Environ. Sci. Technol. 2007, 41, 4711-4714
The Chronic Toxicity of Alcohol Alkoxylate Surfactants on Anaerobic Granular Sludge in the Pulp and Paper Industry
ethyl units of alcohol ethoxylates in addition to its hydrophobic tail. The linear formula of the used alcohol alkoxylate is shown in eq 1. Alcohol ethoxylates have been shown to be toxic to aquatic organisms (1)
S T E V E N W . V A N G I N K E L , * ,§,⊥ S J O N J . M . K O R T E K A A S , †,# A N D J U L E S B . V A N L I E R †,‡ Lettinga Associates Foundation, P.O. Box 500, 6700 AM, Wageningen, The Netherlands, Subdepartment of Environmental Technology, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands, and Department of Civil and Environmental Engineering, Pennsylvania State University, University Park, Pennsylvania, 16801
with R ) H or R ) CH3, and k ) 5-17 units. The degree of methanogenic inhibition exerted by these compounds is dependent on the available cellular surface rather than on the actual concentration of the inhibitor. This phenomenon is referred to as surface proportional toxicity. As such, AAs can have a strong negative impact on the long-term methanogenic conversion capacity of anaerobic reactors in the closed water loops of zero-discharge pulp and paper mills. Surfactants, in general, are amphiphilic and are able to partition in cellular membranes which cause the membranes to leak (2-4). In effect, energy-producing sodium or proton motive forces are uncoupled and maintenance energy requirements are increased (5, 6). Methanogens are less able to maintain a favorable internal environment when ions freely flow across the cellular membrane. The toxicity due to the partitioning of the surfactant depends on the size and the degree of branching of the surfactant (3, 7). The toxic effects are increased during famine conditions since less energy is available for cellular maintenance. Furthermore, methanogenic activity decreases with increasing exposure time since it takes time for the surfactant to be absorbed and partition into the cellular membrane (8, 9). Toxicity relief is only observed when the toxicant is biodegraded or when additional cellular surface (new sludge) is added to the reactor. There is much evidence to suggest that alcohol ethoxylates are degradable in anaerobic treatment processes (10-12). According to Kaluza and Taeger (1996), the biodegradability and ecotoxicity of alcohol ethoxylates depend on the length of the alkyl chain, the extent of branching, and the degree of ethoxylation (13). However, according to Mosche (2004), the inclusion of propylene oxide units in AAs are detrimental to anaerobic degradability (14). Since AAs are used in the manufacture of paper, AAs may persist in pulp and paper mills with closed water cycles, affecting the in-line anaerobic treatment plant. According to the Environmental Risk Assessment Steering Committee (1999), “if a surfactant is rapidly degradable under aerobic conditions, and its transitory presence in anaerobic environments does not affect the function and structure of that environment (e.g. it is not inhibitory), then its anaerobic degradability is of minor importance” (15). However, the presence of AAs is not likely transitory in the anaerobic treatment unit described previously since the AAs are more likely to associate with the more hydrophobic granules of the anaerobic stage rather than the biomass of the secondary aerobic treatment unit (16). In the present paper, research is described elucidating the chronic toxicity of AAs on methanogenic consortia using long-term batch tests under nonfed conditions simulating worst case scenarios within a pulp and paper anaerobic treatment reactor. The aim of this investigation is to observe chronic toxicity due to AAs during famine conditions. Standard methods to assess methanogenic toxicity normally test the “acute toxicity” of a substance. These methods are based on the effect of slug doses of potential inhibitors on methanogenic activity. The inhibition effect is assessed in the presence of the toxicant or after removal of the toxicant from the batch medium. This method does not predict the
The chronic toxicity of an alcohol alkoxylate surfactant used in the pulp and paper industry was observed in methanogenic consortia under unfed conditions. Methanogenic inhibition was not observed until 250 h of famine conditions while in the presence of the surfactant. The delayed onset of inhibition is likely due to the amount of time necessary for the surfactant to partition into the cellular membrane which uncouples cellular energy conservation mechanisms and exhausts internal energy reserves necessary to maintain homeostasis.
