Bacterial Bioassay for Level I
Toxicity Assessment
Oregon State Univ., Corvallis
Prepared for
Corvallis Environmental Research Lab,, OR
Mar 83
PB83-182287
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PB8 3-182257
EPA-600/3-83-017
March 1983
BACTERIAL BIOASSAY FOR LEVEL I TOXICITY ASSESSMENT
by
Kenneth J. Williamson
Peter 0. Nelson
Department of Civil Engineering
Oregon State University
Corvallis, Oregon 97331
Grant No. R806297010
Project Officer
David T. Tingey
Terrestrial Division
Corvallis Environmental Research Laboratory
Corvallis, Oregon 97330
CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
er
NATIONAL TECHNICAL
INFORMATION SERVICE
US. OfPARIVEN! Of COM»£RC£
WRMCflflO. M. 22161
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TECHNICAL REPORT DATA
(Please readiHflnictions on llic rercni1 Injure iunip'.cling)
1. REPORT NO.
EPA-600/3-83-017
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
Bacterial Bioassay for Level 1 Toxicity Assessment
5. RtPORT DATE
March 1983
G. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Kenneth J. Williamson and Peter 0. Nelson
8. PERFORMING ORGANIZATION REPORT NO.
182287
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Civil Engineering
Oregon State University
Corvallis, Oregon 97331
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
R806297010
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis, Oregon 97333
13. TYPE OF REPORT AND PERIOD CCVERFD
final
14. SPONSORING AGENCY CODE
EPA/600/02
15. SUPPLEMENTARY NOTES
.Project Officer: David T. Tingey FTS 420-4521
1S. ABSTRACT
Nitrifying bacteria were tested to determine their applicability as a Level I bioassay
organism. Level 1 testing involves general bioassay and analysis procedures that will
identify the presence of toxicity in a given waste stream.
The toxicity of five metals and three organic toxicants to the nitrifying bacteria
(Nitrobacter and Nitrosomonas) were determined and compared to other common bioassay
organisms. In general, bacterial exhibited somewhat lower sensitivity for general
metabolic toxicants, but dramatically lower sensitivity for specific target-site toxi-
cants.
The application of the bacterial bioassay was shown for two cases of Level I testing:
a field study of a toxic industrial waste and its pre-treatment and an assessment
study of the potential leachate problems for a flue-gas scrubber solid waste.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
l>.IDENTIFIERS/OPEN ENDED TERMS
C. COSATI lidd/C.rOUp
1<3. DISTRIBUTION STATEMENT
Release to public
18. SECURITY CLASS ( This Report)
21. NO. OF PAGtS
87
20. SECURITY CLASS (
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
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DISCLAIMER
Although the research described in this report hab been funded by the United
States Environmental Protection Agency through grant number R806297010 to
Oregon State University* it has not been subjected to the Agency's peer arid
policy review and therefore does not necessarily reflect the views of the
Agency and no official endorsement should be inferred. Mention of trade
names or commercial producst does not constitute endorsement or recommenda-
tion for use.
ii
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ABSTRACT
Nitrifying bacteria were tested to determine their applicability as
Level 1 bioassay organisms. Level 1 testing involves general bioassay and
analysis procedures that will identify the presence of toxicity in a given
waste stream.
The toxicity of five metals and three organic toxicants to the nitrify-
ing bacteria (Nitrobacter and Nitrosomonas) were determined and compared to
other common bToassay organisms. In general, bacteria exhibited somewhat
lower sensitivity for general metabolic toxicants, but dramatically lower
sensitivity for specific target-site toxicants.
The application of the bacterial bioassay was shown for two cases of
Level 1 testing: a field study of a toxic industrial waste and its pre-
treatment and an assessment study of the potential leachate problems for a
flue-gas scrubber solid waste.
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CONTENTS
Abstract . ill
Figures vi
Tables viii
Acknowledgements. ix
1. Introduction 1
Background ,...., 1
Initial Studies 3
2. Conclusions 4
3. Reconmendations 5
4. Research Plan 6
Approach 6
5. Toxicity to Nitrifying Bacteria 8
Toxic Mechanisms to Bacterial Cells...... 8
Kinetic Theory of Toxicity 8
Chenicals Causing Nitrification Inhibition 10
6. Nitrifying Bioassay 13
Bi ochemi stry of Nitrifying Bacteria 13
Procedure 15
Culturing of Nitrobacter 15
Freeze-Drying 15
Bioassay 17
Data Analysis 17
Results 18
Nitrobacter Bioassay 19
Hi trosomona's Bioassay 19
7. Acute Toxicity of Selected Chemicals to Common Oioassay
Organises 26
Lead 26
Cadcri itr.\ 28
Copper 28
Zinc 29
Silver 31
Heptachl or 31
Endosul fan. 35
Pa rat hi on 35
Comparison of Nitrifying Bioassay to Other Test
Or ga n i sins 35
IV
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8. Bacterial Bioassay Application to Industrial Wastewater
Treatment 40
Methods of Measuring Toxicity to Activated Sludge 40
Laboratory Studies with Known Toxicants 41
Laboratory Studies with a Metal Processing Wastewater.... 43
Field Studies with a Fiberboard Mill Wastewater 43
9. Bacterial Bioassay Application to Solid Waste Leachate......... 52
BOF Scrubber Sludge 54
Extraction 54
Bioassays 56
Results 56
10. Continuous-Flow Bacterial Bioassay 60
Procedure 60
Results 61
Interfacing Anions Present 64
Discussion 64
References 70
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FIGURES
Number Page
p-v««^hww» ^M^ fc^fc^
1 Apparatus for Culturing Nitrifying Bacteria 16
2 (a) TCP Toxicity Measured by Reduced Substrate Utilization and
(b) the Relative Metabolism Rate of Nitrobacter 18
3 Relative Metabolism of Nitrobacter vs. Dosage of Lead, Cadmium
Copper, Silver and Zinc 20
4 Relative Metabolism of Nitrobacter vs. Dosage of Organic
Toxicants 21
5 Summary of Relative Metabolism of Toxicants to Nitrobacter. 22
6 Relative Metabolism Rate of Nitrospmonas vs. Dosage of Lead,
Cadmium, Copper, Silver, "and "Zinc.."'. , , 23
7 Relative Metabolism of Nitrosomonas vs. Dosage of Organic
Toxicants. 24
8 Summary of Sensitivities of Toxicants to Nitrosomonas 25
9 Toxicity Ranges (Thatched Bars) of Lead to Various Bioassay
Organisms *(EC$0 for Algae) 27
10 Toxicity Ranges of Cadmium to Various Bioassay Organisms
*(EC50 for Algae) 29
11 Toxicity Ranges of Copper to Various Bioassay Organisms
*(EC50 for Algae) 30
12 Toxicity Ranges of Zinc to Various Bioassay Organisms
*(EC50 for Algae) 32
13 Toxicity Ranges of Silver to Various Bioassay Organisms
*(EC50 for Algae) 33
14 Toxicity Ranges of Heptachlor to Various Bioassay Organisms
*(EC5Q for Algae) 34
15 Toxicity Ranges of Endosulfan to Various Bioassay Organisms 36
16 Toxicity Ranges of Parathion to Various Bioassay Organisms 37
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Number Page
17 COD Efficiency in Response to Zn, TCP Toxicity (a) and Relative
Metabolism Rate of Nitrobacter at 50 nuj/t Zn and 10 mg/fc
TCP (b) 42
18 COD Efficiency in Response to a Toxic Industrial Wastewater
(a) c»nd Relative Metabolism Rate of Nitrobacter vs.
Wastewater Concentration (b) 44
19 Flow Diagram for Fiberboard Manufacturing Operation 45
20 Flow Diagram for Fiberboard Uastewater Treatment Process 46
21 Relative Metabolism Rate of Nitrobacter vs. Concentration of
Wastewater Tested 48
22 Evaluation of .Several Wastewater Treatment Methods Using the
Nitrobacter Bioassay 49
23 Assessment of Fungicide Toxicity Using the Nitrobacter
Bioassay 51
24 Aqueous Leachate Toxicity 57
25 Acetate Leachate Toxicity, pH = 5.0 58
26 EDTA Leachate Toxicity, pH = 5.2 59
27 Continuous-Flow Bioassay Apparatus 62
28 Effects of TCP on the Metabolism of Nitrobacter (triangles
represent restart of regular feed)..... 63
29 Comparison of Batch and Continuous-Flow Toxicity of TCP 65
30 Effects of Cd2+ on the Metabolism of NUrobacter (triangles
represent time at which regular feed restarted) 66
31 Relative Metabolism Rate of Nitrobacter vs. Cd2+ Concentra-
tion 67
32 Monitoring Toxicity in the Presence of an Interfering Anion
(*Based on Electrode Reading) (Triangles represent restart
of regular feed) 68
VI 1
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TABLES
Number Page
1 Level 1 - Bioassay Tests (After 6) 2
2 Various Mechanisms of Toxicity . 9
3 Inhibitory Concentration for Biological Nitrification (22) 11
4 Comparison of Sensitivity of Nitrobacter to Other Rioassay
Organisms. 38
5 Comparison of Sensitivity of Nitrosomonas to Other Bioassay
Organisms .... 39
6 Maximum Permitted Levels of Toxic Contaminants 53
7 Sludge Metals Analysis. 55
Will
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ACKNOWLEDGEMENTS
The contribution of our graduate students, Elizabeth Lundt, Richard
Taug, Motasimur Khan, Diane Johnson, Michel Cotillon, Patricio
Guerreroritiz, David Kessey, and Jeff Chaffee, is gratefully acknowledged.
Or. Norbert Jaworski, Director, U.S. EPA Laboratory, Duluth, Minnesota,
stimulated continued interest in this project end provided crucial review
while on sabbatical leave at Oregon State University.
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SECTION 1
INTRODUCTION
BACKGROUND
Bioassay tests can be used to detect biologically Irrtnful chemicals
whose effects can be manifested as cellular, genetic, behavioral, or meta-
bolic damage. Many different bioassay tests are presently used by health
officials to detect toxic chemicals (1)(2). For example, the Environmental
Protection Agency (EPA) has developed a three-phased approach to performing
assessment of the toxicity of aqueous solutions (including solid waste
leachates); this program is divided into Level 1, 2, and 3 tests
(3)(4)(5). Level 1 involves general bioassay and analysis procedures that
will identify the presence of toxicity in a given waste stream. Level 2
tests are used to identify and quantify specific compounds associated with
the toxicity found in the Level 1 test. Level 3 tests will provide more
detail concerning chronic health and ecological effects of the stream com-
ponents.
The focus in the Level 1 tests is a complementary series of bioassay
tests. These tests and their brief description are listed in Table 1 (6).
These tests j 'ovide no specific identification of the toxicant, but serve as
signals for a wide range of potentially toxic responses.
The primary difficulty with the application of these tests is the com-
plexity that results in high costs. The tests require highly trained per-
sonnel, modern laboratories, and long time periods. A listing of estimated
unit costs for these tests are included in Table 1 (7), Total costs could
exceed $5000 per sample for all the tests. These costs appear reasonable
for a small number of samples. However, in situations where wastew.-«er
compositions vary temporally or with process changes the cost of this as-
sessment program could be prohibitive.
An attractive alternative to the bioassay organisms listed in Table I
for acute toxicity is lower levpl organisms, especially bacteria. Such a
bacterial bioassay would be supplemental to the proposed Level i bioassay
tests and other health and toxicity tests and, hopefully, correlative. The
advantage of ising bacteria as compared to the other Level 1 bioassay organ-
ises for acute toxicity would be greater simplicity, shorter testing times,
and lower cost. Such a test cculd be accomplished within a few hours by
chemical technicians and would involve minimal laboratory facilities.
If a bacterial bioassay could identify the presence of a wide range of
toxic compounds, it could be widely used for routine and continuous monitor-
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TABLE 1. LEVEL 1 - BIOASSAY TESTS (AFTER 6).
