- Email: [email protected]
Element determination in natural biofilms of mine drainage water by total reflection X-ray fluorescence spectrometry
Spectrochimica Acta Part B 61 (2006) 1146 – 1152 www.elsevier.com/locate/sab
Element determination in natural biofilms of mine drainage water by total ref lection X-ray f luorescence spectrometry☆ Margarete Mages ⁎, Wolf von Tümpling jun., Andrea van der Veen, Martina Baborowski UFZ Centre for Environmental Research Leipzig-Halle, Department of River Ecology Magdeburg, Brückstraße 3a, D-39114 Magdeburg, Germany Received 16 December 2005; accepted 2 May 2006 Available online 17 August 2006
Abstract Human impacts like mining activities lead to higher element concentration in surface waters. For different pollution levels, the consequences for aquatic organisms are not yet investigated in detail. Therefore, the aim of this investigation is to determine the influence of mining affected surface waters on biofilms. Elements like heavy metals can be absorbed on cell walls and on polymeric substances or enter the cytoplasm of the cells. Thus, they are important for the optimization of industrial biotechnological processes and the environmental biotechnology. Beyond this, biofilms can also play an important role in wastewater treatment processes and serve as bioindicators in the aquatic environment. The presented total reflection X-ray fluorescence spectroscopic investigation was performed to compare the element accumulation behavior of biofilms grown on natural or on artificial materials of drainage water affected by former copper mining activities. A high salt and heavy metal pollution is characteristic for the drainage water. For an assessment of these results, samples from stream Schlenze upstream the confluence with the drainage water, a small tributary of the Saale River in central Germany, were analyzed, too. © 2006 Elsevier B.V. All rights reserved. Keywords: Heavy metals; Element determination; Element accumulation; Biofilms; Mining; TXRF
1. Introduction Element analysis of different freshwater biota (e.g. biofilm, zooplankton) is an important task in today's environmental sciences. Such investigations help to understand the element uptake mechanisms into the trophic chain [1]. In general, at lowest trophic aquatic level, biofilms are complex, heterogeneous, thin and slimy layers formed by bacteria, fungi or algae, which are attached to a boundary surface. The organisms are embedded in a matrix of extracellular polymeric substances (EPS) (Fig. 1). Because of the adhesion of microorganisms on the EPS, stable synergistic communities (microconsortia) can build up [2]. Since biofilms can settle on nearly all boundary surfaces in the water, they are actively involved in the sorption
and desorption processes of elements. As shown in Table 1, biofilms can have positive as well as negative effects in environment, medicine and industry [3–5]. Biofilms can adsorb elements like heavy metals [6]. For that reason, they can be used as bioindicators in the environmental sciences. The metal ions can be bound to the bacterial extracellular polymeric substances (EPS) or by the cytoplasm, as well as to the cell walls [3,4,7].
☆
This paper was presented at the 11th International Conference on Total Reflection X-ray Fluorescence Spectrometry and Related Methods (TXRF2005), held in Budapest, Hungary, 1822 September 2005, and is published in the special issue of Spectrochimica Acta Part B, dedicated to that conference. ⁎ Corresponding author. E-mail address: [email protected] (M. Mages).
Fig. 1. Scheme biofilm composition.
0584-8547/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.sab.2006.05.007 SAB-03390; No of Pages 7
M. Mages et al. / Spectrochimica Acta Part B 61 (2006) 1146–1152 Table 1 Significance of biofilms
Table 3 Mean values for physico-chemical parameters (May–August 2005, n = 5)
Useful applications
Drawbacks
Self-purification of waters
Bioaccumulation of harmful chemicals in the aquatic biocoenosis Binding and removal of toxic Biogenic collision of materials metals, concrete, etc. Biotechnological uses Biofouling in pipeline systems Removal of organics and N-compounds in Germination of water pipelines wastewater treatments plants Contamination with pathogenic germs in the medicine Table 2 Background information water phase — anions and cations (May–August 2005, n = 5) Adit Schlüsselstollen
NH4-N NO2-N NO3-N SRP Na K Mg Cl SO4
1147
Schlenze
Mean concentration [mg/l]
S.D. [mg/l]
Mean concentration [mg/l]
S.D. [mg/l]
0.20 0.01 4.87 < 0.003 8200 43 143 12,800 1790
0.02 0.01 0.09 – 603 2 5 990 34
0.46 0.32 8.57 0.36 75 16 54 122 455
0.49 0.18 0.44 0.11 1.6 1.1 2 3 13
This study presents element concentrations of biofilms, which grew in a nutrient-limited, high saline (Table 2) and heavy metal contaminated water, the adit named Schlüsselstollen (engl. Key adit), which is influenced by drainage water from former
Parameter [unit]
Adit Schlüsselstollen
Schlenze
pH O2 [mg/l] Dry Substance [mg/l] Water temperature [°C] Conductivity [mS/cm]
7.4 10.4 24.6 11.8 45.8
8.3 8 33.8 15 1.7
mining activities. For comparison, biofilms from the stream Schlenze downstream the confluence with the adit with a lower contamination levels were analyzed. At the same time, the macrostructure of the biofilm was investigated. Elemental analysis of biofilms using conventional atomic spectrometric methods such as atomic absorption spectrometry (AAS) or inductively coupled plasma atomic emission/mass spectrometry (ICP-AES/MS) [8,9] needs large amounts of biological materials in the milligram range (mg range). Therefore, the accumulation of heavy metals in the biofilm matrix was determined with the total reflection X-ray fluorescence spectrometry (TXRF) [10–12], which offers the advantage to analyze even a small amount of a sample material in the microgram range [13]. 2. Experimental 2.1. Sampling and sample preparation 2.1.1. Sampling sites Samplings of water and biofilms were carried out in the adit Schlüsselstollen (total length 31.6 km) and the stream Schlenze. The adit is a dewatering system that drains a former mining
Fig. 2. Sampling sites.
