 2008. Accepted October 2, 2008.

Plant litter and organic matter of aquatic sediments provide a significant sink of soluble inorganic uranium species in contaminated ecosystems. The uranium content in detritus has been observed to increase significantly during decomposition. However, the influence of the decomposer community on uranium fixation remains unclear. In view of this, we investigated the influence of a shredder (the freshwater shrimp Gammarus pulex L.) on uranium fixation and mobilization during the degradation of plant litter. Leaf litter from Alnus glutinosa (L.) Gaertn. with 1152 mg kg-1 U of dry biomass (DM) and without uranium was used in a 14-day laboratory experiment. The uranium concentration in the particulate organic material (POM) at the end of experiment was 1427 mg kg-1 DM. After 14 days of decay, the residues of the leaves show a uranium concentration of 644 mg kg-1 DM. Uranium concentrations in the media initially increased reaching up to 63.9 µg L-1 but finally decreased to an average value of 34.3 µg L-1. At the same time, DOC levels increased from 2.43 mg L-1 up to 11.4 mg L-1 in the course of the experiment. Hence, inorganic uranium fixation onto particulate organic matter was enhanced by the activity of G. pulex.

Introduction Former uranium mining sites of Saxony and Thuringia serve as examples for problems concerning contaminations of water bodies with heavy metals and especially radionuclides. From dumps, tailings, and other remnants of uranium mining contaminants like uranium, other radionuclides and arsenic are discharged into surface and groundwater (1). Uranium concentrations of up to 500 µg L-1 in the water body have been observed (2-4). In water, uranium is dissolved not only at low pH values (predominantly as uranyl cation) but also in a neutral to alkaline milieu (e.g., as calcium-carbonate complexes) (5). It may also occur as organic complex of humic and fulvic acids as part of dissolved organic carbon (DOC) as well as fixed to colloids. In these forms, uranium is readily transported in flowing water systems (6, 7). * Corresponding author phone: 0351 463 31375; fax: 0351 463 31399; e-mail: [email protected]. † Institute of General Ecology and Environmental Protection, Dresden University of Technology. ‡ Institute of Material Sciences, Dresden University of Technology. 10.1021/es801456q CCC: $40.75

Published on Web 11/04/2008

 2008 American Chemical Society

At the same time, uranium may accumulate in or on plant litter, which settles on the bottom of the water body and will eventually decompose. The process of uranium accumulation in fresh leaf litter is induced by free carboxylic functionalities (8). During decomposition of the organic matter, uranium may be fixed to particulate organic material (POM) (7), the emerging DOC and the individuals of the decomposer community and their exudates (Biofilm) (9). The partitioning of uranium fixation between these compartments may be shifting. Once primary microbial degradation (e.g., through hyphomycetes and bacteria) proceeds, litter serves as main food source for invertebrates (10), especially G. pulex L. (11, 12). G. pulex is commonly known as a shredder. Species from the genus Gammarus are dominant invertebrates in most fresh water systems in Europe and Central Asia. Because these species influence the formation of particulate organic matter (13), they may influence the uranium partitioning. However, the impact of shredders like G. pulex on heavy metal fixation on particulate organic material (POM), as well as the influence of G. pulex on the remobilization of fixed heavy metals from POM has not been investigated to our best knowledge. The accumulation potential of pretreated leaf litter with, for example, G. pulex may be used in a semiartificial treatment plant for the removal of uranium and other heavy metals. This is of special interest in view of the expenditure of conventional water treatment plants with partial low efficiency and high costs (up to $10/m3 14, 15). Therefore, the objective of our study was to investigate the influence of G. pulex on heavy metal accumulation and remobilization using uranium and leaf litter as a model system. Furthermore, the effect of DOC on the remobilization of uranium from POM is presented.

