Vol. 51, No. 5APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1986, p. 919-9250099-2240/86/050919-07$02.00/0Copyright C) 1986, American Society for Microbiology

Effect of Salinity Gradients and Heterotrophic Microbial Activity on

Biodegradation of Nitrilotriacetic Acid in Laboratory Simulations ofthe Estuarine Environment

MICHAEL HUNTER, TOM STEPHENSON,t PETER W. W. KIRK, ROGER PERRY, AND JOHN N. LESTER*

Public Health Engineering Laboratory, Imperial College, London SW7 2BU, United Kingdom

Received 26 November 1985/Accepted 31 January 1986

The biodegradation of nitrilotriacetic acid (NTA), a synthetic replacement detergent builder, in the estuarineenvironment was examined by using a laboratory estuarine simulation. Two interdependent microcosms were

used; each of five vessels was equilibrated with a saline gradient between 1.30 and 17.17%o, with the final vesselsubsequently being increased to a maximum salinity of 31.6%o. Each microcosm was seeded simultaneouslywith heterotrophic bacteria from both fresh and saline sources. Viable counts demonstrated the ability of eachmicrocosm to sustain a mixed heterotrophic bacterial community throughout the range of salinities for 183 daysafter a stabilization period. Isolation studies demonstrated that both systems contained four bacterial species,representatives of the genera Vibrio and Flavobacterium and members of the coryneform group and the familyEnterobacteriaceae. Total bacterial numbers and species diversity decreased with increased salinity. NTA was

administered at low and high concentrations, one concentration to each microcosm, initially with the leastamount of saline. Removal of both concentrations of NTA occurred and was attributed to biodegradation aftera period of bacterial acclimatization. Subsequent dosing of NTA to vessels of higher salinity demonstrated thatbiodegradation was incomplete at observed mean salinities of >9.18%o at low influent NTA concentrations and>5.08f%r at high influent NTA concentrations. Therefore, acclimatization was dose dependent. It was concludedthat NTA acclimatization at the higher salinities ceased because of salinity stress-induced failure of NTAcatabolism and not the disappearance of a particular bacterial species.

The use of sodium tripolyphosphate detergent builders hasbeen cited as a major source of the contribution of phospho-rus to eutrophication of water bodies (20). As a consequenceseveral potential substitute detergent builders have beenproposed, of which the most notable is nitrilotriacetic acid(NTA) (15, 23). Currently NTA is in limited use (less than1% by weight of total detergent consumption) in severalEuropean countries such as Sweden, Switzerland, the Fed-eral Republic of Germany, and The Netherlands (23). InNorth America, up to 15% by weight is used in Canada;however, in the United States NTA was withdrawn from usein 1969 at the request of the U.S. Surgeon General on thegrounds that is may be detrimental to public health. Thisposition has recently been relaxed at the federal level, andthere is limited use of NTA in Indiana, although New YorkState has stated its intention to ban the use of detergentformulations containing NTA (23). A variety of toxicologicaleffects have been reported for NTA and its trisodium salt,including tumorigenesis in the rat kidney (7) and carcino-genic properties in rat and mouse urinary tracts (19). Re-cently, in mammalian cell cultures, increased mutagenicityhas been demonstrated by NTA when it is complexed withchromium (33).NTA is biodegradable after a period of acclimatization in

aerobic biological sewage treatment processes (30). How-ever, incomplete removal of NTA during wastewater treat-ment has been demonstrated (9, 31). This will inevitablyoccur and result in discharge of NTA to receiving waters(37). Although NTA biodegradation occurs in freshwaterriverine systems after acclimatization (15, 32), heterotrophic

* Corresponding author.t Present address: North East Biotechnology Centre, Teesside

Polytechnic, Middlesbrough TS1 3BA, United Kingdom.

919

bacteria associated with sewage discharges have been un-able to acclimatize to NTA in the saline environment of amarine simulation, under either aerobic or anoxic conditions(12). Certain estuarine bacteria have demonstrated NTA-degrading potential when it is transferred to a freshwatermedium, but they were unable to demonstrate this undersaline conditions (4).

