Article pubs.acs.org/est
Widespread Microbial Adaptation to L‑Glutamate‑N,N‑diacetate (LGLDA) Following Its Market Introduction in a Consumer Cleaning Product Nina R. Itrich,*,† Kathleen M. McDonough,† Cornelis G. van Ginkel,‡ Ed C. Bisinger,‡ Jim N. LePage,‡ Edward C. Schaefer,§ Jennifer Z. Menzies,† Kenneth D. Casteel,† and Thomas W. Federle† †
Procter and Gamble, 8700 Mason Montgomery Road, Mason, Ohio 45040, United States Akzo Nobel Functional Chemicals, 525 West van Buren Street, Chicago, Illinois 60607, United States § Wildlife International, 8598 Commerce Drive, Easton, Maryland 21601, United States ‡
S Supporting Information *
ABSTRACT: L-Glutamate-N,N-diacetate (L-GLDA) was recently introduced in the United States (U.S.) market as a phosphate replacement in automatic dishwashing detergents (ADW). Prior to introduction, L-GLDA exhibited poor biodegradation in OECD 301B Ready Biodegradation Tests inoculated with sludge from U.S. wastewater treatment plants (WWTPs). However, OECD 303A Activated Sludge WWTP Simulation studies showed that with a lag period to allow for growth (40−50 days) and a solids retention time (SRT) that allows establishment of L-GLDA degraders (>15 days), significant biodegradation (>80% dissolved organic carbon removal) would occur. Corresponding to the ADW market launch, a study was undertaken to monitor changes in the ready biodegradability of L-GLDA using activated sludge samples from various U.S. WWTPs. Initially all sludge inocula showed limited biodegradation ability, but as market introduction progressed, both the rate and extent of degradation increased significantly. Within 22 months, L-GLDA was ready biodegradable using inocula from 12 WWTPs. In an OECD 303A study repeated 18 months post launch, significant and sustained carbon removal (>94%) was observed after a 29-day acclimation period. This study systematically documented field adaptation of a new consumer product chemical across a large geographic region and confirmed the ability of laboratory simulation studies to predict field adaptation.
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carboxylic acid chelating agents, 2,3,11 herbicides,20 and pesticides.10 L-Glutamate-N,N-diacetate (L-GLDA), is an aminocarboxylate chelating agent and builder sold under the commercial name Dissolvine GL-47-S (Akzo Nobel, Amersfoort, The Netherlands). Extensive work by van Ginkel et al. 21 demonstrated the ability of L-GLDA to pass OECD 301D Ready Biodegradation Tests inoculated with activated sludge from a wastewater treatment plant (WWTP) and surface water from the Rhine River, both located in The Netherlands. They further conducted an OECD 303A Activated Sludge WWTP Simulation Test using wastewater and activated sludge from a WWTP in The Netherlands that showed extensive removal of L-GLDA (>90%) after an 11-day lag phase.21 Moreover, they isolated a bacterium from activated sludge identified as a
INTRODUCTION Adaptation in the biodegradation literature refers to a change in a microbial community that results from exposure to a chemical and is manifested as an increase in the rate and extent to which that chemical is biodegraded. The occurrence of this phenomenon has been documented over decades and has been attributed to a combination of factors including the need for induction or derepression of enzymes, gene transfer or mutation, proliferation of a small population of competent degraders, or the absence or presence of other environmental factors affecting biodegradation.1 Occurrence of microbial adaptation is usually demonstrated experimentally or inferred by comparing biodegradation in preexposed versus pristine sites. Adaptation has been seen in a variety of environmental compartments including fresh2 and estuarine water,3 periphyton,4 sediment,5−7 aquifers,8 soil,9,10 activated sludge,11,12 and septic tank systems.13 It has also been observed for a wide range of chemicals including toluene,8 monosubstituted phenols,14 nitrophenols,1,8 aromatic amines,12 polychlorinated biphenyls,15 hydrocarbon mixtures,9,16−18 cationic,4−7,19 nonionic,19 and anionic13,19 surfactants, amino© 2015 American Chemical Society
Received: Revised: Accepted: Published: 13314
July 28, 2015 October 9, 2015 October 14, 2015 October 14, 2015 DOI: 10.1021/acs.est.5b03649 Environ. Sci. Technol. 2015, 49, 13314−13321
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Environmental Science & Technology
Table 1. Results of OECD 301B Ready Biodegradation Tests with L-GLDA Using Inocula from Midwest and Mid-Atlantic Wastewater Treatment Plants (WWTP) Sampled at Various Times Before, During, and After Initial Market Introduction in Early January 2010 WWTP location (map number) Fairfield, OH (1)
Brownsburg, IN (4) Cambridge, MD (12)
Washington, DC (11)
a
influent concentrationa (μg/L)
inoculum collection date
0 0 65 65 65 65 42 20 48 48 29 29 13 13 14 61 40 40
June 2007 Nov 2007 Feb 2010 July 2010 Sept 2010 Nov 2010 July 2011 April 2012 Feb 2010 July 2010 Aug 2010 Sept 2010 March 2011 Sept 2011 May 2012 Sept 2010 March 2011 Sept 2011
CO2 producedb (%)
lag period (days)
met OECD 301B criteria?
