Effect of temperature and dissolved oxygen on biodegradation of nitrilotriacetate

Water Research Vol.15, pp. 615 to 620, 1981 Printed in Great Britain.All rights reserved

0043-1354/81/050615-06502.00/0 Copyright © 1981 PergamonPress Lid

EFFECT OF TEMPERATURE AND DISSOLVED OXYGEN ON BIODEGRADATION OF NITRILOTRIACETATE R. J. LARSON1, G. G. CLINCKEMAILLIE 2 and L, VAn BELLE 2 tEnvironmental Safety Department, lvorydale Technical Center, Procter & Gamble Company, Cincinnati, OH 45217, U.S.A. and 2Professional and Regulatory Services Department, European Technical Center, Procter & Gamble Company, Temselaan 100, B 1820 Strombeek-Bever, Belgium (Received November 1980)

Al~traet--The effect of temperature and dissolved oxygen on the rate of biodegradation of nitrilotriacetate (NTA) was examined in water samples collected from the Rur River. Biodegradation of NTA was first order with respect to NTA concentration over a concentration range of 50-1000 #g 1-1. First order rate constants showed a typical temperature dependency (temperature coefficient, Qlo = 2) and biodegradation of N T A was observed over a temperature range of 2-24°C. The effect of temperature on the rate of NTA biodegradation was described by the Arrhenius equation, with calculated activation energies in the range reported for ordinary enzyme reactions. Biodegradation of NTA was also observed at low dissolved oxygen concentrations (0.3 mg I- t ), although at reduced rates compared to high oxygen concentrations (13 mg l-t). Biodegradation of NTA was oxygen-dependent, suggesting an obligate oxygen requirement for the initial steps in NTA metabolism by natural microbial communities in surface waters. In general, our results indicate that NTA biodegradation will occur in natural waters under conditions of low temperature and low dissolved oxygen and also at low NTA concentrations.

INTRODUCTION A number of studies have established the biodegradability of nitfilotriacetate (NTA) in sewage treatment systems (Bouveng et al., 1970; Warren & Malec, 1972; Walker. 1975: Gudernatsch, 1975; Warren, 1974; Bjomdal et, al., 1972: Forsberg & Lindquist, 1967; Swisher et al., 1967: Renn, 1974: Thompson & Duthie, 1968: Janicke, 1968: Pfeil & Lee, 1968; Bouveng et al., 1968: Shumate et al., 1970: Shannon et al., 1978: Shannon, 1975: Hubly & Cleasby, 1971: Cleasby et al., 1974: Klein, 1974; Rudd & Hamilton, 1972), soils (Tedje & Mason, 1974: Tabatabai & Bremner, 1975: Dunlap et al.. 1971) and surface waters (Warren & Malec, 1972; Shannon et al., 1974: Chau & Shiomi, 1972: Thompson & Duthie, 1968: Warren, 1974). However, there have been conflicting reports about the effect of two important variables,

To address these problem areas, the present study was conducted to determine the effect of temperature and dissolved oxygen on NTA biodegradation in fiver water. Additionally, kinetic and thermodynamic parameters for NTA biodegradation were calculated to compare our results to other systems and to provide an appropriate environmental perspective.

temperature and dissolved oxygen, on NTA biodegradation. In some studies, NTA biodegradation is disproportionately reduced at low temperatures and low oxygen partial pressures (Eden et al., 1972: Rudd & Hamilton, 1972: Klein. 1974: Moore & Barth, 1976). In other studies, temperature effects on NTA biodegradation are less pronounced and biodegradation is observed under both aerobic and anaerobic conditions (Tabatabai & Bremner. 1975: Claesson. 1971: Dunlap et al.. 1971 : Swisher et al., 1973). Although it is difficult to determine with certainty the reasons for the observed discrepancies, two problem areas can be identified: Ill the development, in the laboratory, of acclimated microorganisms with the appropriate temperature optimum: and (2) the maintenance and measurement of truly anaerobic conditions, 615

