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Transformation of organic micropoliutants during river banl<

filtration: laboratory versus field data

C.Bertelkamp^'^ J. Reungoat^ S. Botton", E. Cornelissen", E. Ghadiri^ M. de Jonge^ N. Singhal^ J.P. van der Hoek''', A.R.D. Verliefde''^

^Delft University o f Tecfinology, D e p a r t m e n t o f W a t e r M a n a g e m e n t , PO Box 5048, 2600 GA Delft, The N e t h e r l a n d s (Email: C.Bertelkampmudelft.nl)

^Ghent University, Faculty o f Bioscience Engineering, Particle and Interfacial Technology Group, Coupure Links 653; B-9000 G h e n t , Belgium

^The University of Q u e e n s l a n d , A d v a n c e d W a t e r M a n a g e m e n t Centre ( A W M C ) , QLD 4 0 7 2 , Australia " K W R Watercycle Research I n s t i t u t e , PO Box 1 0 7 2 , 3430 BB N i e u w e g e i n , The Netherlands

^Tehran University, D e p a r t m e n t o f E n v i r o n m e n t a l Engineering, N o . 15, Ghods St., Azadi Ave., Enghelab Sq., T e h r a n , Iran

'^Vitens N.V., PO Box 1090, 8 2 0 0 BB Lelystad, The N e t h e d a n d s

' U n i v e r s i t y o f A u c k l a n d , D e p a r t m e n t of Civil a n d E n v i r o n m e n t a l Engineering, Private Bag 92019, A u c k l a n d 1142, New Zealand

''strategic Centre, W a t e r n e t , Korte O u d e r k e r k e r d i j k 7 , 1 0 9 6 AC A m s t e r d a m , The Netherlands

Abstract This p a p e r i n v e s t i g a t e s t h e d e g r a d a t i o n b e h a v i o r o f 14 o r g a n i c m i c r o p o l i u t a n t s ( O M P s ) , s e l e c t e d f o r t h e i r d i f f e r e n t p h y s i c o - c h e m i c a l p r o p e r t i e s (e.g., m o l e c u l a r w e i g h t , h y d r o p h o b i c i t y a n d c h a r g e ) . In soil c o l u m n s s i m u l a t i n g t h e c o n d i t i o n s p r e v a i l i n g in t h e f i r s t m e t e r o f r i v e r b a n k f i l t r a t i o n (RBF) m e d i a . T h e r e s u l t s f r o m t h e c o l u m n s y s t e m a r e c o m p a r e d t o RBF f i e l d d a t a o b t a i n e d f r o m t h e V i t e n s d r i n k i n g w a t e r c o m p a n y i n T h e N e t h e r l a n d s . The s t u d y e x p l o r e s t h e r o l e o f s o r p t i o n m e d i a (sand f i l l e d c o l u m n s a n d p o l y e t h y l e n e t u b e s ) as c a r r i e r m a t e r i a l f o r t h e b i o m a s s . P o l y e t h y l e n e t u b e s w i t h t h e s a m e specific s u r f a c e area as s a n d in t h e c o l u m n s , w e r e o p e r a t e d u n d e r s i m i l a r c o n d i t i o n s t o c o m p a r e O M P r e m o v a l in t h e t w o s y s t e m s . B o t h t h e c o l u m n a n d f i e l d d a t a i n d i c a t e t h a t n e g a t i v e l y c h a r g e d O M P s w i t h Log D r a n g i n g f r o m 0.65 t o 1.95, p o s i t i v e l y c h a r g e d O M P s w i t h Log D r a n g i n g f r o m - 0 . 5 9 t o 0 . 2 1 a n d n e u t r a l O M P s w i t h Log D (-1.9 t o 1.12) w e r e m o r e s u s c e p t i b l e t o b i o d e g r a d a t i o n . T h e c o m p o u n d s t h a t p e r s i s t e d ( c a r b a m a z e p i n e , a t r a z i n e , p h e n y t o i n , l i n c o m y c i n ) w e r e p o s i t i v e l y c h a r g e d w i t h l o w e r Log D (-1.33) o r n e u t r a l w i t h h i g h e r Log D ( 1 . 5 6 t o 2.64). H y d r o c h l o r o t h i a z i d e s h o w e d p o o r b i o d e g r a d a b i i i t y d e s p i t e b e i n g n e u t r a l a n d h a v i n g a l o w e r l o g D ( - 0 . 7 1 ) ; i t is a n e x c e p t i o n t o t h e a b o v e b e h a v i o r f o r r e a s o n s t h a t h a v e n o t y e t b e e n i d e n t i f i e d . A c o m p a r i s o n o f O M P r e m o v a l in a b i o l o g i c a l l y a c t i v e p o l y e t h y l e n e t u b e w i t h a b i o l o g i c a l l y a c t i v e c o l u m n s h o w e d t h a t t h e b i o m a s s e s t a b l i s h e d in e i t h e r s y s t e m s r e m o v e d t h e s a m e O M P s a n d t o s i m i l a r e x t e n t f o r a m a j o r i t y o f t h e O M P s . This f i n d i n g s u p p o r t s t h e use o f p o l y e t h y l e n e t u b e s as a s i m p l e , c h e a p a n d q u i c k m e t h o d f o r i n v e s t i g a t i n g t h e t r e n d s in O M P r e m o v a l in RBF.

