&EFA
United States
Environmental Protection
Agency
Environmental Research
Laboratory
Duluth MN 55804
EPA
July
600/3-80-050
1980
Research and Development
The Effect of
Nitrilotriacetic Acid
(NTA) on the
Structure and
Functioning of
Aquatic
Communities in
Streams
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
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The nine series are:
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3. Ecological Research
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5. Socioeconomic Environmental Studies
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This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-80-050
July 1980
THE EFFECT OF NITRILOTRIAGETIC ACID (NTA)
ON THE STRUCTURE AND FUNCTIONING OF AQUATIC
COMMUNITIES IN STREAMS
by
Thomas Bott, Ruth Patrick,
Richard Larson, and Charles Rhyne
Academy of Natural Sciences of Philadelphia
Philadelphia, Pennsylvania 19103
Grant No. R-801951
Project Officer
J. Kent Crawford
Environmental Research Laboratory - Duluth
Grosse lie, Michigan 48138
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S, ENVIRONMENTAL PROTECTION AGENCY1
DULUTH, MINNESOTA 55804
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DISCLAIMER
This report has been reviewed by the Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved
for publication. Approval does not signify that the contents
necessarily reflect the views and policies of the U.S. Environ-
mental Protection Agency, nor does mention of trade names or
commercial products constitute endorsement or recommendation for
use.
11
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FOREWORD
The Great Lakes with their large volumes and long reten-
tion times present a unique set of problems when the introduc-
tion of new chemicals is contemplated. Sometimes the substitu-
tion of one chemical for another in an effort to improve water
quality results in additional problems.
This study provides some insight into the ecological conse-
quences of discharging a substitute chemical. The problems
associated with the discharge of phosphorus have long been
documented, however, careful research is required prior to the
wide spread use of a substitute.
J. David Yount
Acting Director
Environmental Research Lab.-Duluth
111
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ABSTRACT
Studies were conducted to determine some of the effects of
nitrilotriacetic acid (NTA) on organisms characteristic of a
natural stream, measure its degradation by these organisms, and
assess some of its possible abiotic chemical reactions under
environmental conditions with particular emphasis on chelation
properties. Near-natural communities were established in micro-
cosms and ecosystem streams in a greenhouse and exposed to NTA
concentrations of 0.02 to 2 mg/1 (10~7 - 10~5 M) , a range includ-
ing and exceeding most expected environmental levels. Higher
concentrations were used in some laboratory and screening ex-
periments .
NTA at 2 and 20 mg/1 had only slight effects on algal com-
munity structure and function, and 2 mg/1 protected organisms from
the toxic effects of approximately 100 yg Cu++/l.
Protection from the toxicity of 30 yg Cu++/l with 2 mg/1 NTA
was also obtained in an experiment lasting 3 months conducted in
ecosystem streams containing natural sediments and more complex
communities. In this experiment, exposure to 2 mg NTA/1 did not
result in increased concentrations of Zn, Fe, Mn, Mg, or Cu in
algae, Anacharis, Lemna, Planaria, and Tubifex spp. Zinc concen-
trations in algae, Anacharis, and Lemna were frequently reduced.
The accumulation of added Cu by tubificids was not prevented by
NTA.
Bacterial communities adapted readily to the presence of 0.02-
20 mg NTA/1 and degraded the compound under aerobic conditions.
Glucose metabolism of non-NTA degrading bacterial communities was
protected from metal ion toxicity when Cu, Zn, Cd, Ni, Pb, and Hg
were complexed with NTA. Glucose metabolism by NTA degrading
bacteria was inhibited, however, presumably as a result of release
of Cd, Cu, and Zn ions from concomitant NTA degradation.
Substantial extraction of metals from sediments occurred at
10-3 M (200 mg/1) but not at 10~5 or 10~7 M NTA.
Although NTA was relatively resistant to chlorination, IDA,
in concentrated solution, reacted rapidly with aqueous chlorine
to produce an unstable product with oxidizing properties, pre-
sumably N-chloro-IDA. Attempts to isolate the product were not
successful.
IV
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Both NTA and IDA could be photooxidized in the presence of
a sensitizer, but the reactions were very slow.
This report was submitted in fulfillment of Grant No. 801951
to the Academy of Natural Sciences of Philadelphia under the
sponsorship of the U.S. Environmental Protection Agency. Work
was completed as of January 31, 1977.
v
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CONTENTS
Abstract iv
Figures viii
Tables xi
Acknowledgment xv
1. Conclusions and Recommendations 1
2. Introduction 4
3. Literature Review 6
4. Methods and Procedures 16
5. Results and Discussion 28
References 65
vix
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FIGURES
Number
1 Structures of NTA (R = CH2COOH) and some related
compounds: IDA, R = H: EDTA, R = N(CH2COOH)2:
MIDA, R = CH3 6
2 Chelates of calcium with tripolyphosphate and
nitrilotriacetate anions 7
3 NTA analytical methods — ranges and limitations . 8
4 GC separation of butyl ester of twenty-five
carboxylic acids 72
5 Standard curve for NTA gas chromatographic
analysis 73
6 Effect of NTA on solubilization of copper from
three stream sediments ..... 74
7 Effect of NTA on solubilization of iron from
three stream sediments 75
8 Effect of NTA on solubilization of manganese from
three stream sediments 76
9 Effect of NTA on solubilization of zinc from three
stream sediments 77
10 Mineralization (14CO2 evolution) of NTA by cell
suspensions of a Pseudomonas sp. known to
degrade NTA 78
11 Semi-log plot of NTA degradation rate (x + s.d.)
by bacterial populations at 9OC 79
12 Semi-log plot of number (x + s.d.) of NTA
degrading bacteria on coverslips exposed to
NTA at the indicated concentrations 80
viii
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FIGURES (cont'd)
Number page
13 Semi-log plot of NTA degradation rate (x + s.d.)
by bacterial populations at 18°C 81
14 Semi-log plot of number of NTA degrading bacteria
(x + s.d.) on coverslips exposed to NTA at
approximately the indicated concentrations .... 82
ISA Changing concentrations of NH3-N, NC^-N, and
15B NO3~N resulting from the degradation of NTA
15C in 87 1 microcosms 83
16 Changing concentrations of C-NTA in an 87
1 microcosm system resulting from dilution
(Theoretical curve) and both dilution and
bacterial decomposition (Empirical curve) .... 85
17 Incorporation of C glucose by washed cells from
a culture of naturally occurring bacteria in
the presence of added metal ions or NTA-metal
chelates 86
18 Incorporation (I = O •, open = NTA exposed, closed
= control) and mineralization (M = A A, open =
NTA exposed, closed = control) of 14C glucose
by suspensions of washed cells harvested from
cultures of mixed natural populations 87
19 Concentration of copper (x + s.d.) in algal com-
munities from ecosystem streams through time ... 88
20 Concentration of iron (x + s.d.) in algal com-
munities from ecosystem stream through time ... 89
21 Concentration of zinc (x + s.d.) in algal com-
munities from ecosystem streams through time ... 90
22 Concentration of manganese (x + s.d.) in algal
communities from ecosystem streams through
time ; . 91
23 Concentration of magnesium (x -^ s.d.) in algal
communities from ecosystem streams through
time 92
24 Concentration of copper (x + s.d.) in Anacharis
from ecosystem streams through time ~. '. I '. T . . 93
IX
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FIGURES (cont'd)
Number Page
25 Concentration of iron (x + s.d.) in Anacharis from
ecosystem streams through time 94
26 Concentration of zinc (x + s.d.) in Anacharis
from ecosystem streams through time 95
27 Concentration of manganese (x + s.d.) in Anacharis
from ecosystem streams through time 96
28 Concentration of magnesium (x + s.d.) in Anacharis
from ecosystem streams through time 97
29 Concentration of copper (x + s.d.) in Lemna from
ecosystem streams through time 98
30 Concentration of iron (x + s.d.) in Lemna from
ecosystem streams through time 99
31 Concentration of zinc (x + s.d.) in Lemna from
ecosystem streams through time 100
32 Concentration of manganese (x + s.d.) in Lemna
from ecosystem streams through time 101
33 Concentration of magnesium (x + s.d.) in Lemna.
from ecosystem streams through time 102
34 Concentration of copper (x + s.d.) in Planaria
from ecosystem streams through time 103
35 Concentration of iron (x + s.d.) in Planaria
from ecosystem streams through time 104
36 Concentration of zinc (X + s.d.) in Planaria
from ecosystem streams through time 105
37 Concentration of manganese (x + s.d.) in Planaria
from ecosystem streams through time 106
38 Concentration of magnesium (x + s.d.) in Planaria
from ecosystem streams through time 107
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TABLES
Number Page
1 Frequency of determination of water chemical
parameters during algae experiments 108
2 Relative retention times (RRT) and relative
molar responses (RMR) of n-butylesters of
twenty-five carboxylic acids 109
3 Evaluation of GC analytical procedure using
carboxyl-labeled 14C-NTA 110
4 Chelation of metals from sediments by 10~3 M
NTA (191 ppm) HI
5 NTA determinations for the period November 14,
1974 - February 13, 1975 112
6 NTA determinations for July 4 - September 2,
1975 experiment 113
7 Water chemistries for the test chambers
(November 14, 1974 - February 13, 1975) 114
8 Water chemistries for the experimental boxes
(June 20, 1975 - September 3, 1975) 115
9 Metals in biomass (mg cation/g Dry Weight)
November 1974 - February 1975) 116
10 Metals in algal biomass - mg cation/gm
July - August 1975 117
11 Slide observations, November 14 - December 10,
1974 - End of seeding 118
12 Biomass, chlorophyll and ^4C determination
(November 14, 1974 to February 13, 1975) 122
13 Detailed analysis of diatom populations by the
truncated normal curve technique 123
XI
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TABLES (cont'd)
Number Page
14 Distribution of the more common diatom species . . .
15 Microscopic examination of communities .......
16 Analysis of diatom population using truncated
normal curve factors ............... 126
17 Distribution of the more common diatom species ...
18 Effects of NTA on algal communities ........ I28
19 Water chemistries for the experimental boxes
(March 14 - April 15, 1975) ........... 129
20 Determinations for NTA and Cu during the period
March 12 - April 15, 1975 ............ 13°
21 Microscopic examination of communities
22 Detailed analysis of diatom populations by the
truncated normal curve technique ......... 132
23 Distribution of the more common species ...... 133
24 Biomass, chlorophyll and C determination ..... 134
25 Metals in algal biomass (mg cation/g) March -
April 1975 ........... ......... 135
26 NTA concentration in microcosms March 12 -
April 24, 1975 .................. 136
27 NTA degradation by bacterial populations exposed
to different NTA concentrations for four
weeks ...................... 137
28 NTA concentration in microcosms July 2 -
August 14, 1975 ................. 138
29 Effect of cadmium, zinc, lead, and nickel ions
and NTA-chelates of the same on the metabolism
of 14C glucose by heterotrophic bacteria from
White Clay Creek ................. 139
30 Effect of copper ions and copper-NTA chelates on
the metabolism of 14C glucose by heterotrophic
bacteria from White Clay Creek .......... 14°
xn
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TABLES (cont'd)
Number Page
31 Effect of 4 mg/1 NTA (probably present as Ca or Fe
chelates) on metabolism of ^C glucose by
heterotrophic bacteria from White Clay Creek .... 141
32 Effect of cadmium, zinc, and copper ions and NTA
chelates of the same on metabolism of 14C
glucose by NTA degrading isolates 142
33 Effect of copper, cadmium, zinc, and mercury ions
and NTA-metal chelates on microbial metabolism . . . 143
34 Concentration of selected chemical species in
ecosystem stream water; July 13-October 6, 1976 . . . 144
35 Concentrations of NTA and copper in ecosystem
stream water; July 13-October 6, 1975 145
36 Comparison of concentration of Cu, Fe, Zn, Mn and
Mg in algal biomass from ecosystem streams at a
given sampling time 146
37 Comparison of concentration of Cu, Fe, Zn, Mn and
Mg in Anacharis from ecosystem streams at a
given sampling time 147
38 Comparison of concentration of Cu, Fe, Zn, Mn and
Mg in Lemna from ecosystem streams at a given
sampling time 148
39 Comparison of concentration of Cu, Fe, Zn, Mn and
Mg in Planaria from ecosystem streams at a
given sampling time 149
40 Grand mean of metal concentrations in Tubificid
worms from collections 2-8 150
41 Cumulative mean concentration of metal ions in the
water in ecosystem streams; August 11 -
October 6, 1976 151
42 Concentration factors (metals in biomass of
indicated samples/metal in water) 152
43 Concentration factors (metal in Planaria/metal
in algae) 153
44 Concentrations of copper and NTA in microcosms
for sediment studies 154
xiii
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TABLES (cont'd)
Number Page
• . ••! - -—. • ! - "*
45 Concentration of copper (ng/g) in sediments
exposed to copper or copper/NTA 155
46 Concentration of iron, manganese, and zinc
(all in yg/g) in sediments on 12/17 after
65 days exposure to copper or copper/NTA 156
xiv
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ACKNOWLEDGMENTS
This project could not have been executed without the
diligent assistance of many co-workers whom we gratefully acknowl-
edge: Dr. R. L. Brunker, J. S. Coles, J. A. Finlay, S. M. Howell,
J. Peirson, S. Roberts, A. L. Rockwell, W. Shaw, E. Siekmann,
P. 0. Stabler, L. L. Wash and J. C. Weston.
The research was made possible by grant #801951 from the
Environmental Protection Agency. We acknowledge with thanks the
constructive help of our project officers, Dr. Donald Mount and
Dr. Kent Crawford.
xv
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SECTION 1
CONCLUSIONS AND RECOMMENDATIONS
The purpose of this study was to determine the effects of NTA
on organisms in streams, and on heavy metals in sediments of the
stream bed. The effects of chlorination on NTA and IDA were also
examined. To accomplish this purpose, we examined effects on the
metabolism of bacteria and algae. We also studied the effects of
NTA on the uptake of heavy metals by organisms living in the sedi-
ments and in the water of a flowing stream.
The experiments were conducted in flowing water microcosms
and ecosystem streams in which many natural conditions were
reproduced. Ecological aspects of NTA exposure were emphasized.
NTA concentrations ranging from 0.02 - 200 mg/1 (10~7 - 10~3 M)
were studied. Usually, concentrations that might be found in
natural habitats (0.02 - 2.00 mg/1) were used, but higher levels
were occasionally used to verify that experimental conditions were
appropriate when effects were not evident at lower concentrations.
Unless other metal ions were deliberately added, dissolved NTA in
these experiments was principally present as its calcium and iron
complexes.
A comparison of various methods of analyses of NTA was made.
It was found that below 2 ppm the Zinc-Zincon method produced
results that averaged somewhat higher than gas chromatographic
results. The mathematical relationship between the two methods
was: mg/1 NTA (zinc - zincon) = 1.036 x mg/1 NTA (g.c.) + 0.356
(R = 0.962).
The presence of 10~ M (1.9 mg/1) or less NTA did not lead
to significantly higher concentrations of metal in water. At
10~3 M concentration of NTA (191 mg/1) there were significant
increases in the water (3x - 15x). At 10"3 M IDA the concentra-
tion of Fe, Zn, and Cu were significantly higher in the water.
In the presence of sewage inoculum, Cu and Zn concentrations
dropped in 10~3 M NTA experiments.
Experiments with chlorinating water containing NTA indicated
that even if N-chloro-IDA was formed it would not persist, as
acidic and basic pathways are available for its degradation.
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Natural bacterial communities degraded NTA under aerobic
conditions at different seasons of the year. During the course
of experiment, populations became acclimatized, and the degrada-
tion of NTA was directly correlated with temperature, length
of exposure time, and concentration of NTA. NTA degradation
under anaerobic conditions has been demonstrated in other studies
(See Literature Review). The redox potential of the sediment
may be an important determinant as to whether this will occur and
additional investigations should address this correlation.
NTA degradation under aerobic conditions resulted in an
increase in NH4-N followed by bacterial oxidation of the
ammonium-N to NO2-N. Nitrate-N concentration increases were not
detected in our system when NTA was present at environmentally
realistic NTA levels, because NO3-N in the stream water was
already 3.0 mg/1, but in other environments the nitrate concen-
tration may increase. The extent to which NTA usage might
accelerate eutrophication through nitrogen additions has been
considered minimal by other workers (See Literature Review) and
has to be weighed against additions of other algal nutrients when
alternative non-NTA containing compounds are used. Studies of
possible effects on detritus production should be considered in
future work since nitrogen supply may affect processing.
NTA had no overt effect on the metabolism of some other
organic compounds (glucose, amino acids) by natural bacterial
populations. NTA protected bacterial metabolism from concentra-
tions of copper, cadmium, zinc, lead, and mercury ions that were
otherwise toxic to varying degrees. However, studies with NTA-
degrading isolates suggested that free ions of zinc, cadmium and
copper could be released in sufficient amounts from concomitant
NTA-chelate degradation to inhibit glucose metabolism under the
conditions of the test. These populations were not specifically
acclimatized to the heavy metals tested and this may be an impor-
tant variable. In nature, sediments or other particulate and
dissolved organic compounds may offer alternative binding sites
for released metals but if inhibition of bacterial activity
should occur, the self-purification capacity of a system would
be impeded. This potential problem should be considered in future
evaluations of environmental effects of NTA.
The results of the algal community experiments indicate that
NTA in concentrations of 2 mg/1 and 20 mg/1 had no adverse effects
on the structure of the diatom communities, and there was no
significant change in the amount of the green algae Stigeoclonium
lubricum or of blue-green algae. The results of the June-
September and November-February were similar except that in the
20 mg/1 experiments of June-September the ash free dry weight was
somewhat less than in the controls. Since 2 mg/1 and 20 mg/1
NTA are higher than present literature indicates occur in natural
bodies of water that have received NTA over considerable periods
of time, these experiments indicate that no significant changes
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would occur in the algal communities at these or lower concen-
trations.
In experiments adding 2 mg/1 NTA with and without the addi-
tion of 30 yg/1 copper, the results indicated that the NTA
chelated the copper both naturally occurring and added, so that
it did not accumulate significantly in the algal cells, and that
their growth and 14c uptake were similar to the controls. In
those experiments with only 30 yg/1 of copper, the algal cells
died.
In a three-month experiment conducted in ecosystem streams
containing natural sediments, 2 mg NTA/1 exhibited no acute or
chronic toxicity to natural communities of algae, two aquatic
plants (Lemna and Anacharis), or the worm genera Planaria and
Tubifex.This NTA concentration also protected organisms from
the toxic effects of 30 yg Cu/1 when added simultaneously. From
macroscopic observations, there were no significant shifts in the
structure of the communities and the species composing them in the
control, NTA, and NTA and copper experiment. Where 30 yg/1 Cu was
added, Anacharis and algae died, but a population of Ulothrix re-
colonized the stream after three weeks. Concentrations of Cu, Zn,
Mn, Mg, or Fe in the tissues of these organisms did not increase
significantly as a consequence of NTA exposure. In fact, the con-
centration of zinc in the biomass of algae, Anacharis and Lemna
tended to be lower when NTA was present, suggesting that NTA-
chelated zinc might be less available to these organisms. Study
in other habitats is necessary to test the generality of this ob-
servation since chelation equilibria depend on the relative con-
centrations of dissolved metal ions and on competition from sorptive
surfaces and other chelating substances, such as naturally occurring
humic materials and secretions from organisms. The presence of NTA
did not reduce the accumulation of copper added to the water column
by sediments; nor did the presence of NTA prevent the accumulation
of added copper by sediment-dwelling tubificid worms.
We have addressed some of the environmental aspects of NTA
usage in this work. The environmental consequences of moderate
NTA usage do not appear to be detrimental. If one wishes to com-
pare the ecosystem effects of NTA with other commercial formula-
tions thorough investigations in systems of different types are
needed. Although some evidence may be gleaned from existing moni-
toring studies in countries where NTA usage is greater than in the
United States, this should not substitute for additional compara-
tive investigations in large scale experimental ecosystems (such
as those used in this study) over long periods or detailed environ-
mental monitoring if greater usage of NTA occurs in this country.
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SECTION 2
INTRODUCTION
The purposes of these experiments were several. One was to
determine the effects on selected freshwater organisms of NTA,
some of its degradation products, and products that might be formed
by the chlorination of NTA. Second was to determine the effects
of NTA on the release of metals from sediments. Third was to
study in a stream ecosystem the effects of NTA on metabolic pro-
cesses of organisms representing some of the stages of energy
transfer in the food web. These studies included its effect on
the uptake of Cu.
Nitrilotriacetic acid (NTA) has been used in industrial pro-
cesses, agriculture, and in some countries in detergent formula-
tions. At the time this program of work was initiated, experi-
ments had been carried out to determine the chemical characteris-
tics of NTA, its degradation (with particular emphasis on
biochemical pathways of selected isolates and rates of decomposi-
tion in sewage treatment facilities), and its acute toxicity had
been reported or was being investigated (See Literature Review).
Little, however, was known of its fate in receiving waters or
the effects on natural communities of chronic exposure. The
studies reported here were conducted to provide information con-
cerning the possible effects of NTA on aquatic communities in
streams when conditions of exposure simulated nature in many
respects. Special attention was also given to the chelation and
possible mobilization of metals by NTA.
It has been predicted that NTA will be present in many natu-
ral environments at low concentrations if the compound is widely
used. Although estimates of discharge from sewage treatment
facilities have ranged in concentration from 1-20 mg/1, most
reported values fall at the lower end of this range. With a 10-
1000 fold or more dilution in the receiving body of water, con-
centrations greater than .02 - 2 mg/1 would seldom occur. The use
of concentrations from .02-2 mg/1 seemed reasonable for studying
the effects in natural bodies of water.
The microbial components of a community are frequently most
responsive to changes in water chemistry. Therefore, in evalu-
ating the environmental effects of NTA in this program, emphasis
was placed on the algal and bacterial populations.
In many aquatic systems, algae are the important primary pro-
ducer organisms, although aquatic macrophytes may assume local
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importance. Algae require for growth macronutrients such as carbon
dioxide, nitrate-N, ammonia-N, phosphates, some metals (Ca, Mg,
K) and a host of organic and inorganic micronutrients such as
vitamins and trace metals. Individual species have exacting re-
quirements for these trace substances. The biomass of algae pro-
duced may be primarily dependent on the supply of macronutrients
but the species of the algae found in a given system may be related
primarily to the supply of micronutrients. For example, Patrick,
Crum, and Coles (48) and Patrick, Bott, Larson (47) have shown
that manganese, vanadium, nickel, and selenium at various concen-
trations altered the dominant algae in a community by fostering the
development either of diatoms or blue-green algae. Shifts in algal
communities brought about by the presence of organic substances
have been shown by Rice (56), Proctor (53) and Keating (35).
Changes in micronutrient concentrations may bring about the
development of communities of algae that are desirable or undesir-
able food species. In the former instance, efficient transfer of
energy from primary producers to higher forms will be enhanced; in
the latter instance, nuisance growths ("algal blooms") may accumu-
late. Many observations indicate that diatoms and some unicellular
green algae are desirable food sources whereas filamentous greens
and blue-green algae are not as desirable food sources (29,46).
In contrast to many other investigations in which the response
of organisms to NTA has been studied in single species laboratory
experiments of relatively short duration, the experiments reported
here were designed to investigate the effects of chronic exposure
of natural communities to the compound. The emphasis in all
instances was on the ecological aspects of exposure rather than on
the more detailed physiological or biochemical questions concerning
exposure. The following aspects were selected for our study:
1. The effects of NTA and NTA-heavy metal chelates on the
community structure of algae and primary productivity.
2. The effects of NTA and NTA-metal chelates on the miner-
alization activity of natural bacterial communities.
3. The degradation of NTA by natural bacterial communities.
4. The behavior of NTA, copper, and NTA-chelated copper in
experimental ecosystem streams with particular emphasis
on the fates of the various compounds, the mobilization
of metals from sediments and the identification of sinks
for metal ions.
Experiments under conditions as natural as possible (con-
tinuous flow, natural light and temperature regimes) and with
natural communities, but in which many variables were controlled
and monitored closely were performed to study the effects of NTA
and NTA + metal ions at different seasons of the year.
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SECTION 3
LITERATURE REVIEW
CHEMICAL PROPERTIES
Nitrilotriacetic acid (NTA, Fig. 1 ) is a synthetic amino
acid having a tertiary nitrogen atom. Its commercial synthesis
affords the trisodium salt monohydrate in a purity of 98%; the
product also contains small amounts of the related compound imino-
diacetic acid (IDA, Fig. 1 ). In the free acid form, NTA ionizes
with successive loss of three protons; the reactions have pKa's
of 1.92, 2.38 and 9.95. Thus, at all pH's between 3.4 and 9.0,
the NTA dianion predominates (42).
HOOGCH,^..^GH2GOOH
N
I
R
Fig. 1. Structures of NTA (R = CI^COOH) and some related com-
pounds: IDA, R = H: EDTA, R = N(CH2COOH)2: MIDA, R=CH3<
Salts of NTA have extremely high water solubility and capac-
ity for metal ion chelation. These properties (among others) have
made NTA one of the prime candidates for introduction as a
phosphate-replacing builder in detergents. Formulations con-
taining NTA were, in fact, available in some parts of the United
States during 1967-1970, but industry withdrew the products
following expressions of concern over possible health and environ-
mental hazards. Nevertheless, NTA continues to be used for this
purpose in several other countries.
Detergent builders are used to maintain in solution alkaline-
earth cations (e.g. Mg++, Ca++), which otherwise form insoluble
curdy precipitates during the washing process. Tripolyphosphate
builders form soluble, chemically stable chelates with three sites
of coordination, whereas, chelates of NTA are tetrahedrally coor-
dinated (Fig. 2).
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o
Fig. 2. Chelates of calcium with tripolyphosphate and nitrilo-
triacetate anions.
Equilibrium constants for the reactions of a large number of
metal ions with NTA have been tabulated by Sillen and Martell (63).
For all di- and trivalent cations studied, these reactions are
very favorable, with equilibrium constants ranging from 6.3 x 104
(Ba++) to 1013 (Cu++). Accordingly, if the rate of metal complex
formation is rapid, the level of uncomplexed NTA in almost all
natural waters would be expected to be infinitesimal. The rela-
tive amounts of chelated NTA species at equilibrium would depend
on the aqueous concentrations of the metal ions (Cu++, Fe+3, Pb++,
Ni++, etc.) which form particularly stable complexes. Because
the rates of formation of the complexes have been shown to be
very fast, predictions based on equilibria probably have environ-
mental usefulness. Childs (14) has calculated that, in Lake
Ontario, the copper complex would predominate at equilibrium at
concentrations of 1.5 mg NTA/1 or less.
ANALYSIS OF NTA
Many methods of NTA analysis have been described. At rela-
tively high concentrations, the colorimetric zinc-Zincon (77)
method has found wide use. Below about 0.2 mg/1, polarographic
(1) and gas chromatographic (6) methods appear most applicable.
In addition, assays based on chemical kinetics (16), chelation
(36), fluorescence (57), and ion-selective electrodes (73) have
appeared, but none of these has been widely tested. The claimed
sensitivity range for each method is summarized in Fig. 3. Some
possible interferences in analysis of waterborne NTA are:
a) Polyvalent metal ions may complex NTA.
b) Natural chelating agents (polycarboxylic acids,
polyphenols, amino acids) may interfere in methods
based on chelation. In addition, IDA and other
possible degradation products have chelating ability.
-------
c) Oxidizable or reducible substances may interfere
in polarographic methods.
d) Chromatographic methods must achieve essentially
complete resolution of the NTA derivative from
interfering substances.
e) Fluorescent, quenching, or colored substances
may interfere with fluorometric or colorimetric
determinations.
Analysis of NTA chelates has received little attention. A
thin-layer chromatographic (TLC) method has been reported; it
requires a minimum of 500 ng of the chelate, in relatively concen-
trated solution (55).
mgl'l
0.0001
0.001
0.01
0.1
10
100
, fluor*
1 (tap \
l gas chroi
\
sscence
rater)
L
r
latography
, polarogrc
1
, kinetic
1 analysis
gas chrome
- no pret
with preti
phic me the
1 zinc-Zii
colorinu
• ion-se!
' electr<
tography
reatment
eatment
ds
icon
stry
.ective i
>de •
Fig. 3. NTA analytical methods — ranges and limitations.
8
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CHEMICAL AND BIOCHEMICAL REACTIONS
Oxidation Reactions
The reactions of NTA are controlled by its high polarity and
its chelating properties. Although it is a compound having con-
siderable intrinsic (thermodynamic) stability, NTA (because of
its similarity to natural amino acids) would be expected to be
relatively susceptible to environmental decomposition reactions
such as photooxidation or metabolic transformations by living
organisms. The literature indicates that this is indeed the
case. Rather harsh conditions are required to effect thermal
oxidation of NTA. In the presence of a palladium catalyst and an
oxygen atmosphere at 90°C, it can be converted in 92% yield to
IDA (74). However, photooxidation of some NTA complexes occurs
readily. Copper or iron complexes of NTA, when exposed to long
wavelength ultraviolet light in the laboratory (37) or summer sun-
light (66) were oxidatively transformed to IDA, formaldehyde, and
C02. The complexes of IDA were relatively stable to further
photochemical destruction. Ligand-to-metal charge transfer com-
plexes appeared to be involved; NTA chelates of some other metal
ions (Pb**, Cd++, Mg++) were not significantly degraded.
Chelation of Metals from Sediments
It has been suggested that NTA may sequester toxic or nutri-
ent metal ions from sediments or suspended material and possibly
facilitate the transport of such metals through biological mem-
branes. Conversely, it is conceivable that NTA could protect
aquatic organisms from the toxic effects of free metal ions, or
deprive them of required metallic nutrients.
Several investigations (8,13,30,50,72) of the interaction of
NTA with natural sediments, employing NTA at concentrations
ranging from 0.2 to 20 mg/1, have been reported in the literature.
In several of the studies, the sediments were shaken with the NTA
solutions. It might be argued that such a design lacks environ-
mental relevance, and data from the studies do not agree fully.
Nevertheless, certain trends may be noted. Significant increased
solubilization of zinc was observed by three research groups; (72,
13,8) iron likewise appeared to be solubilized (8,13,50) although
one report indicated no increased solubility (72). Manganese (72,
50), lead (30), and nickel (72) concentrations may or may not be
increased by NTA. There is general agreement that levels of
cobalt, chromium, copper, and several other metal ions were not
significantly increased by NTA at the concentrations studied.
Interestingly, polyphosphate appeared to be about as effi-
cient as NTA at mobilizing Fe, Zn, and Cu from sediments; EDTA
(Fig. l.'l was superior to both (8). Allen and Boonlayangoor (3)
concluded that at 0.75 mg/1 of NTA the concentration of no metal
would increase more than 10% in the rivers studied. Since this
concentration of NTA is much higher than 0.05 mg/1 found in 95%
of the Canadian rivers studied (83) they concluded that metal con-
centration would not measurably increase with .02 mg/1 NTA.
-------
Biodegradation
Since the early report of Forsberg and Lundquist (27) of
bacteria capable of using NTA as a carbon and nitrogen source, it
has been shown repeatedly that NTA is biodegradable to the inor-
ganic end products CO2 and NH4+. In pure culture, organic
intermediates in the metabolism of NTA have been shown, but these
have not been identified in the environment. A few biochemical
studies with bacteria (primarily Pseudomonas species) in pure
culture have been done to determine degradation pathways. The
evidence suggests that NTA was oxidatively metabolized, initially
with cleavage of a C-N bond, to IDA and a two-carbon fragment,
probably glyoxylic acid (17,26,79). The isolates studied have
been shown to oxidize the compound rapidly, reducing the concen-
tration from 290,000 yg/1 to <50 yg/1 in 45 min. in one instance.
Some NTA-degrading isolates did not metabolize IDA (17), while
others did (26).
As noted above, NTA in solution rarely exists as the free
acid, but rather as metal chelates. Concern has been expressed as
to whether the heavy metal chelates of NTA could be readily
degraded. Firestone and Tiedje (23) reported a series of experi-
ments with an NTA degrading Pseudomonas sp. and found the organism
was capable of degrading the Ca, Mn, Mg, Cu, Ni, Cd, Fe, and Na
chelates at approximately the same rates if the concentration of
freed metal did not become toxic. If this occurred, the addition
of soil allowed continued degradation, presumably by binding free
metals. The nickel chelate, however, was not degraded. However,
as will be discussed below, degradation of the nickel chelate in
some experiments with mixed populations in soil or water has been
observed. The authors hypothesized that in chelate degradation
the metal was not transported into the cell with NTA but rather
was disassociated at the cell surface.
Although NTA degradation was thought originally to occur only
under aerobic conditions, there are recent reports of anaerobic
degradation. Enfors and Molin (20,21) reported the isolation of
a facultatively anaerobic bacterium capable of metabolizing NTA
under anaerobic conditions if nitrate was present in the medium.