1148
M. Mages et al. / Spectrochimica Acta Part B 61 (2006) 1146–1152
Fig. 5. Underwater photo of the biofilms grown on round polycarbonate carrier.
Fig. 3. Biofilms grown on stones below the outlet of the adit.
laboratory, 1 ml of the filtered sample was mixed with an internal standard (100 μg/l Ga), homogenized and analyzed by TXRF. 2.1.3. Biofilms
district (Mansfelder Land, copper shale mining). It enters the stream Schlenze and significantly influences its water quality [14–16]. The Schlenze flows into the Saale River. Besides the Rivers Schwarze Elster, Mulde and Havel, the Saale is one of the main tributaries of the Elbe River (Fig. 2). 2.1.2. Surface water samples All surface water samples from the stream and the adit were taken directly with the sampling bottles (volume of 250 ml). The physico–chemical parameters, pH-value, water temperature, conductivity, dissolved oxygen and redox potential, were measured in situ in the stream and adit, and are shown in Table 3. To discriminate the dissolved and the particulate part of the total element concentration, 50 ml of the samples were filtered through 0.45 μm syringe filters (Minisart, non-pyrogenic), stabilized with HNO3 and stored in polypropylene capes. At the
Fig. 4. Biofilms grown on long polycarbonate slide.
2.1.3.1. Sampling. For a representative sampling and a better preparation of the biofilms for the element analyses by TXRF, artificial supports for biofilm growth were positioned in the adit Schlüsselstollen and the stream Schlenze besides the natural supports like stones (Fig. 3). For comparison, two types of artificial supports were used: (1) polycarbonate slides (Fig. 4), which were already applied successfully in reactor experiments [13], and (2) newly developed round polycarbonate supports (Fig. 5), which can be taken out individually. To have sufficient biofilm material shortly after the start of a time series investigation, supports with a diameter of 5 cm were used for TXRF. Before placement, all polycarbonate slides were washed with a detergent (RBS 50 Roth, Germany), washed in 10% HNO3 and rinsed with ultrapure water. The sampling of the supports was done after different deposition times within 3 months.
Fig. 6. Sample preparation scheme for biofilms.
M. Mages et al. / Spectrochimica Acta Part B 61 (2006) 1146–1152
1149
Fig. 7. a/b Biofilm structure in the dried biofilms from the adit Schlüsselstollen in comparison with the Schlenze River.
2.1.3.2. Preparation. Fig. 6 shows the further handling of the biofilms after sampling. To minimize changes in the biofilm structure, they were transported at a temperature similar to the water temperature of around 11 °C. At the laboratory, under a stereomicroscope (ZEISS, Stemi 11), the samples were cleaned from attached particles (e.g. wood). For the documentation, digital photos were taken from the sample material. To eliminate the strong salt matrix, which can lead to erratic results, the biofilms were rinsed three times with ultrapure water. This was achieved by transferring the biofilm with ceramic tweezers between the subsequent rinsing liquids. Afterwards, the samples were frozen at − 40 °C prior to freeze-drying and finally freezedried at − 20 °C (Alpha 1, Christ, Germany). The dried samples were homogenized in a quartz micromortar followed by the weighing of about 500 μg sample on an ultrafine balance (Sartorius, Ultramicro S4, sensitivity 0.1 μg). For digestion, the weighed sample material was transferred into 7 ml PTFE vials Table 4 Element contents in water samples from the adit Schlüsselstollen and the Schlenze stream (May–August 2005, n = 5) and the enrichment factor of the adit compared to the Schlenze Adit Mean concentration [μg/l] Pb 713 Zn 18,000 Cu 326 Ni 233 Fe 202 Ca 700,000
Schlenze stream S.D. [μg/l]
Mean concentration [μg/l]
50 0.5 1480 28.7 25 31.0 62 29.6 61 57.8 51,200 207,000
S.D. [μg/l]
Enrichment factor adit to Schlenze
0.01 1400 8.7 625 16.4 11 8 8 16.9 3.5 15,800 3.4
(Savillex, USA) with the addition of 500 μl HNO3 and 50 μl H2O2 (all Suprapure grade, Merck, Germany). The internal standard (200 ng Sc) was also added directly to the PTFE vials because pre-investigation indicated that Sc should be preferred instead of Ga due to a line overlap of Ga with Pb in combination with the high Pb content of the samples. The vials were tightly closed with a special key. The digestion took 3 h at 120 °C on a hot plate. Certified standard reference material (BCR CRM 414 plankton) [17] was used to determine the accuracy of the method including the treating and digestion procedure as well as the analyses. Furthermore, each sample was digested and analyzed in duplicate if sufficient sample material was available to determine the precision. Since the biofilm samples as well as the certified reference material was dosed with the internal standard based on the sample weight prior to the digestion, the volume after the digestion is not relevant for quantification and the samples can be diluted independently from the volume. For the TXRF measurements, 10 μl of solution were dropped onto a quartz glass carrier and dried at 80 °C on a ceramic hot plate. To exclude contamination caused by the ambient air effects, the complete preparation was performed in a clean bench. 2.2. Instrumentation For the element determination, the digested biofilm samples as well as the filtered water samples were analyzed by a TXRF spectrometer 8030 C (FEI Company, Munich, Germany). The spectrometer was equipped with an 80 mm2 Si(Li) detector and a resolution of 148 eV at 5.9 keV and a computer-controlled multichannel analyzer system combined with a spectrum
1150
M. Mages et al. / Spectrochimica Acta Part B 61 (2006) 1146–1152
Fig. 8. Element contents in biofilms from the Schlenze stream and the adit (all contents in μg g− 1); ST1 = biofilms long carrier, n = 6; ST2 = biofilms round carrier, n = 4; ST3 = biofilm stones, n = 12; SCHL = Schlenze, n = 7.
