Environ. Sci. Technol. 1994, 28, 1916-1920
Effect of Activated Sludge Microparticles on Pesticide Partitioning Behavior Lucy Villarosa, Malcolm J. McCormick, Peter D. Carpenter,. and Philip J. Marriott Department of Applied Chemistry, Royal Melbourne Institute of Technology, City Campus, GPO Box 2476V, Melbourne, Victoria, 300 1 Australia
Ian M. Russell CSIRO Divlsion of Wool Technology, P.O. Box 21, Belmont, Victoria, 3216 Australia
Solutions prepared by suspending freeze-dried sludge in water were found to contain aggregated particles of about 1000-nmsizeand substantial concentrations of surfactants. The microparticles sorbed significant amounts of the organophosphorus (OP) pesticide diazinon, causing low recoveries and a decrease in observed partition coefficients with an increase in the suspended sludge concentration. The microparticles could be removed by prewashing the sludge, by filtration, or by passing the solution through a C-18 cartridge, which effectively acted as a filter. In contrast to diazinon, the recovery of the more polar OP propetamphos was excellent. Microparticles were not significant in muffled sludge obtained from the same source but were significant in three waste streams at a sewage treatment plant, including the final effluent. This could pose problems when predicting the extent of pesticide removal during sewage treatment.
Introduction The partitioning of solutes between immiscible phases has been an important subject for many years. One of the first studies involving partitioning that led to a theory with predictive capabilities was in 1872 (I). Since then, many investigators have studied the distribution of solutes between phases, such as octanol-water (2-4);solids-water, including clays, sludges, and organic materials (5-7); and even microorganisms (8). Partitioning is usually characterized by using a partition coefficient: the equilibrium ratio of solute concentrations between the two phases. Partition coefficientshave been correlated with many other physicochemical properties such as aqueous solubility (9111,bioconcentration factors (12,13),and even the surface area and boiling point of some compounds (14). Partitioning of organic pollutants between dissolved and particulate phases in aquatic environments is one of the fundamental processes controlling their removal (15). When both phases are well below saturation, the partition coefficient should remain constant over a range of solidphase concentrations (16). However, a number of workers have observed a decrease in partition coefficient with increase in solid-phase concentration ( I 7-21 ). The sorbents used included lake sediments, sands, clays, humic acids, samples high in organic matter, calcium bentonite, and cultures of microorganisms. The decreases in partition coefficients have been attributed to, first, increased solubility of the pesticide in the aqueous phase caused by dissolved humic material (21)or organic matter (19) and, secondly, the presence of micrometer or smaller sized particles in the aqueous phase (22-25) that are thought to originate from the solid sorbent. Organic substances that sorb onto these particles are then analyzed as part of the aqueous-phase concentration. If the presence of the 1916
Environ. Sci. Technol., Voi. 28, No. 11, 1994
particles is not recognized, then as the solid-phase concentration increases, the observed aqueous-phase concentration of organic substance will increase more than the concentration in the other phase, resulting in a net decrease in the observed partition coefficient. Recoveries obtained from mass balance studies will also be affected. In another study, we observed a decrease in the sludgewater partition coefficient (KBw)with an increase in concentration of freeze-dried activated sludge for the organophosphorus (OP) pesticide diazinon, but not for the more polar OP, propetamphos, at aqueous concentrations well below their solubility (26). In searching for a cause for this effect, the presence of microparticles was investigated. This study is unique, as to date no literature has been found describing this effect for pesticides partitioned between the aqueous phase and treated activated sludge. The present work is part of a larger study aiming to quantify the interaction of pesticides with typical sludge matrices that may be present in sewage treatment plants.