Further possible reactions of IDA have been considered by
several authors (17,51,75,82). Because it is a secondary amine,
it may react by pathways unavailable to NTA. The possible forma-
tion of N-nitroso IDA by the reaction of NTA with nitrite ion has
(HOOGCH2)2NH+NO;
10
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attracted attention because of the known carcinogenicitv of many
nitrosamines. N-nitroso IDA was shown to be not degraded in river
water within 52 days at concentrations of 5 of 20 ma/I (82). The
compound has not been detected in the environment, although mixed
soil microorganisms in culture have been reported to synthesize it
from NTA and nitrate (51).
CONCENTRATION AND DECOMPOSITION IN THE ENVIRONMENT
Thayer and Kensler have reviewed monitoring studies of NTA in
tap waters, sewage influent and effluent, and receiving streams
(75). Analysis of 165 samples of Canadian tap water for NTA gave
a mean concentration of 0.006 mg/1; most of the samples had unde-
tectably low concentrations. The data indicate that although
inputs of NTA to sewage plants were relatively high (2-118 mg/1),
considerable degradation occurred within the plant; reported
effluent concentrations did not exceed 4 mg/1 NTA, with usual
values in the 1-2 mg/1 range.
Following the introduction of NTA into detergent formulations
in Canada (initially at 6%, later at 15%) the NTA content of
effluent from primary, trickling filter and activated sludge sewage
treatment plants and of receiving streams was monitored (83) . Con-
centrations in sewage effluent ranged from 0.011 - 10.50 mg/1
(geometric means for primary, trickling filter, and activated
sludge plants were 2.98, 3.22, and 0.60 mg/1 respectively at 15%
NTA content). In receiving streams concentrations ranged from
undetectable to 3.36 mg/1 (the median concentration was 0.05 mg/1
and 97% of all samples contained <0.05 mg/1. Several positive
correlations between metal ion and NTA concentration were observed
but the authors considered these to be the results of other factors
because the total concentration of chelatable metals exceeded NTA
concentration. However, it is noteworthy that the metals involved
were frequently those for which NT£ has hiah affinitv (Cu, Cr, Zn,
and Ni).
Although methods differed, several research groups (12, 50, 69,
77) reported essentially complete degradation in laboratory experi-
ments with activated sludge after acclimatization periods of one
to three weeks. In one studv (12) primary effluent was added as
well as NTA. Following acclimatization 80-85% reduction occurred
at 20°C but only 25% removal took place at 5°C. Different popula-
tions were active at the two temperatures; when the temperature of
the system held at 5°C was elevated to 20°C negligible degradation
occurred until a new population developed.
Shumate, et al. (62) performed a field study at a treatment
plant and reported~~90% removal of 8 mg/1 NTA added to influent
waste and 75% removal when the feed concentration was 16 mg/1 NTA.
Because of variability, it was difficult to assess any seasonal
effects on removal efficiency. Rudd and Hamilton (58) studied
degradation in a model aerated sewage lagoon using an influent
concentration of 14 mg/1 NTA. At 15°C the efficiency of degrada-
tion was 93%; at 5°C, 47%; and at 0.5°C, 22%. In cold weather
-------
(<5°C), 50% or more of incoming NTA might be discharged into the
receiving body of water. Similar data were reported for another
treatment plant; 95% removal in summer, <50% in winter (61).
Further decomposition and dilution will occur in rivers and
most samples taken downstream of NTA-using communities contain
undetectable amounts, with only a few reports of levels exceeding
0.1 mg/1. Warren and Malec (82) used gas chromatography to detect
the degradation of NTA and postulated intermediates added to
unacclimatized water from the Detroit and Meramec Rivers. Degra-
dation of NTA was complete, and intermediates could not be detected.
Only N-nitroso-IDA was not degraded; N-methyl IDA, IDA and sarco-
sine were all degraded, although N-methyl IDA degraded more slowly.
Swisher, et al. (70) studied the biodegradation of 5 mg NTA added
to river water with metal ions added to provide 0.1, 0.3, and 1.0
equivalents. The chelates of Fe, Pb, Cd, Ni, Cu, and Zn were
degraded following lag times of varying duration. Others (10,31,81)
have reported degradation of Ni, Cu, Cd, and Cr chelates to be
inhibited. Tiedje and Mason (78) , however, found that the NTA
chelates of Cu, Ca, Fe, Mn, Zn, Pb and Ni, were degraded rapidly
when added to soil; but the Hg and Cd chelates were degraded more
slowly. Recently, degradation in a receiving stream in Ontario
(61) was studied throughout the year, including periods when stream
temperature was less than 3°C. Under winter conditions, NTA con-
centrations as high as 0.125 mg/1 were found about 0.8 km down-
stream from the input, compared to summer levels of 0.01 mg/1 or
less. Evidence was obtained for biodegradation in the receiving
stream at low temperatures, albeit at reduced rates.
The degradation rate of NTA in soils has been related directly
to organic content of the soil, temperature, and NTA concentration
(78). Degradation occurred under aerobic conditions, was greatly
slowed under microaerophilic conditions, and was non-existent under
anaerobic conditions (argon purged systems). However, degradation
on re-exposure to oxygen was not impeded by the imposed anaerobiosis
(78). Tabatabai and Bremner (71), however, have reported
degradation in soils sparged with He gas.
Thus, the degradation rate in any environment will be depen-
dent on environmental conditions such as temperature, chemical
milieu, degree of aeration, and the rate of acclimatization and
fluctuation of populations.
EFFECTS OF NTA ON ORGANISMS
As will be discussed below, NTA has been screened for possible
toxicity against a variety of aquatic organisms including algae,
invertebrates, and fish. Most reported studies have been 96 hr.
bioassays for acute toxicity. Possible stimulation of algal growth
has also been tested in a few instances. The molecule has been
generally shown to be non-toxic to the organisms tested except at
concentrations greatly exceeding those expected in natural habitats.
Studies with all groups of organisms have demonstrated an^inverse
relationship between water hardness and toxicity. NTA complexes
appear not to be acutely toxic and the compound was only toxic when
-------
present in molar excess of compleking cations. This may strongly
imply that NTA toxicity is rare in nature at ordinarily expected
concentrations. It should be noted that studies rarely included
newly hatched or young organisms — stages that are particularly
sensitive to toxicants.
Bacteria
In one study, growth of a strain of the bacterium, Escher-
ichia coli K 12 that did not metabolize NTA was affected only by
concentrations above 5% NTA (50,000 mg/1), but between 0.5-5.0%
altered cell morphology was noted. NTA exerted no mutagenic
effects. Yellow pigmentation was noted when strains were grown on
agar containing 50 mg/1 NTA. Recombinant formation was inhibited,
but only when cells were grown in NTA prior to mating (65).
Algae
Work has been done with a limited number of species in culture
but studies of effects on algal communities over time are lacking.
Christie (15) noted no toxicity to Chiprella pyrenoidosa even at
275 mg/1. Sturm and Payne (67) found the 96 hr. TL5Q for Navicula
seminulum to be 185 mg/1 in water with a hardness of 60 mg/1 CaCO3
and 477 mg/1 in water with 170 mg/1 hardness. They also tested the
effect of NTA on Selenastrum capricornutum in Algal Assay Procedure
(AAP) bioassays. NTA at 5 mg/1 had no biostimulatory effect when
added alone or with sewage effluent to water from eight U.S. lakes
including some with very soft water (6.34 mg/1 CaC03, lowest
reported hardness value). Slight inhibition was noted in one
instance. Similar results were obtained by these workers with
Anabaena flos-aquae and Microcystis aeruginosa. NTA addition
slowed the rate of or prevented Microcystis die-off, probably by
chelating metals present at toxic levels in some waters.
Otherg(22) investigated the effect of NTA on growth of
Cyclotella nana in 72 hr. laboratory studies. In natural seawater,
concentrations of 1.0, 2.5, and 5.0 mg/1 NTA had an inhibitory
effect on cell yield and l^C bicarbonate incorporation but in
synthetic seawater 20 mg/1 NTA had no effect. The authors attri-
bute this to the more abundant concentrations of trace metals in
the synthetic medium and reiterate the finding of Provasoli (54)
that a chelator: trace metal ratio exceeding 3:1 may cause a trace
metal deficiency. The toxic effects of 50 yg/1 copper added to
natural seawater were nullified by the addition of 0.5 mg/1 NTA.
NTA (10 mg/1) added to enriched seawater medium has been re-
ported to stimulate growth of the red tide dinoflagellate, Gony-
aulax tamerensis (84). Photosynthesis was also stimulated in
several tests but to varying degrees. No effect was noted on the
growth or photosynthesis of Phaeodactylum tricornutum or natural
populations dominated by Skeletonema and Rhizpsolenia, but testing
was not as extensive. Martin (43) cautions that these observa-
tions were obtained under laboratory conditions and may not be
applicable to the natural environment. In other studies (18) , no
stimulation of the Florida red tide organism Gymnodinium breve by
NTA was observed although concentrations up to 10 mg/1 were non-toxic.
a- <3
-------
Christie (15) noted that NTA could serve as the sole nitro-
gen source for Chlorella but was not as effective as equivalent
amounts of NO^-N, but others (28) have reported that NTA could not
serve as a sole nitrogen source for Ankistrodesmus falcatus or
S_. Capricornutum. These observations are pertinent to the sug-
gestion made by some that nutrient enrichment of surface waters
(in particular those in estuaries) with nitrogen from NTA may
lead to nuisance growths. Estimates have been made that the
use of 10% NTA in detergent formulatibns could conceivably increase
the N and C content of sewage effluents by no more than 0.7 and
0.8 percent respectively (67) and that N concentrations in sur-
face waters might be increased 0.35 percent (75). Sturm and
Payne (67) and Forsberg and Wiberg (28) found no stimulation
of S_. capricornutum growth when either NC^-N or bicarbonate-C
were added alone at levels expected from the degradation of
5 mg/1 NTA.
Invertebrates
Flannagan (24) tested the effects of Na3NTA on seventeen
species of macroinvertebrates (Chironomidae, Trichoptera,
Ephemeroptera, Odonata, Amphipoda, and Gastropoda) and two
amphibians (larval salamanders and leopard frog tadpoles) in
96 hr. bioassay tests. Results indicated that the compound was
not directly toxic up to 500 mg/1 in both soft (0-20 mg/1 total
dissolved solids) and hard waters (170 mg/1 total dissolved
solids). Where toxicity was observed, it was attributed to pH
increases in unbuffered waters. The author points out, however,
that sewage effluents tend to be buffered and the pH changes
should be minimal. Arthur, et al. (5) also attributed acute
toxicity of NTA to the amphipod Gammarus pseudolimnaeus Bousfield
to increased pH. The chronic toxicity of NTA to G. pseudolimnaeus
was also tested and a "no-effect" level" of 19 mg/1 was reported.
Biesinger, et al. (9) tested NTA and NTA-metal complexes for
chronic toxicity to Daphnia magna in eight natural waters
ranging in hardness from 22-438 mg/1 CaCC>3. Lethality and
reproductive impairment were evaluated over 21 days. As hardness
increased, NTA toxicity decreased (strong negative correlation;
r = 0.95). The three-week LCsg values (mg/1 Na3NTA) ranged from
100 - 900 in waters with total hardness of approximately 35-450
mg/1. Changes in pH were small (<1 pH unit) throughout the
study and chronic toxicity was attributed to anionic NTA present
in excess of the molar equivalence of metal ions, an unlikely
environmental situation. Reproductive impairment (decrease in
number of young born) was reported to be 16% at concentrations
of 57-70% of the three-week LC5Q concentrations for unammended
and artificially hardened Lake Superior water. Fifty percent
reproductive impairment occurred at concentrations of 71-84%
of the three-week LCso concentrations. It was also noted that
the toxicity of zinc and copper ions was eliminated by chelation
with NTA, confirming an earlier report (64).
14
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Flannagan (25) studied the effect of Na3NTA at concentra-
tions ranging from 6.25 - 100 mg/1 (in water of approximately
117 mg/1 total dissolved solids) on four generations of the
snail Helisoma trivolis. Growth (measured by weight increase)
and fecundity of all animals including the controls decreased
with each succeeding generation. There were no significant
differences in growth between the control or populations exposed
to 6.25 or 12.5 mg/1 NTA. Concentrations of 25, 50, and 100
mg/1 depressed growth and fecundity but even at 100 mg/1, NTA
was not acutely toxic. The depression was most apparent in the
first generation and was reduced in each succeeding generation.
Similar studies with species known to be less adaptable to
environmental change are needed.
Fish
The reader is referred to the review of Thorn (76) for a
critical review of some early studies. In general, little
attention was given to the chemical speciation of NTA in
these tests. Toxicity observed at high concentrations may
reflect uncomplexed NTA. Sprague (64) observed that NTA could
protect the brook trout Salvelinus fontinalis from the acute
toxic effects of copper and zinc when tested in soft water
(14 mg/1 CaCO3). He also noted that NTA at concentrations
of 100 mg/1 was toxic and attributed this to increased pH.
Acute toxicity to the fathead minnow Pimephales promelas^ (96 hr.
TL5Q value 114 mg/1 NTA) was also attributed to high pH (5).
Acute toxicity of NTA to bluegills (Lepomis macrochirus)
was tested in static bioassays (67). The TL5Q was 252 mg/1
in water with hardness of 60 mg/1 CaCC^, and 487 mg/1 in hard
water (170 mg/1 CaCC>3). Dynamic bioassays run in parallel
yielded similar results. Additional static tests with blue-
gills in soft water (35 mg/1 CaCO3) provided 96 hr. TL5Q values
of 198 mg/1 (175-225, 95% confidence interval). In flowing water
bioassays with the fathead minnow (P. promelas) a 96 hr. TLso
of 127 mg/1 (93-170) was obtained and with rainbow trout (Salmo
gairdnerii) 98 mg/1 NTA (72-133).
Macek and Sturm (41) reported the results of 28 day exposure
of bluegills and fathead minnows to NTA concentrations ranging
from 5.6 - 200 mg/1 in continuous flow bioassays using recon-
stituted water of 25 mg/1 CaCO3 hardness. Only at 172 mg/1 was
mortality due to NTA observed. At 96.0 mg/1 mortality was not
greater than in controls. Examination of gill tissue after
exposure to NTA indicated no direct histological change. When
the gills of other fish transferred to clean water following
exposure were examined, NTA apparently induced no predisposition
to pathogenic conditions. In another study with P. promelas
chronic toxicity was not observed at the highest exposure level
(54 mg/1 NTA) (5).
15
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SECTION 4
METHODS AND PROCEDURES
NTA ANALYSIS
Colorimetric Analysis
The zinc-Zincon method of Thompson and Duthie (77) was used
without change. The claimed limit of sensitivity for this method is
0.2 mg/1; significant loss of precision was observed below
0.5 mg/1.
Gas Chromatographic (GC) Analysis
1. General: Methods for processing the samples were similar
to those of Aue, et al. (6). Unfiltered 50-ml samples of NTA
in stream water were acidified, cleaned by. anion exchange (Bio
Rad AG 1-X2), eluted (10 ml of 16 M formic acid) into a screw-cap
test tube, and evaporated to dryness in a Kontes tube concentra-
tor (85°C) .
Pure acids other than NTA, used in screening experiments
designed to show that other commonly occurring acids would not
interfere in the GC analysis, were weighed directly into a test
tube. The pure acid (or the dry residue from evaporation of a
water sample) was suspended in 3 M HC1 in n-butanol (2.0 ml),
heated at 85°C for 30-35 min. and~"stirred Intermittently with a
vortex mixer. The solution was then evaporated to dryness with
a stream of dry air at room temperature. Just before analysis,
dry acetone (0.80 ml) was added and solution was transferred
to a dry sampling vial having a PTFE-faced seal. Dibutyl
phthalate (10 yl of a 10% solution) was added to each vial as
an internal standard.
2. Reagents; The disodium salt (monohydrate) of NTA was
used in most analytical and ecosystem studies. It was purchased
(Aldrich Chemical Company) as a specially purified, "99+%"
material. Analysis of its tributyl ester by GC showed no detect-
able impurities. Further purification of NTA was achieved by
repeated recrystallization of the free acid from 1:1 (v:v)
dimethylformamide:water. Other carboxylic acids were of the
highest available commercial quality. Acetone and n-butanol
-------
were reagent grade, redistilled from MgSO^ in an all-glass
apparatus. Acetone was stored over 4A molecular sieves. Hydro-
gen chloride, generated by adding concentrated H2SO4 to solid
NaCl, was dried by passage through H2S04 in an oven-dry
apparatus before dissolving in n-butanol. The HC1 concentration
was determined by titration with standard NaOH solution to a
phenolphthalein endpoint. (1-14C) NTA was supplied by Dr. R. L.
Downey (Procter and Gamble, Cincinnati, Ohio, U.S.A.) as a
10~3M solution of the trisodium salt containing 3.7 x 106 dpm/ml.
(2-l^c) NTA was synthesized from glycine and (2-14c) bromoacetic
acid and purified by anion exchange chromatography.
3. Gas Chromatographic Conditions; The gas flow rates to
the flame detector were 300 ml/min. of air and 40 ml/min. of
hydrogen. The carrier gas (helium) flow rate was 55 ml/min.
Injection-port and flame-detector temperatures were held at
250°C. Temperature program I, which was used to separate all
the acids as their n-butyl esters, was isothermal at 145°C for
8 min. and then increased 6°/min. to 240°C. Program II, used
for rapid analysis of NTA, was isothermal at 185°C. The
retention time of NTA was ca. 19 min. on program I and 9-10 min.
on program II. The automatic sampler was set to inject 4.5 ul
amounts of test solution.
CHELATION OF METAL IONS FROM SEDIMENTS
Sediment cores (7 cm depth) were taken from the White Clay
and Red Clay Creeks and from near a small municipal sewage
treatment plant also on the Red Clay. Duplicate cores were
sterilized in screw-cap jars and incubated aerobically with
varying (10~7 to 10~3 M) concentrations of sterile trisodium NTA,
sodium citrate and disodium IDA solutions.
Concentrations of sediment-derived Mn, Fe, Zn and Cu in the
overlying water were analysed by atomic absorption together with
a control (no chelating agent added). The samples were removed
aseptically with 10 ml syringes after 1, 15, 16 and 29 days.
Between days 15 and 16 the sample bottles were swirled, dis-
turbing the top of the sediment and exposing fresh material to
the potential chelating activity of the solution.
On the 29th day, samples were removed for sterility testing
and for NTA (citrate, IDA) analysis.
At the conclusion of the 29*day sterile period, each jar was
recharged with firesh NTA solution having the same concentration
as at the start of the experiment. At the same time, a small
portion of non-sterile sediment was added to provide a micro-
fadol0gLcal inoculum* Analyses for Mn/ Fe, Cu and Zn were done
after 1,5,8 and 14 days. The jars were bubbled with air twice
daily to ensure aerobic conditions*
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CHLORINATION OF IDA
Disodium IDA monohydrate (1.00 g, 5.17 mmoles) was dissolved
in 100 ml deionized water and 22.1 ml (15.5 mmoles) of Clorox
(5.25% NaOCl) was added. A sample was taken immediately and run
on TLC using 30/110/40 benzene/ethanol/water. Detection was by
PAN spray (55) or 1% potassium iodide. The reaction mixture
showed a major spot at Rf 0.78 (IDA Rf = 0.62).
Attempts were made to esterify portions of the sample with
dimethyl sulfate, methyl iodide, diazomethane, benzyl chloride,
and thionyl chloride under various conditions but no treatment
showed major peaks other than IDA esters.
Column chromatography and preparative TLC led to extensive
decomposition of the compound.
Precipitation of the crude reaction mixture from aqueous
solution with acetone afforded a white solid containing mostly
IDA. Attempts to recrystallize the material from ethanol led
to decomposition.
In an experiment designed to show whether the compound was
stable at various pH's, IDA disodium salt (250 mg, 1.28 mmoles)
was dissolved in 0.2 M phosphate buffers (pH 2,7, or 12) and
bubbled with chlorine^ for 2 min. followed by flushing for
15 min. with air. The reaction mixture was immediately treated
with excess KI solution and the optical density at 570 nm read.
The values were corrected for a blank with IDA absent. Then
the flasks were stoppered and allowed to stand for 17 hr., when
another reading was taken.
ALGAL EXPERIMENTS
Experiments were conducted in a laboratory greenhouse under
natural solar radiation. Radiation records are available but
were not used in analysis. The diatom dominated communities were
developed on glass slides in test boxes similar to those
described by Patrick (45). For the supply of water, each box
was connected to a 19 liter reservoir. Duplicate experiments
were run for each test concentration. The reservoir for each
box on a test concentration was connected to a 100 liter reser-
voir. During seeding once thru water came directly from a header
tank to the test boxes at a rate of approximately 760 1/hr in
order to establish maximum diverse populations of diatoms.
Previous experiments had shown that a flow in this range for these
boxes was optimum for the development of diverse communities.
When microscopical examination determined that similarly
diverse diatom communities were developed on all slides, the
experiments were started. In order to greatly reduce an^j future
seeding, water was filtered through cellulose fiber filters.
18
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These filters also excluded the entrance of most particulate
matter. The 100 liter reservoir was changed daily so that
variability in the concentration of NTA in the test could be
reduced. The turnover time in each of the 19 liter reservoirs
was about four hours.
The pH and temperature were automatically recorded at
intervals of about 15 minutes. For these tests water was
automatically withdrawn from the 19 liter reservoir and passed
over the probes. The pH probes were calibrated daily with
buffer solution of known pH. The temperature probes were
checked with a calibrated thermometer. The general chemical
characteristics of the water during the tests were determined
at various intervals, Table 1. Previous experiments using White
Clay Creek water have shown these are the frequency of analyses
necessary for maintaining a reliable record of the characteris-
tics of the water. It was sometimes necessary to add small
amounts of Mnf Fe, and PO^, in order to maintain similar ambient
concentrations in all tests. Mn was added as MnSC^'H^OjFe as
FeCl3«H2O and phosphate as NaH2PO4. The NTA was added as
disodium NTA and was determined one or two times a day.
In these experiments it was found that a fairly large
predator pressure might develop on the algal communities. This
predator pressure was mainly that of aquatic insect larvae. For
these reasons, the slides were carefully examined under a micro-
scope several times weekly and herbivores removed. It was, of
course, impossible to remove the protozoan predators.
During the course of the experiment frequent microscopical
observations were made as to. diversity of the algal communities,
species dominance, and condition of the algal cells. Concen-
trations of chlorophyll a, chlorophyll c and phycocyanin
pigments; and biomass were determined at the beginning, during
and at the end of each experiment. Primary production measured
by 14c uptake, and the uptake of various metallic ions was meas-
ured three times during the experiments. The relative dominance
of the more common species of diatoms was determined by cell
counts.
Primary Productivity- 14C Uptake
A single slide was selected from each test unit for deter-
mining primary productivity. The community on one side was
removed and placed in a Petri dish with 35 ml of water from the
test system for dark incubation. The community still intact
on the other side was placed in a second Petri dish with 35 ml
water from the system for light incubation. Isotope (NaRl4co3,
New England Nuclear, Boston, MA, sp. act. 8.4 mCi/mM) was
added to each dish to provide a final concentration of 1 yCi in
35 ml. The sample for dark incubation was covered immediately
with aluminum foil. Incubation was conducted for 1 to 2 hours
19
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at naturally occurring temperature under fluorescent lights
with a spectrum closely approximating solar radiation ("Vita
lite", Duro Test Corporation, North Bergen, NJ) . Incorporation
was terminated by adding 1 ml of 37% formaldehyde.
A sample (10 ml) of the water from the sample incubated in
the light was taken, membrane filtered (0.45 y pore size,
Millipore Corporation, Bedford, MA, at 0.5 atm), acidified with
0.5 ml 0.5 N HC1 to pH 3.0, bubbled for 30 minutes to drive off
unincorporated bicarbonate, and neutralized with 0.5 ml 0.5 N
NaOH. The *-^C remaining in the water as organic material excreted
by the algae was determined by liquid scintillation counting of
a 3 ml subsample in a toluene-Omnifluor (New England Nuclear,
Boston, MA) - Triton X (Beckman Instruments, Fullerton, CA)
cocktail. Water samples were counted with 89% efficiency.
The biomass Was scraped from the slide used for light incu-
bation. The algae and water for each light and dark incubation
were transferred to individual 50 ml plastic test tubes and
centrifuged at 12,000 x gravity for 10 min. at 4 C. The pellet
was resuspended in 0.1 M phosphate buffer (pH 7.0) and brought
to a final volume of 5 ml after which it was homogenized briefly
with a teflon homogenizing pestle driven by a stirring motor.
Five 0.25 ml amounts were removed, membrane filtered at 0.5
atmospheric pressure, and washed with successive rinses of
distilled water. The filters were air dried and exposed to fumes
of concentrated HC1 to remove any adsorbed radioactivity. Incor-
porated radioactivity was determined by liquid scintillation
counting after combusting the samples in a sample oxidizer
(Packard Instruments, Naperville, IL) and collecting the ^CC>2
from combustion as a carbamate compound. These samples were
counted with 62% efficiency.
Five 0.5 ml aliquots from each homogenized community were
transferred to test tubes and 7.0 ml acetone (made basic with the
addition of a pinch of CaC03) was added. The chlorophyll a con-
tent was determined after overnight extraction at refrigerated
temperatures. The samples were centrifuged to pelletize the
algal cells and the chlorophyll a was determined from optical
density readings before and after acidification to correct for
phaeophytin content (40).
The values for the five replicate chlorophyll a determina-
tions for each light or dark incubation were averaged and reported
as yg chlorophyll a for the algae on one side of slide. Radio-
activity determinations for each sample type were corrected for
counting efficiency, averaged and the incorporated radioactivity
value was normalized for chlorophyll a concentration. Values
reported have been corrected for dark adsorption and excretion.
20
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Chlorophyll a and c Extraction and Analysis
Extraction (60). The algae on one-third of one side of each
slide were collected and excess water removed.
The biomass was extracted with 4.0 ml of 80% dimethylsuflox-
ide (DMSO) in 25 or 50 ml flasks with shaking on a wrist-action
shaker. The yellowish extract was filtered through Whatman
No. 1 paper into side-arm test tubes.
The filtrate contained most of the chlorophyll c (in this
work, specific for diatoms) and some chlorophyll a. The residue
on the paper was extracted with 4.0 ml acetone (made basic with
MgCO3) by shaking for fifteen minutes in stoppered flasks. The
samples were filtered as before and the green extract was reserved
in stoppered tubes for analysis of chlorophyll a.
Analys is. The spectrum of each extract was determined from
550 - 750 nm using a Beckman DBGT recording spectrophotometer
(Beckman Instruments, Fullerton, CA). Chlorophyll c_ absorbance
was determined at 570 and 630 nm and chlorophyll a absorbance
at 665 nm. Concentrations were computed from published equations
( 60) .
The a/c ratio was determined by adding the two concentrations
of chlorophyll a in the two extracts and dividing by the
chlorophyll c concentration in the DMSO extract. A low ratio
(8 or less) indicates a high proportion of diatoms; pure cultures
of marine diatoms average a ratio of 4 (33).
Phycocyanin Extraction and Analysis
Extraction. The algal community was scraped from one side of
a slide as before. Excess water was removed and the cells were
suspended in 8 ml of 0.005 M pH 6 phosphate buffer. The suspen-
sion was sonicated in a stirred ice bath in the dark for 12
minutes, and centrifuged at 12,000 rpm for 15 minutes.
Column Chromatography. The columns were prepared by filling
25 ml burets with Bio-Gel HT hydroxyapatite (added from a well-
shaken suspension) until the column was about 40 cm high. The
column was rinsed with 10 ml of 0.005 M phosphate buffer and
the sonicate was added using a Pasteur pipet to minimize disturbing
the top of the column. The sonicate was allowed to run through,
using a slight vacuum; the solution was discarded. Pigments were
eluted into a new collection vessel with 0.25 M phosphate buffer.
The column was run until 7 to 9 ml of concentrated buffer had been
collected or until all visible blue color was completely removed.
The spectrum was run on this fraction (68).
21
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Analysis. The absorption spectrum was determined from 750-
500 nm. The phycocyanin peak appeared at 620 nm. The concentra-
tion of phycocyanin in mg/1 was determined using the equation:
Phycocyanin = O.D. 62o X 1Q00/Ext. Coeff.
The extinction coefficient (7.74) reported by Troxler and Lester,
(80) was used. O-D«g20 values were corrected for turbidity
(O.D.750).
Biomass Determinations
The growth from one side of a slide was carefully scraped
and placed in a crucible weighed according to quantitative
analytical procedures. It was then dried to constant weight at
103°C and the weight was determined. The material was ashed at
600°C, dried to constant weight, and the ash free dry weight
determined.
Extraction and Analysis of Metals
The extraction and atomic absorption analysis (Model 303
spectrophotometer, Perkin Elmer, Fort Washington, PA) of metals
associated with the algal biomass was carried out according to
the methods described by Allan (2).
BACTERIAL EXPERIMENTS
NTA Degradation; Flask Bioassays
Experiments were done with populations developed on cover-
slips held in Plexiglas microcosms. The microcosms held 100
coverslips and were placed in a water jacket through which
stream water was passed once in order to maintain the populations
at near ambient temperature. White Clay Creek water was passed
through each microcosm at a rate of approximately 13.8 1/min.
The microcosms were covered with foil to prevent the entry of
light and thereby reduce algal development on the coverslips.
The microcosms are illustrated and described more fully in reference
11. After the coverslips were seeded with the periphytic popu-
lations , the microcosms were converted to recirculating systems
as described for algal studies.
Water was recycled through the boxes at a rate of approxi-
mately 760 1/hr. NTA was added as the disodium salt to maintain
desired concentrations of approximately 0.02, 0.2, 2.0 and 20.0
mg/1. Prior to NTA exposure and periodically thereafter, cover-
slips with the attached microbial community were removed from
22
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each microcosm for use in studies of NTA degradation.
Measurement of degradation. The evolution of ^4CO2 from
1-14C-NTA was monitored as a measure of degradation activity.
After removing any attached larval forms, three coverslips were
transferred to each of six biometer flasks (52) containing 10 ml
membrane filter sterilized (0.45 ym pore size) stream water.
Five replicate flasks were used in each mineralization assay-
The sixth flask was a control with cells killed by the injection
of Lugol's iodine prior to the introduction of 14C NTA. Analysis
of data from fifteen degradation rate assays showed that five
replicates provided a number + 30% of the mean with 95% confidence
in thirteen instances.
Incubations were started by adding 0.5 pCi C NTA (Amersham-
Searle, Arlington Heights, IL; sp. act. 52.5 mCi/mM) to the
water after which the flasks were stoppered to close the system.
Degradation (evolution of 14CO2) was measured at 0.2 mg/1 for the
populations exposed to approximately 0.02 and 0.2 mg/1 in the
microcosms. This was necessitated by the specific activity of
the isotope. It was not possible to use a lower concentration
of isotope in the assay system and insure that the isotope con-
centration would not be limiting. However, unlabeled NTA was
added to biometer flasks to establish the desired concentrations
for the organisms that were exposed to approximately 2.0 and 20
mg/1 in the microcosms. Additions to the assay system were made
from frozen stock solutions or from serial dilutions of the
same. The NTA concentration in each bioassay flask was not
determined but the concentration of 10X stock solutions were
checked prior to several assays of degradation rate. Four
determinations on the stock used for the 20 mg/1 bioassay indi-
cated concentrations of 255, 251, 258 and 263 mg/1. The stock
used to establish concentrations approximating 0.02 and 2.0 mg/1
in the bioassay flask contained 23.8, 19.3, 24.4 and 26.4 mg/1.
Incubations were conducted in the dark with agitation in a
rotary water bath shaker at a temperature in the natural range for
that time of year. The flasks have a side arm to which 0.5 ml
of a saturated solution of KOH in anhydrous ethanol were added.
Respired 14CO2 from the labeled NTA was trapped in the solution.
Every 16 min. for 64 min. the KOH was removed through the stopper
for assay of radioactivity and replaced with fresh solution.
The side arm was rinsed twice with 1 ml of anhydrous ethanol and
the washings were counted with the 0.5 ml initially removed in
Cocktail T (Beckman Instruction Manual 8.555-E, pgs. 2-11, Beckman
Instruments, 1967) containing 2.5 percent Cab-o-sil. Samples
were counted in a liquid scintillation counter at 89% efficiency.
Data from the linear phase of NTA metabolism were used to
calculate the r~ate of breakdown expressed as ng NTA degraded/hr.
