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Eastern Illinois UniversityThe Keep
Masters Theses Student Theses & Publications
1982
Inhibitors Affecting Bacterial Action in ReaeratedActivated SludgeAlice M. DunnEastern Illinois UniversityThis research is a product of the graduate program in Botany at Eastern Illinois University. Find out moreabout the program.
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Recommended CitationDunn, Alice M., "Inhibitors Affecting Bacterial Action in Reaerated Activated Sludge" (1982). Masters Theses. 2898.https://thekeep.eiu.edu/theses/2898
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m
INHIBITORS AFFECTING BACTERIAL ACTION
IN REAERATED ACTIVATED SLUDGE (TITLE)
BY
ALICE M. DUNN
B.S. , EASTERN ILLINOIS UNIVERSITY, 1 979
THESIS
SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
Master o� Science IN THE GRADUATE SCHOOL, EASTERN ILLINOIS UNIVERSITY
CHARLESTON, ILLINOIS
I HEREBY RECOMMEND THIS THESIS BE ACCEPTED AS FULFILLING
THIS PART OF THE GRADUATE DEGREE CITED ABOVE
�J;/!4fi- ADVISER
1?241i z / 97 ;:;.._ TE C:OMMITTFF M�MRFR
� '.7, /?j,fz.. DATE COMMITTEE MEMBER
-Wl�lrE l � [d DEPAlrtMENT CHAIRPERSON
ABS'?�U .. CT
Dunn, �lice M. M.S., Eastern Illinois University. May 1982. Inhibitors Affecting Bacterial Action in Reaerated Activated Sludge4
Inhibition of reaerated activated sludge was assessed
using reduction of dissolved oxygen uptake by the mic�c�ial
population as a criterion. Oxygen uptake is essential �o
biodeg=adation of the organic components in the cctiva�-�d
sludse process; therefore, a decrease in dissol·.-e:i oxy�·.s::s
upt��e is an indication of reduced efficiency in waste�ater
treatment.
The irihibitors tested were cnd1:iil!m, copper:. 1.8ad,
mercury, silver, 2�4-Dichlorophen�xyacetic acid l�-4-D), Ter·Jc:1c il and Zee tran. The I IC 50, determined by v . .<.. ::;.:8.��.:; 2.:.·r
tion of plotted data, for each inhi�itor in mg/l �as
Hg (20), Cu (50), Ag (43), Cd (115), Pb (550), ara 2,;j. J
( 430). The IIc50 was determined by testi:::g the e fi'ec-:
on oxygen uptake of concentrations rangin� frore J.001 to
100J mg/l of the in�ibitor. With the exception o�
Terbacil and Zectran, whose inhibitory effects were
negligible and wer� deleted from fu�ther study, t�e
rrc50 determined for each inhibitor was utilized in
sub:::�qyent short-term aP.d long-te:-:.:: s tu.dies.
Short-term studies measureJ tae eft��t of the
inhibitors on dissolved oxygen �ptake over a 4-ho�r
period. These experiments were intended to simul��3 the
immediate effect upon the contact stabilization unit ,... .� --'··
a sev.rage treatment facility . In eeneral, the short-te:::-:1
effects of the inhibitors followed a similar pattern; an
initial marked inhibition of dissolved oxygen ur.-t:.ke was
followed by small increases until maximal inhibition,
which occurred after 3 or 4 hours in the presence of the
IIC50• The observed variability in the percent inhibition
of dissolved oxygen uptake was attributed in part to the
inherent variability of different sludge samples.
Long-term studies, extending over a 2-week perio�� ..
were intended to indicate possible inhibitory ef�ects on
dissolved oxygen uptake by an agent which remains within
or is continually introduced into the sewage treatment
facility. A periodicity of inhibition followed ·oy semi-
recovery was exhibited in studies of seve=al in�ititors.
Stimulation of dissolved oxygen uptake osc�rred �uri�g
certain phases of the long-term study for both mercury
and 2,4-D.
It was concluded that the inhibitory effects of th�
heavy matals Hg, Cu, Ag, Cd, and ?b, and o� the herbicide
2,4-TI are significant and will therefore affect the
efficiency of wastewater treatment plant operation.
ACKNO\'/LEDGEMENTS
I wish to thank my adviser Dr. William A. �eiler
for his ideas , enthusiasm, and guidance during this study,
during the manuscript preparation , and during my course
o f graduate study.
I would also like to thank the members of my graauate
committee , Dr . Charles B. Arzeni and Dr . Richard Smith ,
for their constructive criticism i n the preparation o f
this paper, and for their help and encouragement throueh
out my course of study at Eastern Illinois University.
In addition , I wish to express my appreciation to
Dr. :terry Weidner for his initial encouragement i.:.1 my
decision to begin my graduate program.
Thanks is also due to Julia Jansen who assisted i�
the laboratory.
My sincere thanks are extended to rr.y family and
friends for their moral support throughout my graduate
study.
Finally , I extend a special thanks to my pa=ents
for their love , understanding , and encouraGement
throughout my college career.