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
NA NA NA NA 12 13 7 8 NA 11 NA NA NA NA 10 NA 21 8
no no no no yes yes yes yes no yes no no no no yes no yes yes
12 6 4 16 84 88 91 90 21 88 23 14 23 6 96 9 65 100
7 6 6 4 4 4 6 3 16 5 5 1 7 7 7 1 1 1
Annual influent concentration estimated based on shipment data using eq 1. bPercent of theoretical CO2 produced at the end of the 28-day study.
Rhizobium radiobacter species that used L-GLDA as a sole carbon and energy source.22 In 2007 L-GLDA was evaluated as a phosphate replacement for United States (U.S.) automatic dishwashing (ADW) products. Part of this evaluation involved conducting two OECD 301B Ready Biodegradation tests with inoculum from a U.S. WWTP. The results of these studies indicated that LGLDA was not ready biodegradable, as the mean level of degradation was 100 mg/L and 265.7 mg/L, respectively, while an acute toxicity test with rainbow trout (Oncorhynchus mykiss) resulted in an LC50 > 100 mg/L.23 Using standard risk assessment methods,24 the low level of expected exposure from use in ADWs combined with L-GLDA’s low toxicity indicated that L-GLDA posed a negligible risk for the environment regardless of whether there was any loss from biodegradation. Nevertheless, the divergent biodegradation results using inocula from U.S. and Netherlands WWTPs catalyzed a program to investigate the potential for activated sludge microorganisms to undergo adaptation following exposure to L-GLDA in laboratory test systems simulating WWTPs artificially exposed to L-GLDA. Moreover, with L-GLDA’s introduction into ADWs in the U.S. in early January 2010, it was decided to monitor the evolution of adaptation in actual WWTPs distributed across a wide-ranging consumer market region in response to the exposure that took place as a result of LGLDA’s entry into the marketplace. The objectives of this paper are to (1) describe laboratory OECD 303A Activated Sludge WWTP Simulation Tests that were conducted to understand the conditions under which microbial adaptation to L-GLDA might occur, (2) report the results of a series of OECD 301B Ready Biodegradation Tests that were conducted to monitor microbial adaption using inocula obtained from activated sludge WWTPs as L-GLDA entered the market, and (3) report the results of an OECD
303A study conducted 18 months post market launch that confirmed that field-adapted inoculum could remove L-GLDA through biodegradation.
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EXPERIMENTAL SECTION Test Material. The L-GLDA (Supporting Information Figure S1) test material was supplied by Akzo Nobel Functional Chemicals, Amersfoort, The Netherlands. It was received as the commercially available Dissolvine GL-47-S sodium salt in a 47.9% active solution (Lot 00502G2001). It is highly watersoluble (10 000 mg/L) and has a measured organic carbon− water partition coeffient (Koc) of 90% domestic wastewater across specific geographic areas. The selection process utilized data from the Clean Watersheds Needs Survey (2004)26 and the U.S. Environmental Protection Agency (USEPA) permit compliance system.27 Any site with a sludge pollutant measurement (As, Cd, Cu, Pb, Hg, Ni, Se, Zn) above USEPA threshold levels28 was eliminated. Effluents from most of these WWTPs had been previously analyzed for ammonia and total suspended (TSS) as indicators of well operating facilities; levels for both indicators were measured below the discharge limits for all plants sampled.29 Figure S2 contains a map showing the locations of all the WWTPs. Initial OECD 301B studies were conducted at P&G in 2007 using inocula from Fairfield Municipal WWTP. Beginning in January 2010 (concurrent with the ADW product launch) and continuing for the next 7 months, OECD 301B tests were conducted at both P&G and WLI laboratories using inocula collected from four U.S. WWTPs in two regions: the Midwest (Fairfield Municipal, Fairfield, OH, and Brownsburg Municipal, Brownsburg, IN) and the Mid-Atlantic (Blue Plains, Wash13315
DOI: 10.1021/acs.est.5b03649 Environ. Sci. Technol. 2015, 49, 13314−13321
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Table 2. Results of OECD 301B Ready Biodegradation Tests with L-GLDA Using Inocula from Additional Wastewater Treatment Plants Sampled After September 2010 (L-GLDA was First Introduced into the Market in Early January 2010) operating capacity (m3/d)
influent concentrationa (μg/L)
inoculum collection date
Mason, OH (2)
5
Sellersburg, IN (5) Santa Claus, IN (6) Miamisburg, OH (3) Highland, IL (9) Augusta, GA (8) Evans, GA (7) Cayce, SC (8) Baltimore, MD (13)
1.5 0.5 2.5
65 65 34 34 42
Sept 2010 Nov 2010 June 2011 June 2011 June 2011
1.6 30 3 6 140
40 33 33 26 14
June 2011 April 2012 April 2012 April 2012 May 2012
WWTP location (map number)
CO2 producedb (%)
lag period (days)
met OECD 301B criteria?