The decomposition rate for the population on the coverslips was
calculated as follows:
23
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1) DPM mineralized
added - = Percent mineralized
2) (Percent mineralized) x (ng NTA in assay system)
x (k-^) x (k2) = ng/hr. mineralized/cm2
ki = factor to convert rate to hourly rate
k2 = factor to correct for surface area used
Estimation of microbial numbers. At the end of the incubation,
the coverslips and the incubation medium from each flask were
aseptically transferred to a grinding vessel and homogenized at
<4°C. Serial dilutions were prepared and enumeration was done by
the method of Harris and Sommers (32). NTA-agar was that described
by Tiedje, et al . (79) with NTA as the sole carbon and nitrogen
source. Pla~tei~~were incubated at or near the temperature of assay
and were scored at weekly intervals.
NTA Degradation: System Experiments
In some experiments , concentrations of the nitrogen end pro-
duct from complete NTA degradation, ammonia, and its oxidation
products nitrite and nitrate were measured in the recycle microcosm
system. Ammonia-N was determined by the phenol-hypochlorite
method, NO2~N by the sulfanilic acid- a -naphthylamine hydrochloride
procedure, and NO3-N by the chromotropic acid procedure (4).
In another experiment, 8 yCi 1-14C was added to a system
whose volume was 87 1. Each hour, 2.9 1 were replaced; thus, a
predictable loss of 14C NTA would result from dilution alone. The
variance of the actual 14C concentration from the predicted con-
centration would be indicative of the mineralization of NTA in
the system.
Periodically, samples (25 ml) were collected and placed in
Erlenmeyer flasks. The flasks were sealed with serum stoppers
and the samples were acidified to pH 1-2 by the addition of con-
centrated HC1 using a hypodermic needle and syringe. A cotton
swab held in a cup suspended from the serum stopper was saturated
with alcoholic KOH to trap carbon dioxide. The samples were
agitated gently to elaborate 14CC>9 that resulted from the mineral-
ization of the labeled NTA from the aqueous phase. Subsamples of
the water were taken and the radioactivity associated with the
CO2 traps and remaining in the water was determined by liquid
scintillation counting. Water samples were filtered through 0.45 y
membrane filters, but there was little difference in radioactivity
associated with filtered and unfiltered water samples. Only back-
ground levels were associated with the filters . ,,
24
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Effects of NTA-Metal Chelates on Bacterial Activity
NTA-metal chelates were prepared by mixing NTA and metal
salts in a 3:2 molar ratio in distilled deionized water. In all
assays, thin-layer chromatograms (benzene/ethanol/water 30:110:40
as solvent, PAN developed) (55) were used to demonstrate that the
metal ions were NTA chelated. Metals were added as the chloride
salt. Chelates were usually prepared from the chloride salt
except for lead - the lead acetate salt was used.
To avoid difficulties with varying cell inoculum on cover-
slips, cell suspensions were prepared by growing stream organisms
in 0.1% yeast extract-tryptone broth overnight at the temperature
of assay. The inoculum was loose silt or sand from an ecosystem
stream. Some experiments were also done with NTA degrading
organisms; a Pseudomonas strain (T-l) supplied by Dr. James
Tiedje, Michigan State University, and isolate "216" from the
White Clay Creek. These cells were cultured in NTA broth.
Cells were harvested and washed two times with sterile dis-
tilled water and resuspended in membrane filtered (0.45 u pore
size) White Clay Creek water. Cell numbers were determined by the
plate dilution frequency assay (32). Cells (1 ml) were trans-
ferred to biometer flasks containing 22.75 ml membrane filtered
stream water. Each flask received 0.25 ml of either NTA, metal
salt, or NTA-metal chelate; metal ions were present in the assay
flask at 1 mg/1, NTA at 4 mg/1. After a 16 min. pre-incubation,
1.0 ml 14C-glucose (uniformly labeled, sp. act. 234 mCi/mM, New
England Nuclear, Boston, MA) was added to give a final concen-
tration of 1.0 uCi/flask. Incorporation was shown to be a more
sensitive indicator of toxicity than mineralization. Therefore,
cells were collected at 16 min. intervals for 64 min. and filtered
onto membranes (0.45 ym) for determination of incorporated radio-
activity by liquid scintillation spectrophotometry.
Another experimental approach was used to study the effect on
bacterial activity of longer exposure (22 hr.) to metals or
their NTA-chelates. Cells from an inoculum of mixed periphytic
bacteria were grown overnight in 0.1% yeast extract, 0.1%
glucose broth, collected, held in double distilled water for 2 hr.
to deplete cellular reserves, and resuspended in broth to a
final density of 8 x 10^ cells/ml determined by direct microscopic
counts. The respiration rate of 50 ml resuspended cells in 50 ml
Erlenmeyer flasks was determined using a model 5331 dissolved
oxygen probe and meter (Yellow Springs Instrument Company,
Yellow Springs, OH) connected to a recorder (Microcord Model 44,
Photovolt Company). Cultures were stirred continuously while
these measures were made. Additions of metal, NTA, or metal
chelates were made to establish desired concentrations, and
respiration measurements were made immediately after addition,
2.5 hr. and approximately 22 hr. later. At the end of some
experiments, a final respiration rate was measured after the
25
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addition of nutrients which stimulated respiration rate. At
22 hr. cell numbers were determined, and concentrations of test
metal in the supernatant and associated with cells were deter-
mined. Metal concentrations were determined by atomic absorption
spectrophotometry. Chelation of metal was determined by thin-
layer chromatography and showed NTA-metal chelates were stable
through the experiments. However, metals added alone chelated
to some degree with organics in the medium during the experiment.
Metals were used at a high concentration (100 mg/1) to arrest
respiration rapidly (15 sec.) although one experiment was per-
formed with 1 mg/1 metal. Studies with Cu and Hg showed 15 min.
were required to terminate respiratory activity when 1 mg/1 was
used.
EFFECTS AND FATE OF NTA AND COPPER IN ECOSYSTEM STREAMS
This experiment was performed to determine the activity and
fate of NTA and copper when introduced into simulated natural
environments (ecosystem streams) over long time periods either
singly or together. Particular emphasis was placed on the
mobilization and uptake of copper (and other selected metals) in
the presence and absence of NTA.
Four 12 m long experimental stream systems with two riffles,
slack water, and shallow pools containing natural substrates
(silt, sand, gravel, and rocks) were seeded with natural com-
munities for over two years prior to initiating experiments.
Water from the parent stream (White Clay Creek) was passed once
through each stream. Flow rate averaged 169 1/min. (c.v. between
streams was 3.2%) and current velocities in riffle sections
measured 30-42 cm/sec. Each stream was populated by diverse
communities of diatoms and other algae such as Oedogonium,
Vaucheria, and Spirogyra had seasonal population expressions.
Rooted aquatics (e.g. Anacharis), and floating duckweed (Lemna sp.)
were common in pool areas. A diverse invertebrate fauna with
species of caddisflies, mayflies, worms, and snails populated
each stream. Although population sizes varied between streams,
representatives of each common species were found in each stream.
The banks of each stream built up naturally with sediment and
supported a thick growth of herbaceous plants of similar species
composition. Additional description is provided in reference 11.
One stream was designated the control, one received NTA to
maintain a concentration approximating 2.0 mg/1, one received
30 ug/1 copper as CuSO., and the last received both NTA and copper
at the indicated concentrations. NTA and CuSO4 were metered
continuously from concentrated stock solutions (using syringe
pumps, B. B. L., Baltimore, MD) into the header tanks of each
stream where rapid mixing occurred. Initially, water was not
recycled, but 9.5 weeks into the experiment a 50 percent recycle
was used. «•
26
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NTA and copper concentrations in the water were monitored
daily using the zinc-Zincon method and atomic absorption spectro-
photometry respectively. NTA concentrations were also confirmed
by gas chromatographic detection. Concentrations of NH4-N, NC>2-N,
NOo-N, and PO4-P were determined daily and other chemical para-
meters (e.g. total alkalinity, sulfate, chloride, silicate) were
measured one-three times weekly (depending on expected concen-
tration change) using Standard Methods (4) or other appropriate
procedures. Concentration of several metals other than copper
(Ca, Mg, Fe, Mn, Zn) were measured several times weekly by
atomic absorption spectrophotometry.
Prior to the addition of Cu++ and/or NTA and at weekly
intervals thereafter samples of sediment were taken for metal
analyses and less frequently for NTA analysis. Five cores were
taken from each stream at each collection time. Sediment samples
(primarily silt) were collected by inserting a parafilm coated
metal tube (10 cm diameter) into the sediment and filling the
tube with dry ice and ethanol. The probing device had a plastic
funnel tip to facilitate entry into the sediment. The dry ice
froze the sediment for 3-4 cm around the core, and the core with
adhering frozen sediment was removed. Samples of frozen sediment
were removed at selected depths (surface, 2 cm, and 5 cm) by
cutting away from the frozen material. The sediments were dried,
sieved, and the fraction <_ 1 mm was saved for analysis of both
exchangeable heavy metals. Preliminary studies demonstrated
higher metal content in this size fraction compared to larger
size fractions. Levels of exchangeable Cu, Fe, Mn, Mg and Zn
were determined by atomic absorption spectrophotometry after
extracting ten grams of sediment with 20 ml of DTPA (Dimethyl-
enetetramine pentaacetic acid) (39) .
This method of sediment sampling led to mixing of the
unsampled material because sediment from adjacent areas collapsed
into the hole created when the frozen core was removed. Only a
small number of samples could be taken from the limited area of
each pool before this mixing appeared to alter concentration
data at different depths.
Another approach was used to evaluate the potential sorption
or release of metals to or from these sediments resulting from
NTA exposure. Containers (10 cm w x 10 cm 1 x 12 cm d) were
filled with sediments from the parent stream that were mixed to
make them relatively homogenous and put in 22 1 microcosms con-
taining stream water (control), 2 mg/1 NTA + 30 ug/1 Cu++, or
30 yg/1 Cu++ in stream water. The solutions were recycled over
the sediments in the microcosms and the systems were held at
environmentally realistic temperatures in a water jacket through
which stream water was passed continuously. Solutions were
changed daily and concentrations were monitored daily (Cu++ by
atomic absorption spectrophotometry; NTA by zinc-Zincon analysis
(withoccasional gas chromatographic confirmation). Containers
27
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of sediment were removed at intervals and frozen. Samples of the
upper cm, 2 cm, and 5 cm depths were taken by cutting through the
frozen block. The perimeter of each layer was removed and the
central area was used for DTPA extraction and a.a. metal analyses.
In order to determine possible accumulation or depletion of
metals in the biota, samples of algal communities, aquatic
vascular plants and animals were analyzed for concentrations of
Cu, Fe, Zn, Mn, and Mg. Mixed communities of periphytic algae
dominated by Melosira varians (sample size; 30-600 mg dry weight)
Lemna, and Anacharis (sample size; 40-90 mg dry weight, 20-50 mg
dry weight, respectively) were sampled at two locations in each
stream. Anacharis samples were taken at the growing tips of the
plants. Tubificid worms (sediment feeders) were collected from
sediments in pool areas and Planaria sp. from the surfaces of
sediments and plants in the poolsTiample size; 10-40 mg dry
weight for both organisms). Significant differences between
sampling locations within a stream were not obtained.
Samples were dried at 100°C for 24 hr. and desiccated over-
night at room temperature. After weights were taken, the samples
were combusted at 500°C for 2 hr. and organic weights obtained.
Ashed plant material was extracted twice in 20% HCl with
warming. The extracts were pooled and diluted to 50 ml with a 1%
lanthanum oxide solution as a releasing agent to allow for Mg
determination. Metal concentrations were determined by atomic
absorption spectrophotometry.
Ashed animal material was extracted by adding 1 ml of con-
centrated HN03 and 2 ml of deionized water to the crucible. The
extract was brought to a boil and after settling, the extract was
aspirated off and its volume adjusted to 5 ml with a 2.5% solution
of lanthanum oxide. Concentrations of the same five metals were
again determined and reported as for the plant material.
SECTION 5
RESULTS AND DISCUSSION
CHEMICAL STUDIES
Analysis of NTA
Gas chromatography. (38) One of the prime goals of this
study was to develop a method of NTA analysis which was reliable,
reproducible, and free from inorganic and organic interferences
at low NTA concentrations. Because we also wished to be al>le to
look for possible degradation products of NTA, we focused our
28
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attention on gas chromatographic (GC) methods, particularly that
of Aue, et al. (6) because it was demonstrated that NTA could be
freed from chelates and some organic interferences by ion
exchange cleanup. In addition, analyses by this method were
very good down to well below 15 yg/1 (our lower study limit),
and good recovery of added NTA from very hard tap water, and also
from sewage effluent, was shown.
There have been no thorough studies of the interference of
organic substances in NTA analysis. Taylor, et al. (73) showed
that the trimethylsilyl ester of NTA eluted between those of
lauric and myristic acids, but did not report studies with other
interfering substances. Citric acid tributyl ester has been
reported to co-elute with the tributyl ester of NTA on Carbowax
20 M columns. The Aue GC method has been particularly successful
in affording rapid reproducible analysis at concentrations in
the yg/1 range. In particular, citric acid is well resolved from
NTA. However, it requires the use of a special column consisting
of Carbowax 20 M on acid-washed Chromosorb W. The column must
first be strongly heated and then continuously extracted for
prolonged periods, leaving a thin film of unextractable polymer
on the support (7).
The difficulties in this approach prompted us to investigate
the capabilities of more common, commercially available, GC
liquid phases, especially those similar to phases already used
for analysis of esters. After some preliminary screening experi-
ments, we found excellent resolution of NTA tributyl ester and
the structurally similar citric acid ester on a commercial 3%
OV-210 column. The two compounds were separated by approximately
one minute (depending on the individual column) when an iso-
thermal (185°C) run of 10-15 minutes was used. Next, we attempted
to find conditions for the analysis which would separate NTA
from some of its possible degradation products and also from some
potentially interfering, naturally occurring acids.
Initially, weighed amounts of each of thirty-six pure acids
were separately derivatized and injected into the gas chromato-
graph. Eleven acids (oxalic, capric, caproic, caprylic, lauric,
oleic, protocatechuic, salicylic, chlorogenic, caffeic and gallic)
gave no detectable peaks and were not further studied. Peak
areas for the remaining twenty-five acids were electronically
integrated and their molar responses were determined relative to
dibutyl phthalate. Retenrion times were also corrected to that
of dibutyl phthalate. Table 2 summarizes this data.
An equimolar mixture of these acids was dissolved in aqueous
acetone. An aliquot of this mixture, containing 2 umoles of each
acid, was evaporated to dryness, derivatized and analyzed by
temperature program I (145°C for 8 min, then 6°/min to 240°C).
Pig. 4 shows the resulting GC trace; the NTA tributyl ester
peak appeared between those of the butyl esters of arachidic and
29
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sinapic acids. Separation from citric acid tributyl ester was
excellent (ca. 1.0 min) using this program, and also (ca. 0.8
min) using the isothermal program II. Although separation from
arachidic acid butyl ester was incomplete using program II, this
program was adopted for routine analysis of NTA in stream water.
Ion exchange completely separated the two acids, and, further-
more, it seemed unlikely that detectable amounts of arachidic
acid would occur in our stream water.
The analysis of NTA was next studied in detail. First, a
standard curve was constructed from measurements on samples of
water from White Clay Creek to which NTA had been added, giving
concentrations ranging from 0.01 to 200 mg NTA per liter.
Samples (50 ml) were subjected to an ion-exchange and derivatiza-
tion procedure (cf. Methods and Procedures Section 3) and injected
in triplicate into the 3% OV-210 column. Fig. 5 shows the
graph of integrated area vs. NTA concentration. It was possible
using the procedure and a well-conditioned column to analyze
NTA reliably at 10-25 yg/1. As. the columns aged, the NTA peak
broadened and eventually became undetectable. The average life-
time of a newly conditioned column was on the order of 200
injections.
Recovery of NTA by the procedure was studied by use of
NTA. Five replicate samples of a 0.20 mg/1 solution were
taken through the ion-exchange and derivatization steps and
examined periodically for radioactivity. The results (Table 3)
showed that 98% of the starting NTA adhered to the ion-exchange
column, 86% of the total was recovered after elution with 16 M
formic acid, and 75% remained after conversion into the n-butyl
ester. Thin-layer chromatography (silica gel/diethyl ether)
on the radioactive esterification product showed a single spot
containing 95% of the total activity; the remainder was mainly
at the origin.
The data demonstrates that NTA can be recovered, its tri-
butyl ester formed in high yield, and specifically detected by
gas chromatography, starting with a dilute solution of NTA in
stream water.
In screening experiments, the OV-210 column was superior to
columns packed with equivalent amounts of OV-1 or OV-225. On
OV-1, separation from citric acid butyl ester was good, but the
NTA peak was close to those of the syringic and plamitic acid
esters. On OV-225, the NTA and citric acid ester peaks were
close together. We repeated the separation on different OV-210
columns from Applied Science Labs., obtained at different times,
with practically identical results.
A few acids were not resolved by program I. Linolenic,
linoleic and stearic acid ester peaks overlapped almost"**com-
pletely and several others were not resolved to baseline levels.
30
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However, NTA and its likely degradation products, IDA and N-
methyl-imino-diacetic acid (MIDA, Fig. 1 ) were well separated
from many potentially interfering acids; only arachidic acid, a,
relatively uncommon fatty acid, could have caused difficulties
if present in large excess.
A disadvantage of the GC method is its slowness; cleanup,
derivatization, and analysis of 4-12 samples requires over six
hours. In addition, it does not distinguish between Ilfree" and
"complexed" NTA. However, we believe that the potential of the
method for the analysis of complex natural mixtures, and its
freedom from interferences, may make it applicable in a wide
range of situations.
Colorimetry. In samples containing higher (over 0.2 ppm)
concentrations of NTA, we tested other procedures based on
colorimetric measurements of total complexing ability in order
to assess more rapid methods of NTA determination. The zinc-
Zincon procedure is a standard colorimetric method for the
analysis of NTA in detergent formulations and waste waters. The
authors claim sensitivity down to 0.2 ppm NTA (71).
In our hands, the procedure seemed quite reliable at con-
centrations of NTA exceeding 2 ppm. Below 2 ppm, there was
increased scatter, and the method was not highly accurate below
0.5 ppm.
A major drawback of the method is its susceptibility to
interference from other metal-complexing agents. For example, we
showed that IDA, a known metabolic product of NTA is nearly as
good a complexing agent as is NTA; on a molar basis, IDA
maintains copper in solution 70% as well as does NTA. Even
glycine, another possible NTA breakdown product, has significant
complexing ability for copper. It is conceivable that under some
environmental conditions, these products or other significant
complexing agents could accumulate in NTA-containing systems.
Therefore, a method of NTA analysis based on total metal-chelating
capacity could conceivably afford erroneous results.
However, in experiments in biological systems in which NTA
analysis was done using GC and the colorimetric method, we did
not encounter serious discrepancies. Below 2 ppm, the zinc-
Zincon method appeared on the average to give somewhat higher
values than did GC. Regression analysis for 503 samples in
which NTA was determined by both procedures indicated a high
degree (r = 0.962) of correlation between them. The equation
of the line was: mg/1 NTA (zinc-Zincon) = 1.036 x mg/1 NTA (GC)
+ 0.356.
31
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Chelation of Metal Ions
In natural waters, NTA will exist in chelated forms almost
exclusively- A computer simulation for equilibrium conditions was
carried out (by J. Chance, as part of a M.S. thesis at the
University of Pennsylvania) using published data for NTA chelate
stability, concentrations of metal ions in White Clay Creek, and
a 10~6 M NTA concentration. Most (85%) of the NTA was predicted
to occur as a mixture of two iron-containing forms, Fe (OH) NTA
and Fe (OH)2 NTA. Possible contributions by colloidal oxides of
iron were not, however, taken into consideration. The copper
complex made up 8% of the total NTA; almost all of the remainder
was present as the lead (6%) and nickel (1%) chelates. The re-
sults differ from those of Childs, who showed that in Lake
Ontario, copper-NTA would predominate at equilibrium (14) . The
difference largely reflects the low abundance of copper in White
Clay Creek.
Computer and spectroscopic studies on the displacement of
metal ions in NTA chelates by free cupric ion (Chance, unpublished
M.S. thesis, University of Pennsylvania) were done. Reactions
between Cu++ and NTA complexed with Mg, Ca, Zn, Cd and Na were
too fast (<1 sec) to measure spectroscopically. Fast but measur-
able rates were obtained using the Ni, Pb, Fe, and Co complexes.
Detailed study of the NTA-Co + Cu++ reaction showed a rate con-
stant of 35.7/mole/sec.; the data were consistent with an Sn^
displacement mechanism. The reaction was observed to reach 90%
completion within 75 sec. The evidence indicates that attainment
of equilibrium among NTA complexes in a natural water system
would be a fast process relative to their decomposition by
biological or chemical means. An approximate upper limit of four
hours for the equilibration of the system was calculated.
Studies of the extraction of metal ions from sediments using
NTA solutions were undertaken. Stream sediments from three loca-
tions (a relatively unperturbed area of White Clay Creek, Chester
Co., PA; an area on Red Clay Creek, Chester Co., PA, immediately
impacted by agricultural use; and another site on the same
stream immediately below a small municipal sewage plant) were
sterilized in jars containing NTA, citrate, and IDA in concen-
trations ranging from 10"' to 10" 3 M. The jars were allowed to
stand for 29 days, then reopened and" incubated with a small
portion of non-sterile sediment from White Clay Creek. Figs. 6-9
summarize the data on metal ion concentrations.
In general, the results of the aseptic studies indicate that
NTA did not lead to statistically significant* increases in con-
centration of the metal ions investigated at 10~5 M (1.9 mg/1) or
less. In only one case (Red Clay sewage sediments and zinc) was
there a significant increase at 10"^ M.
* P <_ 0.05 using Student's T-test
32
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At 10"3 M (191 mg/1) there were significant increases in
concentration of all metal ions tested ranging from 3 x (Mn,
White Clay) to 15 x (Zn, Red Clay). The increases were not
surprising; the NTA concentration approximated that expected in
a household washing machine using a detergent containing 6%
NTA, and was accordingly not considered to be environmentally
relevant.
Agitation of the sediments at the midpoint of the study gen-
erally led to a slight increase in metal concentrations for all
samples, including the control.
At the 10~3 M level, IDA (a known degradation product of
NTA) increased the concentrations of all four metal ions; these
increases were significant for iron, zinc, and (in one sediment)
copper. Trisodium citrate (at 10"3 M), a natural metabolite
stereochemically similar to NTA, significantly increased the
concentration only of iron. For both IDA and citrate, significant
increases in metal ion concentration were not observed at 10~5 M
or less.
The results are in general agreement with those of previous
studies (8,13,72) indicating no large increases in metal ion
mobilization from sediments at NTA concentrations of less than
10-4 M.
The gradual decline in concentration of solubilized iron
and manganese, especially noticeable at the 10~3 M NTA level,
over the course of the sterile portion of the experiment was
attributed at least partly to photooxidation (37,66). Additional
support for the hypothesis is the finding that NTA showed
decreases in concentration over the study period by amounts
ranging from 36 to 68%. Test for sterility (by inoculating
nutrient agar plates with overlying water from all experimental
flasks) showed no bacterial or fungal contamination at the end of
the 29-day period. Soluble zinc remained essentially constant,
but soluble copper increased three fold (0.04 to 0.12 mg 1~1 at
10~3 M NTA) during the sterile period. Chau and Shiomi (13) have
shown that, at 2 ppm in lake water, the NTA complex of copper is
resistant to biological breakdown. The present results may
indicate a similar chemical unreactivity or gradual release of
strongly chelated copper from the sediments.
In all the non-sterile sediments exposed to 10~3 M" NTA, there
was a- sharp drop in copper concentration. The drop was especially
marked with the sewage plant inoculum; within a day the copper
concentration was below 0.01 ppm and indistinguishable from
control samples.
Manganese concentrations in the 10~3 M NTA experiments
became relatively stable after an initial surge. In the White
Clay and sewage plant samples, the concentrations dropped to
33
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7-8 ppm (approximately what they had been tinder sterile condi-
tions) ; but the Red Clay samples dropped to background levels
(< 0.5 ppm).
Iron concentrations tripled in each 10~3 M NTA sample over
the first 8-day period. A concomitant increase in sheathed iron-
utilizing bacteria was observed microscopically. However, by
the 14th day iron concentrations had dropped back to their
initial levels, except in the case of the sewage plant sediments
where they remained high. Iron concentration in the sewage con-
trols also increased, however.
Zinc in the 10~3 M NTA samples dropped gradually over the
14-day non-sterile period. In the case of the White Clay
sediments, but not in the other samples, there was an initial
surge in concentration from 0.5 to 1.25 ppm.
In an attempt to determine the extent of solubilization of
the metal ions, we analyzed all the sediments for metal content.
An arbitrary assumption was made that the metal ions in the top
centimeter would be available for extraction by NTA.
The resulting data for the 10"3 M NTA experiments is sum-
marized in Table 4. ~~
About 5% of the "available" copper was brought into solution
at the White Clay and upstream Red Clay sites. A somewhat higher
(8.5%) degree of solubilization was observed at the sewage plant
site; the copper content of these sediments was much higher.
Results quite similar to those for copper were observed in
the zinc analyses. The sewage plant sediments likewise were
considerably enriched in zinc and a relatively larger percentage
of zinc was solubilized. It is possible that some of the zinc
and copper at the sewage plant site was in forms more easily
accessible to NTA complexation.
At all sites, about 3-5% of the "available" manganese was
solubilized.
In general, iron was not solubilized to a significant extent;
much less than 1% of the "available" iron was chelated in all
cases. Presumably this reflects the existence of iron in strongly
chelated, otherwise highly insoluble, or oxidized form not avail-
able to NTA chelation.
Chlorination of NTA and Derivatives
Some chlorinated derivatives of hydrocarbons are among the
most toxic of known synthetic compounds. Accordingly, chlorinated
derivatives of NTA or of its degradation products might have a
greater impact upon ecosystems than the parent compounds. Such
34
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chlorination could occur in sewage treatment processes; recent
investigations have shown that reactive organics, such as phenols,
are the precursors of a wide variety of chlorinated compounds (34).
Tertiary amines, such as NTA, can be chlorinated by drastic,
oxidative processes leading to N-chloro compounds and alde-
hydes (19). More likely is the direct chlorination of a
secondary amine such as IDA. This compound might accumulate in
some environmental situations, since some NTA-degrading bacteria
do not metabolize IDA (17).
In preliminary studies designed to test these possibilities,
we observed that 0.01 M (1910 mg/1) NTA disodium salt was not
readily attacked by socTium hypochlorite in 3 x molar excess at
moderate temperatures. NTA persisted in such reaction mixtures
for long periods (days) without appreciable conversion to other
products.
Conversely, IDA disodium salt (0.05 M, 6650 mg/1) reacted
almost instantaneously with 3 x excess (lT,200 mg/1) hypochlorite.
These concentrations are, of course, at least three orders of
magnitude higher than would be expected in sewage treatment
plants. Thin-layer chromatography showed rapid formation of a
less polar product. The material had oxidizing properties
(shown by starch-iodide spray), as would be expected for an
N-chloro derivative. The evidence is consistent with a reaction:
Na OC1
(Na OOCCH2)2 N - H »-(Na OOCCH2)2 N-C1 + NaOH.
Efforts were made to separate the reaction mixture by pre-
parative TLC, column chromatography, and crystallization, but
extensive decomposition occurred. In one preparative TLC
experiment, a small amount of pleasant-smelling oil, giving a
positive 2,4-dinitrophenyl-hydrazine test (indicative of a
carbonyl group) and also having a strong carbonyl band in the
infrared spectrum, was isolated.
Attempts to prepare an ester of the N-chloro compound were
not successful. Methods requiring acid catalysis gave IDA esters;
basic and neutral methods led to extensive decomposition or
considerable recovery of starting material.
Attempts were made to prepare the N-chloro compound at
pH 2,7, and 12. At pH 2 or 7, the compound could be made in
about 50% yield as shown by immediate treatment of the reaction
mixture with KI. At pH 12, only 5% conversion was demonstrated.
However, after standing for 17 hr., the compound was 93-99%
destroyed in all samples.
35
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The instability of the compound and the formation of IDA or
a carbonyl compound on its decomposition can be rationalized by
the following reaction mechanisms:
H
(HOOCGH2)2f/0 ^ilg» |OA
(HOOCCH2)2NCI Cl
[HoO]
HOOGCH2N=GHGOOH ]=-LA+
+ HOOGGHO
The results of the experiments suggest that N-chloro IDA,
even if formed at measurable rates at the far lower concentra-
tions of IDA and available chlorine to be expected in sewage
plants, would probably not persist for long periods in receiving
streams since it has both acidic and basic pathways available for
its decomposition. Other routes not investigated (biological
degradation or reactions with ferrous ion, polyphenols, or other
readily oxidizable substances) might also contribute to its
destruction in natural waters.
Photooxidation of NTA and IDA
Preliminary studies showed that NTA and IDA underwent rose
bengal-sensitized photooxidation. Singlet oxygen, the reactive
intermediate in this reaction, has been shown to be formed upon
irradiation of various naturally occurring pigments such as
chlorophyll, riboflavin, and "dissolved organic matter." (85)
Products identified from the NTA photoreaction were IDA, formalde-
hyde, and glycine; the photooxidation of IDA also produced
formaldehyde and glycine. Some metal -NTA complexes have previously
been shown to undergo photooxidation (without the presence of
sensitizer) , with production of IDA, formaldehyde and CC>2 (37/66).
The rates of sensitized photodestruction of NTA and IDA were quite
slow, with half-lives of 10-15 days. Accordingly, photooxidation
probably does not play a major role in NTA destruction in aqueous
solution.
ALGAL STUDIES
Two series of experiments were carried out in order to deter-
mine the effect of NTA on algal communities. The first series
of experiments was to determine the effect of NTA on the structure
of algal communities and the kinds of species composing thqm;
chlorophyll a/c ratios, which reflect the relative abundance of
diatoms to other algae; 14C uptake (measured as DPM per
36
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microgram of chlorophyll a) to determine photosynthetic activity;
and the effect of NTA on the accumulation of metals in the bio-
mass. These experiments were carried out in the winter and then
repeated during the summer months in order to determine the
effects of natural temperature and light variations.
The second series of experiments was to determine the effects
of NTA on the uptake of heavy metals by algae. The same measure-
ments used above to determine change were again made.
Effects of NTA on Algal Communities
The effects of 2.0 and 20.0 mg/1 of NTA were studied. In the
first experiment in November-February, the amounts of NTA during
the course of the experiment varied considerably (Table 5).
In the June-September experiments, the concentrations were more
nearly constant (Table 6). Average daily minimum and maximum
temperature were: November-February, 5.5-8.4°C; June-September,
18.7-22.2°C; March-April, 6.8-12.1OC. Values for pH fluctuated
between 7.4 and 8.5 in the November-February and June-September
experiments, and 7.3 and 7.9 in March-April.
Chemistry of water. The background chemistry of the water
in these series of tests (Tables 7 and 8) was very similar. In
the November-February and June-September tests, the N-NC>2 and
N-NH3 increased in the NTA experiments, particularly at the
concentration of approximately 20 mg/1. This was probably
due to the degradation of the NTA to NH3 and subsequent oxidation.
Sodium also increased when approximately 20 mg/1 NTA was
used, because it was introduced as a di- or tri-sodium salt.
Zinc seemed to be a little higher in the June-September tests.
Metals in algal biomass. Concentrations of calcium, mag-
nesium, sodium and potassium in the algae showed no consistent
trends between summer and winter experiments whether exposed
to NTA or not (Tables 9 and 10). However, zinc concentrations
tended to increase in the control and decrease in the 2 and 20
mg/1 tests in these experiments. There was no dramatic increase
in copper concentration in NTA exposed algae; whereas this was
observed1 in the controls in the winter and summer experiments.
In these experiments, iron tended to decrease in all treatments
and controls. Iron accumulation may have been affected by the
presence of NTA, however, for the decrease in the control was
not as great. The manganese data, although variable, suggests
that accumulation occurred both in the controls and NTA exposed
communities.
Thus, we see that the trend of decrease in many of the metals
was about the same in 2.0 and 20.0 mg/1 during the course of the
experiments. Likewise, we see the trend of increases was about
the same in the control and the various NTA experiments but
37
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increases are often not as large in the NTA tests as in the
controls.
The irregular accumulation in the biomass of various chem-
icals, even in the controls, is probably largely due to the
different growths of various algae in the experiments.
Characteristics of the Algal Communities
(November 14, 1974 to February 13, 1975)
Microscopic examination of the algal communities on Decem-
ber 10th and December 26th showed a diatom dominance in all
experiments (Table 11). Stigeoclonium protonema was common in
the controls on both dates!the blue-green alga Schizothrix
calcicola became common, and Microcoleus vaginatus was infrequent
to frequent. The conditions in the experiments in which the
average NTA concentration was 1.61 mg/1 showed diatom dominance
similar to the control. The blue-green alga £. calcicola became
common as in the controls. In one of the experiments(Box 1)
Scenedesmus sp. became freqxient to common. At approximately
20 mg/1 of NTA, although diatoms remained dominant, the diversity
was reduced and Achnanthes lanceolata seemed to be in poor
cytological condition on December 10th. The blue-green algae,
as in the control, at the end of the experiments were common.