Bloassay System
Test Oroanism
Purpose of Test
Unit Cost (7)
Microbial nutagenicity
Cytotoxicity
Freshwater and marine
static bioassay
Freshwater and marine
algal assay
Range finding acute
toxici ty
Terrestrial ecology
Nine different strains
of bacteria and one of
yeast.
Rabbit alveolar cells
and Chinese hamster
ovary cells.
Fathead minnow, daph-
nids, sheepshead min-
nows, and grass shrimp.
Freshwater and marine
algae.
Young adult rats.
Soil inicrcorganisms.
To determine if a chemical mutagen
(possibly a carcinogen) is present.
To measure metabolic impairment and
death in mammalian cells.
To detect potential toxicity to
organisms present in aquatic en-
vi ronments.
To detect potential growth inhi-
bition and stimulation effects on
primary producers.
Whole animal test to detect poten-
tial toxic effects to mammals.
To determine potential inhibition and
stimulation effects on so:l micro-
organisms. These data are useful if
the effluent is used for crop irriga-
tion.
$ 350
250
1,250
1,373
330
1,280
TOTAL COST
$5,348
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ing, and assessment of a wide number'of waste streams. It potentially could
be used to survey wastewaters discharged to fresh and marine environments,
to public and private treatment facilities, and to terrestrial environ-
ments. The test could potentially identify industrial wastes requiring
pretreatment and/or compliance with pretreatment standards (8). In addi-
tion, this test could identify hazardous solid wastes by detection of toxi-
city in leachates (9). Due to its low costs, the test could be used to
identify which chemical or physical fraction of a toxic wastewater contained
the toxicant (1). The need definitely exists for a simple, rapid, low cost
bioassay test and a bacterial bioassay potentially could fulfill this
role. Previous researchers (10) (11)(12)(13) have shown the applicability of
bacterial bioassays using nitrifiers, marine luminescent bacteria, and mixed
heterotrophs for a wide range of toxicants.
Initial Studies
Williamson (10) has reported initial development of a bacterial bio-
assay test using Nitrobacter. These organisms are strict aerobes which
obtain their energy from the oxidation of nitrite to nitrate. Carbonate is
their sole carbon source. These organisms were chosen over other heterotro-
phic bacterial species such as used by Bauer, Seidler, and Knittel (13)
since the cells can be grown in a simple aqueous medium of nitrite, oxygen,
carbonates, phosphorous, and basal salts and, therefore, the growth medium
does not contribute organics to the bioassay test. In addition, these auto-
trophic organisms do not metabolize organic toxicants. The growth medium
has low levels of inorganic salts as opposed to the high levels in the bac-
terial luminescence assay developed by Bulich (11) and Buiich, et al. (12)
and thus complexation of metal toxicants is avoided. Methods were developed
to freeze-dry and reconstitute Mitrobacter so that standard organisms could
be used by a wide number of laboratories. The results of this work were
promising enough to warrant further research to determine the applicability
of the autotrophic nitrifying bacteria as test organisms for a Level 1
bacterial bioassay.
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SECTION 2
CONCLUSIONS
Nitrifying bacteria can be used as Level 1 bioassay organisms to detect
acute toxicity of aqueous solutions. The bacterial bioassay is simple,
rapid, and lr-w cost and could be standardized using freeze-dried organ-
isms. It may be useful in cases where comparative toxic levels of a large
number of samples are required, or for field application with limited equip-
ment and personnel.
The bacteria bioassay with either Nitrobacter or Mltrosomonas exhibits
somewhat lower sensitivities for general metabolic toxicants (such as heavy
metals) compared to other common bioassay organisms, but exhibits dramat-
ically lower sensitivity for specific toxicants (such as pesticides).
The bacterial bioassay can be used to determine relative toxicities of
aqueous solutions to verify theoretical models of chemical composition
(e.g., toxicity of cadmium speciation), to optimize treatment of toxic
wastewaters, or to assess toxicity of solid waste leachates under various
leaching conditions. A continuous-flow bacterial bioassay can be used to
continuously monitor for toxic discharges from point sources.
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SECTION 3
RECOMMENDATIONS
The bacterial bioassay should be considered for adoption as a Level 1
bioassay test by the EPA. The EPA could develop facilities to provide
freeze-dried nitrifying organisms to the public for such testing.
The continuous-flow bacterial bioassay should be further developed to
detect discharges of toxic wastes from selected point sources. Such testing
could substantially reduce toxic loads to municipal treatment facilities and
the nation's waterways.
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SECTION 4
RESEARCH PLAN
From the initial studies with Nitrobacter, two important considerations
for further research were identified. First, it was questioned whether the
similar autotrophic organism Nitrosoinonas also could be used as a test or-
ganism in addition to Mitrobacter. Several studies had suggested that some
chemicals that had low toxicity to Nitrobacter had a higher sensitivity to
Nitrosomonas. Second, it was unknown what the relative sensitivity of these
two bacterial genera were to other bioassay organises. A comparable sensi-
tivity to other organisms would be required for adoption of a bacterial
bioassay. t
Rased on these two questions and other consideration for application,
the objectives of this study were developed as:
1. Develop methods to successfully freeze-dry Nitroso;nonas.
2. Determine the toxic concentrations of a wide range of known toxi-
cants to Nitrohacter and Nitrosomonas.
3. Compare the concentrations from Objective 2 to known toxic levels
for other health and ecological bioassays presently used.
4. Develop and describe the use of nitrifying bacteria to estimate
toxicity and the type of toxicant present in complex wastewaters
or leachates.
5. Develop a continuous, on-line monitoring system using nitrifying
bacteria to determine the presence of toxic materials in waste-
water streams and/or compliance with pretreatment standards.
APPROACH
To fulfill the objectives described, the project was divided into five
research tasks:
1. A literature review of the toxic levels to common bioassay organ-
isms of several organic and inorganic toxicants.
2. Determination of the toxic levels of these same organic and inor-
ganic toxicants to Nitrobacter and Nitrosomonas.
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3. Development of freeze-drying techniques for N1trosomonas.
4. determination of toxic response to Mitrobacter of complex waste-
waters of two types of toxicants: an industrial waste from a
fiberboard manufacturer and a simulated leachate from a solid
waste.
.5. Development of a continuous-flow technique using specific ion
electrodes to continuously monitor for a toxic response.
A description of the experimental procedures and results for each of
these phases are contained in the following sections.
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SECTION 5
TOXICITY TO NITRIFYING BACTERIA
Numerous studies have been conducted on the mechanisms of toxicity to
bacteria including the nitrifiers. In this section this information is
reviewed to allow assessment of what.types of toxic chemicals could probably
be detected with the proposed nitrifying bioassay.
TOXIC MECHANISMS TO BACTERIAL CELLS
Several different mechanisms exist by which
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TABLE 2. VARIOUS MECHANISMS OF TOXICITY
Inhibitor
Toxicity Mechanisms
Heavy Metals
(general)
(1) interference with cell wall synthesis
(2) decreased oxidative enzyme activity
(3) deactivation of DNA, RNA, and proteins
Specific Heavy Metals
Hg
Ag
Cu
Zn
(1) reacts with sulfhydryl (SH-) groups of
the cells decreases enzyme activity
(1) formation of metal complexes with
polynucleotides, ONA, RNA
(1) binds with DNA
(2) coagulation of bacterial cell colloid
(1) complexation with essential nutrients
Phenols
(1) disrupt cell membranes
(2) inhibition of oxidase enzymes
(3) protein precipitation inside the cells
Alcohol
(1) inhibition of respiration
(2) inhibition of phosphorylation
(3) damage to the cell membrane
NH^-based Compounds
(1) inhibition of bacterial oxidases
(2) inhibition of dohydrogenase system
(protein denaturation and enzyme
suppression)
Chlorine
(1) attacks sulfhydryl (-SH) groups of
enzymes involved in metabolic pathways
Acid (Hf)
or
Base (OH")
(1) displaces ion species such as Ma+,
Ca++ from absorption sites of the cells
(2) cell wall damage
Organophosphate
Pesticides
Carbanate Pesticides
Organochlorirle
Pesticides
(1) inhibition of acetylcholinesterase
(2) respiratory toxicity, high dermal
toxicity
(1) inhibition of acrtylcholinesterase
()) actions on nerve fibers; activities in
the ncsrves still unknown
(2) inhibits Na"1', K+, Mg1"*, ATP
Chlorinated C.yclodienes
(1) neurotoxicity mechanism unknown
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Enzyme inhibition can ho identified as competitive or noncompetitive.
Competitive inhibition requires the interaction of an inhibiting or toxic
agent, I, with the enzyme and the enzyme-substrate complex, to fonn an en-
zyme-inhibitor complex. The resulting Michaelis-Menten relationship is:
(2)
where K = "half velocity" coefficient with competitive inhibition.
Due to inhibition, K will always be larger than K . With nonconpetitive
inhibition, the inhibitor can react with the enzyme to form an inhibitor-
enzyme complex and with the enzyme-substrate complex to form an enzyme-
substrate-inhibitor complex. The appropriate Michaelis-Menten relationship
= ~kn S+K
where k = maximum rate of substrate utilization with noncompetitive
inhibition. Due to inhibition, kp will always be less
than k.
Typically a toxic agont will result in both competitive and noncompeti-
tive inhibition. In the presence of a toxicant, the nitrifying bioassay
should detect either competitive and/or noncompetitive inhibition since the
Ks values for both NO^-N and NH^-N are less than 1 mg/£ (16).
CHEMICALS CAUSING NITRIFICATION INHIBITION
Nitrifying bacteria can also be significantly affected by toxic sub-
stances in the aqueous environment. Numerous researchers have investigated
the effects of chemicals on biological nitrification (17-24) which is sum-
marized below.
A variety of materials, including heavy metals, chlorates, cyanides,
alkaloids, mercaptans, urethancs, quanidines, methyl amines, nitrouroa,
thiourea, phenolic compounds, cresols. halogonated solvents, chelating
agents, and certain fatty acids, can be toxic to nitrifying organisms
(22}. Inhibitory concentrations of various metals and organics are listed
in Table 3. Specific inhibitor may rosult in death of l.he nitrifying bac-
teria, in temporary reduction of metabolism with resumption of normal nitri-
fication rates after removal of the inhibitor, or in only a decrease in the
grorftr- rate. The effects of inhibitors vary between Nitrosomonas arid
Nitrobacter with Nitrosomonas more sensitive to most toxicants.
10
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TABLE 3. INHIBITORY CONCENTRATION FOR BIOLOGICAL NITRIFICATION (22)
Inhibitor Toxic Level?, (mg/f,)
Cobalt 59.0
Copper 20.0
Chromium 0.25
Mercury 2.0
Nickel 11.7
Silver 0.25
Zinc 3.0
Thiourea 0.076
Thioacetamide 0.53
Aniline 7.7
Guanidine Carbonate 16.5
Phenol 5.6
Sodium Cyanide 0.65
Ally Alcohol 19.5
Methyl Isothiocyanate 0.80
Mercaptobenzothiazole 3.0
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Lees and Simpson (24) studied the effects of chlorates, chlorite and
cyanate on nitrite oxidation by Nitrobacter. They proposed a specific bio-
chemical mechanism for the effects of Inhibitors on nitrite oxidation.
Cyanate inhibited nitrite oxidation, but the effect was reversible.
Chlorate caused a gradual decrease in the rate of nitrite oxidation and was
irreversible. Cytochrome 551, an important nitrite oxidizing enzyme, is
destroyed by chlorite, explaining its inhibitory action.
The effects of copper, nickel, zinc, a fungicide, and a wastewater on
both carbonaceous oxidation and nitrification in activated sludge units are
studied by Green, et al. (18). Significant decreases in nitrification were
noted with the metals, the fungicide, and the wastewater. Jackson and Brown
(20) presented a comprehensive review of metals and organics that are toxic
to aerobic treatment processes. Cadnium was nontoxic at concentrations
below 5 mg/£. They compared toxicity levels of heterotrophic bacteria to
autotrophic nitrifiers and demonstrated the sensitivity of nitrifying bac-
teria to toxic materials.