85 88 78 75 77
± ± ± ± ±
8 4 11 7 5
12 10 9 9 7
yes yes yes yes yes
86 91 73 89 105
± ± ± ± ±
6 7 7 5 7
5 7 13 8 6
yes yes noc yes yes
a
Annual influent concentration estimated based on shipment data using eq 1. bPercent of theoretical CO2 produced at the end of the 28-day study. Two of the three replicates met the ready criteria but the average theoretical CO2 yield was 56% for all treatments at the end of the 10-day window (pass criteria = 60%). c
ington, DC, and Cambridge Municipal, Cambridge, MD). Three of the plants have operating capacities in the 2−5 MGD range, whereas the Blue Plains Plant is one of the largest WWTPs in the U.S. with an operating capacity of 370 MGD. Follow-up OECD 301B studies were conducted until a positive result was obtained. After a positive test result was obtained with inocula from one of the four starting WWTPs, the scope of the monitoring program was increased to sample additional WWTPs. Beginning in September 2010 and through May 2012, inocula from five plants in the Midwest, one in the Mid-Atlantic region, and three in the Southeast were included in the OECD 301B testing program. Tables 1 and 2 show the inoculum collection timeline for each study. The Supporting Information contains further details regarding the OECD 301B experimental design. OECD 303A Activated Sludge WWTP Simulation Tests. Three OECD 303A Activated Sludge WWTP Simulation studies30 were conducted in porous pots test systems at either P&G or WLI under conditions prescribed by the test guideline. Removal was followed by measuring dissolved organic carbon (DOC) in the influent and effluent of the porous pot. Given L-GLDA’s high solubility and lack of sorptivity, significant removal could result only from biodegradation, making removal of DOC an unambiguous indicator of biodegradation. The first test was performed at P&G in 2007 using activated sludge and wastewater from Fairfield Municipal WWTP prior to the introduction of L-GLDA. It was operated with a solids retention time (SRT) of 10 days. The second study was performed at WLI in 2008, also prior to L-GLDA introduction, using wastewater and activated sludge from Cambridge Municipal WWTP. In this study three separate porous pots were operated at SRTs of 10, 15, and 20 days. The third porous pot study was performed at P&G in 2011 using inoculum from Fairfield Municipal WWTP; multiple positive ready biodegrability results had been documented from this plant by this time. For the 2011 study, the test conditions were the same as those of the 2007 P&G porous pot study with the exception of an additional 15-day SRT treatment. The Supporting Information contains further details of the OECD 303A WWTP simulation studies. Calculation of Influent Concentrations. The influent concentration of L-GLDA was calculated per state on an annualized basis using eq 1:
Ci =
V *Cf1 P*Cf2*W
(1)
where Ci is the influent concentration in μg/L, V is the annual volume down the drain based on shipping data for that state, P is population of the state, Cf1 is the conversion factor 109 μg per ton, Cf2 is the conversion factor 365 days per year, and W is daily per capita water use (388 L).31
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RESULTS AND DISCUSSION Prior to any commercial use of L-GLDA in the U.S., two OECD 301B Ready Biodegradation Tests were conducted at P&G in June and November 2007 with sludge from Fairfield Municipal WWTP (Table 1). For the June 2007 study, the average carbon dioxide (CO2) production was 12% ± 7 (n = 4) of theoretical after 28 days, and the analyses of DOC at test termination confirmed the low level of biodegradation indicated by CO2 production. The November 2007 study confirmed the previous finding with the average CO2 production being 6% ± 6 (n = 3). These results were unexpected and divergent from those of van Ginkel et al.21 who showed that L-GLDA was fully biodegradable and passed an 301D Ready Biodegradation Test with inocula from a WWTP in The Netherlands. Given these conflicting data, an OECD 303A WWTP simulation test was conducted at P&G in 2007 to determine if microbial adaptation could occur in activated sludge with extended exposure to L-GLDA, resulting in the ability of the chemical to be biodegraded. As shown in Figure 1, these units exhibited virtually no DOC loss with the exception of a 12-day period (day 40−52) when there was significant removal in one unit. However, this removal was not sustained and was not observed again during the study. Nevertheless, these results indicated that, although not initially abundant, L-GLDA degraders can grow or be adapted for when continuously fed L-GLDA in U.S. wastewater or activated sludge. In this case, they were able to become temporarily established in one unit, but it appears that the growth rate may not have been fast enough in the small test system to sustain the population at the 10-day SRT which resulted in their ultimate washout from the system. On the basis of the hypothesis that L-GLDA degraders were unable to establish a stable community because of washout from the reactor due to the 10-day SRT, another OECD 303A study was conducted at WLI in 2008 under similar test 13316
DOI: 10.1021/acs.est.5b03649 Environ. Sci. Technol. 2015, 49, 13314−13321
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presence of and correspondingly biodegrade L-GLDA given sufficient sludge retention time. Consequently, the investigational focus shifted from laboratory WWTP simulation systems to monitoring this phenomenon in the field concurrent with LGLDA’s market introduction. Adaptation was monitored by conducting OECD 301B Ready Biodegradation Tests using activated sludge inocula from multiple WWTPs in the U.S. concurrent with and following the initial commercial release of L-GLDA in early January 2010. As products containing LGLDA started to ship in the first weeks of January and replaced products already in people’s homes and in retail inventories, levels of L-GLDA in wastewater would be expected to increase slowly over time until full market penetration was reached. Moreover, since introduction began in the Midwest and national expansion occurred gradually, and considering consumer preference variation by region, the first appearance of L-GLDA in wastewater and its levels would be expected to differ with both time and geography. During this phase of the investigation, OECD 301B Ready Biodegradation Tests were conducted using inocula from the four initial WWTPs. The two Midwest WWTPs were first sampled within 1 month of L-GLDA’s market launch, and the two Mid-Atlantic plants were first sampled within 7 months but before any appreciable market expansion occurred in this region. The extent of L-GLDA biodegradation in these first tests ranged from 9 to 21% (Table 1), which was consistent with the negative results measured in the 2007 301B Ready Biodegradation studies. Importantly they confirmed that in the U.S., L-GLDA was not ready biodegradable at the time of market introduction. Tables 1 and 2 contain estimated yearly influent L-GLDA concentrations based on shipping data for each state. Especially during the initial product launch (2010) before full market penetration and before the product made it onto the shelves and into consumer’s homes, these estimates are likely higher than what was actually present in domestic wastewater. The first positive OECD 301B Ready Biodegradation results were