Multistage microcosm investigations have monitored theresponse of bacterial species with respect to certain naturaienvironmental parameters (16, 17, 35; J. W. T. Wimpenny,R. W. Lovitt, and J. P. Coombs, 34th Symposium of theSociety for General Microbiology, p. 67, 1983) and havedemonstrated the effectiveness of interlinked continuousmicrocosm systems. The resonse of bacteria to natural andanthropogenic chemical gradients can be examined by usingsuch a method since separate compartments are provided forcellular growth but transfer of cellular material is inducedalong the gradient (16). Similar multistage systems have beenused previously to study physicochemical aspects of theestuarine environment (1, 2). In this study we examine thepossible recalcitrance of NTA within the estuarine mixingzone, using a multistage laboratory simulation, and theresponse of heterotrophic bacteria in this simulation tochanges in positional discharge of NTA.

MATERIALS AND METHODS

Microcosm design. Two series of five 1-liter borosilicateglass reaction vessels (Coming, Stone, United Kingdom) with500-ml sidearm overflow outlets, as described by Kirk et al.(12), were arranged in a stepped sequence (2). Each vesselwas supported by a magnetic stirrer (Stuart Ltd., LeightonBuzzard, United Kingdom) and contained a 40-mmpolytetrafluoroethylene nonvortex magnetic stirrer bar toachieve complete mixing. The freshwater was delivered from

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Freshwater end memberNTA stock

17.51FIG. 1. Schematic diagram of the estuarine interdependent microcosm layout with NTA dosing to the vessel with a nominal salinity of

8.77%c. Numbers below the vessels are per mille.

a 10-liter glass aspirator via a variable speed peristaltic pump(LKB Instruments, Croydon, United Kingdom) to the topvessel at a rate of 30 ml/h. A saline solution was deliveredfrom another 10-liter aspirator via a constant rate multichan-nel peristaltic pump (model MP-13; Ismatec SA, Zurich,Switzerland) to the bottom vessel at a rate of 30 ml/h. Thesaline gradient was achieved by a pumped upflow of moresaline water at a rate of 30 ml/h and a passive downwardoverflow of less saline water between adjacent vessels (Fig.1) at a rate of 60 ml/h.

Salinity values were modeled by using the pumped influentrates from both fresh and saline end members and theoverflow and recycle rates of the microcosms. The upper-most vessel was designated C1, and subsequent vessels weredesignated C2, C3, C4, and C5. The contents increased insalinity toward C5 because of the freshwater input at C1 andthe saline water input at C5. Assuming initial salinity inputsto C1 and C5 of 0.02%o (mean freshwater) and 35%o (meanseawater), respectively, and input and recycle rates of 30ml/h, the following equations are obtained:

60C1 = 30C2 + 30 x 0.02 (1)9°C2 = 60C1 + 30C3 (2)90C3 = 60C2 + 30C4 (3)90C4 = 60C3 + 30C5 (4)90C5= 60C4 + 30 x 35 (5)

Substitution yields theoretical salinities for C1, C2, C3, C4,and C5 of 1.11, 2.21, 4.39, 8.77, and 17.51%o, respectively.The retention time for each reactor vessel was also calcu-

lated from the influent flow rates and was 8.3 h for C1 and 5.6h for each of C2, C3, C4, and C5, giving a total of 30.6 h permicrocosm.Microcosm operation. The two microcosms were situated

in a darkened laboratory with a controlled temperature of 18± 2°C maintained by a Qualitair air conditioning unit(Chrysler Airtemp Ltd., London, United Kingdom). Fresh-water samples with a salinity of 0.04%o were collected inclean polypropylene containers from the River Thames,Teddington Weir, Kingston-Upon-Thames, United King-dom, and, likewise, surface coastal water of salinity 38.30%6ofrom Madera Drive Beach, Brighton, Sussex, United King-dom. The fresh- and seawaters were filtered through GF/Cmicrofiber filters (retention, 1.2 pLm; Whatman Ltd., Lon-don, United Kingdom) to remove suspended solids andphytoplankton but to retain suspended bacteria. The fresh-water was pumped to C1 vessels and the seawater waspumped to C5 vessels for a period of 7 days to seed themicrocosms. Subsequently, the bacterial populations weremaintained with a weak synthetic sewage solution (12)containing neutralized bacteriological peptone (0.0936g/liter; L34; Oxoid Ltd., London, England); Lab Lemcopowder (0.00624 g/liter; L29; Oxoid) and NH4Cl (0.00288g/liter). This was prepared in 10-liter volumes and sterilizedby autoclaving for 15 min at 121°C, and became the fresh-water end member of salinity 0.02%o (Fig. 1). After seeding,the saline end member contained a synthetic seawater solu-tion of salinity 35%o (18) made up from Analar salts (BDHChemicals Ltd., Poole, England) as follows (in grams per