Introduction The closure of water cycles in a “zero-discharge” paper production process causes the increase of inhibitory and/or non-biodegradable compounds in process waters. The final concentrations of these compounds depend on the specific water consumption (m3/ton paper) and the product flow (ton paper/day) from the industry. On some occasions, the start-up of a full-scale anaerobic system in a closed water loop of a zero-discharge pulp and paper mill took more time than expected. The build-up of surfactants in the anaerobic sludge was recently identified as a possible reason (Wageningen University, internal report). The zero-discharge pulp and paper mill in question was equipped with an anaerobic treatment unit followed by an aerobic treatment unit. The accumulation of volatile fatty acids is of greatest concern to the pulp and paper mills with “closed” water loops since these compounds, such as butyrate and propionate, impart an odor to the finished product. Alcohol alkoxylates (AAs) surfactants are used as bulking agents in the paper making process, and they adsorb onto paper fibers and are likely to absorb onto anaerobic granular sludge. AAs are characterized by having both repeating ethyl and propyl repeating units rather than just the repeating * Corresponding author phone: 513-569-7421; fax: 513-4872543; e-mail:
[email protected]. † Lettinga Associates Foundation. ‡ Wageningen University. § Pennsylvania State University. ⊥ Current address: U.S. Environmental Protection Agency, 26 W Martin Luther King Drive B-5, Cincinnati, OH 45268. # Current address: Board for the authorization of pesticides, POBox 217, 6700 AE Wageningen, The Netherlands. 10.1021/es063046m CCC: $37.00 Published on Web 05/22/2007
2007 American Chemical Society
CH3-(CH2)k-C-O-(CH2-CHR-O)m(CH2-CHR-O)n-H (1)
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TABLE 1. Feeding Schedule for Alcohol Alkoxylate Chronic Toxicity Tests
a
For the first to the fourth feeding times, the feeding times are the hours after the initial feeding.
potential long-term effect, or the “chronic toxicity”, of low concentrations of potential toxicants that might accumulate on the granular sludge in an anaerobic process treating pulp and paper mill effluent. Furthermore, possible adaptation of the biomass to the toxicants is not predicted by the frequently used short-term batch toxicity tests.
Materials and Methods Chronic toxicity tests were conducted to test the effect of an alcohol alkoxylate surfactant on anaerobic granular sludge under different lengths of noeedings or starvation periods. The alcohol alkoxylate consisted of a fatty alcohol chain complemented with ethylene oxide and propylene oxide moieties. Concentrations in the batch assays were in agreement with concentrations that might occur during slug dose spillages in a full scale zero-discharge paper mill at Hoogezand, The Netherlands. Anaerobic sludge from an upflow anaerobic sludge blanket (UASB) reactor at a different pulp and paper mill, which does not use alcohol alkoxylate, was used as the inoculum. The basal medium consisted of (mg L-1): NH4Cl (280), K2HPO4 (250), MgSO4‚7H2O (100) and CaCl2‚2H2O (10) and 1 mL L-1 of trace elements containing (mg L-1): H3BO3 (50), FeCl2‚4H2O (2000), ZnCl2 (50), MnCl2‚ 4H2O (500), CuCl2‚2H2O (38), (NH4)6Mo7O24‚4H2O (50), AlCl3‚ 6H2O (90), CoCl2‚6H2O (2000), NiCl2‚6H2O (92), Na2SeO3‚ 5H2O (162), EDTA (1000) and HCl 36% (1). Startup and Feeding. A total of thirty-two glass serum bottle reactors (100 mL volume) were used in these experiments. The bottles were sealed using gray butyl rubber stoppers and aluminum crimp top caps. The anaerobic granular sludge was washed with tap water and sieved through a wire mesh (2 mm). Approximately 0.6 g of wet sludge (1.52 g VSS/L) and 40 mL of the nutrient solution were added to the bottles. The headspace of each bottle was flushed five times with an 80/20% mixture of nitrogen and carbon dioxide, respectively. Na2S (1.0 mM) was added at to each bottle to scavenge any remaining oxygen. All cultures were initially fed 2 g COD/L of acetic acid (0.08 g COD) and half of