observed with sludge from the Brownsburg WWTP in July 2010. Shortly thereafter, a positive result was obtained with Fairfield WWTP sludge in September 2010. In both cases, LGLDA fulfilled the criteria for being ready biodegradable reaching 60% biodegradation in 28 days and meeting the 10day window (10 to 60% of theoretical CO2 production occurring within 10 days). The lag period for both tests, defined in the OECD 301 guideline as the time it takes to reach 10% degradation, was 11−12 days and the extent of mineralization ranged from 84 to 88%. Analysis of residual DOC at the termination of the tests was consistent with complete degradation of the test material. Notably, Fairfield Municipal WWTP was operating with an SRT of 9 days, indicating that L-GLDA degraders were able to establish themselves in a full-scale system with an SRT shorter than 10 days, which was not the case in the OECD 303A WWTP simulation studies possibly due to the higher level of microbial diversity in the field than in the small-scale laboratory systems. Positive OECD 301B Ready Biodegradation results were not seen from the Mid-Atlantic WWTPs until much later (Table 1). In March 2011, the Blue Plains WWTP exhibited 65% biodegradation after a 21-day lag period. However, inocula from the Cambridge Municipal WWTP failed two additional tests in March and September 2011. Inoculum from Cambridge first met the 301B Ready Biodegradation Test criteria in May 2012, exhibiting 97% CO2 production with a 10-day lag.
Figure 1. Percent of dissolved organic carbon (DOC) removed from the effluent of duplicate OECD 303A porous pots operated with a 10day sludge retention time (SRT). The activated sludge and wastewater were obtained from the Fairfield, OH Municipal WWTP. The study was conducted in 2007 prior to the commercialization of L-GLDA in ADW products.
Figure 2. Percent of dissolved organic carbon (DOC) removed from effluent of two OECD 303A porous pots operated with either a 15- or 20-day sludge retention time (SRT). The activated sludge and wastewater were obtained from Cambridge, MD Municipal WWTP. The study was conducted in 2008 prior to the commercialization of LGLDA in ADW products.
conditions but operated at three different SRTs (10, 15, or 20 days). Similar to the previous study, the 10-day SRT unit showed no appreciable carbon removal over the test period (data not shown). However, the 15- and 20-day SRT units initially exhibited a seesaw pattern of removal, ultimately resulting in consistent and extensive (>80%) removal after 40 days in the 20-day SRT unit and 50 days in the 15-day SRT unit (Figure 2). After 61 days, the sludge wasting rate in the 20day SRT unit was increased to deliver a 10-day SRT to determine if the newly established degrading population could be maintained. The amount of L-GLDA removed was not affected by the change and mirrored the removal in the 15-day SRT unit. These results suggested that once the degrader population was firmly established, the length of the SRT became less critical. The importance of SRT on chemical removal has been observed by others including Birch32 and Cech and Chudoba33 who found that in laboratory-operated WWTP simulation studies different chemicals have different critical SRTs below which significant and sustained removal does not occur. The WWTP simulation studies indicated that U.S. activated sludge microbial communities had the potential to adapt to the 13317
DOI: 10.1021/acs.est.5b03649 Environ. Sci. Technol. 2015, 49, 13314−13321
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Environmental Science & Technology Although inocula from these four initial WWTPs took 6 months to 2 years to exhibit positive biodegradation after LGLDA was first introduced into the market, it is important to note that the sampling schedule for some of the WWTPs was based on availability of resources more than on a set time interval. Therefore, the time frame in which adaptation occurred was potentially shorter than observed from this research for some WWTPs. For example, the interval between the last negative and the first positive ready test was 7 months for Blue Plains and 9 months for Cambridge WWTPs. Hence, accurately pinpointing the chronological progression of adaptation at each WWTP is difficult and further complicated by the national expansion process and different consumer preferences. Nevertheless, the fact that the first positive results were observed in the Midwest region was consistent with the market penetration rates in this region. Early distribution of the product was more concentrated in the Midwest and in 2011 this region continued to receive a higher percentage of the product than other regions in the country. The two Midwestern plants were similarly sized and showed a positive result within 5−7 months of product launch. However, the Brownsburg facility, which had a longer SRT than Fairfield (21 vs 9 days), made the transition faster with a positive ready test result within 5 months after market launch. After positive 301B Ready Biodegradation Test results were observed, the range of WWTPs from which inocula were obtained was expanded to include nine additional WWTPs to confirm the geographic extent of the adaptation. These nine additional WWTPs were evaluated between September 2010 and May 2012. They included five plants in the Midwest, three in the Southeast, and one more in the Mid-Atlantic region. The test results showed that L-GLDA was ready biodegradable using inocula from eight of these additional WWTPs (Table 2). Inocula from the remaining plant in Evans, GA exhibited extensive biodegradation of L-GLDA; two of the three replicates met the ready criteria but the average theoretical CO2 yield for all treatments was 56% at the end of the 10-day window (pass criteria = 60%). By the time most of these additional plants were sampled, shipments had become normalized across the U.S. With the exception of the Cambridge plant (Table 1), no negative biodegradation results were obtained from any treatment plant after September 2010. However, the rural Cambridge WWTP proved to be the outlier and took the longest time to adapt, but it also had one of the lowest predicted wastewater concentrations based upon shipment data (Table 1) and a lack of retail inventory observed from two store checks (Sept 2011 and March 2012) in the area. Moreover, the area contains many summer and weekend homes leading to a fluctuating population. Consequently, the long adaptation period could be explained by low, intermittent, and even nonexistent influent concentrations during this period. Nevertheless, once degradation was observed, the 10-day lag period was within the 6−12 day range observed for other plants. Ready biodegradation of L-GLDA was repeatedly evaluated using Fairfield Municipal WWTP activated sludge between February 2010 and July 2011. Figure 3 shows the CO2 production curves for the various tests, which illustrate the progression of the adaptation response with time. Nearly identical curves were observed in February and July 2010 with total mineralization of less than 15%. In September 2010, LGLDA was extensively mineralized after a 12-day lag period with the time to go from 10 to 60% biodegradation being 8
Figure 3. OECD 301B test results for L-GLDA using activated sludge obtained from Fairfield, OH Municipal WWTP on various dates following its market introduction in early January 2010.