This similarity in general structure of the algal communities
is evidenced by the chlorophyll a/c ratios (Table 12). The
DPM of 14C per microgram of chlorophyll a seemed to be somewhat
less at 20 mg/1 NTA, but because the activity of the replicates
was variable it is difficult to draw any conclusions.
Analyses of the structure of the diatom communities as
indicated by the truncated normal curve model indicates, as did
the direct observations during the course of the experiment, that
the structure of the diatom community was about the same in the
control, approximately 2 mg/1, and approximately 20 mg/1 NTA
experiments. As seen in Table 13, the number of intervals of
the curve covered, sigma squared, the number of observed species,
and the number of species in the mode seemed to be quite similar
and were not significantly different. There did tend to be a
decrease in the number of observed species and the number of
species in the mode, and an increase in sigma squared at the end
of all experiments when compared with the start of the experiments
at approximately 20 mg/1 NTA. In previous experiments this trend
has been observed when the invasion rate is reduced as happened
in these experiments by recycling of water (44) . When one looks
at the more common species (Table 14) it is evident that
Achnanthes lanceolata became a larger percent of the community in
approximately 20 mg/1 NTA when compared with the percent dominance
at the beginning of the experiment than in the control and
approximately 2 mg/1 NTA. It is also evident that Nitzschia
kutzingiana tended to increase at approximately 20 mg/l^pf tfTA as
compared to the control. The growth of Melosira varians seemed
38
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to increase more in the NTA tests than in the control. The
growth of Nitzschia fonticola seemed to be less in approximately
20 mg/1 experiment than in the approximately 2 mg/1 and the
control.
It should be noted that these are only very gross generaliza-
tions, because the sizes of the populations of the species at
the start of the experiment have a significant effect as to
whether that species becomes dominant. It is only the long
length of these experiments that allows one to tentatively draw
these conclusions, as over time the effect of the treatment on
reproduction tends to outweigh the size of the original popula-
tion.
Characteristics of the Algal Communities
(June 20, 1975 to September 3, 1975)
As seen in Table 15, the algal communities in all of the
test boxes were dominated by diatoms at the end of seeding
(July 2nd). The green alga Stigeoclonium lubricum was present in
all of the boxes and was frequent in Boxes 4 and 6. The blue-
green alga Schizothrix calcicola was rare to frequent in all of
the boxes at the beginning of the test experiment. Achnanthes
lanceolata and Melosira varians were common to very common.
Cocconeis placentula was common in the controls and Boxes 4 and
6, but was not noted in Boxes 5 and 7. On July 31st some shifts
in the communities had occurred. In Boxes 5, 7, 4, and 6,
Cocconeis placentula was common but in poor condition. In Boxes
5 and 7 D-.94 mg/1) it was pale in color whereas in Boxes 4 and
6 (19.2 mg/1) it was a deep golden brown, which condition had
been observed previously at high NTA concentrations. Melosira
varians had increased in all tests and the control. Achnanthes
minutissima had become common to very common in Boxes 5, 7, 4,
and 6, but not in the control. At the start of the experiments
it was not common in any of the boxes. Spirogyra sp. was very
common in the control, in poor condition but frequent in Boxes 5
and 7, and not present in Boxes 4 and 6. The blue-green alga
Schizothrix calcicola was common in Boxes 4 and 6, rare in
Boxes 5 and 7, and frequent in the control. These variations in
abundance, particularly in the control and Boxes 5 and 7, are
probably not significant. Microcoleus vaginatus was never common.
Detailed examinations of the diatom communities at the end
of seeding show that all of the communities were fairly similar
as seen in Table 16. Analysis by the end of the experiment,
two months later, indicates a normal sequence of events in which
diversity does drop in all boxes when invasion of new species is
cut off by recycling of water, with no significant differences
occurring between boxes. In all boxes a2 or the variance in
population sizes 'increased as the numbers of species dropped.
39
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The analysis of the diatom communities from the detailed
truncated normal curve studies (Table 17) at the end of seeding
substantiated the observations during the course of the experi-
ment-that is, Achnanthes lanceolata was the most common species.
However, the common occurrence of Cocconeis placentula in the
control and Boxes 4 and 6 was not as evident. It may have been
that these diatoms were not well scraped from the slides, as they
stick very tightly to the surfaces of the slides. Since Navicula
secreta var. apiculata is very hard to identify when it is alive,
it is not surprising that it was not recorded in the cursory
examinations. It is very probable that Gomphonema parvulum and
Cymbella cuspidatum, which were observed to be frequent to common
on some of the slides, may have been lost in the transfer to
boxes and final cleaning. These diatoms were growing on jelly
stalks and were fairly loosely attached to the slides. However,
the general condition of the diatom flora in the detailed readings
is quite similar to that from the observations. This analysis
at the end of the experiment shows normal shifts in the distri-
bution of the more common diatom species. Particularly evident
is the reduction in dominance of Achnanthes lanceolata and the
increase in dominance of A. minutissima. Differences between
boxes at this time are agiTin not significant.
As seen from Table 18, the percent volatile weight was
quite similar in all of the analyses or in all the boxes at the
end of seeding and at the end of the experiment. At 30 days after
the seeding the volatile weight seemed to be a little less in
the test boxes. At the end of the experiments the ash-free dry
weight averaged less in the test boxes. A reduction of the
chlorophyll a/c ratio is evident at the end of the experiment as
compared with the control, which reflects a lesser abundance of
algae other than diatoms. The primary productivity analyses
done at the end of the experiment show high variance between the
duplicate boxes, but the averages would be quite similar. The
phycocyanin evidence of blue-green algae increased most in one
of the control boxes and one of the 2 mg/1 NTA boxes. This was
not in accord with the microscopic examinations. This difference
may be due to some unknown error in the analyses or due to greater
amounts of Microcoleus vaginatus as compared to Schizothrix
calcicola. The former were larger filaments and the cells con-
tained more pigment.
In conclusion, one can say that the results showed similar
trends in the June-September experiments as in the November-
February experiments. In the approximately 20 mg/1 experiments
algal growth as measured by the ash-free dry weight was somewhat
less than in the controls at the end of the June-September experi-
ment. This was not evident in the November-February experiments.
Whereas during the November-February experiment the chloro-
phyll a/c ratios were quite similar in all boxes at the end of
the experiment, there was a decided decrease in the approximately
40
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20 mg/1 experiment in June-September and a smaller but not as low
a ratio of chlorophyll a/c in the approximately 2 mg/1 boxes.
These differences can be attributed to two causes (1) the NTA
concentrations were much more constant in the June-September
experiment than in the November-February experiments and
therefore the algae were consistently exposed to more NTA; and
(2) seasonal effects.
The only time a dramatic shift in species was observed was
in an initial screening experiment conducted to observe the
effects of NTA above the level of 20 mg/1. An achieved concen-
tration of 221.5 mg/1 maintained over 30 days led to the replace-
ment of the inital diatom-dominated community by blue-green algae
(Schizothrix and Microcoleus). This condition was verified by
pigment analyses.Biomass of the 200 mg/1 slides were greatly
reduced as compared to controls and communities exposed to
approximately 2.0 and 20.0 mg/1 NTA.
Experiments with NTA as a Chelator of Heavy Metals
These experiments were carried out in March and April, 1975.
As with the previous group of experiments, the water was medium
soft, rich in nitrogen and phosphorus in concentrations typical
of eutrophic conditions (Table 19). Silica was in abundant
supply. The other trace metals and cations and anions were in
the proportions expected in a soft water rural stream receiving
non-point discharges of nutrients. As seen in Table 20, an
average of about 1.57 mg/1 NTA was maintained in the NTA-Cu
tests, and about 1.37 mg/1 NTA was maintained in the NTA-only
tests. A low level of NTA contamination was found in the control
tests (0.02 mg/1) and in the Cu only test (0.01 mg/1). The
identity of the trace contaminants as NTA was confirmed by
GC-MS analysis. The source of the NTA was probably filters
containing paper made from Canadian pulp.
Microscopic observations of the algal communities showed
that in the control, NTA, and NTA plus copper throughout the
experiment the flora was dominated by diatoms (Table 21).
Navicula viridula var. avenacea was the most common species.
Achnantfies lanceolata and Nitzichia palea were common to very
common in the NTA test and frequent to common in NTA and copper
test. In the control, Microcoleus vaginatus became rare and
Stigeoclonium lubricum became fairly common at the end of the
experiment.In the NTA tests at the end of the experiment,
M. vaginatus was rare and S. lubricum was common. In the copper
alone, we found that the dTatoms were dead by March 22nd, roughly
one week after the start of the experiment, and gradually M.
vaginatus, Schizothrix calcicola, and Stigeoclonium lubricum
constituted the algal flora.
It should be noted that the concentration of copper was that
which previous experimentation indicated would be toxic or almost
41
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completely toxic to diatoms. As can be seen by these results,
the NTA chelated the copper and prevented the shift to blue-
green algal dominance after the death of the diatoms. These
results are also supported by a more detailed study of the diatom
community in which the truncated normal curve was generated. We
see from Table 22 that in all tests except those that had only
copper (in which all diatoms died), the height of the mode was
quite similar as were the number of observed species. Sigma
squared tended to be greater in the NTA and NTA plus copper than
in the control. The number of observed species averaged about
the same, and the intervals covered by the curves were similar
(Table 23). The more common species equal or greater than 10% in
the communities were very similar. Thus there is no evidence
that there was a severe alteration in the structure of the diatom
community so long as copper was chelated by NTA.
When one compared the diatom communities in the NTA tests in
March and April with previous experiments with approximately 2 ppm
NTA, it is evident that the number of species at the end of
seeding was less than in the two previous experiments; also the
height of the mode was less. At the end of the experiment the
characteristics of the curve were quite similar to previous
experiments.
The observations during the experiment and the detailed
readings coincide for A. lanceolata and N. viridula var. avenacea.
For species that were frequent in occurrence, one would not
expect them to be >^ 1.7% of specimens identified. The similar-
ities of the communities in the control, NTA, and copper plus
NTA was also manifested by the chlorophyll a/c ratios (Table 24).
Since the diatoms were dead in the copper experiments, no ratio
could be determined. The percent volatiles were similar on the
control, NTA, and copper plus NTA experiments. It was much less
as one would expect in the copper experiments, where the diatoms
were dead. The -^C uptake was quite similar in the control,
NTA, and copper and NTA experiments being greatly reduced in
those experiments where copper was alone. Thus we see that all
of the parameters used point to the similarity of the communities
in the control, NTA experiments, and copper plus NTA. They also
point to the great difference in those experiments where copper
only was present at approximately 100 yg/1.
Phycocyanin concentration was quite similar in all of the
boxes (Table 24). This probably means that in those tests
where diatoms were dominant the blue-green algae were harder to
see and therefore were not so actively recorded; whereas in the
copper experiments where everything was dead except blue-green
algae they were very evident and thus the observations would
indicate an increase in these boxes. Another explanation may be
due to the fact that Schizothrix calcicola, which became very
abundant, is a very small filament and one could have an increase
many fold in filaments without having much increase in phycocyanin.
42
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Thus, the phycocyanin is a very rough measurement of the number
of filaments present. However, it does indicate that blue-green
algae were in all the boxes as did the direct microscopic obser-
vation.
The ash-free dry weight, as one would expect, is much less
in the tests with copper alone. This weight was little less in
the NTA and NTA plus copper tests than in the control.
The experiments with NTA and copper showed increased growth
and diatoms in good condition in the control, NTA, and NTA plus
copper. In copper alone, the diatoms were in poor condition after
one week's exposure and were dead at the end of the experiment.
However, Schizothrix calcicola was abundant at the end of the
experiment as was a yeast, and in the reduced biomass these were
the dominant forms.
Copper in the biomass (Table 25) in the control was first up
a little, then down; in NTA alone it showed no significant change;
whereas, in the NTA plus copper it increased 2.4 times at the end
of the first week and approximately five times by the end of
seeding. As one would expect in copper alone, it increased 13
times at the end of one week after seeding and 42 times by the end
of the experiment.
In conclusion, NTA greatly influenced the uptake of copper
by the algal populations and prevented the toxic effect of copper
to the algal community. It prevented the decrease of l^c uptake
and maintained the chlorophyll a/c ratios similar to those in
the control. There seemed to be also an effect of the presence
of NTA on the absorption of other metals (one week after the
experiment started). In some cases the absorption was decreased
and in others it would appear that it was enhanced (Table 25).
BACTERIAL STUDIES
Bacteria are the most important organisms in the ecosystem
involved in the degradation of dissolved organic nutrients
with the resultant generation of mineralized end products (for
example, carbon dioxide from organic carbon compounds). Experi-
ments to determine the rate of degradation of NTA by the natural
stream flora at different seasons of the year were therefore done.
Degradation was assayed under aerobic conditions in these experi-
ments. Another focus of experiments conducted under this program
was to see whether the degradation of other substrates would
be affected by the presence of NTA-metal chelates.
NTA Degradation Studies
Experiments w-ith pure cultures of NTA degrading bacteria.
Preliminary experiments to evaluate procedures were conducted with
NTA degrading isolates; one (a Pseudomonas sp.) obtained courtesy
43
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of Dr. James Tiedje (Michigan State University), the other was
isolated from White Clay Creek. Organisms cultured in NTA broth
were harvested while in the log phase of growth, washed twice
in phosphate buffer (0.01 M, pH 7), and transferred to each of
six biometer flasks to provide a final concentration of ca. 1010
cells/ml in 15 ml. NTA mineralization was measured for 3 hr.
and cells were recovered at the end of the experiment by
filtration. The results with one isolate (T-l) are shown in
Fig. 10. Active mineralization was evident although only 10 per-
cent of the added radioactivity (170,000 DPM) was recovered as
l^C-carbon dioxide with this isolate after 3 hr. The White Clay
Creek isolate "216" mineralized 20 percent of the added radio-
activity in 90 min. (ca. 10^ organisms/flask). Negligible
activity was associated with the cells.
Experiments with natural bacterial communities. One experi-
ment was conducted between March 12 and April 24,T975. During
this time, the stream temperature ranged from 2.0 to 15.0°C
(mean + standard deviation 8.6 + 1.8°C). Assays were conducted
at 9°C. NTA concentrations in the microcosms are shown in
Table 26. NTA was detected in seven of 41 control samples: in
six the concentrations were in the range of 0.003-0.016 mg/1,
but the seventh contained 0.092 mg/1. This periodic contamina-
tion was traced to filters which were used to reduce the silt
load and algal cells in incoming water during the recycle
portion of the experiment. NTA was subsequently identified by
gas chromatography-mass spectrometry in the leachate from the
pressed paper filters.
Data from a typical series of assays from this experiment
is shown in Table 27. In most instances there is good replica-
tion among the five experimental flasks. There is a striking
difference in degradation rate between the experimental and dead
cell control flasks for populations exposed to NTA in microcosms.
For populations from the control (unexposed) microcosms, activity
is at background level.
Biodegradation rates before and after exposure to NTA are
shown in Fig. 11. The intermittent exposure by contamination
of populations in the control microcosms to NTA never led to NTA
degradation at an elevated rate such as that occurring in the
other microcosms. The development of degradation capability was
concentration dependent, with more rapid development being
correlated with higher concentrations. At the end of six weeks
there was a 250-fold difference in degradation rate 0.4 - 100 ng/
hr/cm2) over the 1000-fold range at which degradation was tested.
Between six and eight weeks the temperature in the microcosms
rose (mean + standard deviation, for this period 12.3 + 0.95°C).
At the eighth week, the degradation rate leveled off or declined
between 0.2 - 20 mg/1. Slight increases were observed for the
0.02 mg/1 and control populations.
44
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This may represent the achievement of a maximal degradation
rate by the flora. Alternatively, the rising water temperature
may have affected the flora detrimentally. If a flora with higher
temperature optimum was developing, and if the assay temperature
(9°C) was not limiting, the results support the contention that
during a time of rapid ambient temperature change, when organisms
with a new temperature optimum are developing, the ability of
the system to assimilate a given input would be diminished until
the new flora become established (85).
The number of cells capable of NTA degradation increased on
the coverslips through the fourth week of the experiment and
declined into the sixth week, as is shown in Fig. 12. Increases
were observed at some concentrations between the sixth and
eighth weeks. Greatest increases were obtained at the highest
NTA concentrations.
The experiment was repeated in July and August, 1975, when
stream temperatures were in the range of 14.5-22.4°C (mean +
standard deviation, 18.4 + 0.9°C). Assays were performed at
18°C. NTA concentrations in the microcosms are shown in Table 28.
The development of NTA degrading ability was again seen, but
this development was faster and reached a higher level of
activity than in the earlier experiment (Fig. 13). This is
presumably the result of warmer temperature since NTA concentra-
tions were similar. The activity of the populations exposed
to 0.2 - 20 mg/1 (actually 0.17 - 20.1 mg/1) appeared to
plateau by the fifth week of exposure. At the higher concentra-
tions (2.0 and 20 mg/1), there was approximately a tenfold
increase in decomposition rate in the summer experiment when
compared to the early spring experiment, but this was not the
case for the populations exposed to approximately 0.02 or 0.2
mg/1. The populations in the control microcosm were inter-
mittently exposed to NTA, and the exposure appeared sufficient
to bring about an increase in degradation rate by the second
and third weeks of the experiment (Fig. 13). Numbers of NTA
degrading organisms increased more than a hundredfold on exposure
to 2 and 20 mg/1 (Fig. 14). Lesser increases were observed at the
lower concentrations.
The complete degradation of NTA yields carbon dioxide,
ammonia, and water. In a supplemental experiment conducted in
November, 1974, NTA was metered into four microcosm systems to
establish concentrations of approximately 100 mg/1 and the water
was assayed over time for concentration of ammonia-N, nitrite-N,
and nitrate-N. The temperature during the experiment ranged
from 6.9 to 11.2°C.
Ammonia-N concentrations increased owing to the decomposition
of the NTA and subsequently, the nitrite-N concentrations increased
(Fig. ISA). Nitrate-N concentrations did not show much change.
The ambient levels of 2-3 mg/1 probably overwhelmed the maximum
45
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additional amount of 0.1-0.25 mg/1 that would have resulted from
the oxidation of all the nitrite-N. Similar results have been
noted in other experiments. Measurements of ammonia-N and
nitrite-N in the 20 mg/1 NTA system of the March-April, 1975,
decomposition experiment discussed above, showed an increase and
subsequent decline in concentration of ammonia-N and nitrite-N
(Fig. 15B). In another experiment, (August, 1974, 16-22°C)
increases in nitrate-N were also seen but NTA concentration in
this experiment appoximated 200 mg/1 (Fig. 15C).
In another study of NTA degradation, ^- C labeled NTA was
added to a recycle microcosm system in which NTA had been main-
tained at a concentration of approximately 2.0 mg/1 for approxi-
mately 35 days. During the three-day experimental period, the
average concentration was 2.2 mg/1 (n = 5, range 1.3-3.5 mg/1).
Temperature ranged from 8.0-10.2°C. The results are shown in
Fig. 16. Degradation of the 14C-NTA began immediately with the
subsequent release of CO2 to the water. After 12 hours,
approximately 24 percent of the radioactivity introduced into the
system was lost as 14CO2. The slope of the empirical line differs
from the slope of the line showing the theoretical loss from
dilution alone. A semi-logarithmic plot of the data was made,
and regression lines were fit. The difference in the slopes of
these lines was used to calculate a half life of NTA in the
system resulting from bacterial activity (T% = 14.4 hr.). The
degradation estimate from slopes of lines is probably conservative
because any non-volatile products of decomposition would be
considered as NTA.
In summary, these experiments with natural stream bacterial
populations showed (from direct measures of NTA mineralization)that
an NTA degrading flora will develop at different seasons of
the year and degradation will occur under aerobic conditions.
Degradation capability was correlated directly with concentra-
tion of exposure, duration of exposure, and with temperature.
Increasing temperature and concentration shortened the lag time
before degradation occurred at an elevated rate. Our findings are
in keeping with those of others (61*62,70,82) in which NTA
degradation (measured by disappearance) in water was also
demonstrated.
Preliminary experiments in which NTA degradation was followed
under microaerophilic (0.27 + 0.31 mg dissolved oxygen/1) and
oxygen free (<0.01 mg dissolved oxygen/1) were also performed.
Samples were sparged twice daily with helium to lower oxygen
concentrations in the first instance. To provide oxygen-free
conditions samples were incubated under H2-CC>2 in anaerobic jars
and measurements and manipulations were made in a glove bag under
an argon atmosphere. Sediment microbial populations degraded NTA
from initial concentrations of 8 and 140 mg/1 in the presence
of low oxygen but not from 200 mg/1 in its absence. In one
46
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experiment lasting 3.5 months the redox potential (Ecai) in the
oxygen free environment was -380 to -480 mv, conditions under
which N03- would not be present but S04= would be present to some
degree. Enfors and Molin (19,20) noted NTA degradation in an
anaerobic environment when N03~ was present at 1000 mg/1 to
serve as a terminal electron acceptor. Our results, although
preliminary, indicate that the redox potential will be an impor-
tant determinant of NTA degradation in anaerobic environments
and further studies should be done.
NTA-Metal Chelates and Bacterial Activity
Studies were done to examine the effectiveness of NTA in
protecting bacteria from the toxicity of selected toxic metal
ions. Heterotrophic bacterial populations from the White Clay
Creek and NTA degrading isolates were used in these studies. The
metabolism of l^C labeled glucose was used to measure effect.
Data from a typical experiment with stream populations is
presented in Fig. 17. The metal ions tested (zinc, cadmium,
copper) were toxic although to varying degrees. Protection was
afforded by the NTA-metal chelates.
The results with mixed heterotrophic populations cultured
from stream communities showed that the toxicity of cadmium,
zinc, lead and nickel varied considerably from experiment to
experiment with inhibition ranging from 18-84% (Table 29).
Inhibition of glucose incorporation by exposure to metallic ions
was similar whether expressed as a percent of the activity of
controls (metal/control) NTA exposed cells (metal/NTA) or cells
exposed to NTA-chelated metal (metal/chelate) . Because the composi-
tion of the inoculum undoubtedly differed between dates a strong
inverse relationship between inhibition and inoculum level was
not apparent. Copper toxicity, however, was consistently pro-
nounced (Table 30) and inhibition of glucose metabolism ranged
from 75-90%. Exposure to metals as NTA-chelates conferred
protection from toxicity. (Compare metal/control with chelate/
control columns in Tables 29 and 30). In two experiments (lead,
10/28; nickel 9/16) the metal concentrations were increased from
1 mg/1 to 20 and 30 mg/1 respectively, but even at these levels,
protection was obtained when the metal was chelated. Glucose
incorporation by mixed stream organisms in the presence of the
chelate was generally similar to that of the control; in 75% of
the experiments, activity was +25% that of the controls (chelate/
control in Tables 29 and 30). Some additional experiments per-
formed with cells that had been starved prior to glucose exposure
gave results essentially the same as those for unstarved cells
shown here. In experiments with lead chelates the acetate salt
was used. A separate experiment showed no effect on glucose
incorporation from acetate added in the amount that would be freed
on NTA-chelate formation.
47
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Simulation models (14; Chance, unpublished) suggest that 4.0
ppm NTA added to White Clay Creek water without chelation to a
specific metal yielded the iron or calcium chelate. Glucose in-
corporation in the presence of these NTA salts was occasionally
stimulated or repressed, but was + 25% of incorporation in con-
trols in 7 of 18 experiments (Table 31).
Other experiments also showed NTA neither dramatically stimu-
lated nor depressed the utilization of glucose bv mixed popula-
tions. In these assays, ^-^C labeled substrate was added initially
and NTA was added to provide final concentrations of either 2 or
4 mg/1 either initially or after a 48-minute preincubation during
which time a baseline metabolic rate was determined for the com-
munity. With this approach, a change in rate of 14c glucose
metabolism before and after NTA addition would be indicative of
an effect. Both incorporation and mineralization rates were moni-
tored, and the results (Fig. 18) show no striking rate change on
NTA addition compared to controls. Again, NTA was probably present
as the iron or calcium chelates, the predominant form in White
Clay Creek water. Other experiments conducted similarly but with
natural populations colonizing microscope cover glasses suggested
slight stimulation of 14c glycine mineralization by 20 mg/1 NTA
and of 14C glutamic acid mineralization by 2% NTA; but these NTA
concentrations are unexpected in nature.
NTA degrading isolates were used in short-term experiments.
Copper consistently reduced incorporation of ^C glucose by approx-
imately 90% (Table 32). Inhibition of glucose incorporation by
cadmium and zinc ions varied from 41-85% and 8-47% respectively -
The NTA-chelate of zinc protected organisms from toxicity (compare
metal/control with chelate/control) . However.- in one experiment
with cadmium and in those with copper, incorporation in the pres-
ence of the chelate was also considerably reduced compared to the
control. Perhaps metabolism of the chelate by the NTA degrading
isolate released metal ions in concentration sufficient to be
toxic to these populations unacclimatized to copper exposure. It
is also of interest that glucose incorporation by isolate "216"
was reduced when NTA was present as the Ca-or Fe-chelate, suggest-
ing the presence of NTA lowered the utilization of an alternative
energy and nutrient resource.
At the end of the experiment in which stream communities were
exposed to 30 yg Cu/1 and 2 mg NTA/1 or 2 mg NTA/1 with no copper
(pp.49-64), bacterial communities on silt from the ecosystem stream
bottom were tested for their ability to degrade 14C NTA. Popula-
tions from the Cu-NTA stream were tested for ability to degrade
the Cu-chelate and those exposed to NTA for their ability to de-
grade the Fe- or Ca-chelate. Although there were no statistically
significant differences between streams when NTA degradation
activity was normalized for bacterial numbers, activity normalized
for sediment weight was lower for the Cu-chelate than the Ca- and
Fe-chelates. The results of two trials are shown here:
48
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yg NTA degraded/yg sediment (n, x, s.d.)
Exposure Trial 1 Trial 2
Control 5, 145, 137 5, 47, 49
NTA/Cu 5, 110, 49 5, 147, 37
NTA 5, 316, 166 5, 864, 575
Differences in trial 1 were not significant statistically, but
in trial 2 the activity of the NTA exposed population was signif-
icantly greater than the control or the population that had been
exposed to NTA and copper for the previous four months.
Exposure over longer time periods was studied in experi-
ments in which the respiration of cell suspensions was used to
measure effect. Results are presented in Table 33. In all
instances exposure to Cu, Cd, Zn, and Hg ions stopped respira-
tion, but respiration usually resumed by 2.5 hr. A 5:1 NTArmetal
ratio provided protection from the toxic effects of metal ions
in all experiments but the one with mercury. Cell numbers in-
creased during experiments, and after 20 hr., a multiplicity of
types were observed (long and short bacilli, cocci, and some
yeasts) in many flasks. In all instances, the metal concentra-
tions associated with cells at the end of the experiment were
lower when the metal was added as the NTA chelate than when added
alone.
In summary, our studies on the effect of metals or NTA-metal
chelates on the metabolism of other organic compounds by natural
stream bacterial populations demonstrate that NTA afforded
protection from Cd, Cu, Zn, Ni, Pb, and Hg ions which were toxic
to varying degrees at 1.0 mg/1. Accumulation of metals was not
accelerated by NTA in the one experimental series in which this
was measured and additional experiments on the sequestering of
metals by NTA should be done. Additional work with NTA degrading
isolates which may release metal ions in concentrations sufficient
to be toxic and inhibit microbial activity is also needed.
EFFECT OF NTA AND COPPER IN ECOSYSTEM STREAMS
This study was performed to assess the impact of NTA on
entire communities in experimental streams in which natural con-
ditions could'be closely simulated. From our studies in
microcosms and the literature, we did not expect NTA to exert acute
effects on the biota and most emphasis was placed in this
aspect of the program on the chelation of metal ions by NTA. To
emphasize this, 2 mg/1 NTA was added along with 30 yg/1 copper
(as copper sulfate) to one stream (designated stream II), 30 yg/1
copper was added to another (designated stream III), 2 mg/1 NTA
to another (stream-IV) , and one stream was the control, which
received no additions (stream I). The concentration of copper and
49
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four other metals (iron, manganese, magnesium, and zinc) in
plant and animal biomass was monitored in order to examine whether
NTA, introduced at the suspected upper limit of environmentally
realistic concentrations, could alter these over a period of time.
The experiment lasted approximately 3 months, from July 13-
October 6, 1976. Average water temperature during the study
period was 16.9 + 2.00 (x + s.d., n = 86) and ranged from 10.5 -
22.50C.
Concentrations of several dissolved inorganic constituents
during the study period are presented in Table 34. There was
very little difference between streams in concentrations of the
ions measured. Concentrations of NTA and copper are shown in
Table 35. The mean concentrations of both NTA and copper actually
achieved were extremely close to those desired over the three
month experimental period.
Within the first week of the experiment, the community in the
stream receiving copper alone was dramatically affected.
Anacharis populations had a bleached appearance and eventually
disappeared as a result of decomposition and sampling pressure.
Algal populations sloughed off leaving the sediments bare for
approximately two weeks. At a later time, approximately three
weeks into the experiment, the green alga Ulothrix developed
macroscopically visible populations. Planarian worms which were
prevalant on the Anacharis also disappeared in the copper treated
stream. The streams receiving NTA and copper or NTA alone
appeared little different from the control stream, suggesting NTA
had no acute effects and, in addition protected the biota from
copper toxicity.
The concentrations of copper, iron, zinc, manganese and
magnesium ions in the tissue of algal communities, Anacharis sp.,
Lemna sp. and Planaria sp. and Tubifex sp. were monitored through
the experiment. The data for each sample type were used in a
two-way analysis of variance to test for significant difference
between times and streams. The ANOVA's invariably were invali-
dated by highly significant two-way interaction effects. Therefore,
the data were examined for (1) differences between the first
(prior to chemical additions) and subsequent (post treatment)
collections for each stream and (2) significant differences
between streams for individual collections that occurred consist-
ently. The Scheffe multiple range test (P = 0.05) was used where
variances were homogeneous and the Kruskal-Wallis multiple
comparison test (P = 0.05) where variances were non-homogeneous.
Both tests are conservative with respect to revealing significant
differences; the Kruskal-Wallis is more so than the Scheffe.
Statistically significant differences in data from post-treatment
collections in a given stream were also obtained sometimes but
these are not discussed in the interest of clarity. Although we
infer biological significance where statistical significance is
demonstrated, this may not always be the case.
50
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ALGAE
No material was available for collection from the copper
treated stream (III) at sampling times 2 and 3.
Copper (Fig. 19)
Data for collection 5 was excluded from analysis because of
small sample size.
Differences between first and subsequent collections in;
Stream I. Concentrations of copper in algal communities
(Control) decreased through time. Collections 6, 7, and 8
were significantly lower than 1 (Scheffe test).
Stream II. Concentrations decreased through time. Concentra-
(NTA/Cu) tions in collections 7 and 8 were significantly
lower than in 1. (Scheffe test).
Stream III.Concentrations associated with the copper tolerant
(Cu) flora (collections 4-8) were significantly
increased over those in the algae present at the
start of the experiment (Scheffe test).
Stream IV. The concentration in collection 4 was significant-
(NTA) ly greater than in collection 1 but the weight
for collection 4 was smaller than for any other.
The coefficient of variation for the data was
similar to other data sets but the small sample
size may have been a source of error. Concen-
trations at collections 6 and 8 were significantly
lower than at the start. (Kruskal-Wallis test).
Differences between streams at each collection:
Copper concentrations in algae in the streams receiving NTA
and the control differed to a statistically significant extent
only once (Table 36). At collection 4 the algae in stream IV
(NTA) had higher copper levels but as noted above, error may be
associated with this data. Copper concentrations in copper
tolerant algae from stream III (Cu) were greater than in the algae
from the other streams in collections 4, 6, and 7 and in the
single determination at collection 8 although the statistical
tests used did not always reveal significance. Copper concentra-
tions in algae exposed to CU and NTA (stream II) never differed
significantly from the control. This indicates that NTA prevented
the uptake of the added copper, since concentrations in algae in
stream III were much higher.
Iron (Fig. 20)
Differences between first and subsequent collections in;
Stream I. Iron concentrations in the algae showed no signifi-
(Control) cant difference between collection 1 and subse-
51
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quent samples but for collection 2 which was higher
(Kruskal-Wallis test).
Stream II. No significant differences occurred but for
(NTA/Cu) collection 2, which was higher than 1 (Kruskal-
Wallis test).