the cultures (16 bottles) contained an alcohol alkoxylate concentration of 0.2 g/L. Bottles were then depressurized and incubated at 30 °C. Culture Feeding Schedule. Batch tests were conducted to determine the effect of alcohol alkoxylates (0.2 g/L) and the length of the starvation period on the activity of anaerobic granular sludge (Table 1). The lengths of starvation, i.e., the time until the second feeding were 48, 95, 217, and 385 h for the different sets of bottles. All tests were conducted in duplicate. Methane production was monitored on half of the tests (16 bottles) and the consumption of acetic acid was monitored on the other half of tests (16 bottles). During the initial feeding, all cultures, i.e., 32 bottles, were given an initial acetate concentration of 2.0 g COD/L and 4712
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FIGURE 1. Methane production (closed symbols, solid line) and acetate consumption (open symbols, dotted line) after the initial feeding with AA (O, b) and without AA (0, 9). were allowed to incubate for 48 h. After 48 h, cultures of Set 1, both with and without AA, were fed with another 0.08 g COD of acetate and sparged five times with an 80/20% mixture of nitrogen and carbon dioxide mixture as described earlier. Set 1 was then allowed to incubate and methane production and acetate consumption were monitored. During the second feeding of Set 1, the other sets (Sets 2, 3, and 4) were not given a second feeding, but remained unfed in incubation at 30 °C. Sets 2-4 were later given a second feeding, i.e., after 95, 217, and 385 h, respectively, and monitored according to the schedule shown in Table 1. Analytical. Methane production was monitored by measuring the methane concentration in the headspace over time. Gas samples (100 µL) were injected using a gastight syringe (Hamilton) into a gas chromatograph (Chrompack Packard 438S) equipped with a flame ionization detector and compared to standard concentrations of methane. The column was a Porapack Q (80-100 mesh). The injector, detector, and column temperatures were 200, 220, and 60 °C, respectively. Nitrogen was used as the carrier gas (20 mL/min). Liquid samples for monitoring acetate consumption were taken immediately after the bottles were monitored for methane production. Approximately 500 µL was withdrawn from the culture bottles through the bottle septa using a disposable plastic syringe. The samples were mixed immediately with 500 µL of formic acid and centrifuged for 10 min at 5000 rpm. Acetate was measured using a gas chromatograph (HP 5890 series II) equipped with a flame ionization detector. The column was an Alltech AT-Aquawax. The injector, detector, and column temperatures were 250, 300, and 60 °C, respectively. The column temperature was initially set at 80 °C (0 min) and was then increased to 200 °C (0 min) at 25 °C/min. Nitrogen, saturated with formic acid,
FIGURE 2. Methane production (closed symbols, solid line) and acetate consumption (open symbols, dotted line) after the second feedings of Set 1 with AA (O, b) and without AA (0, 9). The x axis designates the length of time after the initial feeding.
FIGURE 4. Methane production (closed symbols, solid line) and acetate consumption (open symbols, dotted line) after the second feedings of Set 4 with AA (O, b) and without AA (0, 9). The x axis designates the length of time after the initial feeding.
FIGURE 3. Methane production (closed symbols, solid line) and acetate consumption (open symbols, dotted line) after the second feedings of Set 3 with AA (O, b) and without AA (0, 9). The x axis designates the length of time after the initial feeding.
FIGURE 5. The methane production rate (closed symbols, solid line) and the acetate consumption rate (open symbols, dotted line) after each second feeding with AA (O, b) and without AA (0, 9). The x axis designates the length of time after the initial feeding.
was used as the carrier gas. Samples (10 µL) were injected using a gastight syringe (Hamilton) and an autosampler. Statistical analyses were conducted using the Data Analysis functions on Microsoft Excel 1997.