deconvolution program. A Mo anode was operated at 50 kV and 55 mA. The measurement live time was 500 s. 3. Results and discussion 3.1. Biofilm structure The biofilms of the adit were very different in relation to other surface water biofilms due to their remarkable structure. Table 5 Description of element concentration ranges measured in biofilm samples (dry weight) from the adit (all concentration ranges in μg g− 1); ST 1 = biofilms long carrier, n = 6; ST 2 = biofilms round carrier, n = 4; ST 3 = biofilm stones, n = 12 Element Adit Schlüsselstollen ST1
Pb Cu Zn Ni Fe Ca
ST2
ST3
Average enrichment factor adit to Schlenze stream
50,900–115,000 359,000–449,000 90,300–377,000 2200 3180–7000 2290–7910 3630–13,900 140 4690–8560 5510–10,600 4690–16,100 18 60–99 229–326 82–250 7 32,000–63,400 18,600–71,000 34,000–104,000 4 11,500–25,500 17,800–19,800 12,400–35,300 0.2
As visible in Fig. 7a, the rust-colored biofilms exhibit a spongelike structure. Further on, it is interesting that the biofilms show gas inclusions, which are similar to vacuoles embedded in EPS. Normally, biofilms forming in rivers have flat structures, which are adapted in consistency and surface to the flow of the river (Fig. 7b). The reason for the different macroscopic behavior is not yet known. 3.2. Results of the water phase For the assessment of the analyzed biofilms and their element accumulation behavior, the element concentrations of the water phase at the biofilm sampling location were analyzed. The results of the analysis are listed in Table 4. In the water of the adit besides high concentrations of Pb and Zn, high concentrations of Cu and Ni were determined. For comparison, the lower values for Zn and Pb of the stream Schlenze are shown, which lie in case of Pb partly below the detection limit (2 μg/l, flagged). Table 4 also contains the enrichment factors in the water samples of the adit compared to the values of the stream Schlenze. All investigated elements are enriched in the adit water of the Schlüsselstollen. The factors for Pb and zinc are extreme. Furthermore, the elements are transported mostly in dissolved form in the adit water.
M. Mages et al. / Spectrochimica Acta Part B 61 (2006) 1146–1152
1151
deposited suspended matters could be not completely eliminated also not by repeated rinsing.
Fig. 9. Microscopy photo from adit biofilm – dried sample – with particle inclusions.
The element enrichment in the water phase of the adit Schlüsselstollen follows the ranking as described below: Pb > Zn > Cu > Ni > Fe > Ca. 3.3. Element accumulation on biofilms 3.3.1. Element contents in biofilms grown on different support materials The element contents of the biofilms in the adit Schlüsselstollen and the stream Schlenze are drawn in Fig. 8. Table 5 lists the element contents of the biofilms from the adit according to the support type. Based on these results, it can be concluded that the support material had no crucial influence on the biofilms of the adit Schlüsselstollen. Exceptions are Pb and Ni from the round supports, where the accumulation was twice as high, while biofilms grown on stones show a marginally enhanced enrichment of Cu and zinc. The relatively high variability of the element contents is pronounced within a sample group. This variance can be assumed to be caused by inhomogeneities of the sample material. Particles of unknown composition can be clearly seen in Fig. 9. The varying occurrence of these particles in the biofilms will certainly influence the element distribution. A further aspect of the inhomogeneities becomes obvious when the dried and homogenized samples are examined under the microscope. The dried biofilms exhibit a strongly netted structure even after homogenization, which interferes with a reproducible composition of the sample. Further on, we assume that the high salt matrix, which accumulates in the outer slime layer of the biofilms, negatively influences the element quantification. The high standard deviations of the element contents in biofilms from the stream Schlenze (Fig. 8) exclusively result from weather-dependent suspended matter transport, which was observed during the sampling campaign. These on biofilms
3.3.2. Element accumulation of the investigated biofilms As it can be seen in Fig. 8, the concentration of all investigated elements in the biofilms from the adit Schlüsselstollen is much higher in comparison to the observed element content in the biofilms from the stream Schlenze excluding calcium. When comparing the element concentrations of the water phase with the element distribution in biofilms, the high Ca concentration in the water of the adit becomes noticeable. The element accumulation of the biofilms considerably depends on the availability of free sorption sites [18]. Regarding all element distributions in biofilms (Fig. 8), it can be assumed that due to the lower metal concentration of the Schlenze more Ca ions can be permanently adsorbed. A further indication can be the variability of diatoms density in the water, which affects the calcium content in biofilms of the stream. Furthermore, it can be assumed that mobilized gypsum particles from regional soils are transported in the stream and cause high variances of the Ca content in biofilms. The accumulation factors of the biofilms in the Schlüsselstollen were calculated based on the element contents of the Schlenze biofilms (Table 5). The following affinity results from this element enrichment: Pb > Cu > Zn > Ni > Fe > Ca. The ranking positions of Cu and Zn have changed in the biofilms compared to the water phase. The affinities of Cu and Zn were also detected in artificially grown biofilms with subsequent dosing of Cu and Zn in a reactor [7]. All other elements display a behavior analog to the water phase. 3.3.3. Quality assurance results The digestion of the certified reference material BCR 414 (Table 6) demonstrated recovery rates ranking from 85.1% to 114.5%, on average 95%. These findings are acceptable regarding the sample weight of around 500 μg. The relative standard deviation of the recoveries ranges between 10% and 20% and the material is certified to be homogeneous for sample weights of more than 1 mg [19]. Table 6 Analytical results and recovery rates of the certified reference material (plankton BCR 414) Certified Element Measured average S.D. [μg g− 1] concentration concentration [μg g− 1] [μg g− 1] K Ca Cr Mn Fe Ni Cu Zn Sr a b
6750 63,500 23.0 269 1870 21.5 30.1 106 222
400 2690 3.4 8 132 3.9 2.5 8.7 7.5
Indicative values. Values for information (n = 10).