TABLE OP COl!TEETS
.ABSTRACT • • • • • • • • • • • • • • • • • • • • • • • • • • .. • • • • • • • • • • • • • • •
ACKt�o�:fLSJJGR�1E}rTS • • • • • • • • • • • • • • • • • • • . • . • • • • • • • • • • • •
i
iii
TP-BLE 01'"' COffTEl�TS. . . • • . • • • • • • • • . • • • • • • • • . • • . • . • • • • iv
LIST 0 F T...t\.BLES . . • . . . . . . . • . . • . . . . . • . . . . . • . • . . . • • � . . v
LIST 0 F FIG lffiE S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi
INTRO-::>UCTIOJ\�. • • . • . . • • • . . . • • . • . . • • • • . . . • . . • . • • . • . . . 1
HISTORICAL REVIEW................................. 3
MAT��HIALS AI·rn METHODS. • • • • • • • • • • • • • • • • • . • • • . . . • . • • 30
RESULTS AHD _DISCUSSIOt�.......................... . • 37
COl·JCLUSIO�S. . . . . . . . . . . . . . . . . . . . . . . . . . • . . . . . . . . . • . . 83 LIT;.]RA TU!tE CI'i'EJ)... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . <35
Table
1
2
3
LIST 0}' �ABLES
Maximum levels of toxic contaminants permitted by the E�vironmental Protection Agency
Inhibitors and their mechanisms of toxicity
�he rrc5 for six inhibitors of the activate� sludge process in a wastewater treatment plant
?age
8
38
Figure
1
2
4
5
6
7
Q '·'
9
10
12
13
14
LIST OF :;:;IGURES
Charleston Wastewater Treatment �lant in Charleston ( Coles County ) Illinois
The effect o f Cadmium or cli:;;solved oxygen uptake in activated s ludge
The effect of Copper o n dissolved oxygen uptake in activated sludge
The effect of Lead on di ssolved oxygen uptake in activated sludge
The effect of Lead on dissolved oxyeen uptake of a diluted ( 25/�) activated sludge
The e ffect of Mercury on dissolved oxygen uptake in activated sludga
The effect o f Silver on d�ssolved oxygen uptake in activ8te� sludge
The effect of 2,4-� on dissolved oxyeen upt�ke in activ�ted slud�e
The e ffect of Terbacil on dissolved oxygen uptake in acti vated s ludge
The effect of Zectran on dissolved oxygen uptake in activated sludge
Short-ter!Tl effects of Cadmium IIC,0 on disso lved oxygen uptake: in ::>
activated sludge
LonCT-term effects of Carlmium rrc50 on dissolved oxyee n uptake in activated sludge ( R eplicat e 1)
Long-terrn effects of Cadmium IICc:;r: a· ... l t k · �v on isso�vec oxyg�n up � c l�
activated sludee ( �eplicnte ?) Short-term effects o f Coppe r rrc50 on disno lved oxycen uptak� in activated sludee
3 1
40
43
44
46
48
50
51
52
53
57
58
58
60
1 5
16
17
18
19
20
21
22
23
24
25
26
27
Long-term effects cf Copper IIC�o on dissolved oxygen uptake in J
activated sludge ( ReplicRte 1)
Long-term effects of Copp�r rrc50 on dissolved oxyeen uptake in activated sludge (Replicate 2)
Short-term effects of Lead IIC�0 on dissolved oxygen uptake in /
activated sludge
Long-term effects of Lead rrc50 on dissolved oxygen uptak� in activated sludge ( Replicate 1)
Long-term effects of Lead IIC50 on dissolved oxygen uptake in activated sludge ( Replicate 2)
Short-term effects of Mercury IICt".0 on dissolved oxygen uptake in � activated sludge
Iiong-term effects of Mercury IIC.- 0 on dissolved oxygen uptake i� 7 activated sludge ( Replicate 1)
LonB-term effects of Mercury rrc50 on dissolved oxygen uptake in activated sludge ( Replicate 2)
Short-term effects of Silver IICh� on dissolved oxygen uptake in Ju
activated sludge
Long-term effects of Silver rrc50 on dissolved oxyg9n uptake in activated sludge ( Replicate 1) Long-term effects of Silver IIC50 on dissolvect oxygen uptake in activated sludge ( Replicate 2)
Short-term effects of 2,4-TI rrc50 on dissolved oxygen uptake in activated sludge
Long-term effects of 2,4-D IIC50 on dissolved oxygen uptake in activated sludge ( Replicate 1)
62
62
65
67
67
69
7(:
70
73
75
75
'i' 7
79
28 Lone-term effects of 2 , 4-D I I C50 on nissolved oxygen uptake in activated sludge (Replicate 2)
79
1
INTRODUCTIO�
I ncoming raw sewage , especially in industrializea
areas may contain inhibitors at levels sufficient to
inhibit bact erial biodegradation in the activated sludge
of a wastewater treatment plant . Significant inhibition
of bacterial biodegradation of the organic material
decreases the effectiveness of the activated sludge
proce s s and , hen c e , results in loss of eff�ciency in the
plan t . I t i s therefore pertinent to determine the e ffects
of different concentrations of various inhibitors ( i . e . ,
heavy metal s , herbicide s , and pesticides ) on the �ctivatcd
sludge process. Subsequent analyses may determine what
constitutes acceptable levels of the inhibitors i� the
incoming raw sewage , which levels would not significantly
alter the effectiveness or efficiency of ·the activated
sludge process in the treatment plant .
The purpose of this study w�s to evaluate the affect
o f various inhibitors on the bacteria in reaerated
activated s ludge . Resultant inhibition was asses sed
using the reduction of dissolved oxygen uptake by the
bacteria and protozoans . Oxygen uptake is essential to
biodegradation o f the orga�i c components in the &ctiv�ted
sluage pro c e s s and, therefore , a decrease in dissolved
oxygen uptake is indicative of reduced efficiency in
wastewater treatment.
2
This study was approached with the following
questions in mind:
(1) What is the magnitude of the resulting
inhibition by different concentrations of various
inhibitors on the dissolved oxygen uptake by bacteria
in reaerated activated sludge?
(2) What is the Initial Inhibitory Concentration
for 50% inhibition of dissolved oxygen uptake (rrc50) during the initial 15-minute period after the addition
of each inhibitor?
( 3 ) What happens to the inhibition level over a
short time period ( 4 hours) after the bacterial
population in the reaerated activated slu�ge is subjected
to that 2-fold dilution of the maximum concentration
applied which is closest to the rrc50?
( 4 ) What happens to the initially inhibited
population over an extended period of time and does
bacterial adaptation to the inhibitor occur during
this time period?
3
HISTORICAL REVIEW
Toxic substances, according to Weber and Sherrard
(1980), present a 2-fold problem: (1) they may exceed
acceptable levels when discha�ged into a receiving water
from a wastewater treatment plant and, therefore, could
damage aquatic and plant life and cause harm to humans
if the concentrations are not reduced to within drinking
water standards, and (2) they may cause a reduction in
the efficiency of a wastewater treatment plant that could
result in a significant increase in oxygen-demanding
carbon and nitrogen compounds being released into the
receiving water.
Heavy metals are one type of toxic substances poten
tially hazardous at low concentrations. Heavy metals
generally include many of the transition series of
metals and some of the metals and metalloids in groups
IIIB, IVB, VB, and VIB of the periodic table (Sterritt
and Lester, 1980). Sources of heavy metals that have the
most direct adverse effect on the environment originate
from the weathering and disintegration of rock and soi�,
precipitation and atmospheric fallout, and industrial
pollution. Two large natural contributors are volccn!�
dust and forest fires , while the steel manufacturing
process is one of the largest man-made sources . �etals
resulting from man's activities include industrial
discharges, acid mine drainage, agricultu�al and 0�ban
4
runoff , and domestic wastewater ( W eber and Sherrard,
1980). C ertain transition heavy metals , such as iro n ,
mangan ese , cobal t , copper, and zin c , are essential for
the me tabolism of many organisms while other s , such as
mercury, lead and cadmium , are not neede d . �hese heavy
metals may enter treatment plants and , since nearly all
munici pal and many industrial wastewater treatment
plants utilize some form of biological tre�tment process ,
toxico logical studies are needed to determine the effects
of the s e substances ( Weber and Sherrard, 1980). As a
result , a new branch o f research called eco logical
toxico logy (ecotoxicology) is developing (Mcintyre and
M i l l s , 1974).