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liter): NaCl, 23.48; Na2SO4, 3.92; MgCl2 - 6H20, 4.98;CaCl2 * 6H20, 1.10; KCl, 0.66; H3BO3, 0.03; KBr, 0.10;SrCl2 - 6H20, 0.02; and NaHCO3, 0.19. This regime wasemployed for a period of 132 days, and subsequent increasesin the saline feed concentrations were used to produce themodeled salinities of 20, 25, and 30,oo in the C5 vessels foranother 51 days.

Addition of NTA. Initially NTA was added to each of thefreshwater end members as the Gold label trisodium saltN(CH2CO2Na)3 * H20 (Aldrich Chemical Co., Inc., Milwau-kee, Wis.) to obtain the low or high modeled NTA concen-tration in the respective C1 vessels. NTA was administereddirectly to the individual vessel (Fig. 1) at a salinity of 2.21%oor above from stock solutions of 87.8 and 877.8 mg ofNa3NTA per liter to achieve the modeled low and high NTAconcentrations, respectively (Tables 1 and 2). The lowerconcentrations ofNTA represented conditions that are likelyto occur in some North American and European surfacewaters (W. Salomons and J. A. van Pagee, Proceedings ofthe International Conference on Heavy Metals in the Envi-ronment, CEP Consultants, p. 694, 1981), whereas thehigher concentrations represented more extreme conditionsunder which little secondary biological treatment and lowdilution occur.Sampling and analysis. Salinity measurements were taken

every 2 days by a conductivity probe method (29). The probewas connected to a PTI-20 digital water analyzer (DataScientific, High Wycombe, United Kingdom) and was cali-brated prior to the recording of each group of readings withsynthetic seawater standards. Simultaneous temperaturemeasurements were taken with a temperature probe con-nected to the same analyzer. Dissolved oxygen concentra-tions were maintained at saturation throughout and deter-mined with a dissolved oxygen probe (Uniprobe, Cardiff,United Kingdom) connected to a portable meter (model 410;Uniprobe). Samples were taken from appropriate vesselsevery 2 days immediately following these routine measure-ments and preserved in 0.5% (vol/vol) Analar formaldehyde(BDH Chemicals) prior to NTA analysis. A differential pulsepolarographic method, as described previously for salineconditions (13), was used for the determination of NTAconcentrations down to 0.05 mg/liter.

Bacterial assessment. Viable counts of bacteria were deter-mined by standard microbial techniques (6, 25). The solidmarine medium of Anderson (la) was used as recommendedpreviously for maximal estuarine bacterial growth (6) andwas corrected to the salinity that was applicable to eachvessel. Viable counts were determined by using a minimum

TABLE 1. Steady-state operating characteristics of the low NTAdosed microcosm

Duration Actual MoeldAcclimati- Mxmm Meanof NTA saled mean nTA zation tA NTAdosing salinity saJinity concen- time tumover removal(das)n O(%) MO tration (d(a1ys)per mg/h (

(mg/liter) ~~per cell)

22 1.11 1.38 0.97 15 3.1 >95a19 2.21 2.71 1.02 15 13.0 >95a34 4.39 5.09 0.98 11 12.7 >95a35 8.77 9.18 0.90 15 12.0 >95a22 17.51 16.92 0.70 7722 20.00 19.83 0.70 5614 25.00 24.10 0.70 7115 30.00 31.60 0.70 48

a Mean NTA removal after acclimatization.