7550 a 65,000 b 23.8 299 1850 a 18.8 29.5 112 261 a
Recovery S.D. [μg g− 1] rate [%] 170 2000 1.2 12 190 0.8 1.3 3 25
89.4 97.7 96.6 90.0 101.0 114.0 102.0 94.6 85.1
1152
M. Mages et al. / Spectrochimica Acta Part B 61 (2006) 1146–1152
4. Conclusions The TXRF is suitable to determine the element content in biofilms grown in adit and drainage waters. The artificial supports, described in the article, can be used to characterize the element accumulation of biofilms in adits or drainage waters. No significant differences were observed between the element accumulations of biofilms grown on natural or artificial supports. First results of element investigation on biofilms from the adit Schlüsselstollen have shown high accumulation rates on lead, copper and zinc, and reflect the high heavy metal concentration in the adit water. Beyond that the following ranking of element enrichment for the biofilm from the adit was found: Pb > Cu > Zn > Ni > Fe > Ca. Acknowledgements We thank Mrs. Andrea Hoff who has worked with us during the sampling campaigns and has done the water analysis, and Mr. Karsten Rahn for taking the underwater photos. References [1] S. Woelf l, M. Mages, S. Mercado, L. Villalobos, M. Óvári, F. Encina, Determination of trace elements in planktonic microcrustaceans using total reflection X-ray fluorescence (XTRF): First results from two Chilean lakes, Anal. Bioanal. Chem. 378 (2004) 1088–1094. [2] W.G. Characklis, K.C. Marshall (Eds.), Biofilms, Wiley, New York, 1990. [3] H.C. Flemming, J. Wingender, Biofilms- the preferred life-time of bacteria, Biol. Z. 31 (2001) 169–180. [4] H.C. Flemming, J. Wingender, What to stick together biofilms, Chem. Z. 36 (2002) 30–42. [5] M.N. Hughes, R.K. Poole (Eds.), Metals and microorganism, Chapman and Hall, London, 1989.
[6] H.C. Flemming, J. Schmitt, K.C. Marshall, Sorption properties of biofilms, in: W. Calmano, U. Förstner (Eds.), Sediments and Toxic Substances, Springer, Berlin Heidelberg New York, 1996. [7] A. Jang, S.M. Kim, S.Y. Kim, S.G. Lee, I.S. Kim, Effect of heavy metals (Cu, Pb and Ni) on the compositions of EPS in biofilms, Water Sci. Technol. 43/6 (2001) 41–48. [8] A. Sanz-Medel, Spectrochim. Acta Part B 53 (1998) 197–211. [9] N.D. Yan, P.G. Welsh, H. Lin, D.J. Taylor, J.M. Filion, R.W. Bachmann, J.R. Jones, R.H. Peters, D.M. Soballe, Lake Reserv. Manage. 11 (1995) 204. [10] K. Friese, R. Frömmichen, M. Mages, K. Wendt-Potthoff, Trace element determination of biological samples used by TXRF, in: B. Welz (Ed.), CANAS '95 Colloquium Analytische Spektroskopie, 1996. [11] K. Friese, M. Mages, K. Wendt-Potthoff, T.R. Neu, Spectrochim. Acta Part B 52 (1997) 1019–1025. [12] K. Kröpfl, Gy. Záray, P. Vladár, M. Mages, E. Ács, Study of biofilm formation by total-reflection X-ray fluorescence spectrometry, Microchem. J. 75 (2) (2003) 133–137. [13] M. Mages, M. Óvári, W.v. Tümpling Jr., K. Kröpfl, Anal. Bioanal. Chem. 378 (2004) 1095–1101. [14] M. Baborowski, M. Mages, C. Hiltscher, J. Matschullat, H. Guhr, Former mining activities influence Uranium concentrations in the Elbe river near Magdeburg, in: B.J. Merkel, A. Hasche-Berger (Eds.), Uranium in the Environment, Mining Impact and Consequences, Springer, Berlin Heidelberg New York, 2005, pp. 585–592. [15] P. Schreck, R. Wennrich, H.J. Stärk, M. Schubert, H. Weiß, Mansfeld—the contribution of a mining-affected catchment area to regional riverine pollution, UFZ-Bericht 18/2004, UFZ Leipzig-Halle GmbH, Leipzig, 2004, pp. 169–170. [16] H. Guhr, Sources of heavy metal pollution in the drainage area of the river Elbe in the former GDR, in: R.D. Wilken, U. Förstner, A. Knöchel (Eds.), Heavy Metals in the Environment. Conference Proceeding, vol. 2, ISBN: 0905941551, 1995, pp. 72–75. [17] P. Quevauviller, K. Vercoutere, H. Muntau, B. Griepink, The Commission of the European Communities, Community Bureau of Reference, BrusselsLuxembourg, (1993) EUR 14558 EN. [18] H.C. Flemming, Sorption sites in biofilms, Water Sci. Technol. 32 (8) (1996) 27–33. [19] P. Quevauviller, K. Vercoutere, H. Muntau, B. Griepink, J. Fresenius, Certified reference material (CRM 414) for the quality control of trace element analysis in plankton, Anal. Chem. 345 (1993) 12–17.