MATERIALS AND METHODS

Analytical

Reagent grade nitrilotriacetate (monohydrate, sodium salt obtained from W. R. Grace, Brussels) was analyzed by the method ofAue et al. (1972). The only modification was the use of 0.5 M HCl/butanol as the derivatizing reagent to form the tri-butyl esters. Gas chromatography was performed with a 1.8 m glass column containing 10~o VCC W-982 on chromosorb at a column temperature of 220°C. The detector was a nitrogen selective alkali bead as described by Williams et al. (1977). Prior to analysis, 40 ng of an internal standard, diethylbarbituric acid, was added to the samples. Sensitivity of the method was about 5 #g 1- t in the original sample, or 10rig injected. All NTA concentrations in this study are reported as H3 NTA. Dissolved oxygen (DO) was measured with an Orbisphere model 2711 high sensitivity DO monitor (Orbisphere Laboratories, Geneva, Switzerland). Specified detection limits of the Orbisphere instrument were 0.5/~g 1- i (accuracy ___2 p g l - l t and the instrument was equipped with a strip recorder for continuous DO monitoring (0--1 V span). Metal concentrations were measured by inductively coupled plasma emission spectroscopy, with a program set for Ca. Mg. Fe. Cu, A1, Ni. Cr. Zn, Mn. Hg and Cd. Viable microorganisms Icolony forming units m l - t CFU ml-t) were enumerated on 2°0 trypticase soy agar spread plates incubated at room temperature.

hi6

R 3 LaRSO',, ,.'! .i;

Dat(l ,mai~,!~

Table t. Characterization data for Rut Rtxer water Parameter pH Temperature (at collectionl Dissolved oxygen Viable counts Total carbon Metals Ca Mg Fe Cu AI Ni Cr Zn Mn Pb

Umt

Value

The rate of decrease ul NT.X v:OllCeil[ratloii LI'.L: 1['!?C .i various initial NTA concentrauon, a a s determined • ,E exponential decay model o1 the lollowing form :

Units C mg I C F U ml-* mg I mg 1

{,.5 ~ I~).5 3 x 104 2!

'Q for x , = ' i(S{, -- Ol[e ~ ] ~ tl where

38.2 IL3 0.8 0.02 0.48 ND* ND 0.30 0.24 0.18

Hg Cd

v t So a k c

= = = = = =

NTA ( m g l - uI: time (days~: initial concentration Img i r): lower asymptote (mg 1- '~: rate constant (days-~!: lag time (da~si

The asymptote and lag time for NTA biodegradation were incorporated to allow rate constants to be determined only during active biotransformation. All parameter estimates (So, a, k and cl were calculated by iterative techniques using a non-linear computer program. Correlation coefficients (r2). standard errors and 95% confidence mtervals were determined for each parameter estimate. 1.96 standard errors equalin~g the 95°,, confidence intervals.

ND ND

• Not detected,

Biodeyradation assays

RESULTS ~ND DISCI_SSION

River water for biodegradation studies was collected during the winter m o n t h s (Feb-Apr) from the Rur River, 10 km below Julich. Rur River water (RRW) was not acclimated to temperature or to N T A prior to the start of testing. Water samples were analyzed for metals, temperature, DO. pH, total carbon and viable microorganisms (Table 1). The N T A was added at various concentrations (50, 100. 500, 1000,ug 1- ~) to 21. of R R W in 4-1. erlenmeyer flasks. Test flasks were incubated at various temperatures and dissolved, oxygen concentrations, with constant mixing. Temperature and D O were measured daily except for low-level D O studies in which D O was maintained at 300/ag 1-1 by sparging with a N2/O~ gas mixture. The D O in low-level studies was analyzed continuously with a strip chart recorder. At specified intervals, 5 0 m l aliquots were withdrawn from the various test flasks, preserved with 1% formalin and analyzed for NTA. Concentrations of NTA in test flasks were corrected for background by subtracting values in blank flasks containing only river water•

Kinetics o f N T A bZodeqradation B i o d e g r a d a t i o n of N T A in R u r River water ( R R W ) was first o r d e r with respect to N T A c o n c e n t r a t i o n over a r a n g e of 50-1000/,tg t- ~. R e p r e s e n t a t i v e d e c a y c u r v e s at the h i g h a n d low e n d of t h e c o n c e n t r a t i o n r a n g e tested are s h o w n in Fig. 1. G o o d a g r e e m e n t betweefi rate c o n s t a n t s was o b s e r v e d in t w o different w a t e r s a m p l e s with e s t i m a t e d half lives (tL2) for N T A in river w a t e r of a b o u t 2 d a y s (Table 2). Rate cons t a n t s for N T A b i o d e g r a d a t i o n a p p e a r e d to increase slightly as initial N T A c o n c e n t r a t i o n s increased. H o w e v e r , a c o n s i s t e n t t r e n d c o u l d n o t be statistically demonstrated, R a t e c o n s t a n t s for N T A b i o d e g r a d a t i o n were m a r k e d l y affected by a c c l i m a t i o n effects. R e s p i k e ex-

i 8o~ ! i

~6o

-_o12c x~. •

z 40 o~

0

......