The inhibitory effects of metall i c ions have been
the focus of numerous studies . Early studies invest
igating ion antagonisms in microorganisms discovered
that c ertain heavy metal inhibitors sxerted toxic effects
upon bacteria by interferen�e in normal magnesium
metabolism (Abelson and Aldous , 1950). It was observed
that low magnesium concentrations led to heightened
toxic ity of the divalent cations of such heavy metals as
cad�ium , cobal t , manganese , nickel , and zin c .
With the advent o f improved sewage treatment
processes and incorporation of biological treatment,
the toxic effects of heavy metals became a matter of
growing concern . Malaney, Sheets, and Quillin l1959)
5 discerned the need to determine the concentration o f each
m e talli c ion which could be permitted in sewage plant
influen t without ups e tting biological treatment . I n
their study the parameter used i n measuring the toxicity
of the metallic ions was the inhibition of oxygen uptake
by the sewage microorganisms in the presence o f various
conc en trations of a selected ion . They comment that the
results revealed a rather surprising abi l i ty o f the sewage
microorganisms to recover from the toxic effec ts of
metallic ions. This recovery phenomenon has sinc e been
labeled acclimation or adaptation and is an important
consideration , n o t only for wastewater treatment plant
operation , but also for natural ecosystems such as the
receiving s treams for wastewater effluents .
The toxic effects of metallic ion inhibitors ,
toxic i ty threshold level s , tolerance levels of t e s t
organ i sms , and mode of ac tion of the inhibitor upon the
organism have been investigated . Tornabene and Edwards
( 1 972) , for example , found that lead in conc entrations
up to 2 . 5 grams/l had no effect on the bacteria
Micro c occus luteus or Azotobacter spp. In another study,
Zwarun ( 1 973 ) showed that 6 mg/l appeared to be the
threshold level for cadmium toxic ity to Escherichia coli.
E. coli was also the t e s t organism utilized in a study
by Bragg and Rainnie ( 1974) on s i lver toxicity. Results
indicated that silver ions reacted with the respiratory
chain at two levels and that 1 �M AgN03 significantly
6
inhibited (@ 80%) respiration . This inhibition of
E. coli metaboli zing glucose occurred , howeve r , after an
initial brief phase of stimulation . This stimulation �as
thought to represent an uncoupling of the s i lver ions .
Similarly , another heavy metal , copper , was found to
stimulate growth at certain low concentrations . Loveless
and Painter (1967) found increasing stimulation by copper
up to an added amount of 0.02 mg/l with a leveline off up
to about 0.06 mg/l and no stimulation at 0 . 48 mg/ l . In
this case the test organism, Nitrosomonas s pp . , which was
iso lated from activated sludge , ut i l i zed the heavy metal
copper as an essential trace e lement involved in its
oxidase system. The pH strongly influenced the toxic ity
of the copper, an observation Loveless and Painter (1567) indicat e d might be true for other heavy metals . Information
was accumulating but much study was sti l l necessary .
Therefore , in addition to the effects of metallic
ions on specific bacterial genera , studi e s were undertaken
to investigate the combined effects of more than one metal
and the immediate ( short-term ) and prolonged ( lon�-term ) effects of each metal in the activated sludge process .
The activated s ludge sewage treatment process was
developed in England during the early 1900's; since that
time , many variations of the originul process have beer.
developed and uti lized in wastewater treatment ( Goodmun ,
1 971 ) . In this proces s , the purification of se\:age is
accomplished by aeration with a flocculent mixed .nic�ob ial
7
culture ( activated sludge). The usual objectives o f
treatment are the removal o f carbonaceous matter and
oxidation of ammonia to nitrite and nitrate (Tomlinson
et al., 1 965). It i s because biological treatment systems
are complex, undefined system� employing many di fferent
microbial specie s that more studies are neede d . Attempts
to quantify a toxic level for a particular metal are
exceedingly difficult, as stres s e d by �eber and Sherrard
( 1980). However, concern for public heal th and .Federal
monitoring has emphasized the need for determin2tion of
acceptable levels of heavy metals. Table 1 shows the
maximum permissable concentrations o f EPA (Environmental
Protection Agency)-regulated toxic substances in leachut0s
such as sewage sludge leachates (Williamson and Nelson,
1981). These concentrations are 1 00 times the acc8ptabla
standard levels o f the inhibitor in drinking water. A
summary of the mechanisms cy which the s e inhibitors ac ��
.·are listed in Table 2 ( Williamson and 5elson, 1 981).
Of the heavy metals investigated by this author,
three have been described in relation to the activated
sludge proces s in previous works, viz., cadmium� mercury,
and copper. The toxic e ffects of lead, silver, and 2,4-D
( 2,4-Dichlorophenoxyacetic acid) upon the activated sludge
process were not explicitly considered within the
literature encountered. However, the herbicide 2,4-D has
been studied extensively with regard to its biodegradation
in soil.
a
Table 1 . Maximum levels of toxic contami�ants permitted
by the Environmental Protection Agency (Williamson and
Nelson, 1 981 . Table 9).
Contaminant
As
Ba
Cd
Cr
}1b
Hg
Se
iig
Bndrin
Lindane
Methoxychlor
Toxaphene
2, 4-D
2,4,5-TP ( Sil vex)
Concentration (mg/l)
5 . 0
1 00.0
1 • 0
5.0
5.0
0.20
1 . 0
5.0
0.02
0 . 40
10.0
0 . 50
1 0. 0
1 .o
9
Table 2 . Inhibitors and �heir mechanisms o f toxicity
(Williamson and l\elson, 1 981 . Table 2).