Element determination in natural biofilms of mine drainage water by total ref lection X-ray f luorescence spectrometry☆ Margarete Mages ⁎, Wolf von Tümpling jun., Andrea van der Veen, Martina Baborowski UFZ Centre for Environmental Research Leipzig-Halle, Department of River Ecology Magdeburg, Brückstraße 3a, D-39114 Magdeburg, Germany Received 16 December 2005; accepted 2 May 2006 Available online 17 August 2006
Abstract Human impacts like mining activities lead to higher element concentration in surface waters. For different pollution levels, the consequences for aquatic organisms are not yet investigated in detail. Therefore, the aim of this investigation is to determine the influence of mining affected surface waters on biofilms. Elements like heavy metals can be absorbed on cell walls and on polymeric substances or enter the cytoplasm of the cells. Thus, they are important for the optimization of industrial biotechnological processes and the environmental biotechnology. Beyond this, biofilms can also play an important role in wastewater treatment processes and serve as bioindicators in the aquatic environment. The presented total reflection X-ray fluorescence spectroscopic investigation was performed to compare the element accumulation behavior of biofilms grown on natural or on artificial materials of drainage water affected by former copper mining activities. A high salt and heavy metal pollution is characteristic for the drainage water. For an assessment of these results, samples from stream Schlenze upstream the confluence with the drainage water, a small tributary of the Saale River in central Germany, were analyzed, too. © 2006 Elsevier B.V. All rights reserved. Keywords: Heavy metals; Element determination; Element accumulation; Biofilms; Mining; TXRF
1. Introduction Element analysis of different freshwater biota (e.g. biofilm, zooplankton) is an important task in today's environmental sciences. Such investigations help to understand the element uptake mechanisms into the trophic chain [1]. In general, at lowest trophic aquatic level, biofilms are complex, heterogeneous, thin and slimy layers formed by bacteria, fungi or algae, which are attached to a boundary surface. The organisms are embedded in a matrix of extracellular polymeric substances (EPS) (Fig. 1). Because of the adhesion of microorganisms on the EPS, stable synergistic communities (microconsortia) can build up [2]. Since biofilms can settle on nearly all boundary surfaces in the water, they are actively involved in the sorption
and desorption processes of elements. As shown in Table 1, biofilms can have positive as well as negative effects in environment, medicine and industry [3–5]. Biofilms can adsorb elements like heavy metals [6]. For that reason, they can be used as bioindicators in the environmental sciences. The metal ions can be bound to the bacterial extracellular polymeric substances (EPS) or by the cytoplasm, as well as to the cell walls [3,4,7].
☆
This paper was presented at the 11th International Conference on Total Reflection X-ray Fluorescence Spectrometry and Related Methods (TXRF2005), held in Budapest, Hungary, 1822 September 2005, and is published in the special issue of Spectrochimica Acta Part B, dedicated to that conference. ⁎ Corresponding author. E-mail address: [email protected] (M. Mages).
Fig. 1. Scheme biofilm composition.
0584-8547/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.sab.2006.05.007 SAB-03390; No of Pages 7
M. Mages et al. / Spectrochimica Acta Part B 61 (2006) 1146–1152 Table 1 Significance of biofilms
Table 3 Mean values for physico-chemical parameters (May–August 2005, n = 5)
Useful applications
Drawbacks
Self-purification of waters
Bioaccumulation of harmful chemicals in the aquatic biocoenosis Binding and removal of toxic Biogenic collision of materials metals, concrete, etc. Biotechnological uses Biofouling in pipeline systems Removal of organics and N-compounds in Germination of water pipelines wastewater treatments plants Contamination with pathogenic germs in the medicine Table 2 Background information water phase — anions and cations (May–August 2005, n = 5) Adit Schlüsselstollen
NH4-N NO2-N NO3-N SRP Na K Mg Cl SO4
1147
Schlenze
Mean concentration [mg/l]
S.D. [mg/l]
Mean concentration [mg/l]
S.D. [mg/l]
0.20 0.01 4.87 < 0.003 8200 43 143 12,800 1790
0.02 0.01 0.09 – 603 2 5 990 34
0.46 0.32 8.57 0.36 75 16 54 122 455
0.49 0.18 0.44 0.11 1.6 1.1 2 3 13
This study presents element concentrations of biofilms, which grew in a nutrient-limited, high saline (Table 2) and heavy metal contaminated water, the adit named Schlüsselstollen (engl. Key adit), which is influenced by drainage water from former
Parameter [unit]
Adit Schlüsselstollen
Schlenze
pH O2 [mg/l] Dry Substance [mg/l] Water temperature [°C] Conductivity [mS/cm]
7.4 10.4 24.6 11.8 45.8
8.3 8 33.8 15 1.7
mining activities. For comparison, biofilms from the stream Schlenze downstream the confluence with the adit with a lower contamination levels were analyzed. At the same time, the macrostructure of the biofilm was investigated. Elemental analysis of biofilms using conventional atomic spectrometric methods such as atomic absorption spectrometry (AAS) or inductively coupled plasma atomic emission/mass spectrometry (ICP-AES/MS) [8,9] needs large amounts of biological materials in the milligram range (mg range). Therefore, the accumulation of heavy metals in the biofilm matrix was determined with the total reflection X-ray fluorescence spectrometry (TXRF) [10–12], which offers the advantage to analyze even a small amount of a sample material in the microgram range [13]. 2. Experimental 2.1. Sampling and sample preparation 2.1.1. Sampling sites Samplings of water and biofilms were carried out in the adit Schlüsselstollen (total length 31.6 km) and the stream Schlenze. The adit is a dewatering system that drains a former mining
Fig. 2. Sampling sites.