Tomlinson, et al. (23) developed a simple short-tenn test for inhibi-
tion of nitrification in activated sludge. This method was applied in
screening numerous organic compounds and several netals for inhibition of
amnonia oxidation, ftetals were (must less toxic to nitrification in acti-
vated sludge cultures than in pure~cuHures. Complexing of metals with
organic matter in sludges is a likely explanation. Downing, et al. (17)
used a similar procedure to screen numerous organics for toxicity to nitri-
fication. Hockenbury and Gra.iy (19) also studied the inhibition of nitrifi-
cation by selected organic compounds. Hitrosononas was much more sensitive
to the compounds tested than Nitrobacter. High ammonia concentrations were
found to be inhibitory to nitrite oxid'ation by Hitrohacter. The effects of
phenols and heterocyclic bases on nitrification were investigated by
Stafford (21). Phenol inhibited ammonia oxidation at 4 to 10 mg/i, but
levels as high as 100 mg/«. failed to affect nitrite oxidation. On the other
hand, pyridine completely inhibited Nitrobacter at 100 mg/?,, whereas
Hitrosomonas was only partially inhilnted. Holland and Green (25) also
reported on~'the inhibition of ammonia oxidation by metals and organics.
In general, these reported results for the toxic response of the nitri-
fiers suggest that these organisms could detect a wide range of both organic
and inorganic toxicants. Few compounds show a specific toxicity to nitri-
fiers that would not induce a toxic response in other organisms, so the
potential for "false-positive" results does not appear significant.
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SECTION 6
NITRIFYING BIOASSAY
The nitrifying bioassay, as proposed, uses cultures of either
Nitrobacter or Nitrosomonas - Kitrobacter as the test organisms. In this
section, the methods for the bioassay test will be described and results
from bioassays using eight chemicals given. The eight, chemicals were chosen
to provide a wide range of inorganic and organic toxicants and included
lead, cadmium, copper, zinc, silver, heptachlor, enciosulfan, and para-
thion. The relative sensitivitiy of the nitrifying organisms to these chem-
icals is compared to other test animals used in standard bioassay tests in
Section 7.
GIOCHEKISTRY OF NITRIFYING BACTERIA
Autotrophic nitrifying bacteria derive their energy from the aerobic
conversion of nitrogen in the form of ammonium to nitrite to nitrate as:
Ni trosomonas
Hj + 3/2 02 > N0~ + 2H4"
Ni trobacter
N0~ +• 1/2 02 .+ N0~ . (5)
Carbon dioxide (or carbonate in the aqueous system) is the sole carbon
source for the organisms. The- equations for energy yielding reactions can
be combined with equations for organism synthesis to form overall synthesis
oxidation relationships. Equations for synthesis-oxidation, using represen
tative iMC'.isiirements of yields to the generalized cell composition of
and oxygen consumptions are (26):
13.
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HI trosomonas
55 NH* + 76 0, f 109 HCOZ > CcH,NO, + 54 NO I + 57 H,0 + 104 H0CO~
4 t ,5 b / £ /: i f. 3
(6)
Nitrobacter
(7)
400 NO + NH + 4 H2C03 + HCO^ * 195 02 *- C5H?N02 + 3 H20 + 400
Experimental cell yield values ranged from 0.04 to 0.13 g volatile
suspended solids (VSS) per g ammonia-nitrogen oxidized for Nitrosomonas, and
from 0.02 to 0.07 g VSS per g of nitrite-nitrogen oxidized 'for Nitrobacter
(26). Thennodynamic calculations produce values of 0.29 and 0.084 for
Nitrosoinonas and Nitrobacter, respectively (26).
The substrjte utilization rate of the nitrifying bacteria have been
found to conform to the Honod relationship (16,?.6,27) as shown in Eq. (1).
Williamson and McCarty (16) measured half velocity coefficients for
riitrohacter and Hitrosomonas - Nitrohacter of 0.07 mg NO? - N/£ and 50 mg
NH^ - N/i, respectively. The maximum substrate utilization rate ranged from
1 to 4 mg fl/mg total suspended solids (TSS) - day.
Because the substrate concentration in a bioassay test will always be
greater than the K value, the substrate utilization rate (r) will be rela-
tively constant ana equal to -kX. The growth rates as shown by the cell
yield values are very low. Therefore, either organism is quite suitable for
batch bioassay tests because of the relatively linear uptake of substrate
over test periods of several hours since X will not change significantly
from metabolism of a few mg/i of nitrogen.
Numerous investigators have studied and reported on the parameters that
affect the inorganic netabulisin of nitrifying bacteria (24,28-32). Lees and
Simpson (24) did the classic study on the biochemistry of nitrite oxidation;
they found that the cytochromes were intimately involved in the oxidation of
nitrite to nitrate. Furthermore, the study suggested that inhibitors act by
destroying the required cytochrome. Painter (29) presented a comprehensive
literature review on inorganic nitrogen metabolism in microorganisms. He
reported that nitrifying bacteria were extremely susceptible to toxicants.
The effects of environmental conditions, such as pH, temperature, and dis-
solved oxygen, were also reported.
Nitrifying organisms prefer alkaline pll and were found to grow best at
pH values ranging from 6.3 to 9.4 (29). Hong-Chong and Loehr (32) found an
optimum pH for Nitr^bacter of 7.3.
-------
Temperature also affects the metabolism of the organisms, although
reported optimum ranges vary widely (29). In general, the optimum growth
temperature is within the range of 25 to 40°C.
Studies on the effect of dissolved oxygen (00) concentration on the
growth of nitrifiers have shown varying results (26,29). Loehr (33) showed
that 00 for nitrification is not limiting above 1.0 mg/£. In the bioassay,
the DO level was maintained above 2 mg/A to assure that oxygen was not lim-
iting.
PROCEDURE
Culturing of Nitrobacter
Enriched oiltures of Nitrohacter and Hitrosononas - Nitrohacter were
grown in a down-flow column similar to the procedure used by Williamson and
McCarty (16) (Figure 1). The 15-cm by 72-crr. columns were packed with poly-
pthylene beads to prevent washout of cells. Feed for the organisms consist-
ed of 30 rog/t nitrite-nitrogen (as NaN02) Or 10 mg/t ammonia-nitrogen (as
NM4SOd), 0.2 ng/Jt phosphorous (as K2MP04), and 20-30 mg/z oxygen in
Corvallis tap water, which supplied the micronutrients and bicarbonate for
growth. The pH of the feed solution was maintained around 7.0 The column
temperature ranged from 20 to 30°C.
Bacteria were removed from the taps on the column in a slurry of organ-
isms and beads which separated readily in a separatory funnel. The organ-
isms flocculated and were concentrated by settling in the bottom of the
funnel; the beads were returned to the column.
Freczn-Drying
Freeze-drying provides a means to preserve organisms indefinitely to
allow storage and shipnent without special handling such as refrigeration.
Successful freeze-drying of a microorganism depends first on the proper
selection of a cryogenic protective agent. Although much work has been done
on freeze-drying bacteria, no work has been reported for Nitrobacter or
Nitrosomonas. The most successful method found was to freeze the organisms
rapidly in a" dry ice-acetone bath in a solution of 10 percent sucrose and 1
percent gelatin. Slower methods apparently lysed the bacteria with large
ice crystals, and consequently no survival was obtained.
The bacteria were removed from the column and washed in glass-distilled
water and centrifugod to remove excess water. They then were suspended in
an approximately equal volume of the cryogenic protective agent of a solu-
tion of 10 percent sucrose and 1 percent gelatin. Two inn of this suspension
then was pipetted into each of several 20 mi glass ampules. The ampules
were then frozen in an acetone-dry ice bath, attached to a freeze-dryer
which removed the water from the frozen suspension, sealed, and stored.
-------
FEED
TANK
n
OXYGENATOR
t
BEADS-
-I FLOWMETER
VALVE TAps
WATER
BATH
EFFLUENT
i
Figure 1 . Apparatus for Culturing Nitrifying Bacteria.
16
-------
Rehydration and resuspension was done with a solution of approximately
5 rog NC^-H/C for Mitrobacter and 5 mg tlH^-N/ft for Nitrosononas. Each am-
pule was broken open ancffmfc of the solution was added dropwise. After the
cells were resuspended, they were washed twice to remove as much of the
protective agent as possible. The cells then were ready to be used for
toxicity testing.
Bioassay
Nitrohacter -- The bioassay procedure for fiitrobacter cultures was as
follows:
1) Fifty rod of the desired concentration of NaN02 (100 mg/« fJO^-N
for long-term test and 15 mg/fc for short-term test) was prepared
with the selected concentrations of toxicants and was placed in a
250 mfc Erlenmeyer flssk. Another 50 mi of the same concentration
of nitrite solution was placed into a similar flask as the con-
trol. The metal toxicants were added as lead nitrate, silver
nitrate, cadmium chloride, copper nitrate, and zinc chloride; the
toxic organics were all analytical grade (Polyscience Corp.). pH
was adjusted to 7.0.
2) An equal volume of Hitrobacter suspension was placed in each flask
and the flasks were shaken at a constant temperature for several
hours. The cells suspension was added to give an initial absorb-
ance reading 'in a 1-cm cuvette of from 1 to 2.
3) One mi of each solution was taken out periodically and measured
for nitrite colorimetrically (34).
4) Dry-weight of the biomass was determined at the end of the experi-
ment by filtration of the solution and by drying at 105°C for one
hour.
Nitrosomonas - Mitrobacter — The bioassay procedure used for
Nitrosoinoinas - NTtrobacteV was similar to that for Nitrohacter except that
the pH of all solutions were maintained at 7.5. In Step 1 the solutions
were prepared with (NH^SO^ stock solutions and in Step 4, NH^ uptake was
measured colorimetricaTly by the brucine method (34).
DATA ANALYSIS
NO^-N or NII^-N concentrations versus time for each flask were plotted
and the slope determined by a best squares fit; an example plot is shown in
Figure 2a for 2,4,5-trichlorophenoI (TCP). A comparison of the calculated
slope of each line with the slope obtained for the control yielded the rela-
tive metabolism rate (relative rate of either UO^-N or NH^-N oxidation) of
the test solution. These rates are plotted versus concentration of the
wastewater or toxicant; a reduced rate of metabolism confirms a toxic re-
sponse from the wastewater sample. This is shown for TCP in Figure 2b.
17
-------
~ o
CT
e
'&
UJ
co 3
UJ
c:
uj 4
o
30 60 90 120
TIME (min)
150
METABOLISM RATE
rnOBACTER (%)
Ul -i C
O 01 C
I! »
< o
_j
UJ
n
—
—
.
.
02^58
TCP CONCENTRATION (ma/I)
Figure 2 . (a) TCP Toxicity Measured by Reduced Substrate
Utilization and' (b) the Relative Metabolism
Rate of Mitrobacter.
18
-------
RESULTS
jfitrobacter Bioassay
The dose-response curves for Nitrobacter from the eight toxicants are
shown in Figures 3 and 4. No significant inFibition was obtained for para-
thion except at values above 20 ;ng/n which is the solubility limit. The
extrapolated 50 percent and 90 percent relative-metabolism levels are shown
in Figure 5; it was judged that these values would represent the range of
maximum sensitivities that could be expected in routine application of the
bacterial bioassay. These ranges varied for the netals from about 10 to c
1(T ug/fc for silver to 1 x 10 to 1 x 10° vg/t for zinc, and from 10 to 10-
for the organics.