Inhibitor
Heavy Metals ( g eneral)
Specific Heavy Metals Hg
Ag
Cu
Zn
Pheno l s
Alcohol
Chlorine
( 1 )
( 2 )
( 3 )
( 1 )
( 1 ) ( 1 ) ( 2 )
( 1 )
Toxicity Mechanisms
interference with cell wall synthesis decreased oxidative en zy:ne activity deactivation of DNA, RNA, and proteins
reacts with SH- groups of the cells decreased e nzyme activity formation of metal comnlex�s with polynucleotides , bNA, RN, binds w i th D�'.A coagulation of bacterial cell colloid complexation with e s sential nutrients
(1 ) disrupt cell membrane ( 2 ) inhibition of oxidase enzymes ( 3 ) protein precipitation inside
the cells
( 1 ) inhibition of respiration (2) inhibition of phosphorylation ( 3 ) �amagc to the cell membrane
(1) inhibi tion of bacterial oxidase s ( 2 ) inhibition of de:11ydrog2nase
system (protein denaturation and enzyme suppression )
(1) attacks sulfhydry l (-SH) groups of en zymes involved in metabo1i c pathways
Acid (H+) or
Base (OH-)
Organophosphates Pesticides
Carbamate Pesticides
Organochloride Pesticides
Chlorinated Cyclodienes
10
( 1 ) di�places i�n species such as Na a nd Ca from absorution sites of the cells
-
( 2 ) cell wall damage
( 1 ) inhibition of acetylcholinesterase
(2) respiratory toxicity; high dermal toxicity
(1) inhibition of acetylcholinesterase
( 1 )
( 2)
( 1 )
actions on nerve fibers; activities in the nerves still unknown + inhibits Na , K+, Mg++, ATP
neurotoxici ty mechanisr1 unknown
1 1
Heavy Metal Interactions in Sewage Treatmen�. The
heavy metal cadmium is emitted to the ecosystem in
significant amoun t s . The metal plating industry and the
burning o f fossil fuel expel the largest quantities ; the
e l ectroplating industry accou�ts for approximately 2 the
cadmium consumed annually in the United State s . Cadmium
coatings are used primarily as protection against corrosion
for ste e l , bras s , copper, and other alloys . Cadmium
pigments are used extensively in coloration o f pla stics
and , due to their stabi lity, they are utilized in fabric s ,
textiles, rubber , glas s , paints, enamel s , and printing
inks (Weber and Sherrard , 1 9 80 ) .
The e ffects o f cadmium on the activated s ludge
process were studied by Weber and Sh�rrard (1980 ) .
I nfluent concentrations as high as 10 mg/l Cd (IIj di� not
influence microbial growth coeffic ients . �urthex-, the
influence o f cadmium on the rate of ni trificatio�i was
significant .
Mercury is one o f the most toxic of all heavy metals .
More than 80 , 000 tons of mercury was consumed in the
United States in the last century. The largest
commercial user in the U . S . i s the chlorine-alkali
industry, which is also the m3jor environmental contributor.
The second largest use of mercury is in the manufacture of
electrical apparatus . This heavy matal is also used
extensively in the production o f fungicid e s , p lastics,
photographic chemical s , and petrochemicals (Gho8h and
12
Zugger, 1 973 ) .
Ghosh and Zugger (1973) investigated the toxic e f!ects
o f mercury on the activated sludge process . Results o f the
investigation indicated that mercury at concentrations less
than 2.5 mg/l had little effe c t on the activity of a mixed
aerobic biomas s , but at 5.0 mg/l or higher definite
inhibition occurred . Thus , the threshold toxic ity level
of mercury seems to lie between 2.5 and 5.0 rng/l . There
was a time lag for the inhibition by mercury to take place .
Furthermore , the inhibitory effe c t of mercury was seen to
be temporary. Ghosh and Zugger (1973) observed that it is
possible for an aerobic biologic al process to acclimate to
a moderately high level of mercury, but that accli�atior.
time i ncreased with increased concentrations of mercury .
After acclimation, the system apparen t ly func tioned quite
well .
Copper could be present in domestic sewage and
industrial waste mixtures in several forms , depending
upon i t s source and subsequent reactions . As copper
sulphate, it is used in the manufacture of copper articles
by deposition from solution ( e le c tro-depositio n ) , for
recovery of copper from ore ( e lectro-refining ) , and for
electroplating . In the most common elec troplating proces s ,
copper is deposited from cyanide baths . Copper is also
used as a catalyst in synthetic chemical manufacturing
operations and may become associated with liquid wastes in
some undetermined form. The petroleuQ re �in ing industry
13
util i z e s copper chloride in mercaptan removal · processes
( McDermott et al . , 1 963 ) .
McDermott e t a l . ( 1 963 ) studied e ffects o f varying
copper concentrations on aerobic biological sewage treat
ment and conc luded that the maximum conc entration that did
not have a detectable effect o n treatment efficiency was
1 mg/l . Slug doses of a few hours duration with up to
50 mg/l Cuso4 had only a s light effec t upon treatment
e fficiency. The 1 00, 2 1 0 , and 4 1 0 mg/l doses of copper
caused severe effects over the first 48 hours , but even
at the highest leve l , the sludge recovered to normal i�
about 100 hours . They estimated that the process is about
6 5% effective in removing a slug dose o f 410 mg /l . Another
observation was tha t increased turbid i ty o c curred with
increased copper concentratio n . It was noted also that at
the 1 mg/l copper leve l , the protozoa and rotifera would
be present in normal numbers . A t sufficiently high copper
concentratio n s , the sludge settled rapidly and it app eared
that the growth o f certain filamentous organisms respo�sible
for sludge bulking was inhibited (r.1cDermo tt � a l ., 1963 ) •
The effect o f copper in combination with chromium,
nickel , and zinc on biological treatment has been
investigated by Barth et al. ( 1 965a) . The results
show that the aeration phase of bio logi cal treatment
can tolerate, in the influent sewage , copper, chronium,
nickel, and zinc , up to a total heavy me tal concentration
of 10 mg/ l , ei ther singly or in combinatio n , �ith about
14
a 5% reduction in overall plant efficiency.
Barth et al. (1965a) make two important generalizations
regarding heavy metals in biological treatment. One such
generalization was that the concentration of metal that
constitutes a harmful slug dose is determined by such
factors as the waste volume, the volume and characteristics
of the dilution water, the specific form of the metal, and
the usage of the stream below the point of effluent
dischargeo Also, they note that, in general, acclimation
of the system to low concentrations of metals did not
offer protection from slug doses. Another important
generalization was that there was no evidence of acclimation
of the nitrifying organisms to the metals.
Inhibition of nitrification is regarded as an
important effect of metal toxicity. According to Barth
et al. (1965a) a plant affected by heavy metal inhibition
would discharge all the inf�uent nitrogen in excess of
that needed for synthesis, predominantly in the form of
ammonia. Such an effluent would use large amounts of
oxygen to transform ammonia to nitrate in the process of
nitrification.
The subject of inhibition of nitrification in the
activated sludBe process was addressed further by
1.romlinson et al. ( 1965), who tested the effects of a
variety of organic and inorganic substances. They found
that the short-term effects of various organic corn9ounds
on the rate of oxidation of ammonia by Ni trosoni:..?j� spp.
1 5
in activated sludge appeared similar to the r eForted
effects on pure cultures of this organism . I n contrast,
the inorganic compounds , such as those with copper,
mercury , and chromium, appeared to affect Nitrosomonas spp.
in activated sludge very much less than in pure cultur e .