After the second feeding of Set 4, there was a clear difference in methane production between the cultures with and without added AA (t test for comparison of means, p ) 0.004) (Figure 4). Approximately, 0.05 g COD of methane was produced after 25 h from the cultures without added AA. In contrast, only 0.01 g COD of methane was produced from the cultures with added AA. There was also a difference in acetate consumption between the two cultures with and without added AA (t test for comparison of means, p ) 0.01). After approximately 50 h after the second feeding of the cultures without added AA, the amount of acetate decreased to an average of 0.001 g COD-HAc, while the amount of acetate of the cultures with added AA decreased to an average of 0.02 g COD-HAc. Overall Methane Production and Acetate Consumption Rates. The differences in methane production rates between the cultures with and without added AA increased along with the length of starvation (Figure 5). The methane production rate remained fairly constant at 2.1 ( 0.1 mg COD/hr among all Sets without added AA. In contrast, among the sets with added AA, the methane production rate decreased from 2.2 mg COD/hr to 0.5 mg COD/hr as the length of starvation increased to 410 h. Strikingly, the difference in the acetate consumption rates between the cultures with and without added AA of all sets were not significant (t test for comparison of means, p ) 0.07) (Figure 5). However, after the second feeding of Set 4, there was a 22% decrease in the acetate consumption rate when AA was added to the cultures.
Results Initial Feeding and Second Feeding of Sets 1 and 2. Methane production and acetate consumption of all cultures after the initial feeding are shown in Figure 1. The data shown are the averages and standard deviations of all 16 bottles monitored for methane production and all 16 bottles monitored for acetate consumption for cultures with and without added AA. As shown, there is little if any difference between cultures with or without added AA. In all cases, methane production was approximately 0.07 g COD-CH4 and the amount of acetate remaining was approximately 0.009 g COD-HAc, indicating that approximately 90% of the acetate had been consumed (Figure 1). After the second feeding of Set 1, there was no difference in methane production between the cultures with or without added AA (Figure 2). The amount of acetate remaining decreased to less than 0.005 g COD-HAc approximately 50 h after the second feeding indicating that approximately 95% of the acetate had been consumed (Figure 2). After the second feeding of Set 2, there also was no difference between the cultures with or without added AA (results not shown). Second Feeding of Sets 3 and 4. After the second feeding of Set 3, there was a distinct difference in methane production between the cultures with and without added AA (t test for comparison of means, p ) 0.009), but there was no difference in acetate consumption between the cultures (t test for comparison of means, p ) 0.22) (Figure 3). In regard to the cultures without added AA, methane production increased to approximately 0.05 g COD after 20 h (Figure 3). In contrast, 20 h after the second feeding, only an average of 0.022 g COD of methane was produced from the cultures with added AA.
Discussion It has been shown from the batch tests conducted that an alcohol alkoxylate surfactant exerted a toxic effect on granular anaerobic sludge, approximately 250 h after its introduction under nonfed conditions. The fact that toxicity effects were not instantaneously observed but only after a minimum required time period, which indicates that the possible VOL. 41, NO. 13, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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partitioning of the surfactants into the archaeal membrane is a slow process. The onset of inhibition could have also been delayed since the methanogens had the initial feeding and, therefore, had the energy necessary to maintain favorable internal conditions. However, after 250 h in the presence of the alcohol alkoxylate and famine conditions, increases in biomass die-off was the likely result and methanogenic activity decreased. For practical purposes, the surface proportional chronic toxicity of alcohol alkoxylates on methanogenic sludge is at least of concern and will most likely lead to changes in the chemical additives in zero-discharge pulp and paper mills. Furthermore, since the closed water loops in pulp and paper mills is generally characterized by having hot water temperatures reaching 60 °C, it is of interest to consider thermophilic anaerobic treatment. From previous research it is known that the long hydrophobic chains characteristic of surfactants are even more toxic under thermophilic conditions than under similar mesophilic conditions (17, 18). The unexpected toxic effects of AA on anaerobic sludge might also lead to changes in operational strategies, i.e., directing the wastewater flow immediately to the aerobic posttreatment when AA spills in the closed water loop are inevitable. From the literature it is known that AA is more degradable under aerobic conditions (14).
