Chapter 7
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Analysis of Biodegradation Intermediates of Ethylenediaminetetraacetate and Nitrilotriacetate by High-Performance Liquid Chromatography Zhiwen Yuan and JeanneM.VanBriesen Department of Civil and Environmental Engineering, Carnegie Mellon University, Pittsburgh, PA 15213
Anthropogenic chelating agents released to the environment can form degradation intermediates that are recalcitrant to further attenuation. Evaluation of the potential for environmental persistence requires new methods to detect these intermediates. Two HPLC methods were developed for measuring the biodegradation intermediates of EDTA and NTA. Ethylenediaminetriacetate (ED3A), Ν,Ν' -ethylenediaminediacetate (N, N'-EDDA) and 3ketopiperazine-N,N'-diacetate (3KP) were measured concurrently by normal phase HPLC using a LiChrospher 100NH column. Iminodiacetate (IDA) and glycine were measured concurrently by reversed phase HPLC after derivatization with 9-fluorenylmethyl chloroformate (FMOC -Cl). These two methods were simple, fast, accurate, specific, and applicable to the analysis of these intermediates in different matrices and their detection limits are all below 1μΜ using U V detector. 2
© 2005 American Chemical Society Nowack and VanBriesen; Biogeochemistry of Chelating Agents ACS Symposium Series; American Chemical Society: Washington, DC, 2005.
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Introduction EDTA and NTA are two important anthropogenic chelating agents able to form stable, water soluble complexes with metals. They are widely used in many industrial and consumer products and processes, such as detergents, photo processing, the paper and pulp industry, nuclear reactor decontamination, and many other industries. The estimated U.S. production of EDTA in 1992 was 7,241 tons (/). European usage of EDTA is much higher with an estimated 35, 000 tons in 1990 (2). European NTA consumption is about 17,350 tons in 1990 (3). The total worldwide use of aminopolycarboxylic acid (EDTA, DTPA and NTA) was 186,000 tons in 1994 (4). The usage of EDTA is expected to increase in the future, especially in the paper and pulp industry due to transition to chlorine free processes mat require additional chelates. Due to minimal biodégradation in natural and engineered systems and the low potential for photodegradation in many turbid waters, EDTA persists in surface waters at concentrations ranging from 1-100 μg/L (2). Aminocarboxylic chelating agents such as EDTA and NTA are generally regarded as nontoxic and used in food products and medicines. However, the effects of low persistent concentrations in the environment are unclear. Chelating agents may enhance eutrophication, remobilize heavy metals, and have long-term toxicological effects on microorganism (5). Biodégradation of EDTA and NTA is a possible solution for their removal from the environment. Several species able to degrade NTA have been isolated. The well studied N T A dégrader, Chelatobacter heintzii ATCC 29600 (C. heintzii), utilizes a monooxygenase pathway to catalyze N T A biodégradation (see Figure 1) (5-9). EDTA degrading organisms are not as thoroughly characterized. Nortemann and coworkers identified a mixed culture (BNC1/BNC2) and isolated a pure culture (BNC1, D S M 6780) that degraded low concentrations of EDTA (μΜ to mM) (10-14). Witschel et al (15) reported another pure strain D S M 9103 that is closely related to BNC1 and utilizes a similar degradation pathway to degrade EDTA. The proposed multi-step degradation pathway of EDTA by BNC1/BNC2 is shown in Figure 2 (10-14, 16-18). Nortemann (10) reported that bacterium BNC1 initiates the breakdown of EDTA but nitrogen-containing intermediates were produced and persistent in the system. These intermediates may still have significant chelating ability. Intermediate persistence was also reported during NTA degradation by C. heintzii (19, 20). If these intermediates persist, they sequester carbon and electrons, reducing cell yield, slowing the rate of removal of chelating agents from the system and continuing to influence the system. As shown in Figure 1, the anticipated intermediates of N T A biodégradation are iminodiacetic acid (IDA), glycine and glyoxylate. As shown in Figure 2, the anticipated intermediates of EDTA biodégradation may include ethylenediaminetriacetate (ED3A), N.N'-ethylenemaminediacetate (Ν,Ν'-EDDA), ethylenediaminemonoacetate (EDMA), ethylenediamine (ED) and glyoxylate. ED3A can spontaneously cyclize to form 3-ketopiperazine-N,N-diacetate (3KP), especially under acidic condition. This reaction is shown in Figure 3. 3KP is
Nowack and VanBriesen; Biogeochemistry of Chelating Agents ACS Symposium Series; American Chemical Society: Washington, DC, 2005.
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NTAmo.iooxyge.iase
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°2
+
2e
IDAdehydrogem.se
"
H0 2
2e 2H +
+
°NTA Ο OH
glyoxylate
Figure 1. NTA degradation pathway by C. heintzii.
EDTAnMnoasygeaase OH Τ
EDTAmDnoaxygenase OH
Oz+te
HP
Τ
Hp
8 1 3 3 4
(Mesylate
0 1 1
Q2+2e"
Τ
Qyoxylate
OH
OH
EDTA IDAfBddase (V2e*
IDAarita
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^
0^+2e"
Hp NH2
I
"
9»
EDM*
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»
"°
Figure 2. Proposed degradation pathway of EDTA by BNC1/BNC2 co-culture.
Nowack and VanBriesen; Biogeochemistry of Chelating Agents ACS Symposium Series; American Chemical Society: Washington, DC, 2005.
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ΌΗ
PH