It was suggested that this phenomenon was due to the
formation of chemical complexes or physical interaction
between the metal and organic matter in the sewage or
sludge . Hence , the complexity of the activated sludge
system becomes more apparent�
Variability in the Activated Sludge Process.
Reference to the complexity and inherent variability has
been mentioned i n nearly every study attempting to address
the problems associated with heavy metal intera�tion i�
the biological treatment procesc . �s a result , there ic
no general consensus regarding the best method of study
or parameter to be measure d . Consequently , quant itative
results often differ among seemingly similar studi e s
(Weber and Sherrard , 1980 ) . Hence , comparisons of results
and conclusions drawn must be approached with an awareness
of the variability and often wide range of experimental
procedures and , perhaps more importantly, the variable
operating conditions which could be encounteredo
This caution is evident in the consideration of
threshold concentrations by Barth et al� \ 1 965a) . They
cautiously indicate that their observed limits were
obtained under carefully controlled laboratory condi t ions
1 6
and that the significance of the threshold c oncentrations
is mainly of academic interest because actual plant
situations are concerned with the p lateau region of �etal
dosage and respons e . It is stressed that to fix these
concentrations accurately would require an inordinate
amount of time and expens e . They therefore conclude that
the b e st estimate o f inhibition by a metal is that concen
tration of the metal which causes an exceptional displace
ment o:f treatment plant performance . Furt�ermore, the
results of their studies showed that for each phase o f
treatment--aerobi c , anaerobi c , and discharge o f final
effluent--there are di fferent bases for judging the
concentrations of metals acceptable in the influent sewage .
For examp le� the plateau-type response o f the aeration
phase showed that concentrations o f metal many tir:?es higher
than the threshold concentration co�ld be received without
greatly reducing e ffic iency. However, in a situation where
removal of organic matter is not critical , Barth et a l .
" ( 1 965a ) suggested that the most sensitive performanc e
criterion might be the ability of the anaerobic digester
to handle the sludges produce d . Finally, in other cases ,
especially where heavy metals in the effluent are monitored ,
the amount o f metal passed through the plant to the
rec eiving stream could be the major factor for determinine
pe�missable metal concentrations in the plant influent.
The degree of complexity and factors limitine
biological wastewater treatment plant performance ara
1 '7
exempli fied by several reports published by the BPA (Eegg e t al . , 1 979 ; Gray et al . , 1 979 ; Keinath e t al . , 1 980 ) . - - -- -
EPA research has addressed the heavy metal problem by the
initiation o f studies to ( 1 ) develop a controlled bioassay
system for measuring toxicity.of heavy metals ( Maney e t al . ,
1 977 ) ; (2) determine the biological e ffects of metals
( Eisler et al . , 1 978) ; ( 3 ) investigate the removal and
recovery of metals in wastewater treatment and sludges
( Scott, 1 980 ; Esmond e t a l . , 1 980 ) ; and ( 4) determine the
applicability o f using bacterial bioassay for heavy metal
toxic i ty ( \'/illiamson and Nelso n , 1 98 1 ) .
I t is not surpris ing , then , tha t numerous approaches
t o the heavy me tal problems exi st and that the scope o!
methodology employ�d has been broad . Blok ( 1 974) presents
an overview of frequently employed respirometric measure
ments made on activated sludg e . Ile indicates that the
most important assumption made in nearly all kinetic views
about activated sludge is that a c omplicated sludge is
regarded as a simple system of monoculture and s ingle
substrate ; this assumption is generally faulty.
Khararjian ( 1 980 ) addressed the use o f oxygen uptake
as a control parameter, and Giona and Annesini (1979 )
presented a thorough review o f the literature pertinent
to the oxygen uptake of the microbial population in an
activated sludge process for both oxidation and
stabilization phase s .
Mowat ( 1976 ) utilized the bio logical oxyger. d�mc..r.·.�
18 ( BOD) t e s t for measurement �f toxicity, a method she
considered perhaps the simpl e s t , most pr�ctical and
available method for any sanitary laboratory . Though its
drawbacks are well documented, its usefulness is also
evid ent. She also stressed the need for development o f
methods using bacteria for the testing o f toxicants and
aptly indicated that bacteria respond more quickly than do
most o ther organisms to changes in their environ�ent and
therefore should be sensitive indicators o� pollution.
Further , she notes that metal pollution effects are
determined at the first stage of toxicity in the biological
cha i n , namely, the microbial level .
At the same level in the bio logical chain as the
bacteria are the protozoans . Reynoldson l1942) suggested
the use of Vorticella as an organism capable of indicatine
the condition of activated sludge . Curds and Cockburn
(1970) did an extensive study o f protozoan species in
biological sewage treatment processes an·d their use as
indicator organsims in the activated sludge proc ess . The
use of the protozoan Vorticella convallaria var similis
in studying the toxic effects of heavy me tals was invest
igated by Sartory and Lloyd (1976) . \'/hether protozoans
or bacteria should be denoted the major indicator
organisms in activated sludee is academic . Pract ical
considerations may, howeve r , dictate the more feasibl3
choice . Bacteria such as gschcrichi?. coll_ dispL:.y a.
doubling time of about 20 minutes unde!" optimal r·:covrth
1 9
conditions at 3 7 ° C , whereas values o n the order of 1 0 hours
are displayed by many protozoa and mammalian tissue cel l s
( Pike e t al . , 1 972) .
Bacterial Reactions to Influent Toxicity. Further
study by Sterrit t and Lester ( 1 970 ) e lucidated the inter-
actions of heavy metals with bacteri a . They conc lud ed
that the major factor determining the toxicity of heavy
metals to bacteria is probably the extent to which they
penetrate the cytoplasm. Also discussed were factors
which influence metal forms and, thus , their potential
toxicity. Two such factors are pH and chloride concen
trati o n , ·which were studied by Hahne and Kroo n t j e ( 1 973 ) .
Other factors include chelation and competi tive inter-
actions . Although the formation o f complexes or chelates
with several metal-binding agents may prevent the metal
from entering the cell , and thus may pro tect the �icrobial
population , their influence on metal uptake may have serious
effects in wastewater treatment processes where immobili za
tion o f ·metals by the biomass is the most important
mechanism of heavy metal removal .
One unique mechanism o f microbial resistance i s that
o f hydrogen sulfide production . Microbial hydrogen
sulfide production often has significant e ffects on metal
toxic i ty , since most heavy me tal s form insoluble sulfides
with H2s . Consequently, H2S-producing oreanisms often
exhibit tolerance to heavy metals ( G add and Griffi ths ,
1 978) . For example , the sulfate reducer
20
Desulfo vibrio desulfuricans produces H2s , grows in high
sulfide concentrations , and may b e unaffe c t e d by the
add i t i o n o f high concentrations o f heavy metals ( Gadd and
Griffi ths , 1 978 ) . Furthermore , i t has beer1 observed that
in some c a s e s sulfide-producing organisms can protect
s e n s i tive organisms from the toxi c i ty of he avy meta l s .