All of the results in Figures 3 and 4 wore for a 4-hour exposure. An
experiment was conducted with cadmium to determine how much the sensitivity
could be increased by longer exposure. The results of increasing the expo-
sure to 24 hours are shown in Figure 3. The GO percent relative metabolism
rate was reduced from 4 x 10 moles/.l for 4 hours to 6 x 10 moles/* for
24 hours. Of course, the increased exposure time would result in greater
cost of test analysis.
jjjtrosonionas Bioassay
The dose-response curve for Mitrosononas for seven of the eight toxi-
cants are shown in Figures 6 and 7. Only a slight inhibition was obtained
for parathion. The 50 percent and 90 percent relative metabolism rates were
extrapolated to estimate the range of maximum sensitivity and are shown in
the bar graphs in Figure 8. The ranges were similar to the values obtained
for Nitrobacter for lead, cadmium, copper, and silver; however, Nitrosomonas
exhibited increased sensitivities for zinc, endosulfan, heptachlor, and
parathion.
19
-------
Lead
ICT'
10
Metal Concenfrafion (moles/I!
Figure 3 . Relative Metabolism of Nitrpbacter versus Dosage of
Lead, Cadmium, Copper, Silver and Zinc.
20
-------
r-o
100
80
o 60
o
a>
cu
a:
20
0
Endosulfan
Heptachlor
Parathion
0 1C 20 30 40 0 !0 20 3O O
Concentration (mg/l)
100 20O 300 400 500
Figure 4 . Relative Metabolism of Nitrobacter versus Dosage of Organic Toxicants.
-------
10'
icr
10'
en
50%
,90%
5O%
-9 O %
50%
90%
50%
/x^
-90%-
50%
50%
90%
PO
ro
a
IG1
50%
10'
LeoJ Cadmium Copper Silver Zinc Endosul/on HepiacMor Paralhion
Figure 5. Sunsnary of Relative Metabolism of Toxicants to Nitrobacter.
-------
Lead
10
-3
10"° !0'J !0"* 10
Metal Concentration (moles/I)
10'
-2
Figure 6 . Relative Metabolism Rate of Nitrpsomonas versus
Dosage of Lead, Cadmium, Copper, Silver, and Zinc.
23
-------
100
E
en
75
JD
o
0)
IS
a>
a>
rr
901-
80
Endosufan
Heptachlor
0
10 20 30 40 0 50
Concentration (mg/l)
100 150
200
*
Figure 7. Relative Metabolism of Nitrosomonas versus Dosaqe of Organic Toxicants
-------
ro
io-
10'
5O%
90%
50 %
5O%
,90%
'90%'
90%
50 %
90 %
UJ
I0
50%
90%:
•>0_
10"
No
toxic
response
Lead Cadmium Copper Silver Zinc Endosulfcn lleptachlor Parathion
Figure 8 . Summary of Sensitivities of Toxicants to Nitrosomonas.
-------
SECTION 7
ACUTE TOXICITY OF SELECTED CHEMICALS TO COMMON BIOASSAY ORGANISMS
In this section the acute toxicity of eight chemicals are summarized
and these values compared to the toxic concentrations measured for the ni-
trifying bacteria (Section (,;. The eight, chemicals were to provide a wide
range of inorganic and organic toxicants and included load, cadmium, copper,
zinc, silver, heptachlor, eridosul fan, and parathion. The majority of the
literature has heen compiled in a series of reports by the Office of Uater
Regulations and Standards, EPA, under the titles "Ambient Water Quality
Criteria for ..." Appropriate citations are made to these references which
are available for all eight chemicals except parathion.
LEAD
The toxicity of lead in water, like that of other heavy metals, is
affected by pH, hardness, organic materiali, and the presence of other
metals. Maximum and minimum range values of the l.C^Q/EC^Q levels for
Daphnia, two vertebrates, freshwater algae, and phytoplahkton are shown by
the thatched bars in Figure 9 (35).
Chapman (36) used three different levels of water hardness for acute
tests with Daphnia inagna. The results showed that daphnids were three times
more sensitive to lead in sofi. water than in hard water. The toxic levels
were approximately 1 mg/n. Several tests (37,38) showed that lead is acute-
ly toxic to rainbow trout in the range of 1,000 to 500,000 ug/£. The wide
range is attributed to complexation of the load by inorganic anions in hard
waters. Chronic tests showed that most of the trout developed spinal de-
formities in hard water with concentrations of measured lead of 850 yg/£ and
above, and in soft water with lead concentrations as low as 30 ijg/c, (37).
Similar to the trout data, Tarzuell and tfcndorson (39) and Pickering and
Henderson (40) showed toxicity of lead with LCcg values from 2.4 to 542 mg/?.
to fathead minnows depends upon the hardness or the water.
Monahan ('".) and Malanchuk and Gruendling (42) reported acute effects
to freshwater algae of from 500 to 26,000 yg/e. dossier (43) showed phyto-
plankton to be equally sensitive with inhibition and death is in the 2,000
to 60,000 ug/a range.
26
-------
ro
10*
10'
ic
o
in
O
_J
10' -
10'
,0
Daphnia Rainbow Fathead Algae
trout minnow
Figure 9. Toxicity Ranges of Lead to Various Bioassay Organisms (*ECSQ for Algae)
-------
CADMIUM
In general, the range of acute toxicity for cadmium to the hioassay
organisms was about 10 to 100,000 ug/?. (44) (Figure 10). A reduction in the
toxicity associated with increased hardness became evident for both the fish
and invertebrate species.
Rainbow trout exhibited acute ranges of 96-hr LCj-n's in both static and
flow-through tests of 1 to 7 ug/£ (38,46-48). A 200-hr LC50 value of 0.7
pg/Jl for rainbow trout was determined by Chapman (45), not significantly
lower than the 96-hr LC^Q values.
After trout, Daphnia magria was found to be the next most sensitive
species tested with LC5Q values" from iO to 140 ug/£ (49,50). Fathead min-
nows were relatively insensitive with LCcn's from 2,000 to 74,000 ug/£
(51,52).
Reduction in growth rate was the major toxic effect observed with a-
quatic algae. EC™ values for freshwater algae ranged from 5 to 250 ug/t
(44). Two species of saltwater phytoplankton had 96-hr EC5Q values of 160
and 175 ug/l based on growth inhibition (53). the values are considerably
above the chronic values for other saltwater animal species tested (44).
COPPER
Copper toxicity is strongly dependent upon organic and inorganic com-
plexation and, as such, varies widely with chemical characteristics of
aqueous solutions (54). The range of copper toxicity for various bioassay
organisms ranged from 5 to 8,000 yg/£ as shown in Figure 11 (55). Low alka-
linity waters consistently showed higher toxicity.
Cairns, et al. (56) indicated that daphnids are more resistance to
copper at low temperatures in acute tests. The LC^Q for Daphnia magna
ranged from 10 to 200 ug/'- (54). Rainbow trout c.re about equally sensitive
as Daphnia with a 'range of LC^0's of 20 to 500 iig/i depending on the hard-
ness of the water (45,57). Chckoumakos, et al. (57) have reported adult
rainbow trout are approximately 2.5 to 3.0 times nore resistance to copper
than juveniles. Brown, et al. (58) showed a quantitative decrease in the
acute toxicity of copper to trout with increases in naturally occurring
organic chelating agonts. The fathead minnow was about equally sensitive to
copper as rainbow trout as shown in Figure 10 (55).
Copper is known for its algicidaJ and herbicidal characteristics.
Concentrations that inhibit growth range fron 1 to 8,000 ixj/t (55). Salt-
water algae appear to be as sensitive as freshv.-ater algae.
-------
PO
icr
10
O 2
JMO
10'
10
0
Daphnia Rainbow Fathead Algae*
trout minnow
Figure 10. Toxicity Ranges of Cadmium to Various Bioassay Organisms (*EC5Q for Algae)
-------
UJ
o
IOV
o
to
O
10'
10
0
Daphnia Rainbow Fathead Algae*
trout minnow
Figure 11. Toxicity Ranges of Copper to Various Bioassay Organisms (*EC -for Algae)
-------
ZINC
Zinc is an essential trace element for all organisms which, at higher
concentrations, can be toxic. The range of acute values for freshwater
organisms is from 30 to 38,000 pg/fc (Figure 12) and has been found to be
similar for fish and invertebrates (59). The toxicity of zinc compounds to
aquatic organisms is modified by several environmental factors, in particu-
lar, water hardness, dissolved oxygen and temperature. The EPA criterion
for zinc in domestic water supplies is less than 5,000 ug/t (60).
The LC^Q value for Dapnnia ranged from 100 to 655 ug/2. depending on the
water hardness (36,61,6277Rainbow trout and fathead minnows showed much
wider ranges of 90 to 4,700 gg/t and 600 to 35,500 yg/fc, respectively
(59). Algae exhibited growth inhibition effects from 30 to 5,100 yg/S, (59).
SILVER
Acute and chronic data for the toxicity of silver to aquatic organisms
shows o wide variation. For the four bioassay groups in Figure 13, the
acute values for silver range from 0.25 ug/fc for the Daphnia magna to 1,000
vg/l for algae (63). The data base showed that the acute toxicity of silver
apparently decreases as hardness increases. Paphnia exhibited an LCgn sen-
sitivity range of 0.25 to 49 ug/2. with most values TTeing in the few micro-
gram per liter range (64). In comparison, rainbow trout data ranged from 5
to 280 ug/£ and fathead minnows from 4 to 230 jjg/ft (64). Various growth
inhibitions to algae occurred in the range of 30 to 1,000 ug/fc with complete
inhibition of growth at a few hundred micrograms per liter (63).
HEPTACHLOR
Heptachlor has been a widely used pesticide for general insect control
throughout the United States. It is a chlorinated hydrocarbon which has
been shown to be toxic to aquatic life, to accumulate in plant and animal
tissues and to remain in aquatic ecosystems (65). In general, LC5Q values
range from about 10 to 1,000 ug/fc as shown in Figure 14. The data on acute
toxicity for static testing in Figure 13 is based on initial dosed levels,
not exposure concentrations. All values in Figure 14 are expressed as tech-
nical grade heptachlor.
The LCrg value for Daphnia was reported as 78 pg/£ (66). Rainbow trout
exhibited LC^g values of 10 to 27 pg/H (67,68) and fathead minnows, 78 to
130 pq/2 (69). EC^Q values for freshwater algae wore determined as 27 to 39
yg/«, .d for saltwater species, 93 to 2,260 ;jg/£ (65).
In general, toxicity increased with time of exposure. The effect of
increase'^ exposure time on LCj-Q values showed greater increases for the
invertebrates than for the vertebrates.
31
-------
CJ
ro
icr
10'
10s
0
to
o
_J
10
0
Daphnia Rainbow Fathead Algae
trouf minnow
Figure 12. Toxicity Ranges of Zinc to Various Bioassay Organisms ( *l:CSQ for Algae)
-------
CO
00
icr
10'
= icr
o I0
in
•10'
10
0
Daphnia Rainbow Fathead Aigae*
trout minnow
Figure 13. Toxicity Ranges of Silver to Various Bioassuy Organisms (*EC-_ for Algae)
-------
OJ
en
O
O
!04
10'
10'
10
0
Seawaier
Freshwater
Daphnia Rainbow Fathead Algae
trout minnow
Figure 14. Toxicity Ranges of Heptachlor to Various Bioassay Organisms (*EC50 for Algae)
-------
ENDOSULFAN
Endosulfan is an insecticide developed in the raid-fifties for insect
control on vegetables, fruits and tobacco. Currently it is on the EPA re-
stricted list limiting its current use (70). Either technical grade endo-
sulfan or mixtures containing such were used for most of the toxicity test-
ing.
The vertebrate (fish) species tested were shown to be more sensitive
than the invertebrates tested (Figure 15) with LC^n values for trout and
fathead minnows in the low vq/t range and Daphnia in the 100's of ug/fc. range
(70). Little data were available for either freshwater or saltwater algae.
Pickering and Henderson (71) studied the effect of water hardness on
toxicity of endosulfan and observed no significant effect. It was generally
shown that the toxicity of endosulfan increased with increasing temperature
(70). Significant differences were noted between nominal and measured expo-
sure concentrations (72),
PARATHION
The toxicity of parathion, an organophosphorus pesticide, ranged fron
less than one ug/fc for sensitive species like Paphnia to about sever.}! ug/fc
for more resistance species like fathead mi nndws (Figure 16). Space (73)
found the three-week LC-g for Daphnia to be O.H ug/H and the 96-hr i.CrQ in
flow-through bioassays to be 0.62 ug/£. LC^Q values were about 1.8 iig/fc for
trout and from 1.6 vg/i for fathead minnows. No data were available for
algae.