Stream III.Iron concentrations in collections 6 and 7 were
(Cu) elevated over collection 1 (Kruskal-Wallis test).
Stream IV. No significant differences between the first and
(NTA) subsequent collections occurred but for collection
2 which was higher (Kruskal-Wallis test).
Iron concentrations at collection 2 were elevated in samples
from all streams, but the coefficients of variation for these
data (9-45%) were not greater than for other collections and so
the data are not excluded.
Differences between streams at each collection:
No significant difference between streams receiving NTA (II
and IV) and the control occurred in six of seven post treatment
collections (Table 36). The exception was collection 7 when algae
in stream II (Cu/NTA) contained more iron than those from the
control, but variance for these samples is large. NTA exposure
appeared to have no effect on the iron content of algae. Con-
centrations of iron in algae in the copper treated stream (III)
tended to be higher and were significantly elevated over the
control in two collections (5 and 7) .
Zinc (Fig. 21)
Data for collection 5 was excluded because small sample
size may have introduced analytical error. Data for stream I
at collection 4 had a coefficient of variation of 166% (compared
with the 8-30% associated with most data) and was also eliminated
from analysis.
Differences between first and subsequent collections in;
Stream I. Zinc concentration in collection 3 algae was
(Control) significantly greater than in collection 1
material (Kruskal-Wallis test).
Stream II. Collections 4, 7, 8, were significantly lower
(NTA/Cu) than collection 1 (Kruskal-Wallis test).
Stream III. No significant difference in concentrations
(Cu) between the first and subsequent collections
(Scheffe test).
Stream IV. At collections 2, 6, 7 and 8, zinc concentra-
(NTA) tions in the algae were significantly lower
52
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than in collection 1 (Scheffe test).
Differences between streams at each collection;
Prior to treatment (collection 1) differences between streams
(I and IV) were observed.
No statistically significant differences in zinc concentra-
tion in algae from the control and NTA treated streams (II and
IV) occurred at collections 3, 6 and 7 (Table 36). When com-
pared to the control, algae in these streams had significantly
lower zinc concentrations in collection 2, and in collection 8 the
algae in stream II did. In collection 7, algae in the copper
treated stream contained significantly more zinc than algae in the
other streams.
Concentrations of zinc in algae in stream IV (NTA treatment)
declined through time and were significantly lower at the end of
the experiment than at the start. Although statistically
significant differences between streams were not observed con-
sistently, the decreasing trend in concentration over time in
streams receiving NTA suggests that zinc concentration in algae
biomass should be monitored in future studies with NTA.
Manganese (Fig. 22)
Differences between first and subsequent collections in:
Stream I. No significant differences in manganese concentra-
(Control) tions in algae were found between collection 1
and subsequent collections (Kruskal-Wallis test).
Stream II. At collections 2 and 6 there was significantly
(NTA/Cu) less manganese in algae than at collection 1
(Kruskal-Wallis test).
Stream III. Concentrations in collections 5 and 6 were
(Cu) significantly lower than in collection 1 (Kruskal-
Wallis test).
Stream IV. At collection 2, manganese concentration was
(NTA) significantly lower than at collection 1 (Kruskal-
Wallis test).
Differences between streams at each collection;
Concentrations in algae in the treated streams were not
significantly different from the control for collections 2, 3, 4,
6, and 7 (Table 36). In two collections (5 and 8) manganese con-
centrations in algae from stream IV (NTA) were significantly
elevated over the control, but in collection 8 the concentration
in algae from the. NTA/Cu stream (II) was significantly lower than
collection 8 in the control. Again, note that differences were
observed prior to treatment; at collection 1 stream I differed
significantly from streams III and IV. The lack of consistent
53
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differences between streams and the similarity of concentration
changes in all streams both suggest that NTA exposure did not
affect manganese concentrations of the algal communities studied
here.
Magnesium (Fig. 23)
Differences between first and subsequent collections in;
Stream I. Magnesium concentrations in algae in collections
(Control) 2 and 5 were significantly higher than in
collection 1 (Kruskal-Wallis test).
Stream II. In collections 2, 5, and 7 concentrations were
(NTA/Cu) significantly higher than in collection 1
(Kruskal-Wallis test).
Stream III. In collections 5 and 7 concentrations were signifi-
(Cu) cantly higher than in collection 1 (Kruskal-
Wallis test).
Stream IV. Concentration of magnesium in algae in collection
(NTA) 5 was significantly higher than in collection 1
(Scheffe test).
Although significant differences in concentration of magnesium
in algae were obtained between the initial and post treatment
collections, these occurred in all streams and there was no
increasing or decreasing trend in concentration through time.
Differences between streams at each collection:
Magnesium concentrations in the algae from the control and
NTA containing streams differed significantly over at collection 7
when algae in stream II (NTA/Cu) had higher levels than algae in
the control (Table 36). Overall, it is unlikely that NTA exposure
affects magnesium concentration in algae. Concentrations in
material from the copper treated stream (III) differed significantly
from the control in collections 6 and 7 (Table 36) although
standard deviations overlapped at collection 6.
ANACHARIS
No material was collected from stream IV at collection 6 or
from stream III following collection 4.
Copper (Fig. 24)
Differences between first and subsequent collections in;
Stream I. At collection 4, Anacharis had significantly more
(Control) copper than at collection 1 (Kruskal-Wallis test).
Stream II. Samples in collection 8 contained significantly
'(NTA/Cu) less copper than material at collection 1.
(Kruskal-Wallis test).
54
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Stream III.Concentration in collection 2 was significantly
(Cu) greater than in collection 1 material (Scheffe
test) and the single determination at collection
4 was elevated.
Stream IV.
(NTA)
Collections 6, 7, and 8 contained significantly
less copper than collection 1. (Kruskal-Wallis
test).
Changes in concentrations of copper in Anacharis through
time were similar in all streams.
Differences between streams at each collection;
Differences between the control (I) and streams receiving
NTA (II and IV) existed in some collections (Table 37) but some-
times concentrations in NTA exposed material were significantly
greater and sometimes significantly less than in material from the
control stream. Overlap of standard deviations occurred in some
instances. Copper concentrations in Anacharis in stream III
(Cu) were elevated over the control in collections 2 and 4 but
not 3 although differences were not statistically significant by
the tests used. Differences existed prior to treatment. Stream
II differed from stream III and IV.
Iron (Fig. 25)
Differences between first and subsequent collections in:
Stream I. Iron concentrations in Anacharis in collections 3
(Control) and 5 were significantly lower than in collection
1 (Kruskal-Wallis test).
Stream II. In collections 3, 5, 7, and 8 concentrations were
(NTA/Cu) significantly lower than in collection 1.
(Kruskal-Wallis test).
Stream III.Concentration in collection 2 was significantly
(Cu) higher than in 1 (Scheffe test).
Stream IV. In collections 2 through 8, Anacharis contained
(NTA) significantly less iron than in collection 1.
(Scheffe test).
Differences between streams at each collection:
Differences existed before chemical additions; at collection
1 stream IV was significantly higher than I (Table 37). Concen-
trations of iron in Anacharis in the copper treatment were
significantly elevated over the control in collection 2 and 3,
and in the single sample available at collection 4. Although
significant differences between one of the NTA treated streams
and the control were observed only twice, (Stream IV, collection
4; stream II, collection 7, Table 37) iron concentration in
55
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material exposed to NTA tended to be lower than in the control.
Iron accumulation in Anacharis should also be more thoroughly
investigated in any future work with NTA and aquatic organisms.
Zinc (Fig. 26)
Differences between first and subsequent collections in:
Stream I. In this stream large variance was associated with
(Control) collection 1 data and only collection 3 differed
from it (Kruskal-Wallis test).
Stream II. Differences between the first and subsequent
(NTA/Cu) collections were not significant (Kruskal-Wallis
test) .
Stream III. Collection 3 was significantly higher than 1
(Cu) (Scheffe test).
Stream IV. In collections 2, 7, and 8, Anacharis contained
(NTA) significantly less zinc than in collection 1; in
collections 3, 4, and 8 it did not (Kruskal-Wallis
test). Five extremely high values (range: 1687-
2977 yg/g were eliminated from collection 6 data
because of suspected contamination.
Differences between streams at each collection:
A significant difference in zinc concentration in Anacharis
from the control stream and either one or both of the streams
receiving NTA was observed in all post-treatment collections but
3 and 6 (Table 37). The tendency was for the concentration of
zinc to be lower in Anacharis exposed to NTA. No significant dif-
ferences between material in the control and copper treated
stream was observed. As with algae, NTA appeared to lower zinc
concentrations in plant tissue and for the Anacharis data,
statistical significance is present.
Manganese (Fig. 27)
Differences between first and subsequent collections in:
Stream I. No significant differences in concentration of
(Control) manganese in Anacharis between the first and
succeeding collections occurred but for collection
2 which was significantly lower (Kruskal-Wallis
test).
Stream II. Concentration in collection 2 was significantly
(NTA/Cu) lower than in collection 1 (Scheffe test).
Stream III. Concentrations in collections 1, 2, and 3 were
(Cu) not significantly different (Scheffe test) but
the single sample at collection 4 had a valxfe of
507 yg/g.
56
-------
Stream IV. No significant difference in manganese concentra-
(NTA) tion in Anacharis occurred between collection 1
and subsequent collections but for collection 2
which was lower (Scheffe test).
Concentrations at collection 2 were low is material from all
streams.
Differences between streams at each collection;
A significant difference between the control and NTA exposed
material was observed only once; in collection 4 concentrations
in Anacharis exposed to NTA were significantly lower (Table 37).
Overall, NTA exposure had no striking effect on manganese con-
centrations in this plant. Differences between the control and
copper treatment were not significant except in the single sample
taken at collection 4 in which the concentration was unusually high.
Magnesium (Fig. 28)
Differences between first and subsequent collections in:
Stream I. Magnesium concentrations in Anacharis were signifi-
(Control) cantly higher at collections 3, 4, and 5 than at
collection 1 (Kruskal-Wallis test).
Stream II. In collection 3, the concentration was signifi-
(NTA/Cu) cantly higher than collection 1; otherwise, there
were no significant differences (Kruskal-Wallis
test).
Stream III. The concentration in collection 3 was significant-
(Cu) ly higher than collection 1 (Scheffe test).
Stream IV. No significant differences between collection 1
(NTA) and subsequent collections occurred (Kruskal-
Wallis test).
Differences between streams at each collection:
A significant difference in magnesium concentration in
Anacharis from either NTA stream and the control was obtained only
in collection 8 (Table 37). Concentration in collection 3 material
from the copper treatment was elevated over the control. Under
the experimental conditions, NTA exposure did not appear to
affect the concentration of magnesium in Anacharis.
LEMNA
No material was available from stream IV at collection 8.
Copper (Fig. 29)
Differences between first and subsequent collections in;
Stream I. Copper concentration in Lernna at collection 8
(Control) was lower than in collection 1 (Kruskal-Wallis
test). 57
-------
Stream II. Concentration in collection 4 was significantly
(NTA/Cu) greater than in collection 1 (Kruskal-Wallis test).
Stream III. Concentrations in pre- and post-treatment material
(Cu) were not different (Kruskal-Wallis test).
Stream IV. Concentrations in material from collections 4 and
(NTA) 5 were significantly greater than at collection 1
(Kruskal-Wallis test).
Changes in concentration through time were similar in all
streams.
Differences between streams at each collection:
Copper concentrations in Lemna from the control and the NTA
treated streams were significantly different only in collection
5 (Table 38). Samples from stream I (Control) and IV (NTA) never
differed significantly, but in collection 5 material from stream
II (NTA/Cu) differed from the control. The Lemna taken from
the copper (III) stream contained significantly more copper than
samples from the control at collection 2 and 4. It is unlikely
the NTA treatments used here affected the concentration of copper
in Lemna. Prior to treatment, streams I and IV differed.
Iron (Fig. 30)
Differences between first and subsequent collections in;
Stream I. Iron concentrations in Lemna in collections 7 and
(Control) 8 were significantly lower than in collection 1
(Kruskal-Wallis test).
Stream II. Iron concentration in collection 7 was signifi-
(NTA/Cu) cantly lower than in collection 1 (Kruskal-Wallis
test).
Stream III. Differences in concentration between collection 1
(Cu) and subsequent collections were not significant
(Kruskal-Wallis test).
Stream IV. Collection 4, 5, and 7 contained significantly
(NTA) lower iron concentrations than collection 1
(Scheffe test).
Concentrations in collection 6 were unusually high in samples
from all streams.
Differences between streams at each collection;
No significant differences in concentration between samples
from control or either NTA stream existed in four post-treatment
collections (2, 3, 5, and 7, Table 38). Concentrations in stream
IV (NTA) material were lower in two collections (4 and 6) and
higher in stream II (NTA/Cu) material at collection 8 although
58
-------
standard deviations overlap in the latter instance. It is
unlikely NTA exposure affected iron concentrations in Lemna.
Zinc (Fig. 31)
Data from collection 3 was excluded from analysis because of
contamination.
Differences between first and subsequent collections in:
Stream I. No significant differences in zinc concentration
(Control) in Lemna occurred between collection 1 and subse-
quent collections (Kruskal-Wallis test) .
Stream II. Concentrations in collections 7 and 8 were signifi-
(NTA/Cu) cantly lower than in collection 1 (Kruskal-Wallis
test).
Stream III. Concentration in collection 8 was significantly
(Cu) lower than in collection 1 (Kruskal-Wallis test).
Stream IV. No significant differences in zinc concentration
(NTA) in Lemna occurred between collection 1 and subse-
quent collections (Scheffe test).
Differences between streams at each collection;
Significant differences in concentration of zinc in Lemna
between the control and one or other of the streams receiving NTA
were observed in all collections and at collections 4 and 7, the
material from both NTA streams differed significantly from the
control (Table 38). The trend was for NTA exposure to lower the
measured concentration of zinc in Lemna biomass. This is the
trend noted with algae and Anacharis. Copper exposure (III)
affected zinc concentration significantly only in collection 2.
Manganese (Fig. 32)
Differences between first and subsequent collections in:
Stream I. Manganese concentrations in Lemna at collections
(Control) 6 and 7 were significantly lower than at collection
1 (Kruskal-Wallis test).
Stream II. Manganese concentration in collection 2 was
(NTA/Cu) significantly higher than in collection 1 and in
collection 6 it was significantly lower (Scheffe
test).
Stream III. Concentrations in all collections but 4 (i.e. 2,
(Cu) 3, 5, 6, 7, and 8) were significantly lower than
in collection 1 (Scheffe test) .
Stream IV. Concentrations in collections 5, 6, and 7 were all
(NTA) significantly lower than in collection 1 (Kruskal-
Wallis test).
59
-------
Differences between streams at each collection;
Concentrations of manganese in Lemna from streams I and III
were significantly different at collection 1. Variable groupings
of data were obtained at each collection time and consistent
differences between streams were not evident (Table 38). One or
both NTA streams differed from the control usually, but the
concentrations in the NTA exposed material were sometimes lower
and sometimes higher. Copper exposure significantly lowered
manganese concentration in Lemna in five of seven post treatment
collections.
Magnesium (Fig. 33)
Differences between first and subsequent collections im
Stream I. Concentrations in collections 3 and 5 were signifi-
(Control) cantly higher than in collection 1 (Scheffe test).
Stream II. Concentrations in collections 2, 3, 5, and 6 were
(NTA/Cu) significantly higher than in collection 1 (Scheffe
test) .
Stream III. In collections 2 and 3 concentrations were signifi-
(Cu) cantly higher than in collection 1 (Kruskal-Wallis
test).
Stream IV. In collections 2, 3, and 5, concentrations were
(NTA) significantly higher than in collection 1 (Kruskal-
Wallis test).
All streams tended to have higher concentrations at collec-
tions 2, 3, 5, and 6 compared to the initial values; some differ-
ences were significant, others were not.
Differences between streams at each collection;
Magnesium concentration at collection 1 was significantly lower
in Lemna from stream IV than stream I (Table 38). Differences be-
tween the control and treated streams existed only in collections
7 and 8 when the Lemna from streams IV and III, respectively, had
lower magnesium concentrations than the control. In conclusion,
NTA exposure did not appear to affect the concentration of mag-
nesium in Lemna.
PLANARIA
No Planaria were found in the copper treated stream (III)
after treatment but for collection 7. NTA conferred protection
from copper toxicity when added simultaneously in stream II, as
samples were available throughout the experiment.
Copper (Fig. 34)
Differences between first and subsequent collections in;
Streams I, II. Copper concentration in Planaria showed no
60
-------
(Control and significant differences between collection 1
NTA/Cu) and subsequent collections (Stream I, Kruskal-
Wallis test; Stream II, Scheffe test).
Stream IV. Concentrations in collections 2, 6, and 8
(NTA) were significantly lower than in 1. There
were no significant differences between any
collections following NTA additions, sug-
gesting that collection 1 was unusually
high (Kruskal-Wallis test).
Differences between streams at each collection:
Significant differences occurred prior to treatment (col-
lection 1); Planaria from stream IV contained significantly more
copper than those in streams I and II. There were no significant
differences between the NTA treated streams and the control after
treatments were initiated (Table 39).
Iron (Fig. 35)
Stream I. In collections 7 and 8, the Planaria had signifi-
(Control) cantly lower iron concentrations than collection
1 (Kruskal-Wallis test).
Stream II. Concentration in collection 8 was significantly
(NTA/Cu) lower than in collection 1 (Kruskal-Wallis test).
Stream IV- No significant differences between first and
(NTA) subsequent collections existed (Scheffe test).
But for collections 4 and 8 there were no statistically
significant differences in iron concentration in Planaria between
streams (Table 39).
Zinc (Fig. 36)
Data for collection 5 were elevated for all streams, and
although the coefficients of variation for the data from each
stream were not unusually great, the data are excluded from
analysis because of suspected contamination.
Differences between first and subsequent collections in stream;
Stream I. Concentrations in collections 3 and 8 were signifi-
(Control) cantly lower than in collection 1 (Scheffe test).
Stream II. No significant differences between collection 1
(NTA/Cu) and subsequent collections existed, but variance in
the data was great in collection 4 (Kruskal-Wallis
test).
Stream IV- No significant differences between the first and
(NTA) subsequent collections were found but variance in
the data was great in collection 4 (Kruskal-Wallis
test). 6i
-------
Differences between streams at each collection:
There was no significant difference between streams for zinc
concentration in Planaria (Table 39). Lowered concentrations in
algae exposed to NTA did not lead to lowered zinc concentrations in
at least one animal that grazes on the algae.
Manganese (Fig. 37)
Differences between first and subsequent collections in stream;
Streams I, II, IV. There were no significant differences between
(Control, NTA/Cu, the first and subsequent collections (Kruskal-
and NTA) Wallis test).
Differences between streams at each collection;
There were no significant differences between streams in man-
ganese concentrations in Planaria at any collection time (Table 39).
Magnesium (Fig. 38)
Differences between first and subsequent collections in streams;
Streams I, II. Collections 5 and 6 were significantly higher
(Control, NTA/Cu) than collection 1 (Scheffe test).
Stream IV. There were no significant differences between
(NTA) the first and subsequent collections
(Kruskal-Wallis test).
Differences between streams at each collection;
There were no significant differences in magnesium concentra-
tion in Planaria between streams at any collection except collection
4 when the concentration in stream II material was significantly
higher than in stream I material, and in collection 6 when the con-
centration in Planaria from stream IV was significantly lower than
in stream I material (Table 39).
Samples of tubificid worms were taken from the sediments of
each stream for metal analyses at collections 3-8. Most times
only a single sample of sufficient dry weight for metal analysis
could be obtained with the sampling effort possible. Therefore,
the data from all collections has been analyzed together.
Copper was the only metal for which statistically significant
differences between streams was obtained (Table 40). Mean con-
centrations in animals from the streams receiving copper (II and
III) were significantly greater than in samples from the control
but differences between the treated streams were not significant.
In another analysis, concentration factors were calculated
for each sample type to assess bioaccumulation. For each metal,
concentration in the biomass from a given stream was divided by
the cumulative mean concentration in the water of that stream *,to
that point in time (ug/g x 1000 '.- yg/1, or ppb * ppb) . These
concentration factors were calculated for each collection from
4-8 and the mean was obtained. Concentrations of metals in the
62
-------
water are shown in Table 41. Where measured concentrations were
below the lower detection limit of the analysis procedure, the
lower detection limit was used as the concentration in calculating
the cumulative mean. Concentration factors and the results of
statistical tests for significant differences are summarized in
Table 42. Variance in the data was large and no significant dif-
ferences between the control stream (I) and any of the experimental
streams were obtained. Usually all streams grouped together
(no significant differences in concentration factor). In the
two instances when they did not (algae, Cu and Planaria, Cu) the
samples from control still grouped with those from each NTA
stream suggesting no effect from NTA exposure on metal accumula-
tion.
The possible biomagnification of metals between Planaria and
algae was examined also (Table 43). Only zinc showed biomagnifi-
cation in the Planaria tissue; the other metals showed reduced
concentrations in the animal tissue compared to the plant tissue.
For zinc there was a significant difference between streams7
the ratio was greater in streams containing NTA than in the
control.
Although samples of sediments were cored from each stream for
metal analyses, the data suggested that after the first few
collections the sediments were mixed, which probably occurred as
samples were removed. The sampling area was limited by the size
of pools in each stream and so samples could not be obtained from
undisturbed areas for the entire experiment. Therefore, another
experiment was performed in recirculating microcosms to simulate
exposure of sediments in a flowing water environment to copper and
NTA over a period of 9 weeks. NTA and copper were added to
provide 30 yg Cu++/l and 2.0 mg NTA/1 in the experimental micro-
cosms (Table 44).
Concentrations of copper were measured at the sediment sur-
face and at 2 and 5 cm depths four times through the experiment
(Table 45). A mean copper concentration was calculated for the
experiment using the mean of each of the four sampling times and
between treatments and depths. Copper concentrations in the
control did not differ with depth but in sediments exposed to Cu
or Cu/NTA, the copper concentration at the surface was signifi-
cantly higher than at 5 cm but not 2 cm. There were no statistically
significant differences in copper concentration as a result of
treatment at the 2 and 5 cm depths. At the surface concentration
was greater in the Cu/NTA and Cu only treatments than in the
control. However, only the Cu/NTA treatment differed significantly
from the control and it did not differ significantly from the
copper treatment. Sanchez and Lee (58), however, noted that NTA
enhanced the adsorption of copper by lake sediments.
At the end of the experiment sediments were also tested for
concentrations of iron, manganese and zinc (Table 46). These
63
-------
did not differ significantly with depth in any treatment (Kruskal-
Wallis test, P = 6.05 for Zn in control and copper treatments
otherwise Scheffe tests, P = 0.05). There were no statistically
significant differences between treatments for manganese and
zinc concentration at any depth or for iron at the surface and
5 cm. At the 2 cm depth, however, iron concentration was
inexplicably high in the Cu and Cu/NTA exposed sediments. Note
that zinc concentrations were near the lower detection limit of
the method used for analysis.
CONCLUSIONS
NTA clearly protected the organisms in stream II from the
toxic effects of copper. Copper concentrations in algae, Anacharis,
Lemna or Planaria. from stream II were not elevated over control
data when copper was added as the NTA chelate. Zinc concentra-
tions in samples of algae, Anacharis, and Lemna exposed to NTA were
lower than concentrations in samples from the control stream in
several collections. Sufficient NTA (2 mg/1, 10~^ M) to com-
pletely chelate the copper (30 yg/1, 4 x 10-7 M) was used which
also would have complexed with some (if not all) the zinc (1 x
10-6 M) present in the White Clay Creek water in stream II. Where
NTA was added alone (stream IV) the probability of zinc-NTA com-
plex formation was all the greater. It is likely that the lower
concentrations in the biota resulted from the chelation of zinc
by NTA and relative inaccessability of the NTA-zinc complex to the
biota compared to the zinc-carbonate complex which predominated in
the control (Chance, unpublished M.S. thesis, University of
Pennsylvania, has studied the chemistry of inorganic ions in White
Clay Creek water). Examination of concentration factors lends
support to this observation. For zinc, these were lower in the NTA
containing streams than those not containing NTA (although statistical
significance was not noted). Concentration of magnesium, manganese
and iron were probably not affected by the presence of NTA
although iron in Anacharis may have been lower owing to the
presence of NTA and should be more thoroughly studied in the
future work with the compound.
Although 2 mg/1 NTA protected the biota from harmful effects
of 30 yg/1 copper, copper accumulation in sediments was not pre-
vented by NTA at this level nor did depletion through extraction
occur. Similarly, the accumulation of Cu in sediment dwelling
worms was also unaffected. Copper concentrations in tubificids
from both streams to which copper was added were elevated over the
values for the control stream even when NTA was added simultaneously.
64
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Nat. Acad. Sci. U.S. 58:1335-1342, 1967.
45. Patrick, R. The structure of diatom communities in similar
ecological conditions. Amer. Nat., 102:173-183, 1968.
46. Patrick, R. Effects of trace metals on aquatic ecosystems.
Am. Scientist, 66:185-191, 1978.
47- Patrick, R., T. Bott, and R. Larson. The role of trace
elements in management of nuisance growths. EPA Report
EPA-660/2-75-008, 1975, 250 pp.
48. Patrick, R., B. Crum and J. Coles. Temperature and manganese
as determining factors in the presence of diatom or blue-
green algal floras in streams. Proc. Nat. Acad. U.S.
64:472-487, 1969.
49. Patrick, R., M. H. Hohn, and J. H. Wallace. A new method for
determining the pattern of the diatom flora. Not. Naturae,
Acad. Nat. Sci. Philadelphia, No. 259, 1954, 12 pp.
68
-------
50. Pfeil, B. H., and G. F. Lee. Biodegradation of nitrilotri-
acetic acid in aerobic systems. Env. Sci. Techn. , 2:543-
546, 1968.
51. Pickaver, A. H. The production of N-nitrosoiminodiacetate
from nitrilotriacetate and nitrate by microorganisms growing
in mixed culture. Soil Biol. Biochem. 8:13-17, 1976.
52. Pramer, D., and R. Bartha. Features of a flask and method
for measuring the persistence and biological effects of
pesticides in soil. Soil Sci., 100:68-70, 1965.
53. Proctor, V. W. Studies of algal antibiosis using Haemato-
coccus and Chlamydomonas. Limnol. Oceanogr., 2:125-139, 1957,
54. Provasoli, L., J. A. A. McLaughlin, and M. R. Droop. The
development of artificial media for marine algae. Arch.
Mikrobiol., 25:392-399, 1957.
55. Rajabalee, F- J. M., M. Patven, and S. Laham. Separation of
NTA and EDTA chelates by thin-layer chromatography. J.
Chromatog., 79:375-379, 1973.
56. Rice, T. R. Biotic influences affecting population growth
of planktonic algae. Fish. Bull., U.S. Fish Wildlife Serv.,
87:227-245, 1954.
57. Robinson, J. L., and P. F. Lott. A fluorometric method for
the determination of nitrilotriacetic acid. Microchem. J.,
18:128-136, 1973.
58. Rudd, J. W. M., and R. D. Hamilton. Biodegradation of tri-
sodium nitrilotriacetate in. a model aerated sewage lagoon.
J. Fish Res. Bd. Canada, 29:1203-1207, 1971.
59. Sanchez, I., and G. F. Lee. Sorption of copper on Lake Monona
sediments. Effect of NTA on copper release from sediments.
Water Res., 7:587-593, 1973.
60. See^y, G. F., J. J. Duncan and W. E. Vidaver. Preparative
and analytical extraction of pigments from brown algae with
dimethyl sulfoxide. Mar. Biol., 12:184-188, 1972.
61. Shannon, E. E., P. J. A. Fowlie, and "R. J. Rush. A study of
nitrilotriacetic acid (NTA) degradation in a receiving
stream. Canada Env. Prot. Serv. Tech. Develop. Report
EPS-4-WP-74-7, 1974, 34 pp.
62. Shumate, K. St., J. E. Thompson, J. O. Brookhart, and C. L.
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Water Pollut. Contr. Fed., 42:631-640, 1970.
69
-------
63. Sillen, L. G., and A. E. Martell. Stability constants of
metal-ion complexes. Spec. Pub., No. 17r Chenu Soc.,
London, 1964.
64. Sprague, J. Promising anti-pollutant: chelating agent NTA
protects fish from copper and zinc. Nature, 220:1345-1346,
1968.
65. Stine, G. J., and A. A. Hardigree. Effect of nitrilotri-
acetic acid on growth and mating in strain of Escherichia
coli K-12. Can. J. Microbiol., 18:1159-1162, 1972.
66. Stolzberg, R. J., and D. N. Hume. Rapid formation of
iminodiacetate from photochemical degradation of Fe(III)
nitrilotriacetate solutions. Environ. Sci. Technol., 9:
654-656, 1975.
67. Sturm, R. N., and A. G. Payne. Environmental testing of
trisodium nitrilotriacetate: Bioassays for aquatic safety
and algal stimulation. In Bioassay techniques and environ-
mental chemistry, Ann Arbor Science Publ., Ann Arbor, 1973,
pp. 403-424.
68. Swingle, S. M., and A. Tiselius. Tricalcium phosphate as an
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48:171-174, 1951.
69. Swisher, R. D., M. M. Crutchfield, and D. W. Caldwell.
Biodegradation of nitrilotriacetate in activated sludge.
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70. Swisher, R. D., T. A. Taulli, and E. J. Malec. Biodegrada-
tion of NTA metal chelates in river waters. In Trace metals
and metal-organic interactions in natural waters, D. C.
Singer (ed.). Ann Arbor Sci. Publ., Ann Arbor, 1973, 364 pp.
71. Tabatabai, M. A., and J. M. Bremner. Decomposition of nitri-
lotriacetate (NTA) in soils. Soil Biol. Biochem., 7:103-106,
1975.
72. Taylor, J. K., R. Alvarez, R. A. Paulson, T. C. Rains, and H.
L. Rook. Interaction of nitrilotriacetic acid with suspended
and bottom material. EPA-WQO Project 16020-GFR-7/71, 1971.
73. Taylor, J. K., W. L. Zielinski, Jr., E. J. Maienthal, R. A.
Durst and R. W. Burke. Development of method for NTA analysis
in raw water. EPA Report EPA-R2-72-057, 1972, 27 pp.
74. Tetenbaum, M. T., and H. Stone. Oxidative cleavage of nitri-
lotriacetic acid to iminodiacetic acid. Chem. Commun./ 1699,
1970.
70
-------
75. Thayer, P. S., and C. J. Kensler. Current status of the
environmental and human safety aspects of nitrilotriacetic
acid (NTA). Grit. Revs. Environ. Contr., 3:375-404, 1973.
76. Thorn, N. S. Nitrilotriacetic acid: a literature survey.
Water Res., 5:391-399, 1971.
77. Thompson, J. E., and J. R. Duthie. The biodegradability and
treatability of NTA. J. Water Pollut. Control Fed., 40:
306-319, 1968.
78. Tiedje, J. M. and B. B. Mason. Biodegradation of nitrilo-
triacetate (NTA) in soils. Proc. Soil Sci. Soc. Am.,
38:278-283, 1974.
79. Tiedje, J. M., B. B. Mason, C. B. Warren and E. J. Malec.
Metabolism of nitrilotriacetate by cells of Pseudomonas
species. Appl. Microbiol., 25:811-818, 1973.
80. Troxler, R. F., and R. Lester. Formation, chromophore com-
position, and labeling specificity of Cyanidium caldarium
phycocyanin. Plant Physiol., 43:1737-1739, 1968.
81. Walter, A. P. Ultimate biodegradation of nitrilotriacetate
in the presence of heavy metals. In_ Proc. 7th Inter. Conf.
on Water Pollut. Res., Paris, Pergamon Press Ltd., London,
1974, pp. 1-9.
82. Warren, C. B., and E. J. Malec. Biodegradation of nitrilo-
triacetic acid and related imino and amino acids in river
water. Sci., 176:277-279, 1972.
83. Woodiwiss, C. R. , R. D. Walker, and F. A. Brownridge. Con-
centrations of nitrilotriacetate and certain metals in
Canadian wastewaters and streams: 1971-1975. Water Res.,
(In Press).
84. Yentsch, C. M., C. S. Yentsch, C. Owen and M. Salvaggio.
Stimulatory effects on growth and photosynthesis of the
toxic red tide dinoflagellate, Gonyaulax tamarensis, with
the addition of nitrilotriacetic acid (NTA). Env. Letters,
6:231-238, 1974.
85. Zeikus, J. G., and T. D. Brock. Effects of thermal additions
fromthe Yellowstone geyser basins on the bacteriology of
the Firehole River. Ecol., 53:283-290, 1972.
86. Zepp, R. G., N. L. Wolfe, G. L. Baughman, and R. C. Hollis.
Singlet oxygen in natural waters. Nature, 267:421-423, 1977.