days. In November 2010, the lag period remained approximately the same, but the time to go from 10 to 60% biodegradation decreased to approximately 5 days, and in July 2011, the time to go from 10 to 60% remained approximately 5 days and the lag period was reduced to only 7 days. The data indicate that, as the duration of L-GLDA exposure increased, the microbial community became more adept at degrading LGLDA, as reflected not only in shorter lags but also in faster degradation rates. Similar improvements in degradative ability were observed with inocula from the Blue Plains WWTP where the lag period decreased from 21 to 8 days and the time to go from 10 to 60% biodegradation decreased from 7.5 to 4 days between March and September 2011. As a concluding experiment, an OECD 303A WWTP simulation study was repeated in September 2011, approximately 18 months after L-GLDA was first introduced into the U.S. market. The experimental conditions, including the inocula source, were identical to those of the 2007 study with the addition of a 15-day SRT unit to the test design. Unlike the two previous porous pot studies, significant and sustained carbon removal was observed in the 10-day SRT unit (Figure 4). Moreover, both the 10- and 15-day SRT units exhibited DOC removal on average exceeding 94%, which was slightly higher
Figure 4. Percent of dissolved organic carbon (DOC) removed from the effluent of two OECD 303A porous pots operated with either a 10or 15-day sludge retention time (SRT). The activated sludge and wastewater were obtained from Fairfield, OH Municipal WWTP. The study was conducted in 2011 after inoculum from this WWTP exhibited a positive result in a 301B Ready Biodegradation test. 13318
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which both grow on NTA, also grow on L-GLDA.21 At the time van Ginkel et al. conducted the 301D studies,21 NTA was widely used across Europe in household laundry detergents (1140 mT annually36) resulting in a continuous loading of NTA into domestic wastewater. Because L-GLDA was not in commerce in Europe, its ready biodegradability potentially involved the recruitment of indigenous NTA degraders, which were relatively abundant in Europe due to the continuous presence of NTA in domestic wastewater. Conversely, during the time of this study, NTA was not used in detergents or other down-the-drain consumer product applications in the U.S. which resulted in no constant loading of NTA into domestic wastewater. If recruitment of indigenous NTA degraders was the reason L-GLDA was biodegradable in Europe, then the absence of NTA in U.S. domestic wastewater, and hence the absence of NTA degraders, could explain the lack of L-GLDA ready biodegradability using inocula from U.S. WWTPs. This hypothesis could also explain the much longer lag periods (29− 50 days) in the OECD 303A studies performed in the U.S. compared to the 12-day lag observed in the Netherland’s study.21 This would not be the first time exposure to one compound leads to adaptation to other related compounds. Shimp and Pfaender14 demonstrated that exposure of a mixed aquatic microbial community to phenol resulted in adaptation to the structurally related aromatic compounds m-cresol, maminophenol, and p-chlorophenol. They observed that the increased biodegradation potential of the phenol-adapted microbial community was accompanied by a concurrent increase in the number of microorganisms able to degrade the other compounds, leading them to conclude that adaptation to a single chemical may increase the assimilative capacity of an aquatic environment for other structurally related chemicals even in the absence of adaptation-inducing levels of those materials. The differences between the U.S. and Europe ready biodegradation test results highlight the fact that the ability of chemicals to pass an OECD 301B Ready Biodegradation Test is dictated by pre-exposure of the inoculum to the chemical itself or related chemicals already in commerce. Hence, a new ̈ inocula unless the population is chemical can fail using naive adapted to a structurally similar chemical that is currently in commerce. The inability to pass a ready test can terminally impact a chemical’s hazard classification due to limitations on the use of higher tiered testing, i.e. OECD 303A WWTP simulation testing is not allowed for hazard classification.37,38 For a persistence evaluation and exposure assessment, this failure can trigger higher tiered tests24,39 which can be appropriate in some cases but are more time-consuming and costly to execute and not nearly as straightforward to interpret. Although the intent is not to devalue the usefulness of the OECD 301 Ready Biodegradation Test for classification nor to argue that pre-exposure should be carte blanche, the science would indicate that biodegradation tests with pre-exposure should be considered in a regulatory context for use and release scenarios for which adaptation is relevant. The relevance of adaptation to environmental exposure varies as a function of the loading scenario and ensuing chemical and microbial residence times in a compartment. In the scenario in which a consumer product chemical is discarded down the drain, the chemical comes to represent a stable food niche and adaptation can result in development of a degrader population for that niche that can effectively biodegrade the chemical, thereby limiting its release to the environment. It is for this scenario