TABLE 2. Steady-state operating characteristics of the high NTAdosed microcosm

Duration Actual Modeled Aclmt-Maximum MeanofNTA Modeled mean NTA Acliati-n NTA NTdosiNA salinity stluritcovcer-time tumover removal(doysin MO saint tration timeys (1010; mg/h (%(days) (%o) ~~(mg/iter) (dy) per cell)

22 1.11 1.30 9.69 21 36.3 >99a19 2.21 2.60 10.16 17 196.0 >99a34 4.39 5.08 9.80 33 178.0 >99a35 8.77 9.37 9.00 6522 17.51 17.17 6.95 322 20.00 20.04 6.95 014 25.00 23.80 6.95 015 30.00 29.20 6.95 0

a Mean NTA removal after acclimatization.

of three replicate samples per vessel on five separate occa-sions during the steady-state operation of the microcosms.Standard techniques (25) were used to identify bacteria tothe genus level after they were subcultured three times foreach genus on two separate occasions in salinity-correctedAnderson broth. During the final viable count, the Shannondiversity index (H) was determined in each vessel as de-scribed by Kaneko et al. (11) to investigate the change ofgenera with salinity. Maximum bacterial turnover ofNTA ineach vessel was also calculated, in terms of milligrams ofNTA per hour per cell, up to the modeled salinities of17.51%o. Turnover rate was taken to be maximal whenacclimatization occurred at >95% removal (30) or whenminimum NTA concentrations were reached if removal was<95%.

RESULTSSalinities. The steady-state operating characteristics of the

two microcosms are presented in Tables 1 and 2. Therecorded salinities up to the modeled 17.51%o vessel aremeans of 77 readings; and the modeled salinities of 20, 25,and 307oo are means of 11, 8, and 9 readings, respectively.Paired Student t tests indicated that there was no significantdifference (P = 0.05) between the corresponding meanobserved salinities of the two microcosms; therefore, thesalinity gradients achieved were comparable. Mean ob-served salinities were regressed against modeled salinities,giving correlation coefficients of 0.998 and 0.999 for the lowand high NTA dosed microcosms, respectively. These coef-ficients were significant (P = 0.01); therefore, the model is arepresentaive description of the salinities produced in themicrocosm vessels.NTA behavior. The operational characteristics, including

acclimatization times, of the bacterial communities to thelow and high concentrations of NTA are presented in Tables1 and 2, respectively. Typically, the vessel to which NTAwas dosed displayed peak concentrations 2 to 3 days afterdosing commenced. When NTA was dosed at the lowersalinities, smaller, often delayed NTA peaks were observedin the adjacent vessels of higher salinity. The delay suggeststhat initial removal is a consequence of dilution rather thandegradation. When acclimatization occurred the NTA con-centrations in the dosed vessels declined on subsequentdays, followed closely by similar reductions in the connectedvessels of higher salinity. A family of related curves of NTAconcentrations was therefore produced when it was dosed atany particular salinity (for example, dosing to 2.21%o nomi-nal salinity; Fig. 2).The acclimatization period (Tables 1 and 2) for the low and

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E

z

2

1

0

8

7

6

5

E

0-z

4

3

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1

2 6 10 14 18

Time (Days)FIG. 2. Acclimatization to NTA in the estuarine microcosm

vessels at nominal salinities of 2.21%c (A); 4.39%a (A); 8.77%c (U),and 17.51%G (O) when dosing low (upper curve) and high (lowercurve) NTA concentrations to the vessel with a nominal salinity of2.21%C.

high dosed vessels, with actual salinities of 1.38 and 1.30%o,were 15 and 21 days, respectively. In the case of the lowerNTA dose this period did not extend until degradation was

incomplete at an actual salinity of 16.92%o (Table 1). How-ever, at the high NTA concentration (Table 2) the acclima-tization period declined by 4 days, from 21 to 17 days, whenthe dosing moved from 1.38 to 2.60%o (actual values), andthen increased by 16 days, from 17 to 33 days, when movedto the vessel with an actual salinity of 5.08%o. The reductionin acclimatization time may be a result of the overflow ofpreviously acclimatized bacteria from the vessel with a

salinity of 2.60%o, whereas the extended period is an indica-tion of the increased stress of the degrading bacteria. Bio-degradation was incomplete at the salinity of 9.37%o whenthe microcosm was dosed at high NTA concentrations. Thefailure to completely degrade NTA at the high concentrationwas therefore observed at a lower salinity, as reflected bythe percent NTA removal, than at the lower NTA concen-