71
-------
NJ
4 ' 4 ' 4
20
MINUTES
Figure 4. GC separation of butyl ester of twenty-five carboxylic
acids. Numbers refer to acids in Table 1. (Reproduced
by permission from J. Chromatography.)
-------
-J
00
0.1
1-0
100 'OO.Q 1POO.O
NTA(MG/L)
lOOO
Figure 5.
Standard curve for NTA gas chromatographic analysis
(Reproduced by permission from J. Chromatography.)
-------
SEWAGE
PLANT
20 30 40
DAYS
20 30 40 50 60
Figure 6.
Effect of NTA on solubilization of copper from three
stream sediments. Squares, 10~3 M (191 mg/1) NTA:
diamonds, 10 b M (1.91 mg/1): circles, controls.
Vertical dotted line indicates end of sterile
portion of experiment.
-------
Ul
90
80
70
O 60
•£ 50
0)
Q.
CL
40
30
20
10
WHITE
CLAY
40 50
>M—
60
RED
CLAY
SEWAGE
PLANT
10
20 30 0
DAYS
10 20
10 20 30 0
Figure 7. Effect of NTA on solubilization of iron from three stream
sediments. Symbols same as in Figure 6 except that diamonds
also include data for 10~7 M (0.02 mg/1) NTA.
-------
WHITE
CLAY
RED
CLAY
SEWAGE
PLANT
18
16
14
*
.E10-
c
•5
8
E
a
a
0 10 20 30 40 50 60 0 10 20 30 40 50 60 0 10 20 30 40 50 60
DAYS
Figure 8. Effect of NTA on solubilization of manganese from three
stream sediments. Symbols same as in Figure 7.
-------
RED
CLAY
SEWAGE
PLANT
WHITE
CLAY
0 10 20 30 40 50 60 0 10 20 30 40 50 60 0 10 20 30 40 50 60
Figure 9. Effect of NTA on solubilization of zinc from three stream
sediments. Symbols same as in Figure 7.
-------
Figure 10,
Mc
INCORPORATION
30
60
90
MINUTES
120
180
Mineralization (14CO2 evolution) of NTA by
cell suspensions of a Pseudomonas sp. known
to degrade NTA. 14c incorporation by cells
also shown.
78
-------
10'
CN
O)
c
0.0—(I)
(18.7)
10
z
OS
O
LU
Q
O
10
10
10
-2
10
-3
I L
4
WEEKS
8
Figure 11.
Semi-log plot of NTA degradation rate (x + s.d.)
by bacterial populations at 9°C. (March-April,
1975). Data at 8 weeks indicated parenthetically
because of temperature rise to 12°C in microcosms,
Mean NTA exposure indicated in parenthesis along
with desired exposure in mg/1.
79
-------
103
Figure 12.
WEEK
Semi-log plot of number (x + s.d.) of NTA
degrading bacteria on coverslips exposed to
NTA at the indicated concentrations (March-
April 1975). NTA exposure shown as in
Figure 11. Data for 8 week sampling
indicated parenthetically as in Figure 11.
80
-------
20.0
(20.1)
Figure 13.
WEEKS
Semi-log plot of NTA degradation rate (x + s.d.)
by bacterial populations at 18°C (July-August,
1975) . Actual concentration of exposure shown
in parenthesis along with desired exposure in
mg/1.
81
-------
107
CM 106
s
u
105
o
z
Of
O
LU
O
iicr
io3
J 1
J L
1 L
I L
2
WEEK
20.1
J I
Figure 14
Semi-log pilot of number of NTA degrading
bacteria (x + s.d.) on coverslips exposed
to NTA at approximately the indicated
concentrations.
82
-------
00
CO
100
i
o
h: 10h
rii
O
§ ,
.01
A. AUTUMN 1974
./I
NTA-GC
NTA-CuSO4
NITRATE
a AMMONIA
• NITRITE
14 0
14 0
DAYS
14 0
14
Figure 15. A, B, C
Changing concentrations of NH-^-N, NC>2-N, and o
resulting from the degradation of NTA in 87 1 micro-
cosms. Note concentrations placed on log scale to
facilitate comparison of rates of appearance and
disappearance of components in widely differing
concentration ranges.
-------
00
1000 r-
100
10
o
u
.1
.01
B. SPRING 1975
NTA
NH
I I I I I
0 1
2 3
WEEKS
C. SUMMER 1974
NTA
N03
4501
2 3
WEEKS
Figure 15. B, C. (Continued)
-------
CO
on
600
500
400
5 300
I
300
200
200
100
0246
72
HOURS
Figure 16. Changing concentrations of ^C-NTA in an 87 1 microcosm
system resulting from dilution (Theoretical curve) and
both dilution and bacterial decomposition (Empirical
curve) (December, 1974).
-------
3000
2000
ee
O
OL
at
O
U
IU
in
O
O
I
^ 1000
5
o.
O
• CuCI2
• CuNTA
* ZnCI2
A ZnNTA
• CdCI2
• CdNTA
oNTA
& Control
Figure 17
Incorporation of C glucose by washed cells from
a culture of naturally occurring bacteria in the
presence of added metal ions or NTA-metal chelates,
NTA probably present as the Ca- and Fe-chelate
when added alone. Assay performed at 9°C.
86
-------
16 32 48
2 PPM
o
•o
400 40
300 30
200 20
100 10
2 PPM
o
ro
400
300
200
'00
16 32 48 64 80 96
MINUTES
16 32 48 64 80 96
10
9
8
7
6
5
4
3
2
1
4 PPM
O
M
S
X
•s.
100 10
90 9
80
70
60
50
40
30
20
X)
4 PPM
10
16 32 48 64 80 96 16 32 48 64 80 96
MINUTES
Figure 18.
Incorporation (I = 0 •, open = NTA exposed,
closed = control) and mineralization (M = A A,
open = NTA exposed, closed = control) of l^C
glucose by suspensions of washed cells harvested
from cultures of mixed natural populations.
NTA (0.25) ml added at arrow to provide indicated
concentrations. Controls inoculated with an
equivalent volume of water.
87
-------
4001
300-
200
100
rfrT'o ""-...
400
300-•
200 ••
UJ 100
CD
<
O3
o
o> 200
100-
400
II
400
300-
'
it
,
sr1*****
•s
L III
JULY
Col lection No. 1 2 3
Fiaure 19.
Concentration of copper (x. +_ s.d.) in algal
communities from ecosystem streams through
time. Stream I-Control, II-NTA/Cu, III-Cu,
IV-NTA. NTA and copper additions to streams
begun at arrows. Small numbers indicate
sample size. Larger numbers indicate x + s.d,
of data off-scale.
88
-------
JULY AUG SEPT OCT
Collection No. 1 2 3 4 5 6 7 8
Figure 20.
Concentration of iron (x + s.d.) in algal
communities from ecosystem streams through
time. Stream I-Controlf II-NTA/Cu, III-Cu,
IV-NTA. NTA and copper additions to streams
begun at arrows. Small numbers_indicate sample
size. Larger numbers indicate x + s.d. of data
off-scale.
89
-------
200-
ISO-
100-
LJJ
200
ISO*
ioa
50-
O) 200+
c
O5
150+
1004
50+
200-
ISO-
100
50-
II
III
IV
JULY AUG SEPT OCT
Collection No. 1 2 3 4 5 6 7 8
Figure 21.
Concentration of zinc (x + s.d.) in algal
communities from ecosystem streams through
time. Stream I-Control, II-NTA/Cu, III-Cu,
IV-NTA. NTA and copper additions to streams
begun at arrows. Small numbers indicate
sample size.
90
-------
562 - ?BO
JULY AUG SEPT OCT
Collection No. 1 2 3 4567 8
Figure 22.
Concentration of manganese (x + s.d.) in
algal communities from ecosystem streams
through time. Stream I-Control, II-NTA/Cu,
III-Cu, IV-NTA. NTA and copper additions
to streams begun at arrows. Small numbers
indicate sample size.- Larger numbers indi-
cate x + s.d. of data off-scale.
91
-------
4000-
3000-
2000
1000-
JULY AU6 SEPT OCT
Collection No. 1 2 3 4 5 6 7 8
Figure 23.
Concentration of magnesium (x + s.d.) in
algal communities from ecosystem streams
through time. Stream I-Control, II-NTA,
III-Cu, IV-NTA. NTA and copper additions
to streams begun at arrows. Small numbers
indicate sample size. Larger numbers indi-
cate x + s.d. of data off-scale.
92
-------
1000+
750+
500+
250+
1000+
SJ213 t 254
JULY
Collection No. 123
AUG SEPT OCT
45678
Figure 24.
Concentration of copper (x + s.d.) in Anacharis
from ecosysteir streams through time. Stream I-
Control, II-NTA/Cu, III-Cu, IV-MTA. NTA and
copper additions to streams begun at arrows.
Small numbers indicate sample size. Larger
numbers indicate x -f s.d. of data off-scale.
93
-------
4000-
3000'
2000-
1000-
O5
1000- •
JULY AUG SEPT OCT
Collection No. 123 45 6 7 8
Figure 25.
Concentration of iron (x + s.d.) in Anacharis
from ecosystem streams through time. Stream I-
Control, II-NTA/Cu, III-Cu, IV-NTA. NTA and
copper additions to streams begun at arrows.
Small numbers indicate sample size. Larger
numbers indicate x + s.d. of data off-scale.
94
-------
,442 i256
100'
JULY
Collection No. 123
AUG SEPT OCT
45678
Figure 26.
Concentration of zinc (x + s.d.) in Anacharis
from ecosystem streams through time. Stream I-
Control, II-NTA/Cu, III-Cu, IV-NTA. NTA and
copper additions to streams begun at arrows.
Small numbers indicate sample size. Larger
numbers indicate x + s.d. of data off-scale.
95
-------
JULY AUG SEPT OCT
Collection No. 123 45 6 7 8
Fiaure 27.
Concentration of manganese (x + s.d.) in
Anacharis from ecosystem streams through
time. Stream I - Control, II-NTA/Cu,
Cu, IV-NTA. NTA and copper additions to
streams begun at arrows. Small numbers
indicate sample size. Larger numbers
indicate x + s.d. of data off-scale.
96
-------
4000- -
3000- •
2000-•
1000- •
3000- •
CO
1000- •
Tio
5917 ± 1198
4000-
3000-
2000-
1000-
Collection No.
Is
fo
1
JULY
1 2 2
»4495
5~~ f \
10 9 N^.
1 lio
AUG SEPT OCT
14567 8
Figure 28.
Concentration of magnesium (x + s.d.) in
Anacharis from ecosystem streams through
time. Stream I-Control, II-NTA/Cu, III-Cu,
IV-NTP. NT^ and copper additions to streams
begun at arrows. Small numbers indicate
sample size. Larger numbers indicate x + s.d;
of "data off-scale.
97
-------
JULY
Collection No. 123
Figure 29.
Concentration of copper (x + s.d.) in Lemna
from ecosystem streams through time. Stream I-
Control, II-NTA/Cu, III-Cu, IV-NTA. NTA and
copper additions begun at arrows. Small numbers
indicate sample size. Larger numbers indicate
x + s.d. of data off-scale.
98
-------
12,888 ± 3085
JULY ' AUG SEPT OCT
Collection No. 1 2 3 4 5 6 7 8
Figure 30.
Concentration of iron (x + s.d.) in Lemna
from ecosystem streams through time. Stream I-
Control, II-NTA/Cu, III-Cuf IV-NTA. NTA and
copper additions to streams begun at arrows.
Small numbers indicate sample size. Larger
numbers indicate x + s.d. of data off-scale.
99
-------
400- •
30O- •
20O •
100- •
400- >
300-
< 200 •
200
100 •
400-
300- •
kj9.
N,
JULY AUG
Collection No. 1 2 3 45
SEPT-1 OCT
678
Figure 31. Concentration of zinc (x + s.d.) in Lemna
from ecosystem streams through time. Stream I-
control, II-NT£/Cu, III-Cu, IV-NTA. NT^ and
copper additions to streams begun at arrows.
Small numbers indicate sample size.
100
-------
100O
500
200O
1500
1000
500
JULY AUG SEPT OCT
Collection No. 1 2 3 4 5 6 7 8
Figure 32.
Concentration of manganese (x + s.d.) in
Lemna from ecosystem streams through time.
Stream I-Control, II-NTA/Cu, III-Cuf IV-NT£.
NTA and copper additions to streams begun at
arrows. Small numbers indicate samole size.
101
-------
5000- •
3750- •
2500- •
1250- •
5000-t
JULY AUG SEPT OCT
Collection No. 123 4 56 7 8
Ficmre 33.
Concentration of magnesium (x + s.d.) in
Lemna from ecosystem streams through time.
Stream I-Control, II-NTA/Cu, III-Cu, IV-NTA.
NTA and copper additions to streams begun at
arrows. Small numbers indicate sample size.
Larger numbers indicate x + s.d. of data
off-scale.
102
-------
JULY AUG SEPT OCT
Collection No. 1 2 3 4 5 6 7 8
Figure 34.
Concentration of copper (x + s.d.) in Planaria
from ecosystem streams through time. Stream I-
Control, II-NTA/Cu, III-Cu, IV-NTA. NTA and
copper additions to streams begun at arrows.
Small numbers indicate sample size. Larger
numbers indicate x + s.d. of data off-scale.
103
-------
oc
0_
^
0>
O5
1000-
750-
500-
250
1000'
750-
50O
250
1000
750
500
250
100O
750-
500-
250
1 4342 ± 2659
4\
r \ I .••-•'""' J\ '
ly^ w-
l3061±aiO ' .t.5346iS39l'
\ T ,-•-""" '"X "
3
* 1 l I
, 4^6628 ±2773
I '"
1 4
* 1 II
•4377 ±31 14 i2461±4271 ,,
\ /\ IV
\ / " ''"'-•
\/
t 1 ,
JULY AUG SEPT OCT
Collection No. 123 45 67 8
Figure 35.
Concentration of iron (x + s.d.) in Planaria
from ecosystem streams through time. Stream I-
Control, II-NT^/Cu, III-Cuf IV-NTA. NTA and
copper additions to streams begun at arrows.
Small numbers indicate sample size. Larger
numbers indicate x + s.d. of data off-scale.
104
-------
800-
600-
400-
200-
800-
600
400-
<
E200
<
Z
<800-
—1
Q_
0,600-
N400-
' & A
• •- /\
'•L ^ \
1
1 1
; i K
+ (
H
, * l
4^nao±982
in
• J.
3.200+
800-
600-
40O-
20O-
k
IV
JULY
Collection No. 123
AUG SEPT ' OCT
45678
Figure 36.
Concentration of zinc (x + s.d.) in Planaria
from ecosystem streams through time. Stream I-
control, II-NTA/Cu, III-Cu, IV-MTA. NTA and
copper additions to streams begun at arrows.
Small numbers indicate sample size. Larger
numbers indicate x + s.d. of data off-scale.
105
-------
100-
75-
50--
25 •
03
100-
75
50-
25-
•
•
*
4
^
4
1
IV
*
t
JULY
Collection No. 123
AUG SEPT OCT
45678
Figure 37.
Concentration of manganese (x + s.d.) in
Planaria from ecosystem -streams through
time. Stream I-Control, II-NTA/Cu, III-Cu,
IV-NT^. NTA and copper additions to streams
begun at arrows. Small numbers indicate
sample size.
106
-------
2000--
1500--
loco-
500-
cc
2000-
1500- •
1000-
500-
II
£—
<2000-
Q_
0,1500-
X.
^»v
O3 1000-
03 500-
2000-
isoa
1000
500-
1 '"
4
4
* 1 1 1
IV
1 "4j 4 i4 ?4
t 1 I 1
JULY AUG SEPT OCT
Collection No. 1 2 3 4 5 6 7 8
Figure 38.
Concentration of magnesium (x ± s.d.) in
Planaria from ecosvstem streams through time.
Stream I-Control, II-NTA/Cu, III-Cu, IV-NTA.
NTA and copper additions to streams begun at
arrows. Small numbers indicate sample size.
107
-------
TABLE 1. Frequency of Determination of Water
Chemcial Parameters during Algae Experiments
o
00
No.
of measurements per week during indicated Experiment:
11/14/74-2/13/75
Iron
Manganese
Zinc
Copper
Magnesium
Sodium
Potassium
Nitrate
Nitrite
Ammonia
Phosphate
Silicate
Sulfate
Chloride
Total Alkalinity
Calcium
3
4
3
1 per month
1
1
1
daily
daily
3
daily
3
1
1
1
—
6/20-10/3/75
4
4
1
1
1
1
1
daily
2
daily
daily
daily
1
-
daily
1
3/14-4/15/75
daily
daily
4
2 per day
1
-
2
daily
daily
daily
daily
daily
1
daily
daily
2
-------
TABLE 2. Relative Retention Times (RRT) and Relative Molar Responses
(RMR) of n-Butylesters of twenty-five Carboxylic Acids.
o
vo
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Malonic
Succinic
Fumaric
Cinnamic
Malic
p-Hydroxy ben zoic
MIDA
IDA
Tartaric
Vanillic
My ri stic
Shikimic
Palmitic
Phthalic
Coumaric
Syringic
Linoleic
Linolenic
Stearic
Ferulic
Citric
Arachidic
NTA
Sinapic
Behenic
No.
runs
7
5
5
4
5
4
20
4
5
5
10
9
5
5
4
10
4
9
10
10
4
7
10
10
RRT_
X
0.17
0.26
0.27
0.32
0.41
0.44
0.50
0.53
0.55
0.60
0.70
0.83
0.94
1.00
1.07
1.12
1.14
1.16
1.17
1.23
1.29
1.31
1.36
1.46
1.50
RRT_
X
+0.02
+0.02
+0.02
+0.01
+0.01
+0.02
+0.04
+0.01
+0.04
+0.04
+0.09
+0.04
+0.00
+0.00
+0.00
+0.02
+0.02
+0.02
+0.03
+0.04
+0.06
+0.05
+0.09
+0.08
RMR_
X
0.40
0.46
0.41
0.69
0.41
0.43
0.76
0.41
0.30
0.66
1.26
0.59
1.13
1.00
0.61
0.73
1.16
1.29
1.50
0.75
0.93
1.78
0.81
0.73
2.09
RMR_
X
+0.16
+0.06
+0.05
+0.07
+0.03
+0.02
+0.16
+0.07
+0.05
+0.13
+0.50
+0.20
+0.10
+0.06
+0.10
+0.29
+0.27
+0.38
+0.16
+0.11
+0.07
+0.11
+0.22
+0.46
RMR std.
error
+0.05
+0.03
+0.02
+0.03
+0.01
+0.01
+0.04
+0.03
+0.02
+0.06
+0.16
+0.07
+0.04
+0.03
+0.05
+0.09
+0.13
+0.12
+0.05
+0.04
+0.04
+0.04
+0.08
+0.15
-------
TABLE 3. Evaluation of GC Analytical Procedure
Using carboxyl-labeled
M
0
Sample 1
Starting
solution 187,800
O. 1M Formic
acid eluate 3,110
16M Formic
acid eluate 185,000
Esterified
sample 84,400
Sample for
GC analysis 119,350
DPM/Sample
2345 Mean %
216,850 207,450 198,050 207,450 203,510 100.0
4,770 4,200 2,970 3,370 3,634 1.8
185,000 170,000 188,000 147,000 175,000 86.0
134,000 166,000 169,300 138,000 139,360 68.5
178,950 208,900 170,700 87,200 153,020 75.2
-------
TABLE 4. Chelation of Metals from Sediments by 10~3M NTA (191 ppm).
No. samples
Density of sediment
ug Fe/g sediment
mg in top cm
ug Cu/g sediment
mg in top cm
ug Zn/g sediment
mg in top cm
ug Mn/g sediment
mg in top cm
Fe - max ug solubilized (NTA)
% "available"
Cu - mag ug solubilized (NTA)
% "available"
Zn - max ug solubilized (NTA)
% "available"
Mn - max ug solubilized (NTA)
% "available"
White
Clay
5
1.40 + 0.05
23400 + 2920
111
7.8 + 1.1
.259
38 + 3.7
1.26
524 + 97
17.4
450
<0.01
12.5
4.8
56
4.4
830
4.8
Red
Clay
(upstream)
6
1.41 + 0.03
19800 + 3400
665
6.1 + 1.7
.204
27.3 + 2.0
.917
810 -1- 230
27.2
400
<0.01
11.1
5.4
67
7.3
1210
4.4
Red Clay
(sewage
plant)
6
1.40
26700
889
14.1
.469
78.3
2.61
690 +
23.0
2500
<0.01
40
8.5
228
8.7
760
3.3
+ .09
+ 2600
+ 3.8
+ 5.3
107
-------
TABLE 5. NTA Determinations for the Period
November 14, 1974 - February 13, 1975
Desired
Concentrations (mg/1)
Microcosm Box #
Zinc-Zincon Method
X
N
Maximum
Minimum
# out of 30% range*
SD
Coef. of var.
SE
SE (%)
m
GC Method
X
N
Maximum
Minimum
# out of 30% range
SD
Coef. of var.
SE
SEm (%)
2.0
1
1.84
60
409
0.3
24
0.844
46.0
0.109
5092
1.54
85
3.68
0.06
43
0.811
5207
0.088
5.71
2
1.67
61
3.6
0.2
22
0.720
43.1
0.092
5.51
1.72
86
4.20
0.20
47
0.822
47.8
0.088
5.12
5
1.49
61
3.1
0.2
30
0.706
47.3
0.090
6.02
1.52
85
4.50
0.20
42
0.760
50.0
0.082
5.39
20.0
3
19.95
61
37.6
0.6
12
6.11
30.6
0.783
3.92
18.7
85
32.4
8.1
18
4.94
26.4
0.535
2.86
6
20.23
61
36.4
4.2
11
5.50
27.2
0.704
3.48
19.3
84
29.5
4U6
11
4.53
23.5
0*494
2.56
7
20.97
61
30.6
6.6
9
5.23
24.9
0.669
3.19
19.6
85
29.5
6.2
12
4.05
20.7
0.439
2.24
*Previous experiments have shown that in the range of many natural variables this
is the degree of variation one may expect under similar conditions.
-------
TABLE 6. NTA Determination for July 4 - September 2, 1975 Experiment
Concentration (mg/1) Control
Microcosm Box # 2 & 8
Zinc-Zincon Method
X
N
Maximum
Min imum
# out of 30% range
SD
Coef. of var.
SE
SEm (%)
m
GC Method
X 0.06
N 27
Maximum 0.81
Min imum 0
# out of 30% range
SD
Coef. of var.
SE
SEm (%)
2.0
5 & 7
1.94
54
2.5
0.4
3
0.39
19.85
0.053
2.70
1.93
52
3.72
0.4
16
0.661
34.22
0.092
4.74
20.0
4 & 6
19.4
57
31.6
2.0
3
4.09
20.58
0.543
2.73
18.94
50
26.3
1.4
8
4.353
22.98
0.616
3.25
-------
TABLE 7. Water Chemistries for the Test Chambers (November 14, 1974
February 13, 1975) Day Length: 10 hr. .03 min. -
9 hr. 21 min. - 10 hr. 38 min.
Fe Hn Zn
NTA (mg/1)(33-35)!(46-49)(32-35)
cu
(4)
Ca
(12)
Na
(13)
K
(12)
M2M
(57-58) (58-59)
PO-)P SiC>2 SO4
<58-59)/33-34) (11)
Cl Tot Alk
(10-11) (14)
Mg
(12)
(36)
14 Control
Max.
Min.
Ave.
<8 Control
Max.
Min.
Ave.
II (2.0)
Max.
Min.
Ave.
»2 (2.0)
Max.
Min.
Ave.
»5 (2.0)
Max.
Min.
Ave.
13 (20.0)
Max.
Min.
Ave.
96 (20.0)
Max.
Min.
Ave.
17 (20.0)
Max.
Min.
Ave.
.46
.'l70
.36
.02
.150
.36
.04
.19
.50
.04
.19
.50
!l84
.46
.02
.194
.61
.02
.222
.51
!223
.850
.'l86
.48
.1384
.610
.'l4
.380
!l8
.570
!l69
.444
.086
.59
.143
.52
!l61
.014
.'003
.066
.007
.048
.013
.063
.014
.039
.009
.050
!oil
.043
.011
.176
!oi7
.010
!o02
.039
.014
.013
.003
.025
.010
8.9
7.2
7.90
8.7
7.0
7.83
8.9
7.1
7.86
8.7
7.2
7.88
8.9
7.1
7.78
8.7
7.1
7.95
8.9
7.1
7.85
8.5
7.1
7.77
23.7
16.5
19.0
25.0
17.0
19.2
22.9
17.3
19.2
24.4
17.2
19.3
23.7
16.5
18.7
25.3
17.0
19.9
24.4
16.0
19.0
25.0
15.5
18.9
6.65
3.90
5.55
90
00
5.75
35
26
6.18
7.02
4.13
6.09
7.47
4.26
6.14
13.74
8.52
11.41
15.74
8.24
11.47
13.90
«.24
11.47
2.70
1.92
2.25
2.50
1.95
2.12
3.06
2.13
2.64
2.78
2.05
2.41
3.44 .021
2.49 .004
3.06 .010
63
95
21
3.40
2.08
2.54
2.80
1.98
2.35
2.79
2.03
2.32
3.52
2.57
3.14
3.48
2.55
2.99
4.20
2.62
3.10
3.50
2.57
3.09
3.66
2.57
3.13
3.48
2.57
3.07
3.56
3!l4
.028
.003
.012
.078
.005
.026
.061
.005
.028
.065
.005
.033
.293
.004
.075
.078
.003
.033
.308
!o69
.060
C."013
.160
.053
.006
.021
16.3 19.4
7.4 15.3
13,0 17.7
.062
.01 .007
.016 .025
.070 .057
.01 .009
.026 .025
.072 .054
.01 .006
.025 .024
.064 .052
.01 .007
.022 .021
.116 .054
.01 .003
.058 .022
.148 .046
.01 <.002
.044 .019
.174 .065
.01 .003
.043 .022
16.5
11.4
14.2
16.3
11.4
14.2
16.5
11.4
13.9
16.0
9.4
13.4
16.3
11.4
14.3
15.8
8.4
13.1
15.4
10.«
13.7
18.7
13.5
16.9
19.0
14.7
17.3
18.8
13.4
17.2
19.2
14.0
17.4
19.0
15.1
17.4
19
15
17.6
18.5
15.'
17.2
13.2
8.3
10.2
12.6
8.3
10.2
13.2
8.3
10.5
12.6
8.3
10.1
12.0
8.3
10.3
12.6
8.9
10.6
13.8
8.3
10.6
13.8
H.9
10.6
54.0
47.0
50.3
56.0
47.0
51.1
57.0
46.0
51.2
54.0
44.0
49.7
56.0
46.0
50.6
55,
47,
50,
54.0
48.0
51.3
56.0
51.4
^•Number of Determinations
-------
TABLE 8. Water Chemistries for the Experimental Boxes
(June 20, 1975 - September 3, 1975)
Day Length: 14 hr. 54 min. - 12 hr. 59 min.
Ui
Test Unit *1
Boxes 2 and 8
Max.
Min.
Ave.
Test Unit D2
Boxes 5 and 7
Max.
Min.
Ave.
Test Unit 13
Boxes 4 and 6
Max.
Min.
Ave.
Mn
(41)1
- Control
.19
<.02
.067
-2.0 mg/1
.22
<.02
.055
-20.0 mg/1
.19
<.02
.068
Pe
(41)
.35
<.05
.14
.34
.05
.14
.38
.05
.17
Zn
(9)
.500
<.01
.069
.418
< .01
.054
.220
<.01
.029
Cu Mg
(8) (8)
.02 7.5
<.01 6.1
<.01 7.1
.014 7.4
<.01 6.3
<.01 7.08
.015 7.4
<.01 6.4
<.01 7.13
Ca
(8)
.21.6
16.0
19.3
20.7
15.9
19.1
20.7
16.3
19.1
Na
(8)
5.81
4.07
5.49
7.70
4.84
6.34
14.60
8.90
11.98
K
(8)
3.05
1.40
2.23
3.16
1.37
2.19
3.30
1.77
2.49
PO4P
(42)
.051
.009
.026
.037
.007
.019
.043
.007
.017
SiO2
(4lf
16.4
4.2
13.7
16.0
4.2
12.6
17.5
3.4
12.2
NO3N
(42)
3.07
0.60
2.64
3.22
0.55
2.76
3.59
0.63
2.81
NO,N
(13-17)
.014
.005 <
.008
.075
.006 <
.030
.890
.006 <
.192
NH3N
(41)
.018
.002
.005
.075
.002
.011
.264
.002
.071
SO4
(8)
20.0
16.2
17.8
18.8
17.1
18.2
19.4
17.2
18.2
Cl
(8)
14.0
6.0
9.3
13.0
6.0
10.0
14.4
8.0
10.4
Alk P.
(38)
0
0
0
0
0
0
0
0
0
Alk M O
(38)
56.0
.30.0
51.1
58.0
45.0
51.7
68.0
46.0
55.0
Number of determinations
-------
TABLE 9. Metals in Biomass (mg cation/g Dry Weight)
November 1974 - February 1975
Kg
CA
K
*Na
*Fe
*Mn
Cu
Zn
November 14, 1975
(end of seeding)
Control 2 mg/1 20
-------
TABLE 10. Metals in Algal Biomass - mg cation/gm
July - August 1975
Mg
Ca
K
Na
Fe
Mn
Zn
Cu
Hi
July 3, 1975
(end of seeding)
Control 2 mg/1 20 mg/1
8.49 8.44 9.8
7.07 7.2 9.66
4.59 4.39 5.61
1.33 0.91 1.56
50.87 52.05 65.1
3.0 3.1 3.49
0.31 0.24 0..33
0.09 0.09 0.13
0.49 0.54 0.48
August 1, 1975
Control 2 mg/1 20 mg/1
3.9 2.77 3.3
8.07 5.68 6.23
8.04 6.93 7.73
2.16 0.88 1.09
14.24 10.71 10.21
29.32 21.23 19.75
0.45 0.13 0.15
0.09 0.06 0.06
0.16 0.09 0.11
August 31, 1975
Control 2 mg/1 20 mg/1
5.60 4.93 4.30
14.38 12.28 11.72
7.19 7.64 7.93
2.32 1.39 1.50
19.94 15.42 13.51
48.65 39.75 49.30
0.61 0.19 0.13
0.13 0.11 0.11
0.184 0.136 0.23(
-------
TABLE 11. Slide Observations, November 14 - December 10, 1974
End of Seeding
00
Box
*
4
8
1
2
5
3
6
7
Approximate Observations (November 14, 1974)
NTA (mg/1)
Control As in il, except diversity less Synedra ulna and
Nitzschia linearis - dominants
Control As in SI, but much less Synedra
2.0 Diatoms - dominant, diversity - good (Cyclptella sp. ,
Achnanthes lanceolate, Synedra ulna, Nitzchia linearis,
N. acicularis) Greens - frequent
Stigeoclbnium protonema - common
Blue-greens - rare
2.0 As in #1
2.0 As in #1, except Chlamydomonas, Cyclotella and
Stigeoclonium - rare
20.0 (19.8) As in #1
20.0 (19.8) As in #5
20.0 (19.8) As in #1
(Continued)
-------
TABLE 11. (Continued)
(4 weeks after Seeding)
Box Approximate Observations, (December 10, 1974)
# NTA (mg/1)
3 20.0 (19.3) Diatoms - dominant, diversity less than f4 and #8
Achnanthes lanceplata, Synedra ulna, Nitzschia linearis
and Melosira varians - dominant. Some indication of
pavement-like growth for Achnanthes along with many
empty frustules of this species. Overall growth
somewhat less than #4 and #8
Greens: Stigeoclonium protonema common
Chlamydomonas frequent
Blue-greens: frequent to common
7
6
20.0 (19.
20.0 (20.
8)
3)
As in #3, fewer empty frustules of Achnanthes
As in #3, but diatom diversity better than #3 and
17
(Continued)
-------
TABLE 11. (Continued)
(4 weeks after Seeding)
Box
It
to
o
Approximate
NTA (mg/1)
Control
Observations, (December 10, 1974)
Diatoms - dominant, diversity good. Melosira varians,
Achnanthes lanceolata, Nitzschia linearis, Cymbella,
Synedra ulna all common to abundant. Cyclotella,
Diatoma and Cocconeis frequent at best.