Experimental Section R e a g e n t s . Diazinon and propetamphos (Ciba Geigy) and fenthion (Bayer) were all technical grade and were used as supplied. Methanol and diethyl ether were HPLC grade (Ajax Chemicals), and hexane was pesticide grade (BDH). Reagent-grade water was prepared using a Milli-Q system (Millipore Corp.). Bond Elut C-18 cartridges (Varian, 500 mg/2.8 mL) were used as the solid-phase extractant. Each Bond Elut cartridge was used only once. Stock solutions of diazinon and propetamphos (10 mg/L) in hexane were used to prepare chromatography standards (0.1-1.0 mg/L). Aqueous standards were prepared from stock solutions (10 mg/L) of the pesticides in methanol
(28). E q u i p m e n t . Reconstituted sludge was centrifuged in 100-mLglass tubes at 3000 rpm. All glassware was cleaned according to the procedure detailed in U.S. EPA Method 614 (27). Solutions were filtered using 0.45-pm filters (HV, Millipore Corp.). Particle size was determined by using a Model 4700 Zetasizer (Malvern Instruments, U.K.). Laser microscopy was performed using a MRC-600 Series instrument (Bio-Rad). A c t i v a t e d S l u d g e . Live activated sludge was collected from the South Eastern Purification Plant (SEPP), Carrum, Victoria, Australia, in 5-L containers. The sludge was sterilized in an autoclave at 121 "C and freeze-dried (28) or spread onto flat baking trays and dried in a muffle furnace at 105 "C for 1 h (29). The muffled sludge was then ground using a mortar and pestle. The dried sludges were stored in amber-colored bottles in the dark. Freezedried sludge was reconstituted in 10.00-mL aliquots of water to give varying initial suspended solids concentra0013-936X/94/0928-1916$04.50/0
0 1994 American Chemical Society
Table 1. Recovery of 1 mg/L Diazinon Spikes from Successive Washings of Freeze-Dried Sludge Initially Present at 1000,3000, and 5000 mg/LP wash no. 1
2 3 a
2o
15
T
I
diazinon recovered from supernatant (%) lOOOb 3O0Ob 5000b 74 96 100
68 95 106
58 97 101
*
Results representan averageof three replicates. Sludge (mg/L).
tions. The suspensions were equilibrated by shaking for 6 h and centrifuged, and the supernatant (otherwise called a washing) was decanted and kept aside. To produce successive washings, the sludge was air-dried for 1h prior to repeating the above process. Pesticide Partitioning. Sludge suspensions or washings were spiked with diazinon (1.0 mg/L) or propetamphos (1.0 mg/L), equilibrated by shaking for 6 h, and then analyzedfor the pesticide (28). Briefly, the aqueous sample was solid-phase extracted using a C-18 cartridge. The pesticide was then eluted with diethyl ether, the solvent was changed to hexane, and the solution was made 1.0 mgjL with fenthion as the internal standard prior to analysis by gas chromatography. Appropriate procedural blanks were used for all experiments. Pesticides were also extracted from 10-mL aliquots of washings and other aqueous samples containing 0.5 g of added sodium chloride using 2 X 2 mL aliquots of hexane, each shaken for 3 min. The hexane was collected in 10.0-mL standard flasks, fenthion was added (to 1.0 mg/L), and the sample was made up to volume prior to chromatographic analysis. Chromatographic Analysis. Analyses were performed using a Hewlett-Packard 5890A gas chromatograph fitted with a nitrogen-phosphorus detector (NPD) as detailed in Villarosaet a1 (28). Mass spectra were obtained on a Kratos MS25RF mass spectrometer, operating in electron impact mode, attached to a Carlo Erba 5160 Mega Series gas chromatograph. A BP1 capillary column (25 m X 0.32 mm i.d.1 was used with the same conditions (28). Other Analyses. Anionic surfactant concentrations were determined by complexation using methylene blue and are reported as methylene blue active substances (MBAS) in milligrams per liter (30). The total organic content of the freeze-dried sludge was determined gravimetrically in triplicate by ashing at 620 "C (31) after drying at 105 "C for 2 h. The water content was calculated from the loss on drying. BET surface areas were determined on freeze-dried sludge (0.5-1.5 g) that was helium degassed at room temperature for 48 h, reweighed, and then measured using nitrogen on an ASAP 2000 surface area analyzer (Micromeritics). Microparticle content in washings was determined as follows. Freeze-dried sludge (1.5 g) was reconstituted in 500 mL of Milli-Q water and washed. Then 100-mLportions of the wash solution were filtered under vacuum through prewashed, dried (120 "C for 1h), and weighed filters (Whatman GFF). The filters were dried, cooled, and reweighed. Blank filtrations using 100 mL of Milli-Q water were also carried out.
Results and Discussion
Sludge Washings. The recovery of diazinon from successive washings of suspended sludge is shown in Table 1. Diazinon recovery was poor from the first washing and
0
2000
4000 6000 Sutpended 80Me IrnglLI
8000
10000
Flgure 1. Variation in anionic surfactant concentration (determinedas mg of MBAS/L) for a range of suspended sludge concentrations.