Keywords: organic micropoliutants, river bank filtration, biodegradation

INTRODUCTION

In t h e Netherlands, 6.5% of t h e t o t a l a m o u n t of drinking w a t e r is obtained f r o m river bank f i l t r a t i o n [1], a process characterized by long residence times (in t h e order of several m o n t h s t o years). These long residence t i m e s result in d e v e l o p m e n t of reduced redox conditions in

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sections o f the RBF system; as some of the OiViPs have shown redox d e p e n d e n t behavior (e.g. sulfamethoxazole) [2] this can result in greater overall OMP removal. The use of RBF as t h e first step in t h e t r e a t m e n t train was not originally intended f o r OMP removal b u t as a p r e t r e a t m e n t step f o r surface w a t e r t r e a t m e n t (removal particles, N O M , pathogens, viruses etc.). However, since the detection of OMPs in the Dutch surface w a t e r s at ng/L t o pg/L levels [3], due t o t r e a t e d e f f l u e n t discharge or r u n o f f f r o m agricultural land and discharges f r o m industries, interest has arisen in exploiting t h e RBF systems capability t o remove OMPs. RBF is a l o w cost, robust process w h i c h does not require chemicals. Past experience w i t h RBF has shown t h a t t h e system can t r a n s f o r m many of the OMPs t o below detection levels, b u t some OMPs (such as carbamazepine) have shown resistance t o degradation [4-6]. M o r e insight is required in t h e t r a n s f o r m a t i o n behavior of OMPs during passage in RBF media t o b e t t e r understand t h e removal mechanisms so t h a t t h e system can serve as an effective barrier against these c o m p o u n d s .

Removal in RBF systems occurs via a combination of sorption and biodegradation. A l t h o u g h t h e c o n t r i b u t i o n of biodegradation cannot be directly measured, it can be estimated by comparing OMP removal in a biologically active (biotic) c o l u m n w i t h an abiotic c o n t r o l . To create abiotic conditions t h e biological activity is inhibited by adding biocides such as mercurychloride, sodiumazide, or copperchloride t o the system.

M o s t studies on t h e biodegradation and sorption of OMPs during soil passage have involved either a small n u m b e r of c o m p o u n d s (three t o f o u r ) [6-9] or c o m p o u n d s f r o m a specific category o f OMPs (e.g. beta blockers) [10]. The behavior of a larger collection of OMPs in soil c o l u m n systems has been investigated only in a f e w studies [ 5 , 1 1 ] , yet still mainly, limited t o negatively charged and neutral c o m p o u n d s thus not covering a w i d e range of physico-chemical properties. A study involving a large collection of OMPs w i t h a w i d e s p e c t r u m of chemical properties is required t o develop relationships b e t w e e n t h e physico-chemical properties and t h e removal behavior of OMPs. Similarly, t h e comparison o f laboratory findings t o field observations for a larger selection of OMPs has been r e p o r t e d in only a single study limited t o neutral and negatively charged OMPs [12]. A comparison of c o l u m n data w i t h field observations is necessary t o delineate the p o t e n t i a l f o r using c o l u m n studies t o mimic the processes o p e r a t i n g in RBF systems. In a d d i t i o n , OMP removal has been studied f o r d i f f e r e n t porous media (sand, clay, activated carbon) but it has never been investigated w h e t h e r a simpler system (e.g. tubes or pipe systems) is capable of showing t h e same t r e n d regarding OMP removal.