Greens: Stigeoclonium protonema common
Chlamydomonas sp. nonmotile palmeloid colonies
- common, other greens - infrequent
Blue-greens: Microcoleus - frequent at best
Schizothrix - frequent
Oscillatoria - infrequent
Protozoa: Vorticella, DTfflugia, ciliates and rotifers
- common
Control
As in #4, but Schizothrix frequent - common; Scenedesmus
infrequent
2.0 (1.7)
Diatoms - dominant, diversity good, similar to #4 and
#8 Greens: Scenedesmus - common otherwise similar to #4
and #8
2.0 (1.7)
As in #1, but growth less than any other NTA level box
Blue-greens: Microcoleus - common
2.0 (1.5)
As in
(Continued)
-------
TABLE 11. (Continued)
(6 weeks after Seeding)
Box Approximate
# NTA (mg/1)
4 Control
Observations, (December 26, 1974)
Diatoms - dominant, growth good, diversity good,
Melosira varians, Achnanthes lanceolata - abundant
Synedra ulna, Cymbella, Nitzschia linearis,
species of Navicula, Cocconeis placentula -
various
all common.
Greens: rare, except Stigeoclonium protonema common
Blue-greens: Schizothrix calcicola common, Microcoleus
8 Control
1 2.0 (1.7)
2 2.0 (1.7)
5 2.0 (1.5)
3 20.0 (19.3)
6 20..0 (19.8)
7 20.0 (20.3)
infrequent
As in #4
Diatom community as in controls, Blue-greens: similar
to controls, Greens: Scenedesmus - frequent to common
Otherwise as in #4 and #8
As in #1
As in #1
Diatoms - dominant, growth good, diversity
as in controls, pavement-like growth habit
Greens and Blue-greens: similar to control
boxes.
As in #3
As in #3
not as good
observed.
and 2.0 mg/1
-------
TABLE 12. Biomass, Chlorophyll and 14C Determination
(November 14, 1974 to February 13, 1975)
K>
Approximate NTA levels (mg/1)
End of Seeding (Nov. 12)
Ash Free dry weight (mg)
Percent of volatile
material
Six weeks after Seeding (Dec
Chlorophyll a/c
35 days after Seeding (Jan.
Ash free dry weight (mg)
Percent of volatile
material
0
29.6
10.3
. 23)
7.4
17)
64.9
27
0 2.0
9.1 11.6
11.7 9.4
6.5 8.8
76.4 58.5
26 29
2.0
10.7
13.0
8.2
62.3
28
2.0
22.9
10.1
6.7
59.9
28
20.0
4.0
13.0
7.1
56.6
34
20.0 20.0
58.3 16.8
8.2 11.1
7.3 7.4
52.6 52.5
27 36
Coef. S.E.
x var. Mean (%)
20.38 85.2 30.1
10.85 15.7 5.5
End of Experiment (Feb. 11-13)
Ash Free dry weight (mg)
Percent of volatile
material
Chlorophyll a/c
DPM/ug Chlorophyll a
53.6
27
7.5
1094
41.2 34.9
25 34
6.8 6.5
1270 1120
40.4
31
6.8
1449
43.1
29
6.9
1451
44.2
37
7.4
608
29.1 75.9
30 33
6.6 5.8
1626 940
-------
TABLE 13. Detailed Analysis of Diatom Populations by the Truncated
Normal Curve Technique^ (November 12, 1974 - February 13, 1975)
H
NJ
00
Approximate NTA
Concentration (mg/1)
Microcosm Box 1
Position of modal
interval
t of intervals
t of observed
species
It of species in
modal interval
Control
4
B A
2-3 3
10 10
129 75
20.9 11.1
Control 2.0
8 1
B A B A
2-33 3 3
10 11 10 11
133 78 114 75
21.1 10.8 17.9 11.1
2.0 2.0 20.0 20.0
2536
BABABABA
3 2-3 2-3 33333
10 11 9 10 10 11 10 10
112 79 122 80 124 71 118 75
18.2 11.0 20.1 12.0 19.9 9.8 19.0 11.7
20.0
7
B A
3 2-3
10 10
132 75
21.0 11.7
o2 (sigma squared) 8.3 10.7 7.5 11.1 7.8 12.2 7.6 13.0 8.2 9.8 8.0 11.0 8.4 8.8 8.2 9.5
Position of the 2.0 2.4 2.0 2.8 2.3 2.2 2.4 2.1 2.0 2.3 2.1 2.7 2.2 2.4 2.1 2.0
mode
The theoretical 153.9 91.0 155 90.2 125.2 97.3 125.3 100.2 146.9 94.0 142.3 81.4 139.3 87.0 151.9 91.2
universe
Total I specimens 4206 4098 4000 4787 4583 4034 3942 4502 3519 3156 4112 3417 3810 3572 4298 3048
counted
»_of species 3233343222342333
> 10%
B - end of seeding - November 12, 1974
A - end of experiment - February 13, 1975
1Patrick, R., M. Hohn and J. Wallace, 1954.
-------
TABLE 14.
Distribution of the more Common Diatom Species
(_> 1.67% of Specimens Counted)
to
Approximate NTA
Concentration ( mg/1)
Microcoam Box 1
Achnanthes lanceolata
Nitzschia linearis
Nitzschia f rust ul urn
v. perminuta
Nitzschia kutzingiana
Achnanthes minutissima
Navicula genovefea
Navicula secreta
v. apiculata
Cyclotella meneghiniana
Synedra ulna
Malosira varians
Nitzschia fonticola
Amphora ovalia
v. pediculus
Suriella angustata
Nitzschia dissapata
Navicula minima
Navicula tripunctata
Navicula aemlnulum
Control
8
B A
12.5 22.6
18.8
5.4
3.9
- 12.7
10.4
9.4 6.2
8.7
6.2 8.5
13.4
2.3
2.1
3.5
Control
B
12.1
16.2
2.6
2.8
9.9
7.5
10.3
5.4
2.4
4
A
17.6
8.3
4.7
7.2
3.1
3.0
5.1
20.4
2.0
4.2
2.0 2
1
BAB
20.3 23.7 15.0
16.5 - 17.4
3.5
3.2
11.9
9.1 - 11.3
9.1 4.3 8.3
7.1 - 7.8
6.4 - 3.1
1.6 11.2 2.3
13.9
6.0
.0 2.0
2 5
ABA
20.7 10.6 8.8
14.1 2.7
7.4 6.6
3.3 10.5
7.6 2.5 5.9
7.5 T
6.3 8.3 8.6
8.4
5.2 2.9
9.9 2.5 7.9
16.1 17.3
4.1
20.0
3
B A
7.0 30.7
18.7
6.6
11.8
9.6
10.8
7.8 6.0
11.9
5.8
10.0
11.2
6.8
2.3
3.5
3.7
20.0 20.
6 7
BAB
11.8 25.4 8.2
14.2 - 15.5
1 •> 1 9 O —
3.8 9.8
7.5 - 13.1
9.9 4.4 8.1
5.8 - 10.4
5.7 3.9 6.2
4.6 2.8
11.0
5.2
0
A
21.7
3.6
5.2
8.1
8.3
12.7
12.5
3.1
4.2
A - after treament-February 12,1975
blank space = not seen
-------
TABLE 15. Microscopic Examination of Communities
July 31
Controlt
Boxes 8 and
U94 mg/1
Boxes S and 7
19x3 mg/1 OTA
Boxes 4 and 6
Achnanthes lanceolata - very common
Helosira varians - ve'ry common
CocconeTs placentula - common
fionema sp. - frequent
ilia cuspidata - frequent +
linearis - common
S t igeoc1onium' 1ubricurn protonema
jichiTOthrlx c'alcicola - frequent
Achnanthes lanceolata - very common
Helpsira varians - ve'ry common
Gomphpneroa parvulum - common
CymbeHa c us pida t a - common
NitzschTa llnearis - very common
Stigeoclonium luSricum - rare
Schizpthrix calcicola - rare
Achnanthes lanceolata - very common
Melosira varians - common
Cocconeis placentula - common
STtzsehia linearis - common
Piatoma vuAgare - frequent +
CyjubelTa cuspT^a ta - common
Navicula viriduli~- frequent +
StigeocTonium lubricum - frequent
Sphaerpcys t is - frequent +
Schizo^hrlx c'alcicola - rare
Melosira varians - abundant
AchnantKes lanceolata - abundant
Cocconeis placentula - abundant
Cymbella cuspidata - very common
Spirogyra sp. - very common
Microcole'us vaginatus - frequent -
Schizothrix calcicola - frequent -
Melosira varians - abundant
Achnantffes Janceolata - very common
Achnanthes minutissima - very common
Cocconeis placentula - very common pale )
Nitzschia linearis - common
Cymbella cuspidata - common
Gomphonema paryu1um - frequent -
Spiroqyra sp,-frequent -
Schizothrix calcieola - rare
Microcoleus vaginatus - rare
Melosira varians - abundant
AchnantKes minutissima - common +
Cymbella cuspidata - frequent
Cocconeis placentula - poor condition
Nitzschia 1inearis — common -
Gpnfphonema sp.-frequent -
Schizofchrix calcicola - common
Microcoleus vaginatus - frequent
-------
TABLE 16. Analysis of Diatom Population Using
Truncated Normal Curve Factors
I-1
NJ
cn
Approximate NTA
Concentration(mg/l)
Microcosm Box 1
Position of mode
1 of intervals
t of observed species
II of species in modal interval
"'(sigma squared)
Position of the mode
The theoretical universe
Total t specimens counted
1 of species >"10%
Control
Control
2
B
3
11
120
18.2
10.6
2.2
148.5
5486
2
A
2-3
12
67
8.6
17.8
2.0
91.7
9261
3
B
3
10
120
18.1
10.7
2.1
150.0
5783
2
8
A
3
13
68
8.6
16.9
2.3
88.5
15091.
2
2
B
2-3
10
118
17.9
10.4
2.1
146.3
5283
1
.0
5
A
3
12
76
10.2
15.5
2.3
100.3
13102
4
2
B
2-3
11
113
17.1
11.8
2.0
148.6
6322
2
.0
7
A
3
13
71
9.1
16.6
2.6
93.1
14959
4
20.0
4
B
3
10
116
17.3
9.4
2.5
132.6
5311
2
A
3
12
55
7.5
12.4
2.7
66.0
6309
3
F
3
10
107
16
9
2
123
5052
2
20.0
6
H
2-3
11
56
.4 7.5
.0 16.1
.6 2.0
.0 76.4
6784
3
B = Before experiment (July 3, 1975)
A = After experiment (September 3, 1975)
-------
TABLE 17. Distribution of the more Common Diatom Species
to
Approximate
NTA Concentration (mg/l)
Microcosm Box 1
Achnanthes lanceolata
Navicula secreta
v. apiculata
Navicula qenovefea
Nitzschia f rustulum
v. perminuta
Navicula rhvnchocephala
v. germainii
Navicula pelliculosa
Navicula minima
Melosira varians
Rhoicosohenia curvata
Nitzschia kutzingiana
Navicula gregaria
Achnanthes minutissima
Nitzschia fonticola
Achnanthes subhudsonis
v. krae.
Cocconeis placentula
v. euglypta
Nitzschia tropica
Comphonema MN3
Others
Control
2
B*~
19.9
11.1
5.4
3.5
3.4
2.1
3.5
8.1
3.2
2.6
1.5
42.1
A
4
20
4
27
17
5
4
4
2
27
8
B
15.6
11.8
7.2
3.8
4.0
3.3
3.7
6.4
3.4
2.0
1.3
51.1
A
1
5
1
46
18
1
3
6
1
16
5
B
17.5
9.4
4.3
2.6
3.5
3.7
3.5
7.1
4.3
4.9
1.7
41.9
2.0 mg/l
A
2
14
20
28
12
1
3
4
2
15
7
B
23.3
11.3
7.0
5.1
4.4
3.8
3.5
2.2
2.8
3.1
1.0
41.5
20.0 mg/l
4
A
2
11
13
28
16
2
3
5
3
19
B
14.2
10.3
4.1
4.5
3.8
3.8
4.5
8.0
4.1
1.7
3.8
43.2
A
4
11
1
49
10
1
3
3
4
15
6
B
15.8
9.5
6. 3
3.2
4.6
1.0
1.6
15.5
3.5
3.4
1.4
41.6
A
5
12
22
28
9
2
3
3
3
11
*B - Before experiment, ^ 3.2% of specimens counted
After experiment,^ 1% of specimens counted
-------
TABLE 18. Effects of NTA on Algal Communities
i-
N>
00
Approximate
NTA Concentrations mcj/1
Microcosm Box 1
July 3 (end of seeding)
As n- free dry weight (mg)
% volatile material
August 1 (30 days after seeding)
Ash-free dry weight (mg)
% volatile material
Chlorophyll a/c
September 3 (60 days after seeding)
Phycocyanin (mg/side of slide)
Chlorophyll a/c
% volatile material
Ash-free dry weight (mg)
Control
2
10.9
21.1
22.9
38.4
18.2
80.0
15.3
43
38.4
DPM/ug chlorophyll a 1019
8
11
17
21
38
17
274
17
44
59
2388
.0
.7
.0
.3
.9
.0
.6
.9
5
15.
15.
19.
35.
19.
60.
12.
43
24.
1242
2
4
2
8
2
7
9
4
9
.0
7
10.6
16.6
19.4
32.1
15.0
125.0
4.4
43
43.7
1846
20.0
4
8.5
18.7
22.4
36.5
14.0
33.3
4.4
45
26.7
1196
6
10
22
16
37
12
36
4
49
14
1832
x coe f . SE_
var. (%)
.0 11.1 20.9 8.5
.1 18.6 14.2 5.8
.7
.6
.7
.1
.6
.0
-------
TABLE 19. Water Chemistries for the Experimental Boxes
(March 14 - April 15, 1975) Data in mg/1
Day Length? 11 hr. 52 min. to 13 hr. 15 min.
to
•4 Control
Max,. T
Min.
Ave.
• 7 OTA
MaX,
Min.
Ave.
•10 NTA
Max.
Min.
Ave.
«2 NTA-Cu
Max.
MiTl.
Ave,
•6 NTA-Cu
Max.
Min.
Ave.
f8 Cu
Max.
19 Cu
Max.
fciivk
Ave.
Mn
C21^22>
.30
<.01
.05*
.32
<.0l
.048
.44
<.01
,045
.48
<.01
.129
.35
<.01
.109
,50
.03
.186
.48
.01
.155
Fft 2n Mg
* (21-22) (15-17) (4-5)
.17
.04
.086
.i«
.13
.091
.21
.01
.110
.25
.05
.-131
.22
.03
.116
.25
.04
.122
.24
.06
.115
.024
<.01
.007
.027
<.01
.009
.112
<.01
.026
.016
<.01
.006
.016
<.0l
.007
.016
<.01
.006
.54
<.01
.020
7.7
6.2
7.24
7.4
6.8
7.2
7.3
6.7
7.03
7.5
7.0
7.30
7.5
6.7
7.2
7.4
6.8
7.2
7.2
6.9
7.10
' Ca
(9-10)
18.0
10.5
15.6
17.8
12.7
15.7
19.2
13.0
15.6
17.8
14.4
15.9
17.8
11.6
15.4
18.0
10.4
15.4
18.0
11.8
15.3
' '^a
(4-5)
5.30
4.47
5.04
5.81
4.95
5.49
5.63
5.30
5.51
5.80
5.23
5.59
5.80
5.22
5.59
5.35
4.79
5.15
5.30
4.66
5.10
Cu
(0-45)*'
no
deter.
<.01
<.01
<.01
<.01
<.01
<.01
.ISO
.058
.102
.132
.070
.098
.134
.020
.075
.122
.037
.073
'k
k (9)
2.49
1.65
1.99
2.25
1.67
1.91
2.15
1.70
1.87
2.90
1.67
2.20
2.34
1.64
1.93
2.05
1.72
1.86
2.09
1.64
1.87
P04P
122-23)
.022
.004
.011
.019
.022
.011
.028
.005
.014
.020
.004
.012
.021
.005
.011
.028
.011
.018
.022
.007
.016
SiO?
(21-?3)
14.5
7.9
12.4
14.0
9.0
12.3
13.9
9.4
12.7
13.9
9.0
12.4
13.8
8.8
12.4
14.2
9.5
13.0
14.0
9.2
12.9
VJOgN
(22-23)
3.57
1.83
3.02
3.37
2.35
3.03
3.34
2.44
3.05
3.43
2.30
2.76
3.46
2.28
3.04
3.46
2.49
3.08
3.44
2.28
3.07
NOoN NH 3N
(22-23) (20)
.034
.007
.013
.050
.011
.020
.021
.007
.014
.030
.007
.015
.037
.009
.017
.020
006
.014
.027
.005
.013
.032
<.005
<.005
.042
<.005
.008
.044
<.005
.006
.023
<.005
<.005
.027
<.005
<.005
.050
<.005
.008
.025
.005
.005
$04 Cl Alk-P 'TotTUk
(4-5) (19-20) (21-22) (21-22)
19.5
16.4
18.2
19.4
17.1
18.4
19.7
17.9
18.9
20.6
16.0
18.7
19.6
17.9
18.9
20.2
15.9
18.5
20.2
17.4
19.1
11.0
8.0
9.6
12.0
9.0
9.8
12.0
8.0
10. 3
12.0
8.0
10.1
12.0
8.0
10. 3
11.0
9.0
10.2
12.0
8.0
10.1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
49.0
35.0
44.5
49.0
38.0
44.9
50.0
37.0
44.7
48.0
37.0
44.4
49.0
38.0
45.0
48.0
41.0
44.7
51 .0
37.0
45.2
* Number of D«ter»inations
** *5 determination where Cu was added, otherwise 8-10.
-------
TABLE 20.
Determinations for NTA and Cu during the Period
March 12 - April 15, 1975
CO
o
Control
Microcosm Box (t 4
NTA, GC technique
X (mg/1) 0.02
N 7
SD 0.03
Coef. of var. 157
SE 0.01
SEm (%) 49.8
Max. 0.09
Min. 0
# out of 30% range
NTA, Zinc-Zincon technique
X (mg/1)
N
SD
Coef. of var.
SE
O 17* / ft \
SEm (%)
Max.
Min.
# out of 30% range
Cu
X T5g/l)
N
SD
Coef. of var.
SE
SEm (%)
Max1.
Min.
If out of 30% range
NTA-Cu
2
1
27
0
27
0
5
2
0
8
1
23
0
30
0
6
2
0
4
102
41
24
23
4
3
150
58
3
.76
.49
.6
.09
.3
.71
.82
.53
.46
.5
.10
.4
.1
.4
.1
.5
6
1.
27
0.
37.
0.
7.
2.
0.
10
1.
23
0.
31.
0.
6.
2.
0.
4
98
41
14.
15.
2.
2.
132
70
0
60
60
3
11
2
70
52
41
44
1
09
5
1
4
7
1
2
3
7
1
27
0
35
0
6
2
0
9
1
23
0
36
0
7
2
0
6
NTA
10
.25
.45
.9
.09
.9
.12
.46
.31
.48
.8
.10
.6
.2
.7
1
26
0
34
0
6
2
0
8
1
23
0
40
0
8
3
0
8
.47
.50
.2
.10
.7
.96
.86
.45
.59
.7
.12
.5
.1
.6
8
0.015
7
0.02
163
0
0
0.07
0
75
41
25.8
34.1
4
5.3
134
20
8
CU
9
0.
7
0.
129
0.
46.
0.
0
73
41
19.
26.
3
4.
122
37
6
006
007
003
3
016
7
8
1
Molar ratio of Cu-NTA
NTA by CC deternination
5.0
5.5
-------
TABLE 21. Microscopic Examination of Communities
March 15
March 22
Control:
Navicula viridula v. avenacea - dominant
Achnanthes lanceolata - common
Nitzschia palea - common
Diatoma vulgare - frequent
Ulothrix zonata - frequent
Stigeoclonium lubricum protonema - frequent
Unicellular green - rare
Microcoleus vaginatus - rare
Control:
Navicula viridula v. avenacea - dominant
Nitzchia
lea - common
Achnanthes lanceolata - frequent +
Melosira varians - frequent +
Synedra ulna - frequent
Stigeoclonium lubricum protonema - frequent +
Ulothrix zonata - frequent +
Microcoleus vaginatus - rare
NTA:
Navicula viridula
v. avenacea - dominant
Nitzschia palea - common
Achnanthes lanceolata - common
Synedra ulna - frequent
Stigeoclonium lubricum protonema - frequent
Unicellular green - frequent
Microcoleus vaginatus - rare
Schizothrix calcicola - rare
NTA-Cu:
Navicula viridula
v. avenacea - dominant
Nitzschia palea - common
Achnanthes lanceolata - common
Diatoma vulgare - frequent
Ulothrix zonata - frequent
Unicellular green - locally common
Stigeoclonium lubricum protonema - frequent
NTA:
Navicula viridula v.
avenacea - dominant
Nitzschia palea - very common
Achnanthes lanceolata - common
Synedra ulna - frequent
Melosira varians - frequent +
Nitzschia linearis - frequent
Stigeoclonium lubricum protonema - common
Ulothrix zonata - frequent
Microcoleus vaqinatus - rare
NTA-Cu:
Navicula viridula v. avenacea - dominant
Nitzsch ia palea - common
Achnanthes lanceolata - frequent
Meridion ci rculare - common
Stigeoclonium lubricum protonema - frequent
Oedogonium sp. - rare
Cu:
Navicula viridula v.
avenacea - dominant
Nitzschia palea - v. common
Achnanthes lancoolata - common
Stigeoclonium lubricum protonema - frequent
Unicellular green - frequent
Microcoleus vaginatus - rare
Cu:
Diatoms in very poor condition
Stigeoclonium lubricum protonema - frequent +•
in good condition
Schizothrix calcicola - frequent
Microcolaus vaginatus - rare to frequent
-------
TABLE 22.
Detailed Analysis of Diatom Populations by the
Truncated Normal Curve Technique
to
to
Tewts
Microcosm Box t
Position of modal interval
t of intervals
1 of observed spp.
t of spp. in model interval
a2 (sigma squared)
Position of the mode
The theoretical universe
Total 1 specimens counted
i spp > 10%
Control
B
3
11
86
10.8
12.4
2.2
95.1
6677
4
4
A
3
11
77
10.8
12.4
2.2
95.1
5640
2
Cu-NTA
2
B
3
11
70
10.0
13.0
2.3
90.3
5290
4
A
3
11
72
10.1
11.8
2.5
86.8
5716
6
B
2-3
12
88
11.6
15.9
2.0
117
10070
3
A
3
11
76
10.2
14.8
2.2
98.1
7418
3
B
2-3
11
72
10.3
13.2
2.1
94.4
4989
4
NTA
7
A
2-3
12
81
11.6
12.4
2.1
103
7124
3
B
3
12
86
12.1
13.1
2.3
109.8
8378
4
x^
A
4
11
62
8.
8.
3.
63.
5610
2
B
1
8
5
* B - before experiment
A - end of experiment
1Zinc tras very high (2.9 x box 7) & probably caused species reduction.
-------
TABLE 23. Distribution of the more Common Species
(_> 1.7% of the Specimens Counted)
Uu
CO
Microcosm Box I
Concentration
Nitzschia diserta
Navicula secreta v. apiculata
Navicula viridula v. avenacea
Achnanthes lanceolata
Nitzschia kutzingiana
Achnanthes ninutissima
Synedra rumpens v. meneghiniana
Synedra rumpens v. familiar is
Navicula pelliculosa
Nitzschia frustulun v. perminuta
Navicula paucivisitata
Meridion circuiare
Fragilaria vaucberiae
Melosira varians
Nitzschia communis v. hyalina
Nitzschia linearia
Suriella minuta
Suriella ovata
Navicula minima
Navicula genovefea
Others
4
Control
B
26.9
11.4
11.1
5.1
11.9
3.3
4.2
3.3
22.9
A
26.7
21.1
8.6
6.3
4.0
2.5
3.5
1.7
1.6
3.4
1.5
1.1
5.4
18.5
B
20.8
16.4
11.7
2.8
14.0
2.2
2.6
3.6
2.9
23.1
2
6
t
Cu-NTA
A
25.8
20.6
8.9
5.9
3.6
2.3
1.0
1.6
2.2
1.2
1.4
7.1
3.4
2.8
19.6
B
13.1
29.0
8.9
3.4
14.1
2.1
1.9
2.5
2.6
22.5
A
22.2
17.6
14,5
3.8
5.1
2.9
3.2
2.6
2.3
3.4
3.7
3.6
1.6
1.4
19.9
B
13.2
22.1
12,6
13.1
3.7
2.8
2.2
2.8
2.2
25.3
t
NTA
A
33.4
21.6
13.9
3.2
1.0
4.6
1.6
1.4
1.7
1.3
2.5
17.5
10
B
27.1
12.2
12.6
3.8
11.5
1.7
4.3
2.0
4.3
4.2
16.7
A
27
25
1
8
3
4
4
3
1
2
2
18
.3
.5
.7
.8
.0
.4
.2
.1
.1
.3
.7
.6
(-) Denotes less than one percent, but present
B - before experiment - March 12, 1975
A - after experiment - April 15, ' °'>c
1975
-------
14
TABLE 24. Biomass, Chlorophyll and C Determination
C
Test
Microcosm Box it
March 13, 1975 (end of
Chlorophyll a/c
Percent volatile material
Ash free dry weight (mg)
March 22, 1975 (9 days
Percent volatile material
Ash free dry weight (mg)
April 14-16, 1975 (end
Chlorophyll a/c
Chlorophyll a (mg/side of
Chlorophyll c
Ash free dry weight (mg)
Percent volatile material
Control
4
seeding)
9'3,
17. 21
64.6
after seedinq)
21.4
39.6
of experiment)
9.1
slide) 0.83
0.09
79.7
30. 3
Phycocyanin (ug/side of slide) 7.8
DPM/iKj chlorophyll a
1613
NTA
7
8.6
15.9
100.6
26.2
46.8
8.1
0.82
0.10
84.5
29.5
7.5
1366
10
9.9
12.9
80.2
19.0
33.5
7.0
0.71
0.10
57.4
31.8
8.0
1605
C u- NTA
2
10.1
16.4
88.0
36.8
30.6
9.1
0.75
0.08
60.3
35.4
7.6
1344
6
10.9
16.8
47.6
19.4
39.0
9.2
0.81
0.09
68.5
33.8
8.0
2446
CD
8
9.6
19.3
48.1
14.8
44.3
2
0.01
0.01
22.1
14.0
8.0
44
9
9.4
20.0
53.3
16.0
59.5
2
0.07
0
4.4
22.0
8.3
0
Considerable silt resulting in low volatile to non-volatile ratio.
Due to poor condition of diatoms ratio not meaningful
-------
TABLE 25. Metals in Algal Biomass (mg cation/g)
March - April 1975
End of Seeding
One week in experiment
End of Experiment
Mg
Ca
Fe
K
Mn
Na
Zn
Cu
Control
5.84
4.53
31.58
3.92
1.86
0.57
0.22
0.12
NTA Cu-NTA
5.95 4.28
3.86 3.54
29.31 22.71
3.93 2.98
1.49 1.18
0.57 0.47
0.18 0.11
0.10 0.09
Cu co,|f. of
6.93 20.1
4.68 12.4
34.32 20.0
5.32 24.3
1.48 20.3
0.74 20.9
0.22 31.6
0.14 18.7
SEm
7.6
4.7
7.2
9.2
7.7
7.9
11.9
7.1
Control
6.50
4.92
44.44
4.48
4.80
0.83
0.29
0.20
NTA
5.21
4.57
25.62
3.98
5.35
0.62
0.13
0.11
Cu-NTA
6.61
5.31
38.79
4.7
3.59
0.73
0.12
0.22
Cu
5.57
4.16
33.63
3.49
3.18
0.58
0.37
1.85
Control
5.18
3.42
20.08
8.58
9.29
0.87
0.39
0.10
NTA Cu-NTA
5.11 5.43
4.09 4.18
17.72 18.98
6.62 8.93
8.34 11.06
0.87 0.91
0.13 0.12
0.08 0.43
Cu
6.56
3.71
35.54
3.91
6.37
1.07
0.29
5.95
U)
Oi
-------
TABLE 26.
NTA Concentration in Microcosms
March 12 - April 24, 1975
Desired NTA
concentration
(mg/1)
Achieved concentration (mg/1)
Mean + Standard Deviation
Range
Number
determi-
nations
Number
Determi-
nations
±30 % mean
u>
cr>
Gas Chromatog_r_ap_hic determination
0.02
0.2
2.0
20
0
(Control)
0.09 + 0.14
0.16 + 0.14
1.20 + 0.50
18.70 + 8.70
0.01 + 0.02
Zinc-Zincon determination
2.0
20
1.4
18.2
+ 0.5
+ 7.5
0
0.07 -
0.23 -
0.10 -
0
0.9 -
0.5 -
063
.76
2.10
41.90
0.09
2.2
32.8
41
34
43
42
13
30
33
6
13
23
21
-------
TABLE 27. NTA degradation by bacterial populations
exposed to different NTA concentrations
for four weeks.
UJ
Desired
NTA
Concentration
for Assay
0
.02
.2
2
20
Degradation Rate
(ng/hr/cm2)
A
0.02
0.26
6.5
13.6
18.4
B
0.02
0.27
2.7
20.1
15.3
EXPERIMENTAL
C
0.02
0.16
3.5
12.7
20.6
FLASK :
D
0.02
0.21
4.3
10.4
10.3
E
0.02
0.27
4.8
6.9
14.4
Dead Cell
Control
0.02
0.02
0.03
0.14
1.9
-------
TABLE 28. NTA Concentration in Microcosms
July 2 - August 14, 1975
00
00
Desired NTA Achieved
Concentration (mg/1)
concentration Mean + Standard
(mg/1) Deviation
Gas Chroma to graphic
0.02
0.2
2.0
20
0 (Control)
Zinc-Zincon Determination
2.0
20
0.06
0.17
1.70
20,10
0.03
1.9
20.3
+ 0.09
+ 0.16
+ 0.68
+ 6.20
+ .09
+ 0.5
+ 5.4
Range
(0
(0.01
(0.62
(14.10
(0
(1.1
(7.0
- 0.38)
- 0.75)
- 3.30)
-26.10)
- 0.39)
- 3.1)
-39.2)
Number
Determi-
nations
39
37
30
29
25
43
38
Number
Determi-
nation
Jl 30 % mean
3
10
15
22
3
32
32
-------
TABLE 29. Effect of cadmium, zinc, lead, and nickel ions and
NTA-chelates of the same on the metabolism of
14C glucose by heterotrophic bacteria from White Clay
Creek. Metal ions present at 1 mg/1, NTA at 4 mg/1.
u>
VD
Date of
Experiment
Cadmium
3/4
3/5
4/11
4/15
4/18
10/2
Zinc
374
3/5
4/11
4/15
4/18
9/30
10/1
Lead
9/10
9/12
9/16
10/7
10/28
Nickel
9/10
9/12
9/16
9/16
Assay
Temperature
(°C)
9
9
18
18
18
15
9
9
18
18
18
15
15
20
20
20
15
20
20
20
20
20
Inoculum
(104 cells/
ml)
3.18
3.18
4.08
1930.00
40.80
31000.00
3.18
3.18
4.08
1930.00
40.80
260.00
200.00
12.00
23.00
400.00
0.68
38.00
12U00
53.00
400.00
92.00
14
C Incorporation as Percent:
metal
chelate
40
39
35
69
69
14
29
34
47
59
63
36
59
82
30
76
50
12
29
32
25
metal
control
46
39
54
61
59
11
33
35
75
71
66
34
54
59
40
13
84
58
47
24
metal
NTA
42
32
35
67
56
23
30
29
49
77
63
39
59
81
40
86
41
14
33
40
33
24
Mean
43
37
41
66
61
16
31
33
57
69
64
36
57
82
43
81
44
13
47
42
38
24
Percent
Inhibition
57
63
59
34
39
84
69
67
43
31
36
64
43
18
57
19
56
87
53
58
62
76
!4C Incor-
poration
as Percent:
Chel ate/control
115
100
154
89
85
79
114
103
159
119
105
94
91
98
194
97
79
105
49
200
147
96
-------
TABLE 30. Effect of copper ions and copper-NTA chelates on the
Metabolism of 14C glucose by heterotrophic bacteria from
White Clay Creek. Metal ions present at 1 mg/1, NTA at 4 mg/1.
Date of Assay Inoculum
Experiment Temperature (10
(°C) cells/ml)
3/4
3/5
4/11
4/15
4/18
9/10
9/12
9/16
9/29
10/17
10/21
10/24
9
9
18
18
18
20
20
20
15
15
20
20
3
3
4
1930
40
12
23
400
960
23
52
92
.18
.18
.08
.00
.80
.00
.00
.00
.00
.00
.00
.00
C Incorporation as Percent:
metal
chelate
13
21
<1
4
4
2
4
42
3
1
2
3
metal
metal
Mean
Percent
Inhibition
control NTA
12
18
<1
4
3
4
4
19
3
1
1
3
11
15
<1
4
3
<1
3
14
3
1
2
2
12
18
<1
4
3
2
4
25
3
1
2
3
88
82
99
96
97
98
96
75
97
99
98
97
J-iC Incor-
poration as
Percent:
chelate/con trol
91
85
1)1
93
77
182
100
46
93
100
62
100
-------
TABLE 31. Effect of 4 mg/1 NTA (probably present as Ca or Fe
chelates) on metabolism of l^C glucose by heterotrophic
bacteria from White Clay Creek.