Materials and Methods Test Organism. Individuals of G. pulex L. were collected from the Prieβnitz, a small river in Dresden. They were kept for ten days in the laboratory (in 40 L of tap water, 12-15 °C, ∼100 lx of light intensity for 14 h per day) before starting the experiment. They were supplied with degrading leaves of alder (Alnus glutinosa). These leaves were certified to contain uranium concentrations below the detection limit prior to feeding G. pulex. To simulate a natural environment, experimental vessels contained 30 g of quartz gravel obtained from a gravel plant (Ottendorf-Okrilla, Germany). Grain size was between 2 and 4 mm. Litter Collection and Preparation. Fresh fallen leaves of Alnus glutinosa were collected from uncontaminated plants at a trial plot at Moritzburg near Dresden (Germany). The leaf litter was divided into two equal parts for pre-experiment treatments. The first part of the leaf litter was filled into bags (nylon mesh bags, 250 µm mesh size) and exposed for 11 days to uranium-contaminated water (pH, 7.5; conductivity, 860 µS cm-1) at a former uranium mining site in NeuensalzMechelgru ¨n (Saxony, Germany). The uranium concentration in the water body is measured to be between 103 to 217 µg L-1 U (median 154 µg L-1) (16). Own samples taken during the exposure of the leaf litter showed on average a uranium concentration of 150 µg L-1(pH 7.5). The exposure was also done to allow colonization by microbes already conditioned to uranium contamination. At the same time these microbes are necessary to initiate microbial degradation of the leaf litter during the experiment. To obtain a higher uranium concentration the leaf litter was further incubated 3 days in a uranium nitrate solution reaching a final uranium concentration of 1152 mg kg-1 in DM of leaf litter (mixed sample from 8 leaves, one leaf disk per leaf, excluding the major vein VOL. 42, NO. 23, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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of the leaf). This value corresponds to maximum values in leaf litter harvested from the same stream used for the exposure of the leaf litter for the experiments (7). The second part of the harvested leaf litter was soaked for 14 days in water (pH 7.0, conductivity 63.4 µS cm-1) collected from a nearby stream (Wilde Weisseritz, Tharandt, Germany) with uranium concentrations lower than the detection limit. This should initiate colonisation of degrading microbes from a natural site. Experimental Design. The experiment was conducted in batch cultures modified from ref 17. Test vessels containing 3 L of tap water at a temperature of 12 °C, 200 leaf discs (20 mm in diameter, excluding the major vein of the leaf), and 30 g of quartz gravel were inoculated with 100 individuals of G. pulex. The gravel was saturated with uranium prior to the experiments using a solution of 400 µg L-1 U at pH 7.5. For a negative control experiment, the uranium was under detection limit. All experiments were conducted at pH 7.5 using NaHCO3 as buffer solution. At pH 7.5 and in the presence of HCO3-, the test solutions contained predominantly soluble uranium carbonate species. The speciation of uranium in the experiment (72.51% UO2(CO3)22-, 24.2% UO2(CO3)34-, 3.24% UO2CO3, 0.05% UO2OH+) were calculated to be similar to uranium speciation in water from former uranium mining sites (72.05% of UO2(CO3)22-, 24.89% of UO2(CO3)34-, 3.04% of UO2CO3, 0.01% of UO2OH+) [5]. Speciation calculations were done with a geochemical modeling software (PhreeqC 2.13.1) with a modified thermodynamic database (18). The experiment was set up both with positive and negative controls: (a) U contaminated litter in vessels with G. pulex in tap water and uranium saturated gravel; (b) As negative control: U contaminated litter in vessels without G. pulex in tap water and saturated gravel; (c) As positive control: uncontaminated litter in vessels with G. pulex in tap water and clean gravel. The experiment was replicated four times. The test duration was 14 days. During the 14 days of experiment (stopped because the temperature was nearly critical point for survival of G. pulex) we used a light intensity (∼100 lx) for 14 h per day and a rate of aeration of 0.5 L per minute. Test vessels were weighed every second day to determine the water loss by evaporation. Any loss was compensated by the addition of an equal amount of water. The pH value, conductivity, and temperature were measured at the beginning, at the eighth day, and the end of the experiment. Sampling and Monitoring. Water samples for element analysis were taken every 24 h, with the exception of the first sample taken 5 h after starting the experiment. Water samples for analyzing dissolved organic carbon (DOC) were collected 5 h, 3 days, and 7 days after starting the experiment as well as at the end of the experiment after 14 days. For element analysis, 10 mL of water was collected and acidified with HNO3. For the determination of dissolved organic carbon (DOC), 20 mL of water was sampled using PE (polyethylene) vessels and immediately frozen at -20 °C. At the beginning of the experiment, eight leaf disks (see above) were collected from each treatment (i.e., contaminated and uncontaminated litter), dried to constant weight before microwave digestion (for procedure, see below). Mineralized samples were analyzed for uranium within 30 days. At the end of the experiment, five leaf disks from leaf in early stages of decay by initial leaf degradation from microbes, but macroscopic intact structure, were selected from each test vessel and dried to constant weight. After drying, these leaf samples were wet digested. Simultaneously, all G. pulex individuals were separated from the gravel and the residual litter and all these compartments 8722