COMPARISON OF NITRIFYING RIOASSAY TO OTHER TfST ORGANISMS
For the Niitrohact e r bioassays the ranges shown in Figure 5 can be com-
pared directly with the LC^Q values shown in Figures 9 through 16. Such
comparisons are shown in Table 4. These comparisons show that in general
Ni trohacter exhibits comparable sensitivity to the metal toxicants, but
TTttle sensitivity to the toxic organics. This would be expected since the
toxic organics operate through specific enzyme mechanisms that are absent in
bacteria.
For the Hitroiouonas bioassays, the ranges shown in Figure 3 can be
conpared directly with the LC^Q values shown in Figures 9 through 16. Such
comparisons are shown in Table 5. These comparisons show that in general
Hitrosomonas exhibited a range from low to high sensitivity for the metal
Toxicants, but loss sensitivity to the toxic organics. The reduced sensiti-
vity for the organics compared to the other bioassay organism would be ex-
pected as statod above. In general, Mitrosomonas showed less sensitivity
than fli trobactor.
35
-------
CO
iov
10'
O |Q2
O
_J
io'
10
.0
Daphnia Rainbov/ Fathead
trout minnow
Figure JS. Toxicity Ranges of Endosulfan to Various Bioassay Organisms.
-------
CO
10'
ICT
o
UD
O
10'
10'
p
10
-I
Daphnia Rainbow Fathead
trout minnow
Figure 16. Toxicity Ranges of Parathion to Various Bioassay Organisms.
-------
TABLE 4. COMPARISON OF SENSITIVITY OF NITKORACTF.R TO OTHER BIOASSAY
ORGANISMS*
Bioassay Organisn
FaTRead"
Toxicant Daphnia Trout Minnow Algae
Lead 0 4- + 0
Cadmium 0
Copper 0
Silver + 0 + +
Zinc -
Endosulfan x
Heptachlor -
Pa rath ion ....
t, more sensitivity; 0, conparable sensitivity; -, less sensitivity;
x, no data available
38
-------
TABLE 5. COMPARISON OF SENSITIVITY OF NITROSOHQHAS TO OTHER BIOASSAY
ORGANISMS*
Bioassay Organism
Toxicant Paphnia
Lead
Cadnium
Copper
Silver +
Zinc
Endosulfan
Heptachlor x
Parathion
Fathead
Trout Minnow Algae
+ + 0
0
_
+ + +
0 0 0
x
_
• «* Y
* +, more sensitivity; 0, comparable sensitivity; -, less sensitivity;
x, no data available
39
-------
SECTION 8
BACTERIAL 8IOASSAY APPLICATION TO INDUSTRIAL HASTEHATER TREATMENT
Toxic industrial wastes threaten both biological waste treatment sys-
tems and the environment of their ultimate disposal. Both of these problems
can be addressed through the use of Levnl 1 bioassays. In this section the
use of Nitrohacter bioassay is described for evaluation of industrial waste
toxicity to biological wastewater treatment, specifically activated sludge.
METHODS OF MEASURING TOXICITY TO ACTIVATED SLUDGE
Many different bioassay methods have been used to measure a toxic re-
sponse in activated sludgo. In this section several of those methods will
be described and their advantages and disadvantages summarized.
The most common method is to use continuously fed reactors. Duplicate
reactors are fed with and without the toxicant and the organic removal rate
is monitored. This technique is expensive to operate and extremely time
consuming. Equipment requirements include reactors and constant-feed pumps,
and a large source of industrial waste is necessary. This method does ap-
proximate the conditions in activated sludge tanks, although the hydraulic
and solids retention times of the activated sludoe treatment are difficult
to maintain. Short-term studies often never approach assumed steady-states
and long-tern studies do not provide information rapidly enough.
Unfortunately, continuously fed reactors are notoriously difficult to
run. Pumping small amounts of influent at a controlled rate is difficult.
Several weeks may be necessary for stabilization of growth to occur. Often
the effects of toxicity are masked by the normal variability in COD or BOD
removal. For those reasons, continuously fed reactors cannot be used as a
standard analysis procedure. Examples of toxicity tests performed by this
method are reported by Moulton and Shumate (74), Ayers, Shumate, and Hanna
(75), and Barth, et al. (76).
Another common toxicity test is the BOD bottle technique. There are
many variations, but this technique essentially consists of measuring the
BOD of a known organic with or without the toxic waste. The method is ex-
tremely simple and can bo accomplished in standard treatment plant labora-
tories. However, the dilution of the waste for the tost may reduce its
concentration below its threshold limit value. In addition, the small range
of only 2 to 7 mg ROD/fc greatly limits its accuracy. Mowat (77) used this
method to determine the toxicity of various metals to mixed heterotrophic
growth from sewage.
40
-------
Most of the difficulties of the BOO bottle technique can be alleviated
by using a hatch-fed technique. This technique involves measurement of the
substrate utilization rate and the cell mass. The most counon method of
determining the substrate utilization rate is nanornetrically as described by
Hnrtroann and taubenberger (15), although the decrease in concentration of
the organics can also be used as described by Bunch and Chambers (78). The
cell mass can be measured as total suspended solids, volatile suspended
solids (79), organic nitrogen (15), or ATP (80).
Batch-fed techniquee typically require more advanced instrumentation
than available at industrial treatment plants. Respironeters are expensive-
and difficult to operate even for highly trained personnel. Specific meas-
ures of organics will require sophisticated instruments or difficult chemi-
cal extractions. The length of the tests using batch-fed techniques ace
usually long compared to th° generation times of the heterotrophs, so the
number and types of organisms can vary significantly between the reactors
with toxicants and the control.
None of the three methods (continuously fed, BOD bottle, batch-fed) are
directly applicable for determining the toxicity of industrial wastes 'by
plant personnel. These tests primarily require too much effort or advanced
instrumentation. The assessment of toxicity by the use of the Nitrobacter
bioassay is shovm helovi.
LABORATORY STUDIES WITH KNOWN TOXICANTS
To determine the ability of the Nitrobacter bioassay to predict toxici-
ty to biological waste treatment, laboratory studies wore undertaken with
two known toxicants. Three 4-liter activated sludge units with internal
clarifiers were used as conplete-mixed reactors. The units were fed primary
clarifier effluent fron the Corvallis Sewage Treatment Plant to provide a
hydraulic detention time of 18 hours; the solids retention time was not
controlled, but c.e'ls were wastprf daily to provide a food to microorganism
ratio of approximately 0.2 mg BOP/nig TSS-day. Treatment efficiencies were
measured as COD removal across the reactors.
After 4 days of operation, 50 nig/?. nf zinc, (*s ZnSO^) was fed with the
sewage to one of the reactors, and 10 mg/?. of tried! orophenol (TCP) was fed
with the sewage to the second reactor. The third reactor was operated as a
control .
The results are shown in Figure 17a. The reactor dosed with zinc exhi
bited a dramatic change of floe characteristics within 24 hours; the floe
becane nore disperse and lighter in color than the control. The floe dosed
with TCP became darker in color than the control. Roth units showed de-
croasod COD removal as compared to the control which clearly indicated a
toxic response.
On the twelfth day of the tests the Ni trohactcr bioassay was conducted
on the two toxic feeds and the control feed"! The relative metabolism rates
41
-------
100 r
(a)
. '/
*D ^X /.*'
• i*f P* v. * •
'..TCP ^. /;
• ^ * •
'**n .j.
LJ Q
Dose
T 1 ! 1 ! 1 ! i I
4 6 8 '10 12
TIME (days)
(b)
I m
UJ
cc.
COIVTROL Zn TCP
(50mg/i) (IOmg/1)
Figure !7 . ron Efficiency in Response to Zn, TCP Toxicity
(3) and Relative Metabolism Rate of Ni trobacter
at 50 mg/f. Zn and 10 mg/J TCP (b).
-------
of Nitrobacter for the 50 ng/Jt zinc and 10 mg/P, TCP with respect to the
control were 40 percent and 16 percent, respectively (Figure 17b).
These results show that the Nitrobacter bioassay test had adequate
sensitivity to detect these two toxic compounds at levels that produced
measurable toxicity to activated sludge.
LABORATORY STUDIES WITH A METAL PROCESSING WASTEUATER
Another series of tests using bench-scale activated sludge reactors
were conducted as described above except a toxic industrial waste was mixed
with the feed at 0 percent (control), 10 percent and 20 percent. The waste-
water sampled was from an industry that processes various metals and was
known to contain a wide range of organics, chlorinated irganics, and metals.
The concentrations of 10 percent and 20 percent of the industrial waste
caused no visible change in the activated sludge floe. However, the COD
efficiencies were significantly lower than the control without the waste-
water addition for the ?.0 percent industrial waste feed 'Figure 18a).
Seven days after dosing the activated sludge units with the industrial
waste, the _N_1trobacter bioassay was run one each feed. The relative rate of
Nitrobacter^ metabo 1ism for the feeds with 10 percent and 20 percent indus-
trial waste" were 50 percent and 4 percent, respectively.
These results showed that the Nitrobacter bioassay could detect un-
specified toxicity in industrial wastewaters. As such, the Nitrobacter test
could be specifically applied as a Level 1 bioassay in the identification of
toxic wastestreams and appropriate treatment and control technologies.
FIELD STUDIES WITH A FIBERBOARO MILL WASTEMATER
The Nitrobacter bioassay was applied to an industrial wastev/ater treat-
ment problem at a fiberboard nanufacturing site. The manufacturing process
at this mill is known as the "wet process" (Figure 19). Wood chips and
scrap fron a neighboring lumber mill are ground into snail particles. The
solids then are treated with high pressure steam that removes the water
soluble material from the wood leaving a fibrous skeleton. The fibers then
are treated with a preservative and bonding resin, and nixed with reuse
water to fom a slurry (0.5 percent solids by weight). The slurry is poured
onto a wire screen that carries, the fibrous mat to a press whe^e the board
is finally formed.
Excess water ranoved during the manufacturing process is discharged to
an on-site treatment facility consisting of settling ponds, a completely
nixed activated sludge basin, and a small clarifier (Figure 20). The waste
solids from the clarifier are land applied and the clarifier effluent is
recycled to the plant as reuse water.
43
-------
lOO
90
< 80
o
2
UJ
Q
O
O
70
60
UJ
oo
CQ
UJ
LU
100
0
(a)
Controi
20%
Dose
i 1 i ! i 1 i
i
G 8 10 12
TIME (days)
14
(b)
0 10 20
WASTEWATER CONCENTRATION (%)
Fiqure 18 . COD Efficiency in Response to a Toxic Industrial
V.'astewater (a) and Relative Iletabolism Rate of
Nitrobacter versus Vlastewater Concentration (b).
44
-------
CHEMICALS
y^
BOARD FORMATION
FINISHED
BOARD
[W
IBO
WHITEWATER
BOX
WASTEWATER
TREATMENT
PRESS
SECTION
Figure 19. Flow Diagram for Fiberboard Manufacturing Operation.
-------
r
CTl
FIBERFJOARD
[ MILL
REUSE WATER STORAGE
PONDS
ANAEROBIC
SETTLING
ACTIVATED
SLUDGE DASIN
LAND
CLARiFIER AP?LICATION
Figure 20 . Flov/ Diagram for Fiberboard Hastewater Treatment Process.
-------
The mill wastewater is punpeu to a primary settling pond at an average
flow rate of 3 x 10 «/d. The wastewater temperature is 43°C with a pH of
4.5. The settling pond has a volume of 1.5 x 10 i and a hydraulic deten-
tion time of about 5 days.