tration, suggesting that acclimatization is apparently doserelated.The maximum turnover rates of NTA after acclimatization

in both microcosms (Tables 1 and 2) peaked at the nominalsalinity of 2.21%o. However, at the lower NTA dosage thepeak turnover was sustained for the subsequent two salinitychanges. In contrast, the microcosm at the higher NTAconcentration displayed a rapid decrease in NTA turnoverwith an increase in salinity, and it was also clear that largeturnover rates occurred under the higher NTA loadingconditions. At the mean observed salinities from 16.92 to31.60%c after acclimatization ceased, NTA removal in thelow NTA dosed microcosm ranged between 48 and 77%(Table 1). At the high NTA dosing level, no removal oc-curred for the observed salinities of 20.04, 23.80, and29.20%Yo.

Bacterial assessment. The first viable bacterial count onday 7 (Fig. 3) was performed 5 days after completion ofbacterial seeding; this would account for the magnitude ofdifference observed in bacterial numbers between the firstand subsequent counts. However, analysis of variance ofthis count, omitting the nominal salinity of 1.11%o, indicatedthat there was a significant inverse relationship (P = 0.001)between bacterial numbers and salinity. A significant differ-ence (P = 0.001) between the microcosms was also evident,with relatively lower numbers being present in the micro-cosm later used for high NTA dosing (Fig. 3). The largeobserved drop in bacterial numbers between the vessels witha nominal salinity of 1.11 and 2.21%oo could be due to severalfactors, including differential retention time and salinity.An analysis of variance performed on the low- and high-

dosed microcosms by using the counts on days 33, 78, and123, respectively (Fig. 3), exhibited no significant difference(P = 0.05) between bacterial numbers in vessels of corre-sponding salinity as NTA dosing was moved to the highersalinities. This indicates that the positional change of NTAaddition had no effect on bacterial numbers. During thesecounts nominal salinities remained at 1.11, 2.21, 4.39, 8.77,and 17.51%o, for C1 through C5, respectively. The final counton day 182 (Fig. 3) was conducted when the salinity in the C5vessel, previously 17.51%o nominally, was elevated to 30%c.The lowest bacterial count was observed in these vessels,regardless of NTA dosing status, indicating that the reduc-tion of bacterial numbers in the Cs vessels was a conse-quence of salinity change as the sewage feed and NTAdosing remained unchanged from the previous count.

Overall analysis of variance encompassing all but theinitial count, the count on day 7 (Fig. 3), and actual salinitiesconfirmed a significant relationship (P = 0.001) betweensalinity and bacterial numbers. There was also a significantdifference (P = 0.001) between bacterial numbers in the highand low NTA dosed systems dosed in the same manner, asindicated in the initial count. This suggests that no suppres-sion of bacterial numbers occurs as a consequence of NTAaddition.

Standard cellular and morphological tests indicated thatduring the viable counts four bacterial species were consist-ently observed in the microcosm. One additional unidenti-fied species was noted occasionally, but this was veryinfrequent and was confined to the vessels with less saline(nominally 1.11 and 2.21%o). The genus Vibrio was the mostubiquitous and appeared in significant numbers in all themicrocosm vessels, regardless of salinity, as did an indeter-minate species of the coryneform group of bacteria. Thesetwo types of bacteria formed the majority of the suspendedheterotrophic bacterial biomass that was present within the

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to0

15

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151Day 7 Day 33

10F

5 5F

0 5 10 15

; Day 78

I5

15

10

5I

10 15

15

5 10 15

Day 123

lo1

5\0- ----\0

5 10 15

Day 182

-A p

5 10 15 20 25

Salinity %oFIG. 3. The distribution of bacterial numbers in the estuarine microcosms used for low (0) and high (0) NTA dosing concentrations; prior

to dosing (day 7) and dosing to nominal salinities of 2.21%o (day 33), 4.399oc (day 78), 17.51%c (day 123), and 30.OMoc (day 182).

microcosms. The two remaining species occurred regularlybut in lower numbers and were members of the genusFlavobacterium and the family Enterobacteriaceae. Themembers of the family Enterobacteriaceae were frequent inthe more saline vessels of the microcosms, especially in themicrocosm dosed at the higher NTA concentration, andabsent in the vessels with mean salinities of 4.15 and 5.05%oand below in the low and high NTA dosed microcosms,respectively. The relative proportions of all the speciesdetermined the diversity change in each microcosm (Fig. 4);the reduction in species diversity was greatest in the ob-served salinity range of 2 to 16%o.