decreased as suspended sludge concentration increased, even up to 10 000 mg/L. In contrast, diazinon recoveries from the second and third wash solutions were close to 100%. The extraction procedure used can give 100% recovery of diazinon from aqueous solutions (28). The low recoveries obtained from the first wash solutions could be first due to high concentrations of surfactants or humic substances in the washings that could help solubilize the pesticide in the aqueous phase and hence pass through the extraction cartridge or compete for sorption on the (2-18cartridge (28,31) or secondly due to the presence of particles that sorb diazinon which are then not measured by the analytical method, either because the particles have passed through the C-18cartridge or are physically retained by the cartridge but are not fully extracted from diazinon by either the (2-18 cartridge or the diethyl ether eluant. The high recovery of diazinon from the second and third wash solutions (Table 1) is consistent with a decrease in the surfactant and/or particle concentration in these washings. A decrease in microparticle concentration with successive washings of sediments was found by other researchers (24). Repeating these experiments with propetamphos, a more polar OP pesticide with a higher aqueous solubility than diazinon [110 compared with 40 mg/L, respectively (3311 gave recoveries from all wash solutions greater than 95%. This shows that surfactant and/or humic substances did not significantly affect propetamphos recovery and so were unlikely to be responsible for the low recovery of the less polar diazinon. However, the high recovery of propetamphos does not indicate that it was not bound to microparticles, but simply that it was less tightly bound than the diazinon and, therefore, able to be adequately extracted with the ether. The anionic surfactant concentrations in the first washings (Figure 1) were found to vary from 1.4 to 17 mg/L (MBAS) for sludge concentrations of 1000-10 000 mg/L but decreased with successive washings and, in the third washing, were 50-70 % of the initial concentrations (Table 2). Thus, the high recoveries of diazinon found for the second and third washings (Table 1)were obtained in the presence of substantial surfactantconcentrations, again suggesting that the low diazinon recovery found for the first washings was not due to surfactant or humic substances, which can act to reduce pesticide recovery in a similar manner (19, 21, 32, 34, 35). The low diazinon recovery from the first washings could have been due to diazinon passing through the C-18 cartridge under the influence of fine particles. To test this, the first washing from a sludge sample (at 3000 mg/ L) was spiked with 1.0 mg/L diazinon and passed through Environ. Sci. Technol., VoI. 28, No. 11, 1994
1017
Table 2. Anionic Surfactant Concentrations (MBAS mg/L) Found in Successive Washings of Freeze-Dried Sludge Initially Present at Various Concentrations
Table 3. Mass Balance of Diazinon, Spiked to 1 mg/L, from Freeze-Dried Activated Sludge Found for Five Separate Sludge Samples at 3000 mg/L
surfactant concn (mdL of MBAS) 1"
2a
diazinon found( %) 3a 4" 5" mean
56 19
48 32 12
47 23
11
52 31 25
86
108
92
86
~~~~
wash no.
1oooa
3000"
10000"
1
1.41 1.31 0.95
15.1 8.01 7.58
17.2 8.7 8.1
2 3
Sludge (mg/L).
a C-18 cartridge, the eluant was extracted with hexane and then directly analyzed by GC, but no diazinon was detected (detection limit 0.01 mg/L). The eluant from a repeat experiment was directly analyzed by GCMS, but again no diazinon was detected (DL 0.01 mg/L). Given that about 30% of the initial 1 mg/L diazinon spike was not recovered (Table 11, it was clear that diazinon had not passed through the cartridge but had been retained and not subsequently eluted by diethyl ether. We concludethat the (2-18 cartridge was, in effect, acting as a filter to remove particles from the wash solution. To confirm this, the first washings from sludge (at 3000 mg/ L) were passed through either a 0.45-pm filter or a C-18 cartridge without first spiking with diazinon. The eluant in both cases was then spiked with diazinon, equilibrated for 6 h, and analyzed. Excellent recoveries (ca. 94%) were obtained, indicating that both the filter and the cartridge were able to retain most of the particles. This is to be expected if the particles are of micrometer dimensions. Particle Characteristics. Particle size and {potential were determined for particles from first washingsof sludge (at 500-10 000 mg/L) that had been spiked with diazinon immediately following separation from the sludge and then equilibrated for either 6 h or 2 weeks. In all cases, the mean particle size was about 1000 nm (SD 115 nm), regardless of the sludge concentration or equilibration time. The { potential of the particles was about -10 mV. This suggests that the particles could be colloidal aggregates, as aggregation can occur when {potential is less than 25 mV (36). After sonication for 30 min, the average particle size dropped to 300 nm, consistent with the 1000nm particles being aggregates. Laser microscopy of particles collected by filtration showed particle clusters approximately 1000 nm in size. The clusters were comprised of smaller particles of about 200 nm. Aggregation of these particles probably occurred during the freezedrying process, during washing of the sludge, or during the first 6-h equilibration time. The nitrogen sorption isotherm of the freeze-dried sludge showed negligible hysteresis, and the BET N2 surface area was 1.73 m2/g,both of which are typical of nonporous or macroporous solids (23). After washing, the sludge had a surface area of 0.27 m2/g. The decrease can be attributed to either microparticles that were removed by washing or a change in available surface area caused by the wet/dry cycle. The organic content of the freeze-dried sludge (on a dry basis) was 82 % w/w. There was no significant change in the organic content after washing. We attempted to gravimetrically measure the concentration of microparticles in a washing prepared from a 3000 mg/L suspension of sludge. We found that 50-60 mL of wash solution noticeably clogged the filters, although no particulates were visible. The remainder of the 100mL aliquots passed through the filter under vacuum at 1918 Environ. Sci. Technol., Vol. 28, No. 11, 1994
phase aqueousphase sludge microparticles total Determination
16
42 24 14 80
49 26 16
SD 5.3
5.5 5.6
91
number.