4

,~

8

,

Pl2 16 Time (d(:lys)

,

20

~

~

6 ( ~ ill "

z

20! o~-

24

q

,

£

16

,

,

24 Time (clays)

Fig. 1. Kinetics of b i o d e g r a d a t i o n of N T A in R R W at initial c o n c e n t r a t i o n s of 10(X)itg 1-t t[2) and 50 ~g 1- L (n). D a t a have been a n a l y z e d by e q u a t i o n ( 1 ). P a r a m e t e r estimates (So. a. k and c. respectively for the 1000 # g 1- ~ c o n c e n t r a t i o n were t 158.0 + 61.9 ,ug 1- ~ 4.9 _4- 55.5 ,.g I ~, 0.35 ___ 0.08 days " t and 0.01 + 0.4 days with an R z = 0.99• Parameter estimates for the 50,ugt ~ c o n c e n t r a t i o n were 60.0 + 6.4,ugl - l , 4.2 + 8.1 # g l - t , 0.11 _+ 0•05 days - t and 0.6 + 1.5 days with an r-" = 0.96. T h e -2 values are the standard errors of the p a r a m e t e r estimates, and the dotted c o n t o u r s represent the 9 5 " confidence intervals of the true mean•

NTA biodegradation Table 2. Rate constants for biodegradation of NTA in different batches of Rur River water

617 Table 3. Rate constants for NTA biodegradation in different natural waters Water

Sample

k* (days- 1)

t_~ (days)

RRW It RRW II~

0.31 + 0.20 0.37 + 0.34

2.2 1.9

Rur River Ohio River Grinestone Creek (Canada) Grand River (Canada)

* Mean value, +SD at initial NTA concentrations of 50, 100, 500 and 1000/28 I-1 . "1"Collected February 1979. :[:Collected April 1979.

k (days- i)

t, (days)

0.34 0.34* 0.53t

2.0 2.0 1.3

0.795

0.9

* From Thompson & Duthie (1968). t From Shannon et al. (1974). From Allen & Thomsen (unpublished data).

periments indicated that biodegradation of NTA was significantly faster after a period of adaptation was allowed (Fig. 2). This faster degradation was thought to reflect shifts in microbial population density or enzyme levels as a result of the batch test system used. To minimize the effects of these shifts, which may or may not be realistic of the environment, microorganisms in test flasks were not exposed to NTA prior to the initiation of testing, Lag periods were not observed for NTA biodegradation in RRW. This indicates that NTA degrading microorganisms were present among the indigenous microflora. Rate constants (k) for NTA biodegradation in the Rur River were comparable to estimates in other natural waters (Table 3). G o o d agreement between rate constants was obtained in areas where NTA has not been used extensively in detergents (Rur and Ohio Rivers), with somewhat higher values being observed in Canadian rivers. Since NTA has been used in detergent formulations in Canada for several years, it is possible that a microbial population highly adapted to NTA has developed in Canadian waters,

Table 4. Effect of temperature on rate constants for NTA biodegradation Temperature (°C)

k (days-~)

2 23

0.07 + 0.02 0.30 + 0.10

*Mean value at 500pgl -~, +SD for two batches of river water collected approx. 60 days apart.

Alternatively, biomass levels in the different rivers (which were not reported) may have been different. The level of total biomass present in the Rur River (Table 1), was 10-fold lower than levels typically found in other eutrophic waters (Wright, 1978).

Effect of temperatureon NTA biodegradation Biodegradation of NTA was observed over a range of temperatures (2-23°C) in samples of RRW collected during the winter months (Table 4). Water

Loc

8(2

if,, Respi ke

K

}

20 n 0

4

8

I

16

20

24

8

Time (doys)

Fig. 2. Biodegradation of NTA in RRW after prior exposure to NTA. River water was spiked with 500 ,ug 1- ~ NTA and allowed to incubate for 8 days before an additional 500 pg 1- 1 was added (respike). Parameter estimates after the respike were So --- 738.2 + 8.2 pgl -~. a = 6.6 + 7.0#g1-1' k = 1.7 + 0.1 days-~, c = 0.0 ± 0.0 with an r-' = 1.00. The __. values and dotted contours are explained in Fig. 1.