DISCUSSIONThe stable bacterial community produced along the salin-

ity gradient of the microcosm system is consistent with thefindings of Bell and Albright (3), which were that variation of

attached and free-floating aquatic bacteria from a widevariety of sources could be accounted for largely by salinityand particulate load changes. The absence of suspendedsolids in the microcosm input enabled the development ofmainly suspended heterotrophic bacteria,, therefore corre-sponding to estuaries exhibiting low particulate loads such asthe Newport River estuary in the United States (21, 22). Themicrocosms can be used as a simplified estuarine analogbecause of the inverse relationship between bacterial num-bers and salinity observed in such an estuary (21).

Species of the genus Vibrio (28), the coryneform group(26), and the genus Flavobacterium (34) and members of thefamily Enterobacteriaceae (5) are all reported in estuarineand saline waters. Vibrio spp., Flavobacterium spp., andcoryneforms have been observed consistently in the marineenvironment (8). Vibrio spp., in particular, has moderatehalophilic tendancies (14); this is evident in its marked

.0

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II 0.5

i OA._

0.4

3 0.2

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_-

_-

2 6 10 14 18 22 26 30

Salinity /ooFIG. 4. Comparison of bacterial diversity in the two microcosms with NTA dosed to the vessels with a nominal salinity of 30c00 at low (0)

and high (0) concentrations.

distribution throughout the microcosm vessels. Coryneformbacteria and certain pigmented genera, including Flavobac-terium, may contribute significantly to the total numbers ofsuspended estuarine bacteria (36), and it is therefore possiblethat the species present in the microcosms could exist insignificant numbers in estuarine systems. Overall diversity inthe microcosms is relatively low, irrespective of salinity; forexample, considerably higher scores have been recorded inarctic seawater samples (11). In natural estuarine environ-ments, continual bacterial input from freshwater, marine,and adjacent land runoff sources encourage a greater speciesdiversity that does not occur in the microcosm vessels.Reduction in diversity with increased salinity, although lessevident in the low NTA dosed microcosm, is a result of lessequal distribution of bacterial numbers between the threemore abundant genera rather than the loss of the member ofthe Enterobacteriaceae. This is unlikely to affect the NTAdegradation, however, because of the maintenance of therelatively high bacterial numbers throughout which are com-parable to those in several estuaries (24). The loss of theEnterobacteriaceae did not significantly affect biodegrada-tion of NTA, as the mean removal of NTA was unchangedwhen the genus was not present in the vessels with nominalsalinities of 1.11 and 2.21%o. The decrease in diversitytoward the more saline vessels is therefore attributable tosalinity stress.The acclimatization times and turnover rates observed in

this study indicate that NTA biodegradation is seriouslyaffected at mean overall salinities above 9.18%o at low NTAconcentrations and above 5.08%o at high NTA concentra-tions. Batch studies have demonstrated a reduced rate ofNTA metabolism above 15%o with concentrations of NTAbetween 20 and 250 mg/liter, which are higher than thoseexpected in natural waters (15, 37; Salomons and van Pagee,Proceedings of the International Conference of Heavy Met-als in the Environment), suggesting catabolic pathway inhi-bition by high concentrations of sodium chloride (4). Similar

effects have been noted in response to low concentrations ofanother chelating agent, EDTA, by the common sewagebacterium Escherichia coli (10). Seeding of the higher salin-ity vessels by previously acclimatized bacteria from adjacentvessels of the microcosm should have improved the initialbiodegradative ability of the community within the seededvessel, with acclimatized bacteria rapidly becoming domi-nant on the introduction of NTA. However, their inability todo so suggests that metabolic inhibition of the degradativepathway occurs at the higher salinities. The increase inimportance of nitrogen as a limiting nutrient in estuarineenvironments (27), and therefore NTA as a source, coupledwith the inability of estuarine bacteria to use NTA as a solecarbon source under saline conditions but not under fresh-water conditions (4), is also consistent with the notion ofmetabolic inhibition rather than the lack of bacterial num-bers or the loss of species. Results of this study indicate thatbacteria are unable to acclimatize to NTA at moderatesalinities and that prior exposure to a proportion of thebacteria will not induce biodegradation at those salinities.NTA will therefore probably persist when discharged intothe estuarine environment.