about 12 drop/min. However, the average mass of microparticles in the five replicate 100-mL samples was only 0.0002 f 0.0004 g. There was no significant change in the masses of the blank filters. Assuming that no microparticle mass was lost during drying, then the maximum microparticle concentration would be about 0.0006 g/100 mL or 6 mg/L. If we attribute the decrease in BET surface area to the loss of microparticles, then the maximum surface area of the microparticles was about 700 m2/g, a realistic value (37). Because of the low microparticle mass, it was not possible for us to determine organic content of these particles. Pesticide Partitioning. In other studies (28), we determined pesticide partitioning by subtracting the observed aqueous concentration (determined by solidphase extraction) from the total spike concentration in order to calculate the solid-phase concentration of the pesticide, allowing for appropriate blanks. The low diazinon recovery from first washings clearly shows that not all of the diazinon can be extracted from the microparticles collected by the C-18 cartridge. However, in order for the partition coefficient to decrease with an increase in the suspended sludge concentration, as observed, some of the diazinon sorbed to the microparticles must be extracted and determined along with the truly dissolved diazinon. The distribution of diazinon between the three phases present (aqueous, sludge, and microparticles) was determined for five different samples of freeze-dried sludge according to the scheme outlined in Figure 2. The results (Table 3) show a mean of 49 % ,26 % , and 16% associated with these phases respectively, giving a total mean recovery of 91 % . However, total recovery varied between 80 and 108% and was dependent upon the sludge sample used. The microparticles were capable of sorbing 12-25 7% of the added diazinon. Clearly, either the microparticles must be properly accounted for when measuring partition coefficients or they must be removed from the system under study. Removing microparticles by prewashing the solid phase gave diazinon recoveries of 90-94 % ,considerably better than the 70 % found previously (28). Partition calculations based on mass and surface area will be described elsewhere (26). The washings from muffled activated sludge (at 3000 mg/L) were not as colored or frothy as those from freezedried sludge, which indicated lower concentrations of humic substances and surfactants. Diazinon recovery from the first washings was greater than 94 % , better than from freeze-dried sludge washings (Table 1). This suggests that muffle furnace preparation does not lead to microparticle formation. Samples were also taken from the SEPP on separate occasions and centrifuged, and the supernatant was then
Pesticide solution 1 .O mglL diazinon
I
Add freeze dried sludge (3,000 rnglLl
Sample
I Equilibrate 6 hours Centrifuge
I
1
Supernatant
Filter through 0.45 urn filter Add 2 mL ether Sonicate 1 hr fbpeat & combine Extract with C-1S cartridge Elute with 2 rnL ether
Add 1 rnL ether Sonicate 1 hr Repeat & combine I
Extract
1
Evaporate under nitrogen Add internal standard Make up to volume
Flgure 2. Separation scheme used to determine the distribution of diazinon between the aqueous phase, sludge, and microparticles.