R .i L.~RS()N..:~,

618

samples collected in winter were used in our studies to ensure that microorganisms adapted to low temperatures would be present. Rate constants for NTA biodegradation in separate experiments showed a typical temperature dependency (Q~o-~ 2) increasing 4-fold over a 21 temperature span (Table 4). Since the temperatures tested in this study cover the ps?chrophilic to mesophilic range, it is possible that NTA biodegradation was mediated by facultative microorganisms. Alternatively, distinct psychrophilic or mesophilic populations could be involved, as has been suggested for soil (Tiedje & Mason. 1974). The presence of microorganisms capable of degrading NTA at both low and medium temperatures in surface waters makes it likely that NTA degradation will occur uninterrupted during all seasons of the year The effect of temperature on rate constants Ikt for NTA biodegradation was described by the Arrhenius equation: k = Ae -ERT

where A = frequency factor (days- ~); E = activation energy (cal mol-~); R = gas constant (1.986 cal deg-~ mol-~): T = absolute temperature (°K). Arrhenius plots of the temperature data showed good agreement in different batches of river water collected at different times (Fig. 3). The slopes of the lines calculated ,by linear regression ( - 5 . 6 and - 5 . 3 x 103 deg.) correslSond to activation energies (E) of 11.1 and 10.5 kcal m o l - ~, respectively. These activation energies are in the range reported for ordinary enzyme reactions and they indicate that degra-

dation of NTA in RRW is not thermodx:nan~lc:dl,. hampered. In biodegradation studies using Grand R~xer ,~atc~ IGRWI from Canada. M l e n & Thomsen !unpublished data) found similar temperature profiles t ~, those reported in this stud,. Their E value ',~as s~mc~hat lower however, at ~.0 kcal tool ~ This reduced f!: value suggests that NTA biodegradation ~s more ther.* modynamically favored in G R W than RRW. and that temperature effects on NTA biodegradation are less severe in GRW. A reduced temperature dependenc~, for NTA biodegradation is also supported b~ cxtcnsive monitoring studies in Canada which showed m, significant difference in NTA concentrations m recei',ing streams during winter xs summer months iWoodiwiss et al.. 1979). Effect o f dissolved oxygen on N T A biodegradation

Biodegradation of NTA was observed at low DO concentrations, albeit at a reduced rate compared to saturation conditions (Fig. 4). This reduced rate can be expected for microbial systems, which have a critical oxygen concentration (C~,) in the 260-d t00 ~lg I-range (Brown, 19701. Although a 5-fold reduction occurred in the rate of NTA degradation as DO concentrations decreased from 1324).3 mg I ~, NTA degration was still relatively rapid at low DO concentrations. Enzyme studies by Cripps & Noble (1973), Firestone & Tiedje 11978) and Firestone et aL (19781 have demonstrated an obligate oxygen requirement for NTA degradation by pure cultures. Our results also support an obligate oxygen requirement for the initial steps in NTA metabolism by mixed cultures in natural waters, at least in flowing rivers where DO

06[ -0.2

-I0 2t2

5 --18

-26

I / T (OK)x I0 -3

Fig. 3. Effect of temperature on NTA biodegradation in separate samples of RRW collected approx. 60 days apart. River water was spiked with 500 ~=gl-i NTA and incubated at 2, 15 and 2TC (0) and 2, 15 and 24C (O). Rate constants for NTA biodegradation were determined by equation (I) and Arrhenius plots were made and analyzed via linear regression. Estimated slopes were - 5.3 + 0.5 × 103 deg and

-5.6 __ I.l x l03 deg, with r 2 values of 0.99 and 0.96, respectively. The +_ values are explained in Fig= I

NTA biodegradation ~4o

619

of part-per-billion levels of citric and nitrilotriacetic acids in tap water and sewage effluents. J. Chromatogr. 72, 259-267. Bjorndal H., Bouveng H. O., Solyom P. & Werner J. (1972) NTA in sewage treatment-lll. Biochemical stability of some metal chelates. Vatten 28, 5-16.