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were removed from the vessels (by manually picking). The entire remains in the vessels were filtered through polyethylene gauze (400 µm mesh size). The filter residues were freeze-dried (Christ, type 1002.00) and analyzed for coarse particulate organic material (POM). The filtrates from 400 µm mesh size were further filtered (0.45 µm pore cellulose acetate filter) before analyzing for uranium and DOC. The test vessels were rinsed with 100 mL of 2% HNO3 until all residues on the vessel wall were removed. The emerging solution was also analyzed to determine the vessels influence by adsorbing uranium. Sample Preparation and Analysis. The determination of uranium and other heavy metals was performed with an inductively coupled plasma mass spectrometer (ICPMS), equipped with a liquid sample introduction system. Water samples were measured directly, and solid samples were subjected to a digestion or an extraction procedure, respectively. Litter samples were digested in a closed vessel microwave system (MARS5 CEM Corp., Matthews, United States) according to (19) using nitric acid and hydrogen peroxide. Gravel samples were extracted with aqua regia in the same microwave system, according to (20), lightly modified. All water samples were acidified and kept at room temperature in conformity with (21). For ICPMS measurement a PQ2+ Instrument (VG Elemental, Winsford, UK) was used according to (22). Calibration functions were recorded from mixed calibration samples, which were prepared from single element solutions (uranium: Ultra Scientific, Kingstown, UK) and multielement solutions (Bernd Kraft, Duisburg, Germany). Calibration validity was confirmed with a standard reference material GBW 7604, poplar leaves (Office of CRM’s China), digested in the same manner as the litter samples. LOD was calculated as the 3-fold standard deviation of instrument blank (acidified water). Dissolved organic carbon (DOC) in water samples was determined with a FORMACS HT TC/TN Analyzer (Skalar, Breda, The Netherlands). The analyzer uses a combustion process and the procedure follows (23). Calibration samples were prepared from commercial standards (Bernd Kraft, Duisburg, Germany). The SAC value in the current study was measured to be at 254nm. In this value organics like polymeric aromatics (a relevant part of DOC from leaf litter) absorb ultraviolet light (24). The water samples were also analyzed for dissolved humic substances in the course of litter degradation using a UV-vis spectrophotometer (UV 2101 PC, Shimadzu, Duisburg, Germany). The spectral absorption coefficient (SAC-254) procedure for the determination of water pollution by complex aromatic carbohydrates and carbonic acids like humic acids was used according to Hu ¨ tter (25). All chemicals used in the experiment were of analytical grade.

Results and Discussion Influence of G. pulex on POM and DOC Formation. The degradation of litter in general is evident by a significant weight loss and the formation of POM (26, 27). G. pulex being abundant has a significant influence on the degradation of Alnus glutinosa litter (28). The formation of POM in this experiment was significantly higher in test vessels inoculated with G. pulex compared to noninoculated vessels (see Figure 2a; ANOVA p < 0.001). Particle sizes of POM range from 0.4 mm to 2 mm. The shredder cuts the litter into small particles and partially ingests and excretes them, forming faeces or distributing undigested litter released as POM (29). Evidence of decomposition comes from the release of soluble polymeric organic carbon, the release of dissolved and gaseous nitrogen and

FIGURE 1. (a) Accumulation of particulate organic matter in the vessels without invertebrate and with uranium (U+G-), with invertebrate but without uranium (U-G+), and vessels with uranium and invertebrates (U+G+) after 14 days of the experiment (median with maximum and minimum value). (b) is the corresponding accumulation of dissolved organic carbon in the three different vessels. (median with maximum and minimum value), n ) 4. * significant difference p < 0.05 in DOC during experiment between treatments with G. pulex (U-G+; U+G+) and treatment without G. pulex (U+G-) tested with ANOVA.