Acknowledgments This research was supported in part by the Penn State Biogeochemical Research Initiative for Education (BRIE) sponsored by NSF (IGERT) grant DGE-9972759. We acknowledge the grant EET 96.003 of SENTER, Ministry of Economic Affairs, The Netherlands.
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(5) Escher, B. I.; Schwarzenbach, R. P. Mechanistic studies on baseline toxicity and uncoupling of organic compounds as a basis for modeling effective membrane concentrations in aquatic organisms. Aquat. Sci. 2002, 64 (1), 20-35. (6) Lolkema, J. S.; Speelmans, G.; Konings, W. N. Na+-coupled versus H+ -coupled energy transduction in bacteria. Biochim. Biophys. Acta 1994, 1187 (2), 211-215. (7) Kravetz, L.; Salanitro, J. P.; Dorn, P. B.; Guin, K. F. Influence of hydrophobe type and extent of branching on environmental response factors of nonionic surfactants. J. Am. Oil Chem. Soc. 1991, 68 (8), 610-618. (8) Mosche, M.; Meyer, U. Toxicity of linear alkylbenzene sulfonate in anaerobic digestion:influence of exposure time. Water Res. 2002, 36 (13), 3253-3260. (9) Rosen, M. J.; Fei, L.; Zhu, Y. P.; Morrall, S. W. The relationship of the environmental effect of surfactants to their interfacial properties. J. Surfactants Deterg. 1999, 2 (3), 343-347. (10) Steber, J.; Wierich, P. The anaerobic degradation of detergent range fatty alcohol ethoxylates. Studies with super(14) C-labelled model surfactants. Water Res. 1987, 21 (6), 661-667. (11) Salanitro, J. P.; Diaz, L. A. Anaerobic biodegradability testing of surfactants. Chemosphere. 1995, 30 (5), 813-830. (12) Huber, M.; Meyer, U.; Rys, P. Biodegradation mechanisms of linear alcohol alkoxylates under anaerobic conditions. Environ. Sci. Tech. 2000, 34 (9), 1737-1741. (13) Kaluza, U.; Taeger, K. Einfluβ der chemischen Struktur auf o¨kotoxikologische Eigenschaften von Alkanol-Ethoxylaten. Tenside, Surfactants, Deterg. 1996, 33 (1), 46-51. (14) Mosche, M. Anaerobic degradability of alcohol ethoxylates and related non-ionic Surfactants. Biodegradation 2004, 15 (5), 327336. (15) Environmental Risk Assessment Steering Committee. European committee of surfactants and their organic intermediates. 1999. http://www.cefic.be/files/Publications/cesio_2.pdf. (16) Grotenhuis, J. T. C.; Plugge, C. M.; Stams, A. J. M.; Zehnder, A. J. B. Hydrophobicities and electrophoretic mobilities of anaerobic bacterial isolates from methanogenic granular sludge. Appl. Environ. Microbiol. 1992, 58 (3), 1054-1056. (17) Hwu, C.-S.; van Lier, J. B.; Lettinga, G. In Proceedings of Forum for Applied Biotechnology, 25-26 September, 1997, Gent, Belgium. (18) Hwu, C.-S.; Molenaar, G.; Garthoff, J.; van Lier, J. B.; Lettinga, G. Thermophilic high-rate anaerobic treatment of wastewater containing long-chain fatty acids: impact of reactor hydrodynamics. Biotechnol. Lett. 1997, 19, 447-451.
Received for review December 21, 2006. Revised manuscript received March 24, 2007. Accepted April 4, 2007. ES063046M