1148
M. Mages et al. / Spectrochimica Acta Part B 61 (2006) 1146–1152
Fig. 5. Underwater photo of the biofilms grown on round polycarbonate carrier.
Fig. 3. Biofilms grown on stones below the outlet of the adit.
laboratory, 1 ml of the filtered sample was mixed with an internal standard (100 μg/l Ga), homogenized and analyzed by TXRF. 2.1.3. Biofilms
district (Mansfelder Land, copper shale mining). It enters the stream Schlenze and significantly influences its water quality [14–16]. The Schlenze flows into the Saale River. Besides the Rivers Schwarze Elster, Mulde and Havel, the Saale is one of the main tributaries of the Elbe River (Fig. 2). 2.1.2. Surface water samples All surface water samples from the stream and the adit were taken directly with the sampling bottles (volume of 250 ml). The physico–chemical parameters, pH-value, water temperature, conductivity, dissolved oxygen and redox potential, were measured in situ in the stream and adit, and are shown in Table 3. To discriminate the dissolved and the particulate part of the total element concentration, 50 ml of the samples were filtered through 0.45 μm syringe filters (Minisart, non-pyrogenic), stabilized with HNO3 and stored in polypropylene capes. At the
Fig. 4. Biofilms grown on long polycarbonate slide.
2.1.3.1. Sampling. For a representative sampling and a better preparation of the biofilms for the element analyses by TXRF, artificial supports for biofilm growth were positioned in the adit Schlüsselstollen and the stream Schlenze besides the natural supports like stones (Fig. 3). For comparison, two types of artificial supports were used: (1) polycarbonate slides (Fig. 4), which were already applied successfully in reactor experiments [13], and (2) newly developed round polycarbonate supports (Fig. 5), which can be taken out individually. To have sufficient biofilm material shortly after the start of a time series investigation, supports with a diameter of 5 cm were used for TXRF. Before placement, all polycarbonate slides were washed with a detergent (RBS 50 Roth, Germany), washed in 10% HNO3 and rinsed with ultrapure water. The sampling of the supports was done after different deposition times within 3 months.
Fig. 6. Sample preparation scheme for biofilms.
M. Mages et al. / Spectrochimica Acta Part B 61 (2006) 1146–1152
1149
Fig. 7. a/b Biofilm structure in the dried biofilms from the adit Schlüsselstollen in comparison with the Schlenze River.
2.1.3.2. Preparation. Fig. 6 shows the further handling of the biofilms after sampling. To minimize changes in the biofilm structure, they were transported at a temperature similar to the water temperature of around 11 °C. At the laboratory, under a stereomicroscope (ZEISS, Stemi 11), the samples were cleaned from attached particles (e.g. wood). For the documentation, digital photos were taken from the sample material. To eliminate the strong salt matrix, which can lead to erratic results, the biofilms were rinsed three times with ultrapure water. This was achieved by transferring the biofilm with ceramic tweezers between the subsequent rinsing liquids. Afterwards, the samples were frozen at − 40 °C prior to freeze-drying and finally freezedried at − 20 °C (Alpha 1, Christ, Germany). The dried samples were homogenized in a quartz micromortar followed by the weighing of about 500 μg sample on an ultrafine balance (Sartorius, Ultramicro S4, sensitivity 0.1 μg). For digestion, the weighed sample material was transferred into 7 ml PTFE vials Table 4 Element contents in water samples from the adit Schlüsselstollen and the Schlenze stream (May–August 2005, n = 5) and the enrichment factor of the adit compared to the Schlenze Adit Mean concentration [μg/l] Pb 713 Zn 18,000 Cu 326 Ni 233 Fe 202 Ca 700,000
Schlenze stream S.D. [μg/l]
Mean concentration [μg/l]
50 0.5 1480 28.7 25 31.0 62 29.6 61 57.8 51,200 207,000
S.D. [μg/l]
Enrichment factor adit to Schlenze
0.01 1400 8.7 625 16.4 11 8 8 16.9 3.5 15,800 3.4
(Savillex, USA) with the addition of 500 μl HNO3 and 50 μl H2O2 (all Suprapure grade, Merck, Germany). The internal standard (200 ng Sc) was also added directly to the PTFE vials because pre-investigation indicated that Sc should be preferred instead of Ga due to a line overlap of Ga with Pb in combination with the high Pb content of the samples. The vials were tightly closed with a special key. The digestion took 3 h at 120 °C on a hot plate. Certified standard reference material (BCR CRM 414 plankton) [17] was used to determine the accuracy of the method including the treating and digestion procedure as well as the analyses. Furthermore, each sample was digested and analyzed in duplicate if sufficient sample material was available to determine the precision. Since the biofilm samples as well as the certified reference material was dosed with the internal standard based on the sample weight prior to the digestion, the volume after the digestion is not relevant for quantification and the samples can be diluted independently from the volume. For the TXRF measurements, 10 μl of solution were dropped onto a quartz glass carrier and dried at 80 °C on a ceramic hot plate. To exclude contamination caused by the ambient air effects, the complete preparation was performed in a clean bench. 2.2. Instrumentation For the element determination, the digested biofilm samples as well as the filtered water samples were analyzed by a TXRF spectrometer 8030 C (FEI Company, Munich, Germany). The spectrometer was equipped with an 80 mm2 Si(Li) detector and a resolution of 148 eV at 5.9 keV and a computer-controlled multichannel analyzer system combined with a spectrum
1150
M. Mages et al. / Spectrochimica Acta Part B 61 (2006) 1146–1152
Fig. 8. Element contents in biofilms from the Schlenze stream and the adit (all contents in μg g− 1); ST1 = biofilms long carrier, n = 6; ST2 = biofilms round carrier, n = 4; ST3 = biofilm stones, n = 12; SCHL = Schlenze, n = 7.