FIGURE 2. (a) Uranium concentration in water during the experiment. U+G- refers to vessels without the invertebrates, while U+G+ refers to vessels with the invertebrates. The values are median of four replication and error bars. n ) 4. (b) SAC at 254 nm for all experimental variations (U+G-, variant with uranium, without G. pulex; U+G+, variant with uranium and G. pulex; U-G+, version without uranium, with G. pulex) and from water at the beginning of experiments (W), median with maximum and minimum value, n ) 4. carbon, and in later stages, the production of humic substances (30). Figure 1b reveals that DOC content increased in all three variants. There is a significant difference between the treatment with and without G. pulex (ANOVA p < 0.01, logarithmic transformation of the data). After 14 days the vessels without uranium but with G. pulex (U-G+) show the highest DOC concentrations with a median of 11.9 (11.7-12.5) mg L-1. DOC values at the beginning of the experiment were 2.43 (1.83-2.87) mg L-1, which is in the range of most oligotrophic to weak eutrophic waters (31). The vessels with uranium and with G. pulex (U+G+) show a median DOC concentration of 11.4 (11.2-11.8) mg L-1, which is in the range of most eutrophic waters (32, 33). Lowest DOC concentrations of 8.13 (6.40-9.04) mg L-1 were recorded in uncontaminated vessels without G. pulex. This confirms that G. pulex has a significant influence on DOC release and relevantly accelerates litter degradation. However, it can not be ascertained whether DOC is mainly exuded by G. pulex, whether it emerges by leaching of degrading leaves or whether other processes are involved. The spectral absorption coefficient (SAC) values were used to confirm the existence of leaf-derived polymeric (aromatic) carbon compounds in water samples. Furthermore SAC values at the beginning of the experiment were 0.04 (Figure 2b), which are also within the range of oligotrophic waters (32). DOC values especially in the vessels with G. pulex at the end of the tests were in

the range of eutrophic waters (32, 33). However, the overall decomposition of litter is dependent on its association with the microbial decomposer community (34). The decomposition of primary particles is independent of the particle size, but dependent on the existence of bacteria and fungi (35, 36). Bacteria of the genus Pseudomonas and fungi from the group of Hyphomycetes are most important in litter decomposition (37). Futhermore, the formation of POM (by G. pulex) with large surfaces and therefore possible large amounts of microbial decomposers during degradation of the litter has an advantage for uranium fixation in aquatic system. This is because POM has a very high adsorption capacity for heavy metals depending on its quality and physical structure (38, 39). Although G. pulex was collected from a site without uranium contamination there is no significant difference in mortality between the individuals fed with uncontaminated compared to contaminated litter, containing 1026-499 mg kg-1 of uranium in DM (data not shown). Effects of G. pulex on Uranium Remobilization. The remobilization of uranium by elution from the contaminated leaf litter was relatively low in the current experiment. Figure 2a shows that uranium remobilisation was not very different during the experiment neither in vessels with nor without G. pulex. For instance, in the medium in vessels with contaminated litter and without G. pulex, a median concentration in the first three days of 36.6 (34.3-47.5) µg L-1 of uranium was measured, whereas the vessels with VOL. 42, NO. 23, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Uranium concentrations in the leaf samples at the beginning of the experiment (U+, leaf version with uranium; U-, leaf version without uranium) and leaf residues at the end of experiments. (U+G- refers to variant with uranium, without G. pulex; U+G+ variant with uranium and G. pulex; U-G+, experimental version without uranium, with G. pulex). The source material (U+, U-) consisted of one composite sample. Furthermore the uranium concentration in POM (u+g- variant with uranium, without G. pulex; u+g+ variant with uranium and G. pulex, and u-g+ variant without uranium but with G. pulex) after 14 days experiment without or under the influence of G. pulex L, median with maximum and minimum value is shown. Asterisk (*) represents a significant difference p