than the 87% average removal measured in the 15- and 20-day SRT porous pot units in 2008 at WLI. Furthermore, while the 15-day SRT unit in the WLI study did not exhibit a sustained high level of removal until day 50, the 15-day SRT unit in this study reached a high level of sustained removal by day 32. The shorter lag periods, the higher overall sustained removals, and the new-found ability for a porous pot with a 10-day SRT to effectively remove L-GLDA, all provide evidence of an increase in L-GLDA degraders that resulted from exposure in wastewater. However, likely due to the low L-GLDA wastewater concentrations in the field, this population size appears to remain small as evidenced by the 29-day lag. Notably, this lag is still longer than the 11-day lag reported by Van Ginkel et al.21 for an OECD 303A simulation study fed 50 mg/L of L-GLDA with activated sludge and wastewater from a WWTP in The Netherlands. As a final follow-up experiment, activated sludge from the OECD 303A study was used as inoculum for a subsequent OECD 301B test to evaluate the effect of laboratory pre-exposure at 301B relevant test concentrations of L-GLDA on lag and the time needed to meet the 10-day window. The OECD 303A porous pot had been dosed at approximately the same test concentration as the OECD 301B (41 mg/L LGLDA) compared to the freshly collected field sludge samples that were exposed to 42 μg/L. The lag period for the inocula from the porous pot pre-exposed sludge was reduced to 2−3 days compared to 7−8 days for freshly collected inocula, however, the time to go from 10 to 60% CO2 production was the same for both innocula (5 days). The results of this work raise four key issues (1) the ability of activated sludge microbes to adapt to very low (μg/L) levels of L-GLDA, (2) why L-GLDA was ready biodegradable in Europe but not in the U.S., (3) the importance of the 301 Ready Biodegradation Test in regulatory decision making and the implications associated with restricting the use of pre-exposed inocula, and (4) the appropriateness of laboratory simulation test systems for predicting real-world adaptation to new-to-theworld chemical technologies. Following the introduction of LGLDA, estimated wastewater concentrations ranged from 13 to 65 μg/L (Tables 1 and 2). Despite the low estimated influent concentrations, adaptation occurred and manifested itself in the form of passing OECD 301B Ready Biodegradation studies at test concentrations of 41 mg/L. Although reports in the literature indicate that some organic chemicals may have a concentration threshold below which adaption cannot occur or biodegradation becomes limited,34,35 there is no evidence from this study that such a threshold exists for L-GLDA. Prior work has shown a similar lack of threshold for nitrilotriacetate (NTA), another builder, whose biodegradation also has been shown to involve adaptation.2,3,11,13 For example, Larson and Davidson demonstrated that bacteria in river water could adapt to concentrations of NTA as low as 5 μg/L and, once adapted, their first-order rate constants for biodegradation did not vary greatly over a 1000-fold (1−1000 μg/L) concentration range.2 Rhizobium radiobacter strain BG1, which was isolated from activated sludge, uses both L-GLDA and NTA as a sole carbon and energy source.22 It produces an inducible monoxygenase that sequentially removes carboxymethyl groups from the nitrogen in the process releasing two glyoxylates to form Lglutamate.21,22 NTA biodegradation also involves this same successive removal of carboxymethyl groups from its N moiety, and cell free extracts from strain BG1 produced glyoxylate, when incubated with NTA.22 Furthermore, pure cultures of Aminobacter aminovorans and Chelatococcus asaccharovorans, 13319
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that the use of laboratory pre-exposed inocula could open the door to innovation and the commercialization of new chemicals that may in some cases have better environmental profiles than those they replace. If pre-exposure were allowed, it should have high fidelity to actual exposure conditions in the environment. These principles are articulated in the OECD 314 guideline40 for simulation tests to assess the biodegradability of chemicals discharged in wastewater which contains pre-exposure protocols based upon the OECD 303A guideline. The use of these OECD 303A simulation systems accurately and conservatively predicted the adaptation to L-GLDA in U.S. WWTPs that ultimately was realized in the field. This study demonstrated the importance of adaptation in the biodegradability assessment for new-to-the-market chemicals. The introduction of L-GLDA into a down-the-drain ADW detergent allowed the systematic monitoring of widespread adaptation of wastewater microorganisms to a new chemical. LGLDA had failed multiple ready biodegrability tests using innocula from U.S. WWTPs prior to and early in the product launch period because of the limited opportunity for acclimation to occur in the OECD 301B batch system. However, the realistic OECD 303A simulation studies predicted significant removal of L-GLDA in WWTPs with longer residence times (greater than 15 days). Monitoring the adaptive response of activated sludge microbes to degrade LGLDA in ready tests during the early phases of ADW market introduction provided insight into the length of time and amount of chemical needed in wastewater to produce this effect. Within 27 months after market launch of L-GLDA, positive 301B studies were observed using innocula from 13 WWTPs across different regions in the U.S. Correspondingly, 18 months after market launch of L-GLDA a confirmatory OECD 303A porous pot study showed significant (>94%) and sustained removal of DOC. This work demonstrated that a benchtop wastewater treatment simulation system operated under field-relevant conditions can accurately predict field adaptation. It also demonstrated the importance of understanding residence time impact on OECD 303A studies and brings to question the need for adequate acclimation approaches that can be used to generate innocula for the OECD 301 Ready Biodegradation Tests, especially for new-tothe-market chemicals.