deconvolution program. A Mo anode was operated at 50 kV and 55 mA. The measurement live time was 500 s. 3. Results and discussion 3.1. Biofilm structure The biofilms of the adit were very different in relation to other surface water biofilms due to their remarkable structure. Table 5 Description of element concentration ranges measured in biofilm samples (dry weight) from the adit (all concentration ranges in μg g− 1); ST 1 = biofilms long carrier, n = 6; ST 2 = biofilms round carrier, n = 4; ST 3 = biofilm stones, n = 12 Element Adit Schlüsselstollen ST1
Pb Cu Zn Ni Fe Ca
ST2
ST3
Average enrichment factor adit to Schlenze stream
50,900–115,000 359,000–449,000 90,300–377,000 2200 3180–7000 2290–7910 3630–13,900 140 4690–8560 5510–10,600 4690–16,100 18 60–99 229–326 82–250 7 32,000–63,400 18,600–71,000 34,000–104,000 4 11,500–25,500 17,800–19,800 12,400–35,300 0.2
As visible in Fig. 7a, the rust-colored biofilms exhibit a spongelike structure. Further on, it is interesting that the biofilms show gas inclusions, which are similar to vacuoles embedded in EPS. Normally, biofilms forming in rivers have flat structures, which are adapted in consistency and surface to the flow of the river (Fig. 7b). The reason for the different macroscopic behavior is not yet known. 3.2. Results of the water phase For the assessment of the analyzed biofilms and their element accumulation behavior, the element concentrations of the water phase at the biofilm sampling location were analyzed. The results of the analysis are listed in Table 4. In the water of the adit besides high concentrations of Pb and Zn, high concentrations of Cu and Ni were determined. For comparison, the lower values for Zn and Pb of the stream Schlenze are shown, which lie in case of Pb partly below the detection limit (2 μg/l, flagged). Table 4 also contains the enrichment factors in the water samples of the adit compared to the values of the stream Schlenze. All investigated elements are enriched in the adit water of the Schlüsselstollen. The factors for Pb and zinc are extreme. Furthermore, the elements are transported mostly in dissolved form in the adit water.
M. Mages et al. / Spectrochimica Acta Part B 61 (2006) 1146–1152
1151
deposited suspended matters could be not completely eliminated also not by repeated rinsing.
Fig. 9. Microscopy photo from adit biofilm – dried sample – with particle inclusions.
The element enrichment in the water phase of the adit Schlüsselstollen follows the ranking as described below: Pb > Zn > Cu > Ni > Fe > Ca. 3.3. Element accumulation on biofilms 3.3.1. Element contents in biofilms grown on different support materials The element contents of the biofilms in the adit Schlüsselstollen and the stream Schlenze are drawn in Fig. 8. Table 5 lists the element contents of the biofilms from the adit according to the support type. Based on these results, it can be concluded that the support material had no crucial influence on the biofilms of the adit Schlüsselstollen. Exceptions are Pb and Ni from the round supports, where the accumulation was twice as high, while biofilms grown on stones show a marginally enhanced enrichment of Cu and zinc. The relatively high variability of the element contents is pronounced within a sample group. This variance can be assumed to be caused by inhomogeneities of the sample material. Particles of unknown composition can be clearly seen in Fig. 9. The varying occurrence of these particles in the biofilms will certainly influence the element distribution. A further aspect of the inhomogeneities becomes obvious when the dried and homogenized samples are examined under the microscope. The dried biofilms exhibit a strongly netted structure even after homogenization, which interferes with a reproducible composition of the sample. Further on, we assume that the high salt matrix, which accumulates in the outer slime layer of the biofilms, negatively influences the element quantification. The high standard deviations of the element contents in biofilms from the stream Schlenze (Fig. 8) exclusively result from weather-dependent suspended matter transport, which was observed during the sampling campaign. These on biofilms
3.3.2. Element accumulation of the investigated biofilms As it can be seen in Fig. 8, the concentration of all investigated elements in the biofilms from the adit Schlüsselstollen is much higher in comparison to the observed element content in the biofilms from the stream Schlenze excluding calcium. When comparing the element concentrations of the water phase with the element distribution in biofilms, the high Ca concentration in the water of the adit becomes noticeable. The element accumulation of the biofilms considerably depends on the availability of free sorption sites [18]. Regarding all element distributions in biofilms (Fig. 8), it can be assumed that due to the lower metal concentration of the Schlenze more Ca ions can be permanently adsorbed. A further indication can be the variability of diatoms density in the water, which affects the calcium content in biofilms of the stream. Furthermore, it can be assumed that mobilized gypsum particles from regional soils are transported in the stream and cause high variances of the Ca content in biofilms. The accumulation factors of the biofilms in the Schlüsselstollen were calculated based on the element contents of the Schlenze biofilms (Table 5). The following affinity results from this element enrichment: Pb > Cu > Zn > Ni > Fe > Ca. The ranking positions of Cu and Zn have changed in the biofilms compared to the water phase. The affinities of Cu and Zn were also detected in artificially grown biofilms with subsequent dosing of Cu and Zn in a reactor [7]. All other elements display a behavior analog to the water phase. 3.3.3. Quality assurance results The digestion of the certified reference material BCR 414 (Table 6) demonstrated recovery rates ranking from 85.1% to 114.5%, on average 95%. These findings are acceptable regarding the sample weight of around 500 μg. The relative standard deviation of the recoveries ranges between 10% and 20% and the material is certified to be homogeneous for sample weights of more than 1 mg [19]. Table 6 Analytical results and recovery rates of the certified reference material (plankton BCR 414) Certified Element Measured average S.D. [μg g− 1] concentration concentration [μg g− 1] [μg g− 1] K Ca Cr Mn Fe Ni Cu Zn Sr a b
6750 63,500 23.0 269 1870 21.5 30.1 106 222
400 2690 3.4 8 132 3.9 2.5 8.7 7.5
Indicative values. Values for information (n = 10).