This was exemplified when Desulfovibrio desulfuricans �as
grown i n mixed culture with a metal-sensi tive strain o f
Pseudomonas aerugino s a ; the latter oreanism could t o l erate
higher concentrations of mercurials than when i t was grown
in pure culture , and the H2S produced was the responsible
factor ( Gadd and Griffiths , 1 978) .
Sterritt and Le s t e r ( 1 97 9 ) also d i s cuss the environ-
mentally important phenomenon of bacterial r e s i s tan c e to
metal toxi c i t y . They state that i t may o c cur from non
specific mechanisms , such as impermeabi l i t y o f tne c e l l ,
o r i t may be due to s p e c i fi c r e s i s tance transfer fac tors
( RTF ) .
Resistance transfer factors to a number o f heavy
metals have o c curred in recent years in the same way that
the introduction of an t i b i o t i c s has l e d to the appearance
o f s p e c i f i c RTF ' s to these agents in several bac teria.
These factors are d e termined by extrachrortJo somal gene t i c
material ( plasmids ) . Such g e n e t i c e lements are s � l f
replicating and may b e transferred to bacteria o f the
same and similar specie s . Sterritt and Le s t e r ( 1 97 9 )
indicate that speci fic r e s i stanc e s to m A t al s , wh i c h arc
2 1
o ften linked to antibiotic resistan c e s , can make the
bacteria which possess them resistant to as much as 1 00
times the concentration causing inhibition o f sensitive
strain s . They sugee st that resistanc e s may be co-selected
for , and , therefore , that heavy metal po llution may exert
a selec tion pressure for antibiotic resistance s . Thus ,
this fac t further increases their c lincial importance
( Sterritt and Lester, 1979).
This resistan c e chara cter may be one reason that
adaptation or acclimation can occur. Lester et �l . ( 1 979 )
note that acclimation is a complex process involving
adaptation within species , selectio n o f mutants within
species and selec tion between species , and they further
cite several cases o f known RTF ' s . In add itio n , �mith
( 1967) demonstrated the importance of RTF ' s when he found
resistance for up to four metals controlled by a single RTF.
Adaptation by the microbial population is n o t , howdver ,
limited to heavy me�als . The effects of the persistent
chemical DCP ( 3 , 5-Dichloropheno l ) were shown to d � c line
and were large ly overcome within a few weeks by the
bacteria in an activated sludBe ( Broecker and Zahn , 1976) .
In addition to the occurrence o f adaptation by a
m icrobial population, the organisms in a ctivated sludge
have been demonstrated to utilize or transform certain
chemicals . DiGeronimo et al . ( 1 979) conducted a study
which showed util i za tion of chlorobenzoates , which are
often present in soil because o f their use as h�r�ici� � s ,
22
by microbial populations in sewage . � pseudomonad isolated
from sewage was shown to utilize m-chlorobenzoate as its
sole carbon and energy sourc e . Furthermore , i t was
observed that if decomposition occurred and the substrate
di sappeared , populations capable of growing on the compounds
formed from biodegradation proliferated . This phenomenon,
in which microorganisms may modify chemicals that they
cannot util i ze as sources of energy or as nutrients , was
also observed by Jacobson e t a l . ( 1 980 ) . rhe process is
known as cometabol i sm or, i f the reaction invo lves an
oxidative sequence , cooxidation. The accumulation o f
metabolic products derived from toxic chemicals is o f
e cological concern because the product may still h�ve the
properties o f the precursor that were resnonsible for the
toxi c i t y . Jacobson e t a l . (1980) studied cometabolism in
sewage and found the process to be enhanced by merely
increasing the number of organisms , or by the addition o f
chemicals o f dissimilar structure . If either approach
could be made practical , it3 application could po ssibly
increase the rate of destruction of at least certain
toxic chemicals within the sewage treatment process .
For example , microbiological systems have been
evaluated for their potential value in e l ininating 'r::T
( 2 , 4 , 6-Trinitrotoluene ) present in wastewater frc�
munitions-manufacturing facilities (Carpenter e t �� . ,
1977 ) . I t is stated that the degradation o f T�T e n d
related ni tro aroma tic co:-npounds i s d i fficul t , i · r:o t
23 imposs ible . However, the ability o f biological syste�� to
e ffec t the disappearance of TJ.i'.I h?.5 bro ueht under inve st
igation the microbial transformation of T1T in an activated
s ludge system. The results were unlike those of degradation
o f chlorobenzoate s , where less complex products were
produced . Instead , find ines by Carpenter e t a l . ( 197'1)
suggested that polymer formation may have o ccurred .
G enerally i t i s customary to think o f final prod�cts in
pollution abatement processes as simpler compounds rather
than as products o f a more complex nature . However,
accord ing to Carpenter et al . ( 1 977 ) , polymer formation
may be a more common o c c urrence in the bio transformation
react i ons of aromatic compounds than o therhise suspe c t e d .
They indicate the importance o f considering polym�r
formation in any investigation of the fate of organic
compounds sub je c ted to bioloeical treatment proce�se s ,
since the polymers formed may be persi stent in the
environment and of unknown impact on an ecosystem or the
r e c e i ving water of a wastewater treatment plan t .
Bunch and Chambers ( 1 96 7 ) indicated the need for
determining whether a toxi c substance can be degraded
or assimilated e fficiently by wastewater treatment plants .
They wisely s tressed that i t is no t enougt to know only
that a compound is degradable ; the degradntion rate i s
equally importan t , since this information i s valuable for
predicting the behavior of a compound in a wastewater
treatment plant and in receiving waters .
24
Different approaches to the s tudy o f biodegradation
exi s t . The common herbicide 2 , 4-D may enter wastewater
treatment plants ; however, the study of i t s biodegradation
appears limited to soil studies . Although i t is generally
accepted that 2 , 4-D is short-lived in soil ( Foster and
McKercher, 1 97 3 ) the diversity o f environmental and
experimental conditions have shown variations in degrada
tion times ranging from 7 to 98 days ( Altom and Stritzke ,
1 9 73 ) . Such variability in the de termination o f the
biodegradability o f organic compounds was addressed by
Liu e t al . ( 1 981 ) and a s tandard procedure and new apparatus
for determining the relative biodegradability of both
water-soluble and insoluble organic compounds was preser.ted .