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REFERENCES
(1) Wiggins, B. A.; Jones, S. H.; Alexander, M. Explanations for the acclimation period preceding the mineralization of organic chemicals in aquatic environments. Appl. Environ. Microbiol. 1987, 53 (4), 791− 796. (2) Larson, R. J.; Davidson, D. H. Acclimation to and biodegradation of nitrilotriacetate (NTA) at trace concentrations in natural waters. Water Res. 1982, 16, 1597−1604. (3) Pfaender, F. K.; Shimp, R. J.; Larson, R. J. Adaptation of estuarine ecosystems to the biodegradation of nitriloacetic acid: effects of preexposure. Environ. Toxicol. Chem. 1985, 4, 587−593. (4) Schwab, B. S.; Maruscik, D. A.; Ventullo, R. M.; Palmisano, A. C. Adaptation of periphytic communities in model streams to a quaternary ammonium surfactant. Environ. Toxicol. Chem. 1992, 11, 1169−1177. (5) Shimp, R. J. Adaptation to quaternary ammonium surfactant in aquatic sediment microcosms. Environ. Toxicol. Chem. 1989, 8, 201− 208. (6) Tezel, U.; Pavlostathis, S. G. Quaternary ammonium disinfectants: microbial adaptation, degradation and ecology. Curr. Opin. Biotechnol. 2015, 33, 296−304. (7) Oh, S.; Tandukar, M.; Pavlostathis, S. G.; Chain, P.; Konstantinidis, K. T. Microbial community adaptation to quaternary ammonium biocides as revealed by metagenomics. Environ. Microbiol. 2013, 15 (10), 2850−2864. (8) Aelion, C. M.; Dobbins, D. C.; Pfaender, F. K. Adaptation of aquifer microbial communities to biodegradation of xenobiotic compounds: influence of substrate concentration and preexposure. Environ. Toxicol. Chem. 1989, 8, 75−86. (9) Hendry, K. M.; Richardson, C. J. Soil biodegradation of carbofuran and furathiocarb following soil pretreatment with these pesticides. Environ. Toxicol. Chem. 1988, 7, 763−774. (10) Johnsen, A. R.; Karlson, U. PAH degradation capacity of soil microbial communities − Does it depend on PAH exposure? Microb. Ecol. 2005, 50, 488−495. (11) Stephenson, T.; Lester, J. N.; Perry, R. Acclimatisation to nitrilotriacetate acid in the activated sludge process. Chemosphere 1984, 13, 1033−1040. (12) Watson, H. M. A comparison of the effect of two methods of acclimation on aerobic biodegradation. Environ. Toxicol. Chem. 1993, 12, 2023−2030. (13) Shimp, R. J.; Lapsins, E. V.; Ventullo, R. M. Chemical fate and transport in a domestic septic system: Biodegradation of linear alkyl benzene sulfonate (LAS) and nitrilotriacetate acid (NTA). Environ. Toxicol. Chem. 1994, 13 (2), 205−212. (14) Shimp, R. J.; Pfaender, F. K. Effect of adaptation to phenol on biodegradation of monosubstituted phenols by aquatic microbial communities. Appl. Environ. Microbiol. 1987, 53 (7), 1496−1499. (15) Macedo, A. J.; Neu, T. R.; Kuhlicke, U.; Abraham, W. R. Adaptation of microbial communities in soil contaminated with polychlorinated biphenyls, leading to the transformation of more highly chlorinated congeners in biofilm. Biofilms 2006, 3 (1), 37−46. (16) Wilson, J. T.; McNabb, J. F.; Cochran, J. W.; Wang, T. H.; Tomson, M. B.; Bedient, P. B. Biodegradation of chemicals in the subsurface environment: Influence of microbial adaptation on the fate of organic pollutants in ground water. Environ. Toxicol. Chem. 1985, 4, 721−726. (17) Aamand, J.; Jorgensen, C.; Arvin, E.; Jensen, B. K. Microbial adaptation to degradation of hydrocarbons in polluted and unpolluted groundwater. J. Contam. Hydrol. 1989, 4, 299−312. (18) Korotkevych, O.; Josefiova, J.; Praveckova, M.; Cajthaml, T.; Stavelova, M.; Brennerova, M. V. Functional adaptation of communities from jet fuel contaminated soil under bioremediation treatment: simulation of pollutant rebound. FEMS Microbiol. Ecol. 2011, 78, 137−149. (19) Federle, T. W.; Pastwa, G. M. Biodegradation of surfactants in saturated subsurface sediments: a field study. Groundwater 1988, 26 (6), 761−770.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b03649. Details of OECD 301B and 303A experimental conditions; structure of L-glutamate-N,N-diacetate (Figure S1); locations of wastewater treatment plants (WWTPs) sampled for OECD 301B inocula (Figure S2); operational parameters of initial four Midwest and Mid-Atlantic WWTPs (Table S1) (PDF).