I~ ":

BrOWnsewageD E . (1970) Aeration in the submerged culture of microorganisms. In Methods in Microbiology (Edited by Norris J. R. & Ribbons D. W.), pp. 124-174. Academic .~ Press, London. = Bouveng H. O., Davisson G. & Steinberg E. M. (1968) 5 NTA in sewage treatment. Vatten 24, 348-359. Bouveng H. O., Solyom P. & Werner J. (1970) NTA in treatment-II. Degradation of NTA in a trickling .~ filter and an oxidation pond. Vanen 24, 389-402. Chau Y. K. & Shiomi M. T. (1972) Complex(rig properties of nitrilotriacetic acid in the lake environment. War. Air Soil Pollut. Dordrecht 1, 149-164. f u . ~ . . . ...... " Claesson A. (1971) Anaerobic bacterial degradation of 2o . ~ nitrilotriacetate NTA. Vatten 27, 410--411. ........... i/ ............ i_ Cleasby J. L., Hubly D. W., Ladd T. A. & Sehon E. A. ........... (1974) Treatment of waste containing NTA in a trickling o , , , , , , filter. J. War. Pollut. Control Fed. 46, 1873-1887. o s io 15 2o 2s no Cripps R. E. & Noble A. S. (1973) Metabolism of nitriloTime (doys) triacetate by a pseudomonad. Biochem. d. 136, Fig. 4. Effect of dissolved oxygen concentration on NTA 1059-1068. biodegradation in RRW. River water was spiked with Dunlap W. J., Crosby R. L., McNabb J. F., Bledsoe B. E. & 500 #g I- 1 NTA and incubated at DO levels of 0.3 mg I- 1 Scalf M. R. (1971) Investigation concerning probable ira10) and 13.2 mg 1- l (1). Degradation over time was calcupact of nitrilotriacetic acid on ground waters. Environlated on a percentage basis and analyzed by equation ( 1 ) . mental Protection Agency, Ada, Oklahoma. U.S. Gov. Parameter estimates for the 0.3mg1-1 DO concentration Printing Office, Washington, DC. Project No. 16060, Nov. 1971, 55 pp. (So, a, k, and c, respectively) were 100.0+7.2~, 9.! + 25.40~ 0.05 + 0.03 days- ~ and 0.9 + 2.7 days with Eden G. E., Culley G. E. & Rootham R. C. (1972) Effect of an r: = 0.95. Parameter estimates for the 13.2mg1-1 DO temperature on the removal of NTA (nitrilotriacetic concentration were 99.9 + 2.2°0, 11.6 + 1.5%, 0.24 + 0.04 acid) during sewage treatment. Water Res. 6, 877-883. days- ~ and 0.0 + 0.6 days with an r 2 = 0.99. The + values Firestone M. K. & Tiedje J. M. (1978) Pathway of degradaand dotted contours are explained in Fig. 1. tion of nitrilotriacetate by a Pseudomonas species. Appl. envir. Microbiol. 35, 955-961. Firestone M. K., Aust S. D. & Tiedje J. M. (1978) A nitri-. liotriacetic acid monooxygenase with conditional NADH-oxidase activity. Archs Biochem. Biophys. 190, levels are normally high. By contrast, an obligate oxy617-623. gen requirement for N T A degradation has not been Forsberg C. & Lindquist G. (1967) Experimental studies on demonstrated in systems where D O levels are norbacterial degradation of NTA. Vatten 23, 265-277. mally low (Moore & Barth, 1976). Gudernatsch H. (1975) Biological degradation of heavy metal complexes of nitrilotriacetic acid in laboratory activated sludge plants. Gass. Wasserfach. 116, 512CONCLUSIONS 517. Hubly D. W. & Cleasby J. L. (1971) Treatment of waste Based on the results of our studies, the following containing NTA in a trickling filter. Report of Eng. Res. conclusions can be drawn: Inst., I~wa State University, Project 849-S. Janicke W. (1968) Uber das biologische Abbauverhatten 1. NTA biodegradation in surface waters occurs at yon Nitrilotriessigsaure. 2 Mitteilung uber des Verhatten low initial NTA concentrations (50 #g 1-1), low temsyunthetischer organischer Stoffe bei der Abwasserbeperatures (2:C) and low D O concentrations handlung. Gass. Wasserfach. 109, 1181-1184. Klein S. A. (1974) NTA removal in septic tank and oxi(300 ~g 1-~). dation pond systems. J. Wat. Pollut. Control Fed. 46, 2. The tem]aerature dependency (Q~o) of NTA 78-88. degradation is typical of biological systems, with a Moore L. & Barth E. F. (1976) Degradation of NTA durtemperature coefficient of 2 and an activation energy ing anaerobic digestion. J. War. Pollut. Control Fed. 48, of 11 kcal mol - ~. 2406-2409. Pfeil B. H. & Lee G. F. (1968) Biodegradation of nitrilo3. Initial steps in NTA biodegradation by natural triacetic acid in aerobic systems. Envir. Sci. Technol. 2, microbial populations are oxygen-dependent, and 543-546. NTA degradation occurs under low D O conditions. Renn C. E. (1974) Biodegradation of NTA detergents in although at a reduced rate compared to saturation wastewater treatment system, d. War. Pollut. Control Fed. 46, 2363-2371. conditions. Rudd J. W. & Hamilton R. D. (1972) Biodegradation of trisodium nitrilotriacetate in a model aerated sewage REFERENCES lagoon. J. Frsh. Res. Bd. Can. 29, 1203-1208. Shannon E. E. (1975) Effect of detergent formulation on Aue W. A.. Hastings C. R.. Gerhardt K. O.. Pierce J.O.. wastewater characterization and Treatment. J. Wat. PolHill H. H. & Moseman R F. (1972) The determination lut. Control Fed. 47, 2371-2383. .