This paper investigates t h e degradation behavior of a cocktail o f OMPs spanning a w i d e range of physico-chemical properties (molecular weight, h y d r o p h o b i c i t y and charge) in soil columns representative of t h e first m e t e r of soil passage under oxic conditions. The results f r o m t h e c o l u m n system w e r e c o m p a r e d t o RBF field data f r o m t h e drinking w a t e r c o m p a n y Vitens, The Netherlands. The study explores t h e role of sorption media (sand filled columns and polyethylene tubes) as carrier material f o r t h e biomass. Polyethylene tubes w i t h t h e

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same specific surface area as sand in the columns, w e r e o p e r a t e d under similar conditions t o c o m p a r e OMP removal in the t w o systems.

MATERIALS AND METHODS

Columns and Tubes Set-up and Operation

The experimental set-up consists o f t h r e e transparent PVC columns (L = 1 m, D = 36 m m ) filled w i t h technical grade sand (1.4 - 2 m m , Filcom, The Netherlands) and t w o black polyethylene tubes (PLN-10xl.5-SW, Festo, The Netherlands) (L = 97 m, Di = 7 m m ) as shown in Figure 1.

r

t e c h n i c a l g r a d e s a n d A l A I B I C O O u m X u ro c ro U O O u rr, X O + .2'

ro

ro c ro U p o l y e t h y l e n e t u b e

Figure 1 Experimental set-up

A f l o w f r o m b o t t o m t o t o p was m a i n t a i n e d in all columns and tubes. The columns and tubes w e r e o p e r a t e d in a t e m p e r a t u r e controlled r o o m at 20°C. Columns ( l A and I B ) and t u b e {2A) w e r e fed w i t h Schie Canal w a t e r along w i t h 200 pg/L sodium acetate (CH3COONa.3H20, M e r c k , Germany) t o s t i m u l a t e biological g r o w t h . DOC removal stabilized after an acclimation period of 4 m o n t h s , indicating t h e establishment of a stable biomass p o p u l a t i o n w i t h i n t h e sand columns ( l A and I B ) and t h e t u b e (2A). Column (IC) and t u b e (2C) w e r e fed w i t h Schie Canal w a t e r along w i t h 400 mg/L sodium azide (NaNs, Sigma-Aldrich, The Netherlands) f r o m t h e start of the e x p e r i m e n t t o suppress biological activity. Dissolved organic carbon (DOC) r e m o v a l was measured f o r all columns and tubes t o verify t h a t {a)biotic conditions w e r e o b t a i n e d . The columns and tubes w e r e fed f r o m 20L jerrycans t h a t w e r e replaced t h r e e t i m e s a week t o prevent biological degradation in t h e f e e d . A f t e r every r e p l a c e m e n t the jerrycans w e r e washed w i t h a 3% NaOH solution f o l l o w e d by a 3% HCI solution and flushed w i t h demineralized w a t e r t o prevent biofilm f o r m a t i o n in t h e jerrycans.

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The feed solutions w e r e p u m p e d t h r o u g h t h e columns and tubes by a peristaltic multichannel p u m p (205S, W a t s o n M a r l o w , The Netherlands) using Marprene® p u m p t u b i n g (d = 0.63 m m , d = 1.65 m m ) . The p u m p tubes w e r e connected by dark polyethylene t u b i n g (di = 4 m m , Festo, The Netherlands) t o t h e c o l u m n s / t u b e s . The hydraulic loading rate applied on t h e columns was 1 L/d and on the tubes was 6 L/d.

The pore velocities in the t h r e e columns w e r e d e t e r m i n e d using a tracer (5 g/L, NaCl), whose c o n c e n t r a t i o n was measured w i t h a conductivity m e t e r (Tetracon probe, Cond340i, W T W , Germany). Soil porosity was estimated using t h e tracer test. Porosity varied b e t w e e n 0.31 and 0.40, w h i l e pore velocity ranged f r o m 2.5 - 3.2 m / d .

OMPs

A cocktail of 14 OMPs (200 ng/L) was dosed into t h e feed solutions of t h e columns and tubes. Table 1 lists these OMPs and t h e i r physico-chemical properties. All c o m p o u n d s used w e r e o f analytical grade and purchased f r o m Sigma Aldrich, The Netherlands.