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Article
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]; phone: 513 622-0245. Notes
The authors declare no competing financial interest. 13320
DOI: 10.1021/acs.est.5b03649 Environ. Sci. Technol. 2015, 49, 13314−13321
Article
Environmental Science & Technology
Simulation Tests to Assess the Biodegrability of Chemicals Discharged to Wastewater; Paris, 2008.
(20) Spain, J. C.; van Veld, P. A. Adaptation of natural microbial communities to degradation of xenobiotic compounds; effects of concentration, exposure time, inoculum and chemical structure. Appl. Environ. Microbiol. 1983, 45 (2), 428−435. (21) van Ginkel, C. G.; Geerts, R.; Nguyen, P. D. Biodegradation of L-glutamatediacetate by mixed cultures and an isolate. Biogeochemistry of Chelating Agents. Am. Chem. Soc. Symposium Series 2005, 910, 183− 194. (22) van Ginkel, C. G.; Geerts, R.; Nguyen, P. D.; Plugge, C. M. Biodegradation of L-glutamatediacetate by Rhizobium radiobacter strain BG-1. Int. Biodeterior. Biodegrad. 2008, 62, 31−37. (23) European Chemicals Agency (ECHA). Tetrasodium N,Nbis(carboxylatomethyl)-L-glutamate; 2013. http://apps.echa.europa.eu/ registered/data/dossiers/DISS-9eaa34da-6ac8-619a-e04400144f67d031/DISS-9eaa34da-6ac8-619a-e044-00144f67d031_DISS9eaa34da-6ac8-619a-e044-00144f67d031.html. Accessed July 6, 2015. (24) European Chemicals Bureau Institute for Health and Consumer Protection. Technical Guidance Document on Risk Assessment in Support of Commission Directive 93/67/EEC on Risk Assessment for new notified substances. Commission Regulation (EC) No 1488/94 on Risk Assessment for existing substances, Directive 98/8/EC of the European Parliament and of the Council concerning the placing of biocidal products on the market Part II; European Communities: Brussels, 2013. (25) Organization for Economic Cooperation and Development. OECD Guidelines for the Testing of Chemicals. OECD TG 301 A-F: Ready Biodegradability; Paris, 1992. (26) U.S. Environmental Protection Agency. Clean Watersheds Needs Survey (CWTS). http://water.epa.gov/scitech/datait/databases/cwns/ 2004dictionary.cfm. (27) U.S. Environmental Protection Agency. Permit Compliance Systems 2009 data pull. http://www.epa.gov/enviro/facts/pcs-icis/. (28) U.S. Environmental Protection Agency. Standards for the Use or Disposal of Sewage Sludge, Final Rules. 40 CFR Part 257; Washington, DC, Feb 19, 1993; p 288. (29) Sun, P.; Casteel, K.; Dai, H.; Wehmeyer, K. R.; Kiel, B.; Federle, T. Distributions of polycyclic musk fragrance in wastewater treatment plant (WWTP) effluents and sludges in the United States. Sci. Total Environ. 2014, 493 (15), 1073−1078. (30) Organization for Economic Cooperation and Development. OECD Guidelines for the Testing of Chemicals. OECD TG 303: Simulation Test. Aerobic Sewage Treatment; Paris, 2001. (31) U.S. Environmental Protection Agency. Exposure and Fate Assessment Screening Tool (E-FAST) Version 2.0. Documentation Manual. Version 2.0; 2007. http://www.epa.gov/oppt/exposure/ pubs/efast2man.pdf. Accessed Sept 23, 2015. (32) Birch, R. R. Prediction of the fate of detergent chemicals during sewage treatment. J. Chem. Technol. Biotechnol. 1991, 50, 411−422. (33) Cech, J. S.; Chudoba, J. Effect of the solids retention time on the rate of biodegradation of organic compounds. Acta Hydrochim. Hydrobiol. 1988, 16 (3), 313−323. (34) Boethling, R.; Alexander, M. Effect of concentration of organic chemicals on their biodegradation by natural microbial communities. Appl. Environ. Microbiol. 1979, 37, 1211−1216. (35) Spain, J. C.; Pritchard, P. H.; Bourquin, A. W. Effects of adaptation on biodegradation rates in sediment/water cores from estuarine and freshwater environments. Appl. Environ. Microbiol. 1980, 40, 726−734. (36) Bland, A.; Davis, S.; Kishi, A. Chemical Economics Handbook, Chelating Agents; IHS: Englewood, CO, 2013. (37) United Nations. Globally Harmonized System of Classification and Labeling of Chemicals (GHS), 4th rev ed.; New York and Geneva, 2011. (38) European Chemicals Agency (ECHA). Guidance on the Application of the CLP Criteria, Regulation (EC) No 1272/2008. Version 3; Helsinki, Nov 2012. (39) European Chemicals Agency (ECHA). Guidance on Information Requirements and Chemical Safety Assessment. Chapter R.11: PBT Assessment. Version 1.1; Helsinki, Nov 2012. (40) Organization for Economic Cooperation and Development. OECD Guidelines for the Testing of Chemicals. OECD TG 314: 13321
DOI: 10.1021/acs.est.5b03649 Environ. Sci. Technol. 2015, 49, 13314−13321