ACKNOWLEDGMENTS

We acknowledge support for this work from the Centre EuropeenD'Etudes des Polyphosphates E.V. M.H. is the recipient ofa Scienceand Engineering Research Council Studentship.

LITERATURE CITED1. Abbot, W. 1967. Microcosm studies on estuarine waters. II. The

effect of single doses of nitrates and phosphate. J. Water. Pollut.Control Fed. 41:1748-1751.

la.Anderson, J. W. 1962. Studies on micrococci isolated from theNorth Sea. J. Appl. Bacteriol. 25:362-368.

2. Bale, A. J., and A. W. Morris. 1981. Laboratory simulation ofchemical processes induced by estuarine mixing: the behaviourof iron and phosphate in estuaries. Estuarine Coastal Shelf Sci.

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13: 1-10.3. Bell, C. R., and L. J. Albright. 1982. Attached and free-floating

bacteria in a diverse selection of water bodies. Appl. Environ.Microbiol. 43:1227-1237.

4. Bourquin, A. W., and V. A. Przybyszewski. 1977. Distribution ofbacteria with nitrilotriacetate-degrading potential in an estuarineenvironment. Appl. Environ. Microbiol. 34:411-418.

5. Cowan, S. T. 1974. Family 1 Enterobacteriaceae, p. 290. InR. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual ofdeterminative bacteriology, 8th ed. The Williams & WilkinsCo., Baltimore.

6. Goulder, R. 1976. Evaluation of media for counting viablebacteria in estuaries. J. Appl. Bacteriol. 41:351-355.

7. Goyer, R. A., H. L. Falk, M. Hogan, D. D. Feldman, and W.Richter. 1981. Renal tumors in rats given trisodium nitri-lotriacetic acid in drinking water for two years. JNCI 66:869-880.

8. Grieg, M. A., M. S. Hendrie, and J. M. Shewan. 1970. Furtherstudies on long term preservation of marine bacteria. J. AppI.Bacteriol. 33:528-532.

9. Hunter, M., T. Stephenson, J. N. Lester, and R. Perry. 1985. Theinfluence of transient phenomena on the biodegradation ofnitrilotriacetic acid in the activated sludge process. I. Variationsin hydraulic loading and sewage strength. Water Air Soil Pollut.25:415-430.

10. Jones, G. R. 1964. Effects of chelating agents on the growth ofEscherichia coli in seawater. J. Bacteriol. 87:483-499.

11. Kaneko, T., J. Hauxhurst, M. Krichevsky, and R. M. Atlas.1978. Numerical taxonomic studies of bacteria isolated fromarctic and subarctic marine environments, p. 26-30. In M. W.Loutit and J. A. R. Miles (ed.), Microbial ecology, Proceedingsin Life Sciences. Springer-Verlag, Berlin.

12. Kirk, P. W. W., J. N. Lester, and R. Perry. 1983. Amenability ofnitrilotriacetic acid to biodegradation in a marine simulation.Mar. Pollut. Bull. 14:88-93.

13. Kirk, P. W. W., R. Perry, and J. N. Lester. 1982. Determinationof nitrilotriacetic acid in waters and waste waters by gas-liquidchromatography, differential pulse polarography and a colori-metric method. Int. J. Environ. Anal. Chem. 12:293-309.

14. Kushner, D. J. 1978. Microbial life in extreme environments, p.318-357. Academic Press, Inc., London.

15. Larson, R. J., and D. H. Davidson. 1982. Acclimation to andbiodegradation of nitrilotriacetic acid (NTA) at trace concentra-tions in natural waters. Water Res. 16:1597-1604.

16. Lovitt, R. W., and J. W. T. Wimpenny. 1981. The gradostat: abiodirectional compound chemostat and its application in mi-crobiological research. J. Gen. Microbiol. 127:261-268.

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