Table 1 OMP cocktail used in the experiment and their physico-chemical properties

Name M W pKa Charge at pH 7 Log D at pH 7

Propranolol 259.2 9.24 1 0.21 Metoprolol 267.2 9.68 1 -0.59 Lincomycin 406.2 7.6 1 -1.33 Atrazin 215.1 1.7 0 2.25 Phenytoin 252.1 8.33 0 1.56 Carbamazepine 236.1 1; 13.9 0 2.64 Trimethoprim 290.1 3.2; 7.1 0 0.98 Acetaminophen 151.1 9.38 0 0.86 Hydrochlorothiazide 297.0 9.76 0 -0.71 Caffeine 194.1 10.4 0 -0.57 Gemfibrozil 250.2 4.9 -1 1.95 Ibuprofen 206.1 4.91/4.53 -1 1.77 Ketoprofen 254.1 4.35 -1 0.82 Sulfamethoxazole 253.1 1.8;5.6 - 1 0.65

A stock solution of 2 mg/L of OMPs was prepared by adding 20 mg of each c o m p o u n d t o 10 L tap w a t e r . The OMPs w e r e dissolved in the stock solution by mixing f o r a m i n i m u m of t h r e e days b e f o r e use as feed solution in t h e experiments. Samples of 200 mL of t h e influent and e f f l u e n t f r o m t h e columns and tubes w e r e collected in bottles. The OMPs in solution w e r e extracted using Oasis HLB cartridges (200 mL, 6cc) (Waters, USA), w h i c h had been pre-t r e a pre-t e d w i pre-t h m e pre-t h a n o l (&gpre-t; 99.9%, Sigma Aldrich) and demineralized wapre-ter. The OMPs on each cartridge w e r e eluted w i t h 2x5 mL of m e t h a n o l and 2x5 mL of hexane/acetone ( 1 / 1 , v / v ) and t h e extract was gently blown d o w n t o dryness under n i t r o g e n . The extraction recovery was d e t e r m i n e d by spiking some samples at 100 ng/L. The extract was reconstituted in 1 mL MeOH/HzO ( 2 5 / 7 5 , v/v) and spiked w i t h 50 pL of a 200 pg/L mix o f labeled internal standards. A v o l u m e of 20 pL of extract was injected in a Shimadzu UFLC connected t o an AB

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Sciex 4000QTrap QLIT-iVIS equipped w i t f i a Turbo Spray source. Tlie analysis parameters w e r e as described in Reungoat et al. (2012) [13]. The OIVIPs w e r e quantified by an internal calibration r e n e w e d f o r each batch of samples. Quality control standards w e r e injected regularly d u r i n g t h e run t o ensure t h e signal intensity did not vary by m o r e t h a n 10%. The final result was corrected using the recovery o f t h e extraction m e t h o d .

Other analyses

The dissolved organic carbon (DOC) concentrations w e r e measured w i t h a Shimadzu TOC-Analyser after f i l t e r i n g the aqueous samples t h r o u g h 0.45 u m filters ( W h a t m a n , Germany); these filters w e r e flushed t w i c e w i t h demineralized w a t e r prior t o use. U V 2 5 4 absorbance was measured using a UV-Vis s p e c t r o p h o t o m e t e r (Thermo scientific, Genesys 6) and a 1 cm quartz cuvette. Oxygen and t e m p e r a t u r e w e r e measured w i t h an oxygen m e t e r (Cellox 325 probe, Oxi340i, W T W , Germany) and pH was measured w i t h a m u l t i m e t e r (Sentix 4 1 probe, M u l t i 340i, W T W , Germany) in a f l o w t h r o u g h cell c o n n e c t e d t o the i n f l u e n t and e f f l u e n t tubes o f t h e columns and tubes.

Field data

The field data w e r e o b t a i n e d f r o m t h e drinking w a t e r company Vitens. Vitens uses river bank f i l t r a t e as a source f o r t h e i r drinking w a t e r supply at p r o d u c t i o n location Engelse W e r k , close t o Zwolle, The Netherlands. OMP concentrations w e r e measured in the w e l l closest t o t h e river IJssel (distance river t o well = 10 meter, residence t i m e 1 - 3 m o n t h s ) at t w o d i f f e r e n t depths (3.6±0.1 m and 8.6±0.1 m). OMP concentrations measured in t h e river w e r e taken f r o m t h e RIWA year r e p o r t "Jaarrapport 2010 De Rijn" [14]. The RIWA report gives an overview of the measured OMP concentrations at f o u r d i f f e r e n t river locations in t h e Netherlands.