A major herbicide , 2 , 4-D has been used intensively
for macrophyte control in lakes and pond s , and to control
weeds along highways and electrical transmission line s .
Liu e t al . ( 1 981 ) also state that the persistence o f 2 , 4-D
in the environment is quite variable and suggest that this
refle c t s the multitude of conditions which may influence
the degradation processes and which are undoub t edly
present in varying degrees in field s i tuations . They stress
that there is often a great tendency to underestimate the
complexity o f biological processes involved in the
biodegradation o f an organic compoun d . Consequently,
when laboratory data alone are used to predi c t a substance ' s
behavior in the natural ( or another) environment , errors
may o ccur . Therefore, many investigators feel that the
25
best way to test biodegradability is to duplicate i n the
laboratory the conditions as they o ccur in nature or
duplicate the treatment that sewage normally receive s .
This i s evidenced by s tudies using small-scale laboratory
s i zed continuous activated slu.dge uni t s ( Barth e t al . ,
1 965a; Tomlinson e t al . , 1 9 6 6 ; Wheatland e t al·. , 1 971 ;
Pike e t al . , 197 2 ; Weber tl al . , 1 980 ) and by investigators
who chose a field survey method utili zing municipal waste
water treatment plants ( Barth e t al. , 1 9 6 5b. ; Brown � �· ,
1 973 ) . The pursuit o f reliable information which may be
practically applied in wastewater treatment plant operation
is an important goal of such research .
Aspects of Biological Treatment Processe s . I�vest-
igation of the various aspects o f the biological treatment
process of activated sludge has encompassed several areas .
I n addi tion to studies on the activated sludge process
itself, one done by Mosey ( 1 976) assessed the maximum
heavy metal concentration which would not inhibit the
pro c e s s of anaerobic dige stion . Thus , i t appears that
there i s also a need to monitor the heavy metal c c �tent
of sludge sent from the activated sludge process to the
anaerobic diges ter, in order to avoid further treatment
problems .
Jackson e t al. ( 1 970) and Gre en e t al . ( 1 97 5 ) also - - - -
investigated the effects of heavy metals upon anaerobic
digestion and upon other wastewater treatment processe s .
These s tudi e s detected several e ffects o f toxic wastes on
26
receiving waters and their possible e ffects on the aquatic
organisms inhabiting the watercourse s . Green e t a l . ( 1 97 5 )
comment that a measure o f vigilance i s necessary, n o t only
to protect the treatment plant processe s , but also to
monitor important e ffluents w�th a view toward protecting
fish, o ther aquatic life , and the many uses of the water.
Not only is there a need to monitor effluent leaving
the treatment plan t , but also the wastewater s ludge which
has found use i n agriculture . The effect o f heavy metals
in sludge on agricultural crops was studied by Hyde et al .
( 1 979 ) who investigated both the composition o f the sludge ,
and the accumulation o f the metals in the soil and the
resulting concentrations i n plants . An increased effi c iency
i n heavy metal removal by the treatment plant could
facilitate the agricultural use of sludge .
Heavy metal removal by the activated sludge process ,
the e fficiency o f remova l , and factors affecting uptake
have been the topic o f several stud i e s . Cheng et al. - -
( 1 9 7 5 ) investigated heavy metal uptake by activated sludge ;
Neufeld and Hermann ( 1 97 5 ) studied the use o f acclimated
activated sludge for heavy metal removal . I n an invest
�gation of heavy metals-induced deflocculation of activated
s ludge , Neufeld ( 1 976) states that exocellular polymers are
responsible for bioflocculation and that heavy metals
induce deflocculatio n . In addition , he notes that the
exocellular polymers also have the capability o f adsorbing
significant quantities of trace heavy metals from aqueous
27
solutions . The role o f bacterial extracellular polymers in
heavy metal removal in activated sludge is further elucidated
by Brown and Lester ( 1 979 ) , according to whom the extra
cellular polymers are mainly of a polysaccharide nature ,
although protein and nucleic qCid from autolysis are
constituents o f . the polymer matrix .
The influence of certain o ther factors upon heavy
metal removal in the activated s ludge process has been
recently investigated. Sterritt and Le ster ( 1 981 ) invest
igated the influence of sludge age on heavy metal removal .
I n general , most o f the metals studied ( including cadmium ,
c o pper , and lead ) showed maximum removal at an older
s ludge age . I t was suggested that the maximum affinity
o f the metals for the biomass at a s ludge age o f 9-1 2 days
might indicate the presence of specific adsorption sites
on the sludge flo e , the numbers o f which sites decrease
at longer sludge age s . However, the effect o f sludge age
on the affinity of silver for the floes contrasted with
the general trend . The affinity of silver for the sludge
continued to increase and maximum metal concentration
occurred at the 1 8-day sludge age . One possible reason
�uggested was that, unlike the o ther metals , silver forms
a monovalent cation and , henc e , the binding sites for
silver differ from those of the other meta l s . The order
of heavy metal uptake was reported by Berth ( 1 978) to be
Pb> Cu>Ag>Cd> Zn> Hg .
The effec t o f detergent builners on heavy metal
28
removal by activated sludge also has been investigated.
Carrondo � al . ( 1 981 ) studied the Type A zeolite detergent
builders and found them to have no adverse e ffects on heavy
metal removal . The effects o f another detergent builder ,
NTA ( n itrilotriacetic acid) , w�re investigated by Stoveland
et al . ( 1 979 ) with opposite resul t s . They found that
unless NTA were to be biodegraded or otherwise broken down
prior to entering the sewerage system, or were to do so
during i t s passage to the treatment plan t , there would be
a significant reduction in heavy metal removal at the
primary ( mechanical) stage of sewage treatment . At plants
e quipped with secondary ( biological) treatmen t , such as an
activated sludge plan t , the plant would have to tolerate
higher heavy metal loading in addition to an increased
chemical oxygen demand from the NTA. Because of the
problem NTA may present , further study o n i t s biodegradation
has been done ( Warre n , 1 979 ) .
The effi c iency of heavy metal removal has been
s tudied by Brown et .£1• ( 1 97 3 ) in activated sludge plants
o f municipalities ranging f�om a small agricultural
community to an industrial city. Oliver and Cosgrove
( 1 973 ) $tudied a conventional activated sludge treatment
plant which discharges 6 . 5 million gallons of effluent
per day i n to Lake Ontario . Other investigations have
encompassed analyses of heavy metal concen trations in
sludge ranging from a single treatment plan t , such as at
the Oxford ( England) sewage treatment plant ( Lester a t �l . ,
29
1 979 ) , to a survey o f 1 82 sewage treatment plants in
England and Wales ( Williams , 1 97 5 ) . I n add i tion , the
municipal sewage sludges o f sixteen American cities were
analyzed by Furr !:.! al . ( 1 97 6 ) for several elements
( including heavy metals ) and chlorinated hydrocarbons
over the period of a year.