The aeration basin has a 1.5 x 10" l capacity and a 5 day hydraulic
detention time. Aeration is supplied by five surface aerators that maintain
4 to 6 mg/l of dissolved oxygen. Ammonium nitrate and diammoniun phosphate
are added as nutrients. Tbe pH is raised to 6.8 by biological activity.
The clarifier holds 4 x l(r a with a hydraulic dentention time of about 1.5
hours.
The main component of the wastewater are the wood sugars that results
from the steaming process. Some of the. wood used for mill feed has been
treated by the supplier with a fungicide known to contain pentacnloro-
phenol. Phenyl formaldehyde is added to the cleaned wood chips as a preser-
vative; alumina, a "waxy emulsion" and "defoamer" are edded to aid in the
board formation. No slimicides are added during the process, although slime
appears to have been a problem in the past (81).
In 1971, this company developed a completely closed w-istewalor system
to minimize any possible stream pollution. Treatment efficiency of the
activated sludge unit has dropped considerably.from 1975 to 1978. BOO re-
moval has been reduced to nearly 50 percent, while the suspended solids of
the system had increased to over 6000 mg/Ji. The Nitrobacter bioassay was
performed on the activated slu.ige influent and effluent as a Level 1 phase
of investigation. A 10 percent solution of the activated sludge influent
showed a 10 percent relative metabolism rate of the Nitrobacter which indi-
cated significant toxicHy (Figure 21). A 10 percent solution of the acti-
vated sludge effluent showed a 40 percent relative metabolism rate of
Nitrobacter. This indicated that the toxicity was not being significantly
removed by the biological waste treatment.
In an attempt to pinpoint the source of the toxicity, additional sam-
ples were taken at various point sources along the production line. No
particular toxic discharge sites were isolated. A general increase of toxi-
city in the wastewater seened to occur during the process with the toxicant
being recycled through the wastewater treatment system.
Several physiochemcal wastewater treatment methods were applied to the
mill effluent in an effort to identify the optimal method to remove the
toxicity. These include filtration with jlass fiber filters, dissolved air
flotation at 40 psig pressurization, followed by 10 minutes of mixing and 20
minutes of flotation and powdered activated carbon addition at 50 ng/£. A
20 percent solution of the mill effluent was found to exhibit a 63 percent
relative inetaholisn fate of Nitrohactir. Treatment of this 20 percent solu-
tion with either filtration or dissolved air flotation showod only a small
increase in the relative metabolism rate of Mi trohacter, while powdered
activated carbon removed almost all of the observed toxicity (Figure 22).
47
-------
100
OO
0
Activated Sludge.
Effluent
Activated Sludge
Influent
CONCENTRATION OF WASTEWATER TESTED
(% by volume)
Figure 21 . Relative Metabolism Rate of NUrobacter versus Concentration
of Wastewater Tested. :
-------
LL)
"c£
100
90
ft:
<0
_J
LLJ
70
60
50
o
ft:
h-
z:
o
o
UJ
^1
21
o
UJ -
u.
O _J
ljj<
0
o x
<0
Figure 22 . Evaluation of Several Wastewater Treatment Methods Using the
Nitrobacter Bioassay.
-------
Level 1 analysis using the HUrobacter bioassay identified the waste-
water from this plant as being toxic to the activated sludge operation. To
date, this problem has caused no noticeable deterioration in the quality of
the finished fiberboard; however, the continued failure of the treatment
system has caused company officials some concern with the assurance of con-
tinued quality. A Level 2 analysis was conducted to identify possible
causes for the toxicity observed. Pentachlorophenol, used as a preservative
by the wood chip supplier, was identified in the plant effluent at levels of
2 to 3 ppb. Pentachlorophenol is known to be toxic to bacteria by uncou-
pling oxidative phosphorylation and also to denaturing proteins. The cur-
rent literature values for acute toxicity levels for pentachlorophenol are
listed as 25.0 ppb for invertebrates, 14.0 ppb for freshwater fish and 7.5
ppb for algae (82). Although it is not believed that pcntachlorophenol
accounts for all of the observed toxicity, it is considered to contribute to
the toxicity problem. A Hjtrqbacter bioassay verified fungicide toxicity
(Figure 23). Other possible wastewater constituents known to be present
that might exhibit a toxic effect are 2,4,6-trichlorophenol, phenyl formal-
dehyde, and resin acids.
50
-------
100
5 10
FUNGICIDE CONCENTRATION (mg/l)
Figure 23. Assessment of Fungicide Toxicity using the Hitrobacter
Bioassay.
-------
SECTION 9
BACTERIAL 8IOASSAY APPLICATION TO SOLID WASTE 1.EACHATE
In the Resource Conservation and Recovery Act of 1976, Congress direct-
ed the Environmental Protection Agency (EPA) to develop a regulatory program
to manage and control the country's hazardous solid wastes from generation
to final disposal. The EPA has formulated rules to implement this program
(105), including identification and listing of hazardous v/astes, and con-
taining criteria for determining whether a waste is hazardous. Consequent-
ly, the EPA has proposed the Extraction Procedure (EP) for toxicity evalua-
tion; this procedure is a specified 24-hour extraction of the solid waste
using acetic acid. No bioassay evaluation is required.
The fjoal of the EP is to model the fate of a poorly managed hazardous
waste; that is, a toxic waste that is disposed of in a municipal landfill or
open dunp located over an aquifer used for drinking water. The leachates
generated by the RP are analyzed for the toxic components listed in Table 6
with their corresponding maximum permissible concentrations; these limits
are 100 times the EPA national Interim Primary Drinking Water Standards
(83).
Although the F.PA rules do not require hioassay techniques in the evalu-
ation of a waste for toxicity, their inclusion in the rules may occur in the
future. Toward this end the EPA has conducted bioassay tests to evaluate
the use of EP loachate in determining acute and chronic aquatic toxicity,
phytotoxicity, and mutag'jnccity (830. Bioassay tests which have already
been used in leachate toxicity tests include acute toxicity to Daphnia
magna; radicle elongation studies using radish, soryhun, wheat, and soybean
plants; and bacterial gone mutation, fungal microorganism mutation, and
bacterial DflA repair studies (84). Leachates from a number of wastes have
been studied, including municipal sewage sludges (85,86), textile wastes
(37}, fly ash (88,K9) ,~jm si ti£ coal gasification byproducts (90), flue gas
desulfurization scrubber sTudges (91), plastics wastes (92), radioactive
wastes (93), zinc-carbon battery wastes (94), zinc sneltimj wastes (95),
coal mining spoils (96), municipal landfill washes (97,98,99,100), and steel
and alum'nun manufacturing wastes (101)^ The EPA has studied leachates from
such wastes as soybean process cake, platers waste, retorted oil shale, dye
waste, municipal swage sludge, power generation ashes and sludges, and
gasification wastes (10?).
Many of the parameters in extraction procedures have been set at values
considered to be typical of conditions found within municipal landfills.
Snail differences in such variables as complexing liyand concenrations and
pH may cause a wide variation in the solubilities of metals. Theis, pt al.
-------
TABLE 6. MAXIMUM PERMITTED LEVELS OF TOXIC CONTAMINANTS
Contaminant
As
Ba
Cd
Cr
Pb
Hg
Se
Ag
l-ndrin
Lindane
Methyoxychlor
Toxaphene
2,4-D
2,4,5-TP (Silvex)
Concentration
(mg/0
5.0
100.0
1.0
5.0
5.0
0.20
1.0
5.0
0.02
0.40
10.0
0.50
10.0
1.0-
53
-------
(88) found that pH was important in determining the solubilities of metals
in fly ash, while Ham, et al. (103) found that increasing leachate acidity
released higher quantities of inorganic ions from a number of industrial
wastes. Other investigators have determined that pM, conplexing ligand
concentration, and adsorbing surface availability are important in determin-
ing the total solubility, speciation, and distribution of metals in solution
(104-110).
The EP specifies acidification of a sludge suspension to pH 5.0 with
acetic acid. The final acetate concentration may not exceed 0.1 M which may
limit the final pll value to higher than this. Leachates from actual land-
fills have exceeded these limits. Chian and DeWalle (100) found a pH range
of 3.7 to 8.5 and a range of volatile acids concentrations from 0.03 M to
0.3 M in municipal landfill leachates, and Burrows and Rowe (97) found up to
0.8 M volatile acids in municipal landfill leachates. Leachates from land-
fills are also known to contain such substances as humic or fulvic acids and
amino acids which can act as powerful complexing agents and increase the
total solubilities of metals in the leachates (99). The EP does not include
complexing ligands other than acetate, which forms only relatively weak
complexes with most metals.
In this section the use of the Nitrobacter bioassay is described for
evaluation of the leachate from the EPA extraction procedure for a solid
waste. As part of additional studies, the chemical composition of the ex-
tractant was varied to determine its effects of the chemical characteristics
of the leachate; as such, bioassays wore conducted on leachates extracted
with distilled water, EDTA, and acetate. A basic oxygon furnace scrubber
sludge was chosen as the solid waste.
BOF SCRUBBER SLUDGE
Basic oxygen furnace (BOF) scrubber sludges are composed mainly of iron
oxides, but also contain large amounts of calcium and magnesium from lime
used for pH control. BOF scrubber sludges often contain significant quanti-
ties of heavy metals, including zinc, load, chromium, cadmium, and manga-
nese. These metals originate from the me of scrap notal in the ROF
charge. A preliminary analysis of the sludge usod in this research revealed
elevated levels of zinc, lead, and manganese (sec Table 7). The total
solids concentration of the sludge was 72.4 percent and the alkalinity was
114 mg/g dry. The pH was 12.5.
EXTRACTION
The throe leaching solutions used wore distilled water (aqueous solu-
tion), 0.15 M riceUte solution, and 0.0375 M (O.Hi N) CDTA solution. Dis-
tilled water w.is used to study the effects of pH alone, acetate was chosen
to study the effects of a mild complexing ligand commonly found in municipal
landfill leachates, ard EDTA was chosen to study the effect of a very strong
complexing ligand.
54
-------
TABLE 7. SLUDGE METALS ANALYSIS
Metal
Ca
MG.
Sr
R
Fe
Al
fin
Zn
Cu
Cr
Ni
Cd
Pb
As
Mo
Se
Zr
Sb
Co
V
Concentration
in Sludge, pg/g dry wt.
21685
5428
15
285
98108
3094
2942
29420
77
232
26
51
3108
26
552
140
14
83
28
28
Ma x i ir.um
Solubil ity*
mg/t
1570
393
1.1
20.6
7100
224
213
2130
5.6
16.8
1.9
3.7
225
1.9
40
10
1
6
2
2
-
*Btised on 10 in?, leachate per gram of sludge, the dilution factor in leaching
tests.
55
-------
Sludge was extracted by adding 10 mf. of leaching solution for each gran
of sludge. The solutions were shaken in a wrist-shaker for ?4 hours.
BIOASSAYS
The hioassays were conducted as described in Section 5. Leachates were
added at levels of 0 to 90 percent of the bioassay solution. The pH was
varied by the addition of HC1 from 5 to 8 but held constant throughout each
test.
RESULTS
The relative activities obtained in each toxicity test were plotted for
each leachate as shown in Figures 24, 25, and 26. Controls for pH near 5
and 6 are also included; the controls at pH 7 and 8 foe acetate and EOTA
were checked and shown to result in negligible reduction of the metabolism
rate. At equal dilution and pH, the toxicities of the three types of leach-
ates were greatest for acetate, followed by EDTA and then distilled water.
However, if the influence of the complexing agent is accounted by substrat-
ing the relative activity of the controls then the greatest toxicity is
associated with EDTA, followed by distilled watsr and then acetate. This is
probably due to the strong complexing of the released metals by acetate.
For each leaching solution there is a trend of increasing toxicity with
decreasing pH. This v/ould be expected since metals are known to be less
adsorbed at lower pH's and should then be in greater concentrations in the
leachate.
At pH 6 and snail dilutions, both biostinulation was observed in cer-
tain cases. The reason for this is not known although the generation of
trace metal nutrients is most probable.