RESULTS AND DISCUSSION Column data versus field data

The removal of OMPs in t h e c o l u m n , t u b e and t h e field is presented in Table 2. Due t o differences in conditions ( w a t e r c o m p o s i t i o n , bacterial populations, porous media grading and composition etc.) in t h e field and t h e laboratory study t h e comparison allows only f o r comparing the p a t t e r n of OMPs biodegradation in the t w o systems.

In general t h e d e p t h did not have a significant influence on OMP removal, suggesting t h a t effect of t h e differences in t h e hydraulic residence t i m e and the e n v i r o n m e n t a l conditions f o r t h e t w o depths is minor. However, some OMPs do show differences in the a m o u n t s degraded at t h e t w o locations: removals of b o t h caffeine and iopamidol at 3.6 m d e p t h are small in comparison t o those at 8.6 m d e p t h , w h i l e carbendazim s h o w e d a decrease in r e m o v a l at t h e 8.6 m d e p t h .

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Table 2 OMP removal measured in the column, tube, and the well closest to the IJssel river at depths of 3.6±0.1 and 8.6±0.1 m

Compound Column Tube Field Field LogD Charge

(L = l m ) (L = 97 m) (d = 3.6±0.1m ) (d = 8.6±0.1 m) a t p H 7 at p H 7 Removal [%] Removal [%] Removal [%] Removal [%]

Gemfibrozil 100 94 NA NA 250.2 1.95 -Ibuprofen > 9 9 100 > 5 7 > 5 7 205.1 1.77 -Diclofenac NA NA > 7 8 > 7 8 295.0 1.57

-Bezafibrate NA NA >43^ > 4 3 361.1 1.20

-Ketoprofen > 9 9 99 NA NA 254.1 0.82 -Sulfamethoxazole 12 5 NA NA 253.1 0.55 -Phenazone NA NA > 6 6 > 6 6 188.1 1.12 0 Trimethoprim 100 89 NA NA 290.1 0.98 0 Acetaminophen > 9 5 100 NA NA 151.1 0.85 0 lopromide NA NA > 9 3 > 9 3 790.9 -0.36 0 Caffeine 88 95 0 > 2 6 194.1 -0.57 0 Iopamidol NA NA 7 57 776.9 -0.66 0 lomeprol NA NA > 9 2 > 9 2 776.9 -1.41 0 lohexol NA NA > 6 8 > 6 8 820.9 -1.90 0 Propanolol 42 50 NA NA 259.2 0.21 + Metoprolol 17 17 > 8 1 > 8 1 267.2 -0.59 + Carbamazepine 0 0 0 0 236.1 2.54 0 Atrazin 0 0 NA NA 215.1 2.25 0 Phenytoin 0 0 NA NA 252.1 1.56 0 Lincomycin 0 0 NA NA 405.2 -1.33 + Carbendazim NA NA 75^ 50 191.1 2.02 0 Hydrochlorothiazide 0 0 NA NA 297.0 -0.71 0

^ M W , Log K o „ a n d charge were obtained f r o m chemspider.com, except f o r the Log Ko„ o f t h e X-ray contrast agents which were obtained f r o m Violon et al., 1999. ^ The > sign Indicates the compound is removed below detection level

^ At 3.6±0.1m, carbendazim was removed till 0.01 p g / L while at 8.5±0.1m carbendazim was removed till 0.02 p g / L The difference in removal can therefore be attributed t o the sensitivity of the analysis rather than a production of carbendazim during subsurface passage.