7550 a 65,000 b 23.8 299 1850 a 18.8 29.5 112 261 a
Recovery S.D. [μg g− 1] rate [%] 170 2000 1.2 12 190 0.8 1.3 3 25
89.4 97.7 96.6 90.0 101.0 114.0 102.0 94.6 85.1
1152
M. Mages et al. / Spectrochimica Acta Part B 61 (2006) 1146–1152
4. Conclusions The TXRF is suitable to determine the element content in biofilms grown in adit and drainage waters. The artificial supports, described in the article, can be used to characterize the element accumulation of biofilms in adits or drainage waters. No significant differences were observed between the element accumulations of biofilms grown on natural or artificial supports. First results of element investigation on biofilms from the adit Schlüsselstollen have shown high accumulation rates on lead, copper and zinc, and reflect the high heavy metal concentration in the adit water. Beyond that the following ranking of element enrichment for the biofilm from the adit was found: Pb > Cu > Zn > Ni > Fe > Ca. Acknowledgements We thank Mrs. Andrea Hoff who has worked with us during the sampling campaigns and has done the water analysis, and Mr. Karsten Rahn for taking the underwater photos. References [1] S. Woelf l, M. Mages, S. Mercado, L. Villalobos, M. Óvári, F. Encina, Determination of trace elements in planktonic microcrustaceans using total reflection X-ray fluorescence (XTRF): First results from two Chilean lakes, Anal. Bioanal. Chem. 378 (2004) 1088–1094. [2] W.G. Characklis, K.C. Marshall (Eds.), Biofilms, Wiley, New York, 1990. [3] H.C. Flemming, J. Wingender, Biofilms- the preferred life-time of bacteria, Biol. Z. 31 (2001) 169–180. [4] H.C. Flemming, J. Wingender, What to stick together biofilms, Chem. Z. 36 (2002) 30–42. [5] M.N. Hughes, R.K. Poole (Eds.), Metals and microorganism, Chapman and Hall, London, 1989.
[6] H.C. Flemming, J. Schmitt, K.C. Marshall, Sorption properties of biofilms, in: W. Calmano, U. Förstner (Eds.), Sediments and Toxic Substances, Springer, Berlin Heidelberg New York, 1996. [7] A. Jang, S.M. Kim, S.Y. Kim, S.G. Lee, I.S. Kim, Effect of heavy metals (Cu, Pb and Ni) on the compositions of EPS in biofilms, Water Sci. Technol. 43/6 (2001) 41–48. [8] A. Sanz-Medel, Spectrochim. Acta Part B 53 (1998) 197–211. [9] N.D. Yan, P.G. Welsh, H. Lin, D.J. Taylor, J.M. Filion, R.W. Bachmann, J.R. Jones, R.H. Peters, D.M. Soballe, Lake Reserv. Manage. 11 (1995) 204. [10] K. Friese, R. Frömmichen, M. Mages, K. Wendt-Potthoff, Trace element determination of biological samples used by TXRF, in: B. Welz (Ed.), CANAS '95 Colloquium Analytische Spektroskopie, 1996. [11] K. Friese, M. Mages, K. Wendt-Potthoff, T.R. Neu, Spectrochim. Acta Part B 52 (1997) 1019–1025. [12] K. Kröpfl, Gy. Záray, P. Vladár, M. Mages, E. Ács, Study of biofilm formation by total-reflection X-ray fluorescence spectrometry, Microchem. J. 75 (2) (2003) 133–137. [13] M. Mages, M. Óvári, W.v. Tümpling Jr., K. Kröpfl, Anal. Bioanal. Chem. 378 (2004) 1095–1101. [14] M. Baborowski, M. Mages, C. Hiltscher, J. Matschullat, H. Guhr, Former mining activities influence Uranium concentrations in the Elbe river near Magdeburg, in: B.J. Merkel, A. Hasche-Berger (Eds.), Uranium in the Environment, Mining Impact and Consequences, Springer, Berlin Heidelberg New York, 2005, pp. 585–592. [15] P. Schreck, R. Wennrich, H.J. Stärk, M. Schubert, H. Weiß, Mansfeld—the contribution of a mining-affected catchment area to regional riverine pollution, UFZ-Bericht 18/2004, UFZ Leipzig-Halle GmbH, Leipzig, 2004, pp. 169–170. [16] H. Guhr, Sources of heavy metal pollution in the drainage area of the river Elbe in the former GDR, in: R.D. Wilken, U. Förstner, A. Knöchel (Eds.), Heavy Metals in the Environment. Conference Proceeding, vol. 2, ISBN: 0905941551, 1995, pp. 72–75. [17] P. Quevauviller, K. Vercoutere, H. Muntau, B. Griepink, The Commission of the European Communities, Community Bureau of Reference, BrusselsLuxembourg, (1993) EUR 14558 EN. [18] H.C. Flemming, Sorption sites in biofilms, Water Sci. Technol. 32 (8) (1996) 27–33. [19] P. Quevauviller, K. Vercoutere, H. Muntau, B. Griepink, J. Fresenius, Certified reference material (CRM 414) for the quality control of trace element analysis in plankton, Anal. Chem. 345 (1993) 12–17.