56
-------
tn
0
20
40 60
Leachate (%)
80
Figure 24 . Aqueous Leachate Toxicity.
-------
en
co
• , » Controls
o,a,A,O With leachate
0
40 60
Leachate (%)
80
Figure 25. Acetate Leachate Toxicity, pH = 5.0.
-------
• ,« Controls
o,D,A,O With leachate
100 <
CD
•«—
_O
CL>
50
0
0
20
40 60
Leachate (%)
80
Figure 26. EDTA Leachate Toxicity, pH = 5.2.
-------
SECTION 10
CONTINUOUS-FLOW BACTERIAL BIOASSAY
A need exists to develop a continuous bioassay to assess toxicity in
wastewater effluents in streams and rivers. Several investigators (111-113)
have developed such systems using fish; however, these systems are complex,
expensive and offer little potential for wide-scale use.
Poels (111) lists eight criteria for a continuous monitoring system as:
1. reasonable sensitivity to a wide number of toxicants,
2. continuous and automatic measuring,
3. avoidance of false alarms,
4. stability of organisms without toxicant,
5. no effect of external changes,
6. continuous flow of water,
7. easy availability of bioassay organisms, and
8. simple monitoring.
It was judged that these criteria could be net with the MUrobacter bioassay
using a nitrate specific ion electrode to monitor metabo 1i sin rates. In this
section, the development of a continuous-flow bicassay is described and the
results using 2,4,5-trichlorophenol (TCP) and cadmium.
PROCEDURE
The design of the continuous bioassoy system relies upon the ability of
Nitrohacter to convert nitrogen in the form of nitrite (NO^) to nitrate
"([.'O-j}; ion electrode (Orion Model 93-07) was chosen to continuously monitor
the nitrate concentration. Response of the organism to toxicants was deter-
mined by monitoring the amount of conversion, which is directly related to
the metabolic rate.
The continuous-flow system includes 1 liter containers for the feed and
toxicant solutions, a Masterflex (No. 7014) peristaltic pump, a bacterial
support column, a holder for the NO^ and reference electrodes, and a meter
60
-------
for measuring the electrode potential in millivolts (Figure 27). A glass
tube (2.0 cm by 14 cm) full of polyethylene heads was used as the bacterial
column. A plug of glass wool was placed in the tube to prevent wasiiout of
beads and bacteria. Two functions were served by the beads: attachment
sites for the organisms, and dispersion of the incoming flow sufficiently to
approximate plug flow conditions. The probe holder was machined from a
Plexiglas block; it allowed contact between the electrodes and the flowing
solution. 0-ring seals around the electrodes prevented leakage.
Procedures outlined in "Standard Methods" (34) (Method 419B) and the
Orion Instruction Manual (114) were used to calibrate the detection sys-
ten. Sodium sulfate (Na^SO^) was used as the ionic strength adjuster in-
stead of the recommended ammonium sulfate because the ammonium ion inhibited
the metabolism of the organisms. Calibration curves were prepared before
each bioassay test.
Sodium nitrite (NaNO^) at a concentration of 30 m
-------
Calibration
lonanalyzer
Ref.
Bacterial
Column
h- 1
Probe
Holder
Waste
Peristaltic
Toxic
Feed
Regular
Feed
Figure 27 . Continuous Flow Bioassay Apparatus.
62
-------
U)
2.5
2mg/i
4mq/l
Regular
Feed
i
0 25 50 75 100 125
Time (minutes)
I5O
175 20O
Figure 28. Effects of TCP on the Metabolism of Nitrobacter
(triangles represent re-start of regular feed)
-------
nitrate-nitrogen (MO^-N) produced by Mitrobacter are plotted over time to
evaluate the effects of varying levels of TCP.Arrows indicate when the
toxic feed was added and when the systert was returned to regular feed;
stable conversion rates (NO^ concentration) were obtained before switching
feeds. Data for the plots were generated by monitoring the meter readout in
millivolts at specified time intervals (approximately 5 minutes). Millivolt
readings were converted to nitrate as nitrogen (m
-------
100 A-
80
° 60
20
0
0
1
A Batch
§1 Continuous-flow
2468
Trichlorophenol (mg/1)
10
Figure 29. Comparison of Batch and Continuous Flow Toxicity of TCP.
65
-------
0
uosoge img/i;
10
25
50
75 100 125 150
Time (minutes)
!75
200 225
Figure 30. Effects of Cd2* on the Metabolism Rate of Nitrobacter (triangles
represent time at which regular feed re-started)
-------
O—O Continuous Flow
Bioassay
A—A Batch Bioassay
Figure
Log Cd Concentration (moles/1)
2+
Relative Metabolism Rate of Nitrobacter versus Cd Concentration
-------
13.5
cr>
CO
4.0
O—Olg/| NaCI
A—A500mg/l Cd'
-*-1g/l NaCI
Regular Feed
) 25
50
75
1 00
I25
ISO
I75
200
225
Time (minutes)
Figure-32. Monitoring Toxicity in the Presence of an Interfering Anion - .,
(*Based on Electrode Reading), (triangles represent re-start of regular feed)
-------
The response of the fli trobacter test culture to cadmium indicated lit-
tle or no toxicity at 10 mg/fc, and relative increases in inhibition up to
2000 ng/fc which caused nearly complete inhibition. Decreases in NO-i-N were
caused by Cd concentrations of 50 mg/fc and greater (Figure 30). Return to
straight feed solutions showed that none of the short-term doses were
lethal.
The plot of the relative metabolism rate of Ni trobacter when dosed at
different cadmium concentrations (Figure 31) faciTTtatecT comparison of toxi-
city data for the continuous-flow bioassay with results of batch bio-
assays. These data indicated that the continuous-flow bioassay can detect
toxicity at a sensitivity similar to that obtained with the batch bioassay
method described in Section f».
Feed solutions containing chlorides showed the effect of ar. interfering
anion, with the resumption of stable conversion rates following removal.
Similar feed solutions with both cadmium (500 mg/fc) and chlorides also pro-
duced interference, but return to regular feed showed a decrease in the
conversion rate when compared to the initial rate of 5.5 ng/£ NO^-N
(Figure 32). Data showed an increase in the production of NO^-N with time,
which further substantiates a toxic response. These data showed that the
continuous-flow system is capable of detecting toxicity in the presence of
an interfering anion(s) by pulse loading the system with a noninterfering
feed solution and testing for a change in the conversion rates before and
after dosage of the toxicant with the interfering anion.
69
-------
REFERENCES
1. "How Hazardous are Effluents from Today's Technologies?",
Envi ronmental Science and Technology , 12, 5, 509 (1978).
2. Cairns, <)., K.L. Hickson and R.F. Vlestlake, 61 ol oqi c_a 1 Hon i tor i ng [_and
Water and Effluent Quality, ASTM Special Publication 667'"("1976) .
3. Duke, K.M,, M.E. Davis and A.J. Dennis, I ERL-RTP Procedures Ma nua 1 :
Level 1 Environmental Assessment Biological Tests for 'Pilot Studies,
"Environmental Protection AGency, EPA 60U/7-77-043 (1977).
4. Tucker, W.G. , et a!., IERL/RTP Environnental Assessment Guideline
Docunent, Environmental Protection Agency, ResearcFTTriangle Park , NC
~
5. "Process Measurements," Environmental Science and Technology, 12, 5
513 (1978).
6. Rowl ings, G.D. , Source Assessment: Textile Plant Wastewater Toxics
Study - Phase 1 , L'nvfroniTiental Protection Agency, Research Triangle
Park, NC (1978).
7. Jaworski, N. , Director, U.S. Environmental Protection Agency Research
Laboratory, Duluth, MN, Personal Communication (1978).
8. U.S. Government, "Water Pollution Control Act," as amended in 1976
(P.L. 92-500) (1977).
9. EPA, "Criteria, Identification, and Listing of Hazardous Wastes,"
Title 40, Chapter 1, Part 250, Subpart A (under review) (1978).
10. Nil Hanson, K..)., "A Bioassay to Assess Wastewater Toxicity to
Aerobic Biological Treatment," Water Resources Institute, Oregon
State University (1976).
11. Bulich, A. A., "Use of Luminescent Bacteria for Determining Toxicity
in Aquatic Environments," in: Aquatic Toxicology (L.L. Marking and
R.A. Kimerle, eds.) ASTM SpeciTi Publication t>6T, American Society
for Testing and MHerials, Phi Iddelphia, PA, p. 98, 1979.
70
-------
12. Bulich, A. A., M.W. Greene, and D.L. Isenberg, "Reliability of the
Bacteria! Luminescence Assay for Determination of the Toxlclty of
Pure Compounds and Complex Effluents," in: Aquatic Toxicology and
Hazard Assessment (D.R. Branson and K.L. D f c k so n , art s . ) As TM Spe c i a 1
Publication 7377~American Society for Testing and Materials, Phila-
delphia, PA, p. 338 (1981).
13. Bauer, N.J., R.J. Seidler, and M.D. Knittel, "A Simple, Rapid
Bioassay for Detecting Effects of Pollutants on Bacteria," Bull.
Environm. Contain. Toxicol., 27, 577 (1981).
14. Cassarett, L.O. and J. Doul 1 , fids., Toxicology; The Basic Science of
Poisons, Macmillan, New York, NY (197577
15. Hartmann, L. and G. Laubenberger, "Toxicity Measurements in Activated
Sludge," J. of Sanitary Engineering Division, ASCE. 94, SA2, 247-256
(1968). ~~
16. Williamson, K.J. and P.I. McCarty, "Verification Studies of the
Biofilm Model for Bacterial Substrate Utilization," J. of Water Poll.
Control Fed., 48. 2, 281-296 (1976).
17. Downing, A. 1., T.G. Tomlinson and G.A. True.Jale, "Effects of
Inhibitors on Nitrification in the Activated Sludge Procp .s," J. and
Proc. of Inst. of Sewage Purification, Pt. 6, 537-554
18. Green, M.B., et al,, "Applications of Toxicity Testing to Sewage
Treatment Processes," Hater Pollution Control, 74, 1, 40-54 (1975).
19. Hockenbury, M. and C.P.L. Grady, Jr., "Inhibition of flitri fication-
Effects of Selected Organic Compounds," J. of Water Poll. Control
Fed.. <4£, 768-777 (1977).
20. Jackson, S. and V.M. Brown, "Effect of Toxic Vlastes on Treatment
Processes and Watercourses, K Hater Pol lution Control. 69, 3, 292-313
(1970).
21. Stafford, D.A., "The Effect of Phenols and Heterocyclic Bases on
Nitrification in Activated Sludge," J. of Applied Bacteriology, 37,
1, 75-32 (1974).
22. Stensel, H.D. , C.S. McDowell and E.f). Ritter, "An Automated
Biological Nitrification Toxicity Test," in Water and I Uastewater
Manuf. , Assoc. ,, 3rd Annua] Conf . , Langworthy, V.W. (ed.), /tan Arbor,
pp. 6/5-695 ("19757:
23. tomlinson, T.G. , A.G. Boon and G.N.A. Trotman, "Inhibition of
nitrification in the Activated Slud<;:' rrocess of Sewritje Disposal," J_._
of Applied Bacteriology, 29, 2, 266 ( 966).
71
-------
24. Lees, H. and J.R. Simpson, "The Biochemistry of Nitrifying
Organisms: Nitrite Oxidation by Nitrohacter," Biochemical Journal,
6J5, 2, 297-305 (1957).
25. Holland, G.J. and A. Green, "Development of a Gross Pollution
Detector: Laboratory Studies," Water Treatment and Examination, 24,
2, 81-99 (1975).
26. EPA, Process Design Manuel for Nitrogen Control, Technology Transfer,
Washington, D.C. (1975).
27. Poduska, R.A. and J.F. Andrews, "Dynamics of Nitrification in the
Activated Sludge Process," 29th Purdue Ind. Haste Conf._, pp. 1005-
1025 (1974).