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Table 2 shows t h a t t h e degradation behavior of caffeine and carbamazepine was similar in t h e c o l u m n and t h e field - caffeine was degraded t o below detection at t h e 8.6 m d e p t h in t h e w e l l and its removal in c o l u m n was similarly high (88%); No carbamazepine removal was observed in t h e field o r the columns. However, i b u p r o f e n and m e t o p r o l o l show high removal (> 57 and > 8 1 % , respectively) in t h e well at 3.6 m d e p t h , while t h e c o l u m n study shows high ibuprofen removal (> 99%) but low m e t o p r o l o l removal (17%). The i n t e r p r e t a t i o n and reconciliation of such discrepancies is made difficult by t h e overlap of only a small n u m b e r of OMPs b e t w e e n t h e c o l u m n study and those detected in IJssel river w a t e r . This issue is f u r t h e r complicated by t h e detection of OMPs in the river w a t e r at concentrations t h a t w e r e close t o t h e detection limit of 0.01 pg/L.

To make meaningful i n t e r p r e t a t i o n s , t h e p a t t e r n f o r OMPs degradation was related t o their physico-chemical properties, and comparisons t h e n made f o r field and laboratory data t o evaluate t h e similarities and differences in t h e i r degradation patterns. From t h e column results, it appeared t h a t negatively charged OMPs w i t h higher log D (0.65 t o 1.95), positively charged w i t h lower log D (-0.59 t o 0.21) and neutral OMPs w i t h log D ranging f r o m -0.59 t o 0.98 w e r e m o r e susceptible t o biodegradation. The persistent c o m p o u n d s (carbamazepine, atrazine, p h e n y t o i n and lincomycin) w e r e either positively charged w i t h low Log D values (¬ 1.33) or neutral w i t h higher Log D values (1.56 t o 2.64). The exception was hydrochlorothiazide w h i c h was neutral w i t h l o w e r Log D (-0.71) b u t showed poor degradability. The field data gave a similar t r e n d : charged OMPs w i t h log D ranging f r o m -0.59 t o 1.77 ( i b u p r o f e n , diclofenac, bezafibrate and m e t o p r o l o l ) and neutral OMPs w i t h Log D ranging f r o m -0.59 t o 1.12 (phenazone, caffeine) w e r e susceptible t o b i o d e g r a d a t i o n . In a d d i t i o n , neutral OMPs ( i o p r o m i d e , i o p a m i d o l , i o m e p r o l and iohexol) w i t h very low Log D values (-0.36 t o -1.90) w e r e biodegraded, all f o u r characterized by larger molecular weights (776.9 - 820.9 g / m o l ) . An exception is carbendazim w h i c h showed biodegradation in t h e f i e l d , b u t is neutral and characterized by a Log D of 2.02. Since the concentration observed in t h e field was very small and close t o t h e detection limit, it is expected t h a t this removal is mainly caused by d i l u t i o n . This reasoning is s u p p o r t e d by o t h e r studies w h o r e p o r t e d half-lives of carbendazim ranging f r o m 120 days t o 6 m o n t h s [ 1 5 , 1 6 ] .

Column versus tube

Following f o u r weeks of feed input t h e biologically active c o l u m n and t u b e s h o w e d removals exceeding 5 0 % f o r same c o m p o u n d s (caffeine, t r i m e t h o p r i m , a c e t a m i n o p h e n , gemfibrozil, i b u p r o f e n , and k e t o p r o f e n ) . F u r t h e r m o r e , t h e difference in the removals in t h e c o l u m n and t u b e f o r most c o m p o u n d s was insignificant (<7%); t r i m e t h o p r i m showed lower removal in t h e t u b e (11%) w h i l e propranolol s h o w e d lower removal in t h e column (8%).

CONCLUSIONS

The column and field data show t h a t w i t h f e w exceptions negatively charged OMPs w i t h Log D ranging f r o m 0.65 t o 1.95, positively charged OMPs w i t h Log D ranging f r o m -0.59 t o 0.21 and neutral OMPs w i t h Log D ranging f r o m -1.9 t o 1.12 w e r e m o r e susceptible t o

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b i o d e g r a d a t i o n . Tfie c o m p o u n d s t h a t persisted (carbamazepine, atrazine, p h e n y t o i n , lincomycin) w e r e positively charged w i t h lower Log D (-1.33) or neutral w i t h higher Log D (1.56 t o 2.64). Similarly, a comparison of OMP removals in the biologically active t u b e and c o l u m n s h o w e d t h a t biomass established in b o t h systems r e m o v e d t h e same OMPs and t h a t most OMPs w e r e removed t o similar extent. Thus, polyethylene tubes can be used as a simple, cheap and quick m e t h o d f o r investigating OMP removal.

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