620

R. ]. l-ARSON {'g Ot

Shannon E. E., Fowlie P. J. A.. Rush R. J. {19741A study ot nitrilotriacetie acid (NTA) degradation in a receiving stream. Technology Development Report No. EPS 4-WP-74-7. Water Pollution Control Directorate. Environmental Protection Service. Environmental Canada. Ottawa. Shannon E. E., Schmidtke N. W. & M o n a g h a n B. A. 1t978) Activated sludge degradation of nitrilotriacetic acid lNTAJ-metal complexes. EPS-4-WP-78-5. 16 pp. Swisher R D., Crutchfield M R. & Caldwetl D. W. {19671 Biodegradation of N T A in activated sludge. Envir Sci. Technol. 1,820-827 Swisher R D., Taulli T. A. & Malec E. J. 119731 Biodegradation of NTA metal chelates in river water. Trace .'i4etals and Metal-Organic Interactions in .Vatural Waters (Edited by Singer P. C.). Chap S, pp. 237-263. Ann Arbor Science Publishers, Ann Arbor. M I Shumate K. S., T h o m p s o n J. E., Brookhart J. O, & Dean C L. t1970) NTA removal by activated sludge: Field s t u d y J Wat~ Pollut. Control Fed. 42, 631-640. Tabatabai M. R. & Bremner J. M. (1975'p Decomposition of nitrilotriacetate (NTA) in soils Soil Biol. Biochem. 7. 103--106. T h o m p s o n J. E. & Duthie J. R. {1968) The biodegradability and treatability of NTA. J. War, Pollut. Control Fed. 40, 306-3 l 9.

Fiedje J M & Mason B B. (Iq"4i Biodegrada[~,m :,l ~;~1~Iotriacetate (NT~i it~ soils S,,i No,. So~ b,: Y ~ , 38. 27S 2~2 Walker -X. P. (l~a~5~ Ultimate b~odegradation ol nitrilotria cetate in the presence of hea~x metals. Pro~t ~t ~: 7, ,.I.. ~lol 7. 555 560 Warren C. B. {I9741 Biodegradation of nitrilotrlaceuc acid and NTA-metal ion complexes: {The C h e m o Biologtcal Life Cycle of a Detergent Builderl In Surrical i,~ r'~i~ Encironments iEdited by Khan \ I A Q & Bederka J P.), pp. 473M.96 .Academic Press, New York Warren C. B, & Malec E. J. (1972) Biodegradation o1 ruthlotriacetic acid and related imino and amino acids in river water Science 176. 277 270 Williams D. I-. Benoit F . Muzika K. & O'Grad~ R. ilt~7ri Gas chromatographic determination of nitrilotriacetic acid using a nitrogen-selective detector. J ('l~r,matour 136, 423-42 .7 Woodiwiss C R., Walker R. D & Brownridge F \ i i 9 7 ~ Concentrations of nitrilotriacetate and certain metals m Canadian wastewaters and streams: 1971 [975. W~ater Res. 13. 599-612. Wright R 1- (1978) Measurement and sigmficance ot specific activity in the heterotrophic bacteria of natural waters, Appl. envir. .'vlicrohiol. 39. 297-305.