28. Fry, B.A., The Nitrogen Metabolism of Microorganisms, John Wiley and
Sons, New York, NY (i955]T
29. Painter, H.A., "A Review of the Literature on Inorganic Nitrogen
Metabolism in Microorganisms," Uater Research, 4_, 6, 393-450 (1970).
30. Walker, N. (ed.), Soil Microbiology, John Wiley and Sons, New York,
NY, 123-146 (1975)":
31. Wang, L.K., et al., ''Chemistry of the Nitrificfltion-nenitrification
Process," J. of Environ. Sciences, 21, 6, 23 (1978).
32. Wong-thong, G.M. and R.C. Loehr, "Kinetics of Microhial
Nitrification: Nitrite-Nitrogen Oxidation," Water Research, 12, 8,
605-609 (1973).
33. Loehr, R.C.,-Pollution Control for Agriculture, Academic Press, New
York (1977).
34. Standard Methods for the Examination of Water and Wastewaters,
APHA-AWWA-WPCF, 14th Edition (1975).
35. EPA, "Aribient Water Quality Criteria for Load," EPA-440/5-80-057,
Washington, D.C. (1980).
36. Chapnan, G.A., et al.. "Effects of Water Hardness on the Toxicity of
Metals to Daphnia magga." U.S. EPA, Environ. Res. Lab., Corvallis, OR
(unpublished irianuscrTpt) (1980).
37. navies, P.M., .3.P. Goettl, J.R..Sinley and N.F. Smith, "Acute and
Chronic Toxicity of Lead to Rainbow Trout (Salino quirdneri) in Hard
and Soft Hater," Water Research. _1£, 199 (1976]7
38. Hale, J.G. , "Toxicity of Metal Mining Wastes," Bui ). Enyi ron.__Cor:tj_'j_._
.' ' 66
72
-------
39. Tarzwell, C.M. and C. Henderson, "Toxicity of Less Common Metals to
Fishes." Ind. Hastes, 5, 12 (1960).
40. Pickering, Q.H. and C. Henderson, "The Acute Toxicity of Some Heavy
Metals to Different Species of Warmwater Fishes," Air Water. Pollut.
Int. J.. _!£, 453 (1966).
41, Monahan, T.O., "Lead Inhibition of Chlorophycean Microalgae," J.
Psycho!.. 12. 358 (1976).
42. Malanchuk, O.L. and G.K. Gruendling, "Toxicity of Lead Nitrate to
Algae." Mater Air and Soil Pollut., 2. 181 (1973).
43. Hessler, A., "Effects of Lead on Algae: Mutagenesis Experiments on
Platymonas sjjbcordi formis (Chiorophyta:valvocales)," Mutat. Res., 31,
43 (1975)."
44. EPA, "Ambient Water Quality Criteria for Cadmium," EPA-440/5-80-025,
Washington, D.C. (1980),
45. Chapnan, G.A., "Toxicities of Cadmiurn, Copper, and Zinc to Four
Juvenile Stages of Chinook Salmon and Steelhead," Trans. Am. Fish.
Soc.. 107. 341 (1978).
46. Kumada, H., et al.s "Accumulation and Biological Effects of Cadmium
in Rainbow Trout," Bull. Jap. Soc. Sci. Fish., 46, 97 (1980).
47. Kumada, H., et 3!., "Acute and Chronic Toxicity, Uptake and Retention
of Cadmium in Freshwater Organisms," Bull. Freshwater Fish. Res.
JL_ab._, 22, 157 (1973).
48. Davies, P.H. , "Use of Dialysis Tubing in Defining the Toxic Fractions
of Heavy Metals in Natural Waters," in: Toxicity to Biota of Metal
Forms in Natural Water, Proc. Workshop, Duluth, MM, October 7-8,
1975, p. 110 (1976).
49. Cliapman, G.Al, "Effects of Water Hardness on the Toxicity of Metals
to Daphnia rcagna," U.S. EPA, Corvallis, OR (unpublished manuscript).
50. Canton, J.H. and D.M.M. Adema, "Reproducibility of Short-Term and
Reproduction Toxicity Experiments with Daphnia magna and Comparison
of the Sensitivity of Daphnia magna with Daphnia pul'ex and Daphnia
cucullata in Short-Term Experiment," HyciroFTol.. 59, 135 (1978).
51. Pickering, Q.H. and C. Henderson, "The Acute Toxitity of Some Heavy
Metals to Different Species of Uarniwater Fishes," Ai r Water Pol lut.
Int. ,).. 10, 453 (19G6).
52. Pickering, Q.H. and M.H. Oast, "Acute and Chronic Toxicity of Cadmium
to the Fathead Mir; now (Pii'iephales propel as)," J. Fish. Res. Board
_Ca_n._, j?9_, 1099 (1972).
73
-------
53. EPA, unpublished laboratory data, Environ. Res. Lab., Narragansett,
RI (1980).
54. Chapman, G.A. and O.K. McCrady, "Copper Toxicity: A Question of
Form," Recent Advances in Fish Toxicology, a Symposium, EPA 600/3-77-
085, U.S. EPA, p. 132 (1977).
55. EPA, "Ambient Water Quality Criteria for Copper," EPA-440/5-80-036,
Washington, D.C. (1980).
56. Cairns, J., et al., "Effects of Temperature on Aquatic Organism
Sensitivity to Selected Chemicals," Bull. 106 - Virginia Water
Resour. Res. Center. Blacksburg, VA (1978);
57. Chakoumakos, C., et al., "The Toxicity of Copper to Rainbow and
Cutthroat Trouts under Different Conditions of Alkalinity, pH and
Hardness," Env. Sci. Tech., H. 213 (1979).
58. Brown, V.M., et al., "Aspects of Water Quality and Toxicity of Copper
to Rainbow Trout," Water Res.. 8_, 797 (1974).
59. U.S. EPA, "Ambient Water Quality Criteria for Zinc," EPA-440/5-80-
079,'Washington, D.C. (1980).
60. U.S. EPA, "Quality Criteria for Water," EPA-440/9-76-023 (1976).
61. Biesinger, K.E. and G.M. Christensen, "Effects of Various Metals on
Survival, Growth, Reproduction, and Metabolism of Daphnla magna," 0.
Fish. Res. Board Can., 29_, 1691 (1972).
62. Cairns, 0. Jr., et al., "Effects of Temperature on Aquatic Organism
Sensitivity to Selected Chemicals," Bull. 106, Virginia Water Resour.
Res. Ctr., Blacksburg, VA (19.3).
63. U.S. EPA, "Ambient Water Quality Criteria for Silver," EPA-440/5-80-
, Washington, D.C. (1980).
64. Lemke, A.E., "Comprehensive Report - Interlaboratory Comparison:
Acute Testing Set," Environmental Protection Agency (unpublished
manuscript).
65. EPA, "Ambient Water Quality Criteria for Heptachlor," EPA-440/5-80-.
052, Washington, D.C. (1980).
66. Macek, K.J., ot al., "Toxicity of Four Pesticides to Water Fleas and
Fathead Minnows," EPA--600/3-76-099, U.S. EPA (1980).
67. Katz, M., "/"cute Toxicity of Some Organic 'nsecticides to Three
Species of Salnonids and to the Threespine Stickleback," Trans. Am.
Fish. Soc. . 90, 264 (1961).
74
-------
68. Macek, K..J., et al., "The effects of Temperature on the
Susceptibility of Bluegills and Rainbow Trout to Selected
Pesticides," Bull. Environ. Contain. To.xico1.. 4, 174 (1969).
69. Henderson, C., et al., "Relative Toxicity of Ten Chlorinated
Hydrocarbon Insecticides to Four Species of Fish," Trens. Am. Fish.
Soc., JM, 23 (1959).
70. EPA, "Ambient Water Qualtiy Criteria for Endosulfan," EPA-440/5-80-
046, Washington, D.C. (1980).
71. Pickering, Q.H. and C. Henderson, "The Acute Toxicity of Some
Pesticides to Fish," Ohio J. Sci., 66_, 508 (1966).
72, Lemke, A.E., "Comprehensive Report -. Interlaboratory Comparison Acute
Testing Set," EPA, Environ. Res. Lab., Duluth, MN (1980).
73. Spacie, A., "Acute and Chronic Parathion Toxicity to Fish and
Invertebrates," National Water Quality Lab., Duluth, MN (1976).
74. Moulton, E.Q. and K.S. Shumate, "The Physical and Riological Effects
of Copper on Aerobic Biological Waste Treatment Processes," 18th
Purdue Ind. Waste Conf., pp. 602-615 (1963).
75. Ayers, K.C., K.S. Shumate and G.P. Hanna, "Toxicity of Copper to
Activated Sludge," 2Qth Purdue Ind. Waste Conf., pp. 516-524 (1965).
76. Barth, E.F., et al., "Field Survey of Four Municipal Hastewater
Treatment Plants Receiving Metallic Wastes," J. of Water Poll.
Control Fed.. 37. 8, 1101-1121 (1965).
77. Mowat, A., "Measurement of Metal Toxicity by Biochemical Oxygen
Demand," 0. of Water Poll. Control Fed., 48_, 5, 853-366 (1976).
78. Bunch, R.L. and C.W. Chambers, "A Biodegradabi1ity Test for Organic
Compounds." J. of Water Poll. Control Fed., 39. 181-187 (1967).
79. Neufeld, R.H. and E.R. Hermann, "Heavy Metal Removal by Acclimated
Activated Sludge," J. of Water Poll. Control Fed.,
-------
82. EPA, "Ambient Water Quality Criteria for Pentachlorophenol," EPA
440/5-80-065, Washington, D.C. (1980).
83. EPA, "Hazardous Waste Management System: Identification and Listing
of Hazardous Waste (40 CFR Part 261)," Federal Register 45(98) :33084
(May 19, 1980).
84. Tirsch, F.S., et al., "Copper and Cadmium Reactions with Soils in
Land Applications," J. Water Pol 1. Control Fed. ,51, 2649 (1979).
85. Hyde, H.C., et al., "Effect of Heavy Metals in Wastewater Sludge on
Agricultural Crops," 0. Water Poll. Control Fed., _5_1_, 2475 (1979).
86. Connor, J.R., "Disposal of Concentrated Wastes from the Textile
Industry," Industrial Water Engineering, 14(4):6 (1978).
87. Thefs, T.L., et al., "Field Investigation of Trace Metals in
Groundwater from Fly Ash Disposal," J. Wa t c r P o 11. Con t r o 1 Fe d., jO.
2457 (1978).
*'
88. Talbot, R.W., et al., "Qualitative Model of Heterogeneous Equilibria
in a Fly Ash Pond," Environmental Science and Technology, 12, 9, 1056
(1978). ~
89. Hunienick, M.J. and C.F. Mattox, "Groundwoter Pollution from
Underground Coal Gasification," Water Research, 12, 7, 463 (1978).
90. Chu, T.Y.J., et al., "Characteristics of Stabilized Scrubber
Sludges," .). Environmental Engineering Div.. ASCE, 104, 4, 731
(1978). " ~
91. Stonn, D.L. , "Leachate from Plastic Wastes in Landfills," California
;V Vector Views, _24_, 9-10, 33 (1977).
92. Means, J.L. , et al., "Radionuclide Adsorption by Manganese Oxides and
•,: Implications for Radioactive Waste Disposal," Nature, 2_7_4_, 5666, 44
i' (1978). ' -
93. Houle, M..J. and n.L'. Long, "Accelerated Testing of Waste Leachability
. • and Contamination Movement in Soils," Environmental Protection Agency
Publication EPA-600/9-78-016 (1978).
94. Gibb, J.P., "Field Verification of Hazardous Waste Migration from
Land Disposal Sites," Environmental Protection Agency, EPA-600/9-7J-
: 016 (1078).
95. Harrison, A.P., Jr., "Hicrobial Successive and Mineral Leaching in an
Artificial Coa 1 Spoi'l ," Appl i ed and Envi ronnontal Mi crobi_3J_gc|y_, 3_6 ,
6, 861 (1978).
76
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