Table 4. Recovery of Diazinon from Supernatant of Samples from Various Areas in SEPP' treatment process activated sludge settling tank final effluent a
recovery of diazinon ( % ) at sample no. 3 4 5 1 2 75 58 52
99 97 98
78 70 75
44 63 57
90 83 85
Results obtained from five separate determinations.
spiked with diazinon to determine if the microparticles existed in live activated sludge, in the settling tank prior to chlorination, or in the final effluent. Recoveries of the pesticide ranged from 44 to 99%, indicating the presence of microparticles at concentrations which fluctuated considerably with time (Table 4). This is vital information if the behavior of pesticides during sewage treatment and the amounts of pesticide discharged in the final effluent are to be predicted. The presence of microparticles in the final effluent indicates that a strategy should be adopted to either completely remove the microparticles from the final effluent or effectively degrade or otherwise treat pesticides received by the plant. Conclusions Solutions prepared by suspending freeze-dried sludge in water were found to contain aggregated particles of about 1000-nm size and substantial concentrations of surfactants. The microparticles sorbed significant amounts of added diazinon, which was only partially extracted by the analytical technique used, causing low recoveries. Microparticles were found to be the cause of the observed decrease in partition coefficient with an increase in suspended sludge concentration (28). These particles can be removed by prewashing the sludge once, by filtration, or by passing the solution through a C-18 cartridge, which
effectively acts as a filter. The effect of microparticles was a function of the polarity of the pesticide as recovery of the more polar propetamphos was excellent, suggesting that it is less tightly bound to the particles and, hence, more effectively extracted by ether. Microparticles were not significant in muffled sludge obtained from the same source but were significant in three waste streams at a sewage treatment plant, including the final effluent. Pesticides such as diazinon and propetamphos will tend to be sorbed to the microparticles and could be released into the environment with the final effluent if the treatment process does not adequately remove microparticles or pesticides. This could pose problems when predictions of pesticide removal during sewage treatment are to be made. Acknowledgments The authors wish to acknowledgethe valuable help from Mr Bill Scott, South Eastern Purification Plant, Victoria, for obtaining sewage samples; the Australian Wool Research and Development Corporation for their financial support; and the helpful comments made by the reviewers. Literature Cited (1) Leo, A.; Hansch, C.; Elkins, D. Chem. Rev. 1971, 71, 525554. (2) Mirrlees, M. S.; Moulton, S. J.; Murphy, C. T.; Taylor, P. J. J.Med. Chem. 1976,19,615-619. (3) Chiou, C. T.;Freed, V. H.; Schmedding, D. W.; Kohnert, R. L. Enuiron. Sci. Technol. 1977,11, 475-478. (4) Chiou, C. T.;Schmedding, D. W.; Manes, M. Enuiron. Sci. Technol. 1982,16,4-10. (5) Blackburn, J. W.; Troxler, W. L. Enuiron. Prog. 1984, 3, 163-175. (6) Bell, J. P.; Tsezos, M. J. Water Pollut. Control Fed. 1987, 59, 191-198. Environ. Sci. Technol., Vol. 28, No. 11, 1994 1919
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(26) Villarosa, L.; McCormick, M. J.; Carpenter, P. D.; Marriott, P. J.; Russell, I. M. Manuscript in preparation. (27) The Determination of Organophosphorus Pesticides in Industrial and Municipal Wastewaters;U.S. EPA Method 614; Environmental Monitoring and Support Laboratory: Cincinnati, OH, 1982. (28) Villarosa, L.; McCormick, M. J.; Carpenter, P. D.; Marriott, P. J. Int. J. Enuiron. Anal. Chem. 1994,54, 93-103. (29) Tsezos, M.; Bell, J. P. Water Res. 1989, 23, 561-568. (30) Greenberg, A. E.; Connors, J. J.; Jenkins, D. In Standard Methods for the Examination of Water and Wastewater, 16th ed.;AmericanPublicHealth Association: Washington, DC, 1985. (31) Rump, H. H.; Krist, H. Laboratory Manual for the Examination of Water, Wastewater and Soil; VCH Publishers: New York, 1988; pp 106-108. (32) Johnson, W. E.;Fendinger, N. J.;Plimmer, J. R.Ana1. Chem. 1991,63, 1510-1513. (33) The Pesticide Manual, 8th ed.; The British Crop Protection Council; Lavernham Press Ltd.: Lavernham, Suffolk, U.K., 1987; pp 248 and 711. (34) Chiou, C. T.; Malcolm, R. L.; Brinton, T. I.; Kite, D. E. Enuiron. Sci. Technol. 1986, 20, 502-508. (35) Chiou, C. T.; Kite, D. E.; Brinton, T. I.; Malcolm, R. L.; Leenheer, J. A. Enuiron. Sci. Technol. 1987,21,1231-1234. (36) Ross, S.,Ed. Chemistry andphysics oflnterfaces;American Chemical Society: Washington DC, 1971; p 189. (37) Bailey, G. W.; White, J. L. J. Agric. Food Chem. 1964, 12, 324-328.
Received for review January 31, 1994. Revised manuscript received June 3, 1994. @
Abstract published in Advance ACS Abstracts,August 1,1994.