Date of
Experiment
3/4
3/5
4/11
4/15
4/18
9/10
9/12
9/16
9/16
9/29
9/30
10/1
10/2
10/7
10/17
10/21
10/24
10/28
Assay
Temperature
(°C)
9
9
18
18
18
20
20
20
20
15
15
15
15
15
15
20
20
20
Inoculum
(104 cells/ml)
3.18
3.18
4.08
1930.00
48.00
12.00
23.00
400.00
92.00
960.00
260.00
200.00
31000.00
0.68
23.00
52.00
92000
38.00
14 C Incorporation
as Percent:
NTA/ control
111
122
153
92
105
255
157
136
100
100
87
92
48
98
100
50
150
93
-------
TABLE 32. Effect of cadmium, zinc, and copper ions and NTA Chelates of
the same on Metabolism of ^-^C glucose by NTA degrading
isolates. Metal ions present as 1 mg/1; NTA at 4 mg/1.
All assays at 18C.
to
Isolate
Cadmium
NTA-T1
216
216
Zinc
NTA-T1
216
216
Copper
NTA-T1
216
216
Inoculum
(104 cells/
ml)
0.69
54G.OO
9) .60
0.69
548.00
91.60
0.69
548.00
91.60
1 4
C Incorporation
Metal
chelate
57
177
21
62
81
118
14
11
17
Metal
control
49
59
15
49
83
92
8
6
10
as Percent:
Metal
NTA
49
98
28
49
137
179
8
10
19
Mean
52
59*
15*
53
82 +
92*
10
9
10*
Percent
Inhibition
48
41
85
47
18
8
90
91
90
14C Incorporation
as Percent:
Chelate
control
86
34
70
79
103
78
58
54
58
NTA
control
100
61
51
100
61
51
100
61
51
* Percent control only
+ NTA omitted from calculation
-------
TABLE 33. Effect of copper, cadmium, zinc, and mercury ions and
NTA-metal chelates on Microbial Metabolism. (Three
Replicates per Treatment, all data as x, s.d.).
CO
Metals ( u
Exposure
100 mg/1 Metal,
500 mg/1 NTA
Cu
Cu/NTA
NTA
Control
Cd
Cd/NTA
NTA
Control
Zn
Zn/NTA
NTA
Control
Hg
Hg/NTA
NTA
Control
1 mg/1 Metal,
5 mg/1 NTA
Cu
Cu/NTA
NTA
Control
Cell number Respiration (% 0? depletion/min. )
at 22 hr
(x 106)
26,
158,
122,
QC
85,
483,
486,
302,
280,
370,
153,
402,
577,
20,
11,
580,
185,
32,
26,
60,
44,
2
41
23
138
29
167
60
89
58
207
264
4
6
183
21
7
3
19
6
0 (Before 0 (After 2.5 hr.
exposure) exposure)
1.1,
1.0,
1.1,
1A
.4,
1.9,
2.3,
2.4,
1.8,
0.9,
1.2,
1.2,
1.1,
2.9,
2.7,
2.8,
3.3,
0.8,
1.2,
2.1,
0.8,
0.2
0
0.1
0>5
. £.
0.4
0.3
0.1
0.2
0.1
0.2
0. 3
0.1
0.2
0.1
0.2
0.3
0.1
0.4
0.2
0.2
0
1.0, 0
1.1, 0.1
0,
2.1, 0.3
2.4, 0.1
0
1.2, 0.2
1.3, 0.3
0
0
2.8, 0.2
0
1.3, 0.4
1.4, 1.1
0.8,
2.4,
2.7,
20
.8,
0.5,
2.5,
3.3,
2.3,
0.3,
16.6,
9.4,
5.0,
0
0
2.8,
3.4,
0.5,
2.2,
3.7,
2.3,
0.1
1.3
0. 3
0.1
0.5
0.2
0.4
0.1
0.5
3.1
0.5
0.5
0.4
0.2
0.2
0.5
0.1
at indicated
22 hr.
3.0
40.0
38.0
19 f\
j£ , U
5.9
8.2
2.8
4.8
3.8
24.2
12.2
20.0
0.1
0.2
4. 3
6.4
0.4
3.3
4.2
2.7
, 1.2
, 3.8
, 4.0
, 1.8
, 2.0
, 0.2
, 1.5
, 0.8a
, 6.0a
, 7.8a
,10.83
, 0.1
, 0.1
, 0.7
, 0.9
, 0.1
, 0.8
, 0.5
, 0.8
time:
Supernatant
22 hr.*
6.2,
9.2,
17.8,
15.7,
0.1,
0.2,
7.4,
9.0,
1.7,
3.8,
6.8,
4.7,
2.0
2.5
0.8
3.4
0.1
2.2
0.5
0.2
0.7
1.5
3.5
0
78.
78.
44.
44.
76.
76.
24.
24.
78.
78.
22 hr.
6
6
5
5
4
4
9
9
0
0
54 .
49.
13.
15.
28.
66.
16.
20.
44.
74.
3, 6.2
6, 2.8
4, 2.0
9, 0.2
0, 3.2a
6, 4.9a
2, 2.7
2,
7,11.0
9, 4.7
mole)
Cells
22 hr.
1.7,
1.1,
0.25
0.09
66.5,
13.4,
4.6,
0.8,
7.5,
2.6,
0. 1
0.1
,0.03
,0
3.3a
l.la
0.6
0.9b
2.3<=
a65 hr.
bmg dry wgt cells = 1.4, 0.2; u moles metal/mg dry weight = 5.40, 0.77
cmg dry wgt cells = 3,6, 0.2; u moles metal/mg dry weight = 1.09, 0.19
*following nutrient addition
-------
TABLE 34.
Concentration of Selected Cheirdcal Species in Ecosystem
Stream Water; Julv 13-October 6, 1976. Data in mg/1 but
for Mn, Fe, 7n which are ug/1. Stream I-Control, II-NTa/Cu,
III-Cu, IV-NTA.
CONCENTRATION
Stream
I
max
min
X
n
max
min
X
n
III
max
min
X
n
max
man
X
n
NO3-N
3.14
1.79
2.74
78
3.19
1.79
2.78
78
3.19
1.79
2.78
78
3.19
1.79
2.80
78
NO2-N
.012
.002
.006
77
.014
.002
.006
77
.011
.002
.006
77
.012
.002
.006
77
NH3-M
.078
.002
.014
61
.073
.002
.010
61
.076
.002
.009
61
.076
.002
.011
61
P04-P Si02
.07
.02
.04
75
.07
.02
.04
75
.07
.02
.04
75
.07
.02
.04
75
18.
15.
17.
18
19.
15.
17.
18
19.
14.
17.
18
19.
15.
17.
18
7
2
1
1
4
2
0
8
2
0
2
0
pH
7.8
7.5
7.6
28
8.1
7.5
7.7
27
7.8
7.6
7.8
28
7.8
7.0
7.7
28
30^= Cl- Total Ca
Alkal in ity
17.9
14.1
15.4
18
18.0
14.6
15.8
18
17.8
14.2
15.5
18
17.8
13.2
15.4
18
12.0
8.0
9.4
19
11.0
8.0
9.8
18
11.0
8.0
9.8
18
12.0
9.0
10.2
19
7]
56
60
19
64
54
60
19
66
57
61
19
65
56
61
19
27
1 I
Hi
21
28
11
17
21
26
1.1
17
21
25
11
16
21
.2
.2
.9
.1
. J
. 3
.9
.1
.2
.5
. 3
.7
Mq
19
5
10
21
15
5
10
21
19
5
10
21
19
5
10
21
.5
.4
.4
.2
.5
. 3
.6
.5
.6
.6
.5
.4
Mn
28.
0
16.
21
29.
0
16.
21
29.
0
18.
21
26.
0
15.
21
3
6
2
9
2
0
8
8
Ke
591
87
221
21
590
91
215
21
642
81
228
21
607
34
221
21
Zn
9.3
0
1 .4
21
''. 3
0
2.2
.'A
10.0
0
4.0
21
11.6
0
2 .4
21
-------
TABLE 35.
Concentrations of NTA and copper in ecosystem
stream water; July 13-October 6, 1976. NTA
reported in mg/1; cooper in ug/1. Stream I-
Control, II-NTA/Cu, III-Cu, IV-NTA.
Compound
Concentration in Stream
II
III
IV
NTA (GC analysis)
n 8
x 0.06
s.d. 0.07
c_._v. 116
Sx 0.02
Sx(% mean) 41
max. 0.19
min. 0.01
NTA (Zinc-Zincon)
n
x
s.d.
c^_v.
Sx
Sx(% mean)
max.
min.
Copper
n 21
x 3.6
s.d. 3.8
c.v. 105
Sx 0.8
Sx(% mean) 21.8
max. 15.0
min. 0
60
1.99
0.75
38
0.10
5
4.10
0.22
81
1.88
0.44
23
0005
3
2.65
0
20
31.1
10.7
35
2.4
7.7
49.0
0
9
0.06
0.05
93
0.02
31
0.15
0.01
20
29.2
13.3
46
3.0
10.2
55.4
0
60
2.07
1.00
49
0.13
6
4.25
0.02
77
1.86
0.63
34
0.07
4
3.30
20
3.9
3.8
99
0.9
21.8
9.3
0
145
-------
TABLE 36.
Comparison of Concentration of Cu, Fe, Zn, Fn, and Fg in
Algal Biomass from Fcosvstem Streams at a given Sampling
Time. Results of Scheffe Test (S) or Kurskal-Wallis Test
(K), both at P = 0.05, are indicated by grouping streams
between which no significant differences existed in
parenthesis. Stream I-Control, II-NTA/Cu, III-Cu, IV-NTA,
(Stream between
Collection
& Date
] 7/13
2 7/20
3 7/28
4 b/11
5 0/24
6 9/7
7 9 /1!1
8 10/6
Cu
(I, IT., Ill, IV)
(I, II, IV)
(1,11,1V)
(1,11, III) ,
(III, IV)
(I, II, IV) (II,
HI)
(I, II, IV) III
(I, II, IV)
S
S
S
K
K
S
S
(1,11,
(1,11,
(I, II,
(t.il.
(I, II,
'ill
(I, IV)
(1,11,
Fe
III, IV) S
IV) S
IV) S
III, IV) S
1V)III S
IV) (T,
, IV) K
(II,III)S
.IV) K
(I,
which no significant difference exists):
Zn
11,111) (II, III, IV)
KII.TV)
(I,
(II
(I,
(I,
(I,
II, IV)
,111) (III, IV)
II, III) (I, II, IV)
II, IV) ,111
IV) (11,1V)
S
S
S
S
K
K
K
Mn
(I, II) (II, III, IV)
(1,11 , IV)
(I, II, IV)
(I, II, III) (I, II,
IV)
(I, IT) (1,111) ,
(11, IV)
(I, II, III, IV)
(1,11, III, IV)
I, 11, IV
K
S
S
K
K
S
K
S
Mg
(I, II, III, IV)
(I, II, IV)
(I, II, IV)
(I, II) (1,111,
(I, II, III, IV)
(I, II, IV) (III,
(I, IV) (II, III)
(II, IV)
(I, II) (I, IV)
K
S
S
IV) S
S
IV) S
K
K
-------
TABLE 37. Comparison of Concentration of Cu, Fe, Zn, Mn and Mg om
Anacharis from Ecosystem Streams at a given Sampling Time.
Results
both
at
which no
Stream I
Collection
& Date
1 7/13
2 7/20
3 7/28
4 8/11
5 U/24
6 9/7
7 9/21
8 10/6
of Scheffe
P = 0.05
,
Test (S) or
are indicated
Kruskal-Wallis Test
by
grouping
significant differences existed in
-Control
r
II-NTA/Cu, III-Cu
(Streams between which no significant
Cu
(I, II) (I, III, IV) K
(I, II, IV) (I, III) K
(I, III) (II, III,
IV) K
(I, IV) (II, IV) K
(I, II) S
(I,IV)II S
(I.IVJII S
(I, II) (I, IV) K
(I, II,
IV)
(I, II,
(I, II,
(I, II)
(I, II)
(1,11,
(I, IV)
(1,11,
Fe
III) (11,111-,
IV) III
IV) III
(II, IV)
IV)
(II, IV)
IV)
S
S
S
S
S
S
S
K
Zn
(I, II, III) (I, III,
IV)
(I, II, III, IV)
KII, IV)
I, II
(I, II IV)
KII, iv)
(I, IV) (II, IV)
, IV-NTA
streams
(K) ,
between
parenthesis.
difference exists) :
K
K
K
K
S
K
S
K
Mn
(I, II, III, IV) K
(I, II, IV) (I, III,
IV)
(1,11,1V) (I,
KII, IV)
(I, II)
(I, II, IV)
(I, II, IV)
(I, II, IV)
S
III) S
S
K
S
S
K
(i,u,
(i, ii.
(i, ii,
(i, ii,
(i, ID
(i, ii,
(i, ii,
(I, IV)
Mg
III, IV)
III, IV)
IV) III
IV)
IV)
IV)
(II, IV)
S
K
S
S
S
K
S
S
-------
00
TABLE 38. Comparison of Concentrations of Cu, Fe, Zn, Mn, and Mg
in Lemna from Ecosystem Streams at a given Sampling Time.
Results of Scheffe Test (S) or Kruskal-Wallis Test (K),
both at P = 0.05, are indicated by grouping streams
between v/hich no significant differences existed in
parenthesis. Stream I-Control, II-NTA/Cu, III-Cu, IV-NTA.
Collection
& Date
1 7/1 J
2 7/20
3 7/28
4 8/11
5 8/24
6 9/7
7 9/21
8 10/6
(Streams between which no significant difference exists):
Cu
(I, II, III) (II, III, IV)
(I, II) (11,111) (I, IV)
(I, II, III, IV)
(I, II, IV) (II, III, IV)
(I, III, IV) (II, III)
(I, II, III, IV)
(I, II, III, IV)
(I, II) (1,111)
K
S
S
K
S
S
K
K
(I, II,
(I, II,
(1,11.
(I, II,
IV)
(I, II,
(Mill
(I, II,
Fe
III, IV)
IV) (III, IV)
IV)III
III) (11,111
111,1V)
KII.III, IV,
111,1V)
(I, III) (II.III)
s
K
K
K
K
S
S
K
Zn
(I, II, III,
(I, ID (II,
(I, III) (II
IV)
(I, III, IV)
(1,11,111)
IV)
(I, III) (II
IV)
KII.III)
IV) K
III, IV) S
,111,
s
(11, 11 I) K
(ii.ru,
s
,111,
K
S
Mn
(I, II) (I, IV) (III, IV)
(I, II, IV) (III, IV)
(I, II, IV) (11,111)
(I, II, III) (II, III,
IV)
(I, II) (III, IV)
1,111(11 ,IV)
(I, IV) II, III
(1,111) (II, III)
S
S
K
K
S
S
S
S
Mg
(I, II, III) IV
(I, II, III, IV)
(1,11,111) (1,
II ,IV)
(I, II, lit) (I,
II, IV)
(I, II , III ,IV)
(I, II, IV) (I,
III)
(1 ,11, III) (II
III ,IV)
(I, II) III
s
s
s
K
S
S
,
K
S
-------
IO
TABLE 39. Comparison of Concentrations of Cu, Fe, Zn, Mn, and Mg in
Planaria from Ecosystem Streams at a given Sampling Time.
Results of Scheffe Test (S) or Kruskal-Wallis Test (K),
both at P=0.05, are indicated by grouping streams between
which no significant differences existed in parenthesis.
Stream I-Control, II-NTA/Cu, III-Cu, IV-NTA.
Collection
& Date
1
2
3
4
5
6
7
8
7/13
7/20
7/28
8/11
8/24
9/7
9/21
10/6
(Streams between which no
Cu
(I, II, III) (III, IV)
(I,II,IV)(I,II)(II,
IV)
(I, II, IV)
(I, II, IV)
(I, II, IV)
(I, II, IV)
(I, III)
(I, II, IV)
Fe
K
S
S
K
S
K
S
S
(I, II,
(I, II,
(I, ID
(I, II,
III, IV)
—
IV)
IV
—
IV)
(I, III)
(I>II)
(II, IV)
s
K
S
K
S
K
(I,
(I,
(II
(I,
(I,
(I,
significant difference exists) :
Zn
II, III, IV)
II, IV)
,iv>
—
II, IV)
III)
11,1V)
s
s
s
s
s
s
(I
(I
(I
(I
(I
(I
(I
Mn
,11, III, IV)
,11, IV)
,11, IV)
,11, IV)
,11, IV)
,111)
,11, IV)
s
s
s
s
s
s
s
(I, II,
(I, II,
(I, IV)
(I, II,
(1, 1 1)
Mg
III, IV)
IV)
II
IV)
(II, IV)
(1,111)
(I, II,
IV)
s
s
K
S
S
K
S
-------
T£BLE 40. Grand Mean of Metal Concentrations in Tubificid Worms
from Collections 3-8.
Ul
o
Metal
Concentrations (|jg/g; n, x + s.
I
Control
Cu
Fe
Zn
Mn
Mg
10,
9,
8,
8,
8,
44±
18
5704*2816
219i
227±
1377±
247
132
563
11,
10,
8,
10,
12,
II
Cu/NTA
96±
49
5052±3043
358±
236i
1624±
277
144
420
Statistical test results
,d.)for Material from Stream: (streams between which
III
Cu
10, 142±
111
9,5676±5167
7, 335±
10, 199±
9,1562±
285
174
644
4,
5,
5,
4,
4,
IV
NTA
56±
40
15266±17723
186±
238±
1050±
108
133
231
no significant
difference exists)
(I, IV) (II, III, IV)
(I,II,III,IV)
(I, II, III, IV)
(I, II, III, IV)
(I, II, III, IV)
K*
K
S**
S
S
*K = Kruskal-Wallis test
**S = Scheffe test
-------
TABLE 41. Cumulative Mean Concentration of Metal Ions in the
Water in Ecosystem Streams; August 11 - October 6, 1976.
Data in ug/1 for all elements but Mg which is in mg/1.
Ol
Col-
Metal lec-
tion
Cu* 4
5
6
7
8
Fe 4
5
6
7
8
Zn* 4
5
6
7
8
Mn* 4
5
6
7
8
Mg 4
5
6
7
8
Cumulative Concentration in water (x
n
I
II
Control
4
8
14
16
19
4
8
14
15
18
4
8
14
16
19
4
8
14
16
19
4
8
14
16
19
8.25
6.63
5.93
6.44
6.45
247.00
255.93
225.53
229.01
211.54
3.00
4.73
4.19
4.13
3.95
23.23
18.73
18.95
17.79
16.66
10.51
12.42
10.96
10.81
10.48
±
±
+
±
±
+
±
+
±
±
±
±
+
+
+
+
±
±
+
+
±
±
+
+
±
6.50
4.60
3.47
3.97
3.72
57.46
44.90
55.99
55.61
65.53
0.00
3.21
2.54
2.39
2.22
4.87
7.35
7.21
7.62
7.63
2.56
4.35
4.04
3.79
3.58
28
29
26
28
30
236
244
218
223
204
4
5
4
4
4
22
18
18
17
16
10
12
11
10
10
Cu/NTA
.51 ±
.30 ±
.90 ±
.80 ±
.14 ±
.75 ±
.60 ±
.39 +
.43 +
.14 i
.71 ±
.83 +
.71 ±
.56 ±
.31 ±
.51 ±
.33 ±
.59 ±
.33 ±
.70 ±
.41 ±
.23 ±
.05 ±
.92 ±
.50 ±
8.76
6.38
9.36
10.46
10.59
29.34
33.35
45.46
47.96
62.67
3.07
4.04
3.27
3.03
2.87
4.06
7.29
6.95
7.44
7.00
2.37
4.15
3.80
3U57
3.42
± s.d.)
in Stream:
III
Cu
21.09
19.22
21.15
23.32
26.42
248.90
246.58
233.30
238.01
218.40
4.01
5.16
4.23
4.17
3.98
23.36
19.34
19.15
18.22
17.90
10.14
12.81
11.19
11.07
10.62
+
+
+
+
±
±
+
±
±
±
±
±
±
±
+
±
±
±
±
±
±
+
+
±
±
6.28
10.86
10.41
11.37
13.40
27.18
41.03
65.11
65.34
75.52
1.98
2.69
2.26
2.12
1.99
5.58
8.09
7.51
7.83
7.23
2.10
4.99
4.58
4.29
4.06
IV
NTA
8.14
6.07
5.61
6.18
6.22
276.06
254.52
226.27
230.71
211.93
5.52
5.95
4.84
4.72
4.34
21.37
17.18
17.79
16.68
16.26
9.46
11.78
10,91
10.74
10.39
+
±
±
+
±
±
±
±
±
+
+
±
±
+
+
±
±
±
±
±
±
±
±
±
±
6.29
4.85
3.60
4.15
3.88
85.29
63.90
62.49
62.63
72.37
4.13
3.45
2.91
2.75
2.69
7.65
7.81
7.29
7.57
7.13
2.23
4.63
4.00
3.75
3.57
-------
TABLE 42.
Concentration Factors [Petals in Biomass of indicated
Sample/Metal in Water]. Data shown are averages of
3-5 collections for which factors were calculated.
Ul
to
Sample
and
Metal
Sample PPB/watei: PPB
I
Control
ALGAE
Cu
Fe
Zn
Mn
Mcj
ANACHARIS
Cu
Fe
Zn
Mn
Mg
LEMNA
Cu
Fe
Zn
Mil
Mi)
PLANARIA
Cu
Fe
Zn
Mn
My
TUUIFKX
Cu
Fe
Zn
Mn
Mg
4,
5,
3,
5,
5,
5,
5,
•>,
5,
5,
5,
5,
5,
5,
5,
5,
4,
3,
5,
5,
5,
5,
4,
4,
4,
9449±
44494±
214301
59052*
2080021
75384*
10808±
71193±
12465±
2959081
915971
210721
665221
466471
5908
23024
5693
32061
65566
58548
1684
47424
1365
24486
75213
16645
28899
6547
278492H22209
75961
26271
989351
2255*
852151
61671
251811
565091
145831
146901
3660
1158
40564
1104
44395
2966
9773
62589
5198
36473
4,
5,
4,
5,
5,
5,
5,
5,
5,
5,
5,
5,
5,
5,
5,
4,
3,
3,
4,
4,
5,
5,
4,
4,
5,
II
(r\j x
± S.D.)
for stream:
III
Cu/NTA
29031 1945
41!>68i 21720
12794
66238
i 5530
L 26671
231367±109119
16783
9050
32871
10722
283717
16992
19839
25884
58173
311459
3995
9666
70527
3057
128992
3400
22130
79702
J.4563
157204
± 9699
i 2133
i 20114
i 2299
± 30258
i 11750
l 9433
± 17468
1 10230
i 33592
i 1791
± 12837
l 10507
t 1579
i 24849
± 1517
i 10646
i 57900
± 7096
i 20156
4,
5,
3,
5,
5,
5,
5,
5,
5,
5,
5,
5,
3,
4,
5,
Cu
2293!
52270!
241791
44501i
2680091
106686
19719
8119'/
21706
—
315071
17538*
36586'
376441
8110
17335
5246
20609
70456
20509
10911
20761
16502
3382531112813
3428
2096
283211
1754
98284
6878±
21258+
881801
126351
1354201
2884
16864
54438
0150
42813
4,
5,
4,
5,
5,
4,
4,
4,
4,
4,
4,
4,
4,
4,
4,
4,
3,
3,
4,
4,
3,
4,
3,
2,
3,
IV
JJXA.
31719! 46946
34636+10057
140101 3000
96982M9452
270312177072
50464141766
8820* 1-134
408011 3341 J
12416' 2J50
332534166311
108210171787
407301 33239
31811U2179
53554U1115
374494*97617
13495-1 7198
1973'- 1486
7719'{115343
23131 697
117597-' 2918
87101 4782
42070149169
29407123588
18472 i 9484
109952*14832
Statistical test results:*
(streams between which
no significant difference
exi sts)
(1,11,
(I, II,
(I, II,
(I, II,
(I, II,
(I, II,
(1,11,
(1,11,
(1,11,
(I, II,
(1,11,
(I, II,
(I, II,
(I, II,
(I, II,
(I, II)
(1,11,
(I, II,
(I, II,
(I, II,
(I, II,
(I, II,
(I, II,
(I, II,
(I, II,
IV) (I ,111, IV)
III, IV)
III, IV)
III, IV)
111,1V)
IV)
IV)
IV)
IV)
IV)
III, IV)
III, IV)
III, IV)
III, IV)
111,1V)
(I, IV)
IV)
IV)
IV)
IV)
III, IV)
III, IV)
III, IV)
III, IV)
1H,IV)
K
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
*Scheffe multiple range test used except for Algae-Copper, where the
Kruskal-Wallis test was used.
-------
TABLE 43. Concentration Factors [Metal in Planaria/Metal in £lgae].
Data shown are averages of 4-7 collections for which
factors were calculated.
en
Planaria PPB /Algae PPB
Metal
Cu
Fe
Zn
Mn
Mg
7,
5,
4,
6,
6,
I
Control
0.93+0.
0.07±0.
4.44±3.
.05± .
0.62+0.
97
03
15
03
43
5,
4,
4,
5,
5,
II
Cu/NTA
0.93+0
0.20+0
6.05+2
.05±
0.74±0
(n, x
.41
.24
.80
.02
.37
±s.d.) for Stream:
III
Cu
0.16
0.02
9.49
.03
0.25
5,
4,
4,
5,
5,
IV
NTA
0.69+0
0.13±0
5.30+1
.03 +
0.46+0
.33
.17
.27
.02
.16
Scheffe Test Results:
(Streams between which
no significant
differences exists)
(I, II, IV)
(I, II, IV)
I, (II, IV)
(I, II, IV)
(I, II, IV)
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TABLE 44. Concentrations of Copper and NTA in
Microcosms for Sediment Studies (10/13-12/17/76)
Concentrations: x ± s.d. (n)
Microcosm Cu (ug/1)NTA (mg/1)
M Control 4.16 ± 3.56 (38)
(j\
Copper 23.88 ± 10.92 (41)
Copper/NTA 30.20 ± 7.60 (41) 1.77 ± 0.33 (37)
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TABLE 45.
Concentration of Copper (ng/g) in Sediments
Exposed to Copper or Copper/NTA.
Ln
ui
Depth
Surface
2 cm
5 cm
Surface
2 cm
5 cm
Date
10/29
11/8
11/24
12/17
10/29
11/8
11/24
12/17
10/29
11/8
11/24
12/17
For the
n
2
2
2
4
2
2
2
4
2
2
2
4
Control
X
356.5
401.0
376.5
329.2
237.5
413.5
208.5
142.4
205.5
339.5
282.5
214.0
Copper
s.d.
42.5
7.0
195.9
308.0
84.1
178.9
28.3
39.7
115.3
3.5
48.8
93.7
n
2
2
2
4
2
2
2
4
2
2
2
4
X
539.5
450.5
453.0
590.2
448.0
564.5
267.5
324.8
344.0
314.5
250.5
254.0
s.d.
238.3
62.9
59.4
280.0
87.7
307.6
27.6
112.0
80.6
31.8
3.5
131.0
n
2
2
2
3
2
2
2
4
2
2
2
4
Results of Statistical
Copper/NTA Tests: (Treatments
x~
371.
819.
636.
661.
393.
440.
309.
406.
354.
346.
299.
343.
5
5
0
0
5
5
5
0
5
5
5
0
between which no
s.d. significant
difference exists)
70.0
731.2
2.8
255.1
143.5
146.4
38.9
60.0
129.4
62.9
3.5
126.0
Experiment:
4
4
4
365.8
250.4
260.4
30.4
115.8
63.0
4
4
4
508.5
401.2
290.8
68.4
132.4
46.1
4
4
4
622.
387.
335.
0
4
9
185.3 (C,Cu) (Cu, Cu/NTA)*
55.6 (C, Cu, Cu/NTA)*
24.7 (C, Cu, Cu/NTA)*
Statistical Test Results:
(Depths between
which no signifi-
cant differences
exists)
(S, 2,5)*
(S,2)(2,5)*
(S,2) (2,5)**
* Scheffe Test, P = 0.05
** Kruskal-Wallis Test, P = 0.05
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TABLE 46. Concentration of Iron, Manganese, and Zinc
(all in pg/g) in Sediments on 12/17 after 65 days
Exposure to Copper or Copper/NTA.
CTl
Control
Depth
Iron
Surface
2 cm
5 cm
Manganese
Surface
2 cm
5 cm
Zinc
Surface
2 cm
5 cm
n
4
4
4
4
4
4
4
4
4
X
27.6
26.8
43.3
45.9
28.0
42.6
5.56
2.84
3.98
s.d.
16.6
11.4
21.1
21.8
4,4
14.8
2.83
0.91
0.86
n
4
4
4
4
4
4
4
4
4
Copper
X
43.
60.
40.
34.
38.
38.
5.
4.
4.
5
0
8
5
8
0
20
47
59
s.d.
14.8
21.2
27.6
11.9
8.4
6.9
0.84
1.16
4.50
Copper/NTA
n
3
4
4
3
4
4
3
4
4
X
46.6
63.8
52.3
26.5
33.2
32.5
4.92
4.07
2.91
s.d.
23.5
13.8
22.3
14.2
11.3
12.0
1.70
0.87
0.75
Results of Statistical
Tests: (Treatments
between which no
significant differences
existed)
(C,Cu,Cu/NTA) *
C (Cu,Cu/NTA)*
(C,Cu,Cu/NTA) *
(C,Cu,Cu/NTA)*
(C,Cu,Cu/NTA) *
(C,Cu,Cu/NTA)*
(C,Cu,Cu/NTA)*
(C,Cu,Cu/NTA)*
(C,Cu,Cu/NTA)**
* Scheffe Test, P = 0.05
** Kruskal-Wallis Test, P = 0.05
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-80-050
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
The Effect of Nitrilotriacetic Acid (NTA) on the Struc-
ture and Functioning of Aquatic Communities in Streams
5. REPORT DATE
July 1980
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Thomas L. Bott, Ruth Patrick, Richard Larson, and
Charles Rhyne
8. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Academy of Natural Sciences of Philadelphia
19th and the Parkway
Philadelphia, PA 19103
10. PROGRAM ELEMENT NO.
lBA608a
11. CONTRACT/GRANT NO.
R-801951
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory - Duluth MN
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
13. TYPE OF REPORT AND PERIOD COVERED
Final Report
14. SPONSORING AGENCY CODE
EPA/600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT Communities established in microcosms and ecosystem streams in a greenhouse
were exposed to .02-2 mg/1 NTA, a range including most expected environmental levels.
Higher concentrations were used in some laboratory and screening experiments. NTA at 2
and 20 mg/1 had only slight effects on algal community structure and function and 2 mg/1
protected organisms from the toxic effects of approximately 100 yg Cu++/l. Protection
from the toxicity of 30 yg Cu++/l was also obtained in a 3 month experiment conducted in
ecosystem streams with natural sediments and more complex communities. NTA at 2 mg/1 did
not result in increased concentrations of Zn, Fe, Mn, Mg or Cu in algae, Anacharis,
Lemna, Planaria, and Tubifex spp.; Zn concentrations in algae, Anacharis and Lemna were
frequently reduced; accumulation of added Cu by tubificids was not prevented by NTA.
Bacterial communities adapted to 0.02-20 mg NTA/1 and degraded the compound under aerobic
conditions. Glucose metabolism of non-NTA degrading bacterial communities measured in_
vitro was protected from metal ion toxicity when Cu,Zn,Cd,Mi,Pb, and Hg were complexed
with NTA. Glucose metabolism by NTA degrading bacteria was inhibited, however, presum-
ably as a result of a release of Cd,Cu, and Zn ions from concomitant NTA degradation.
Substantial extraction of metals from sediments occurred at 10~3M (200 mg/1) but not at
10~5 or 10~7lM NTA. Although NTA was relatively resistant to chlorination, IDA, in con-
centrated solution, reacted rapidly with aqueous chlorine to produce an unstable product
with oxidizing properties, presumably N-chloro IDA. Both NTA and IDA could be photo-
oxidized in the presence of a sensitizer, but the reactions were very slow.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Nitrilotriacetic Acid (NTA), aquatic
communities, algae, bacteria, ecosystem
streams, chelation
Nitrilotriacetic Acid
Copper
Photooxidation
06F
8. DISTRIBUTION STATEMENT
RELEASE TO THE PUBLIC
19. SECURITY CLASS (This Report;
unclassified
21. NO. OF PAGES
173
20. SECURITY CLASS (Thispage)
unclassified
22. PRICE
EP-A Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
OUSGPO: 1980 — 657-146/0504
157
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