I t is readily apparent that the broad scope of heavy
metal interaction i n activated sludge will require additional
study in several scientific areas . As the need for
regulations on heavy metal concentrations in sludge and
wastewater effluent increases , more attempts to determine
the toxic effects and inhibitory concentrations of heavy
metals will be made . The results o f this study are one
such attemp t .
30
MATERIALS AND METHODS
Description of Sampling Site
A wastewater treatment facility in Charleston ( Coles
County) I llinois was used in this s tudy . The Charleston
wastewater treatment plant ( Figure 1 ) was completed in
1 971 and is capable of treating wastewater from a population
equivalent of up to 20 , 000 people with a hydraulic capacity
for treatment o f 2 to 3 million gallons pe� day.
Sample Collection
One to 2 liters o f aerated activated sludge were
obtained periodically from the Charle s ton wastewater
treatment facility by lowering a sludge collection device
into the aerated activated sludge chamber of the contact
s tabili zation unit ( * in Figure 1 ) . The sludge was placed
into a clean plastic container, capped , and transported
to the Water Quality Laboratory of Eastern I llinois
University, Charleston , I llinoi s . Transport time was
approximately 1 5 minut e s .
Materials
Equipment included a YSI Mo del 54 dissolved oxygen
( DO ) meter ( Yellow Springs Instrument Co . , Inc . ) and an
Omniscribe B-5 2 1 7R-1 5 Strip Chart Recorder ( Houston
Instruments , a Division of Bausch and Lomb) . Other
materials incorporated were a NAPCO Model 2 1 0 water bath
and a Biological Oxygen Demand ( BOD) probe .
All inhibitors were o f reagent-gr�de quality or o i
3 1
' To "C a s s e l l Creek
N
I �
...._
P
_
o
_
n
_
d _
_
N
_
o
_
._,\_J
0 �����-�� ./ I Pond No . 1 )
'---·--_..,_�-----
1 Servi c e Building 2 Aerated Grit Chamber 3 Digestion Tanks 4 Clarifiers 5 Diversi on Structure 6 Parshall Flume · 7 Sludge Drying Beds 8 Chlorination Building 9 Chlorination S e c t i o n
== Waste Sludge """"""' ..... Sewage-Wastev1ater Flow
* Activated Sludge C o l l e c t i o P.
Emergen c::{ �>J_ow
?oi:nt
Rout2
� � Furn.ping � Stat i o n
D ( In�_st)
Figure 1 . Charleston Wastewater Treatment Plant i n Char l e s to n ( C o l e s County) I l l inois .
32
the highest quality availab l e . All solutions were
prepared using water which had been processed by a Milli-Q
( Mi llipore ) Reagent-Grade Water System ( laboratory-pure
water ) . Oxygenated Milli-Q water was kept in a 20° C
BOD incubator.
Methods
General Methods . Upon arrival to the laboratory, the
sludge was transferred to clean Erlenmeyer flasks and a
suffici ent volume of a 1 0% glucose solutio� was added to
give an effective 1 . 0% final glucose concentrat i o n .
Aeration was begun and continued for a t least 1 hour prior
to the beginning of the tes ting period. Aeration was
continued throughout the course o f the experimentation .
The DO meter was calibrated prior to testing each
series according to standard procedure (American Public
Health Associat ion , 1 976) . Preparation of the Omniscribe
strip chart recorder was accomplished during this calibra
tion procedure i n order to render the base line congruent
with the initial meter output . A chart rate of 0 . 25 cm/
minute was used .
Each 300-ml BOD bottle was prepared by adding either
3 . 0 ml of Milli-Q water ( control ) or 3 . 0 ml of the
appropriate concentration o f the inhibitor being tested.
The reaerated activated sludge was swirled vigorously
to insure adequate mixing o f solids and supernatant , and
then poured into the 300-ml BOD bottle as required. The
flange o f the bottle was filled with water to ensure an
33
air-tight seal. The filled BOD bottle was then immediately
placed in the water bath at ambient temperature ( 1 9- 2 1 ° C )
and the BOD probe was inserted • . Gentle swirling or light
tapping brought any air bubbles to the top o f the BOD
bottle ; these were freed and the BOD probe was then
reinserted.
As soon as the DO meter peaked, the time and DO
concentration were recorded. Oxygen uptake , in general ,
appeared linear over time . After about 1 5 _minutes of
linear oxygen uptake, a second reading o f time and DO
concentration was made. ( Minimum time between readings
was 1 5 minutes unle s s the slope o f the line changed,
curved , or leveled o ff significantly . ) In the case where
a s pecific concentration of the inhibitor caus ed complete
inhibition o f dissolved oxygen uptake by the bacteria , the
strip chart continued to record for the minimal time period.
Dissolved Oxygen Uptake . Dissolved oxygen upt��e
was measured by reco rding the initial and final DO concen
trations and taking the difference o f these values divided
by the time elapsed between initial and final readings.
Calculations followed the formula:
[Do] I - [DO] �' 60 min mg/l ------- x = o f DO uptake
T min 1 hour hour
where [no] I is the initial DO concentration, and [Do] F
is the final DO concentration. Time ( T) is in minute s .
Percent Inhibition. Percent inhibition was calcu-
34
lated using the control DO uptake as the standard for
compari son . The control was run both before and after
each series of test runs . Thus , . in the following formula
�� - represents the average DO uptake for two control c
runs . The initial DO concentration in the presence of the
inhibitor is represented by [no] Ii ; similarly, [Do] Fi
represents the final DO concentration in the presence of
the inhibitor. The formula used is :
[noJ I i - [DO] Fi
[no]_ c
X 100 = % Inhibition
r r c50 • The rrc50 i s the initial inhibitory concen
tration which causes 50% inhibition of dissolved oxygen
uptake during the initial 1 5-minute period follow i.ng the
addition of the inhibitor. Each rrc50 was determined by
extrapolation of plotted data. Each inhibitor was tested
at various concentrations ; the percent inhibition was
calculated , and the percent inhibition results for 3
replicates were averaged . The inhibitor concentration
which matched or most closely approximated the rrc 50 was
chosen for subsequent use in the short- and long-term
studies .
The inhibitors tested with their test concentrations
in mg/l included silver ( as AgN03) , 0 . 001 , 0 . 0 1 , 0 . 10 , 1 . O ,
10 , 100 , 1 2 5 , 2 5 0 , 500 , 1 000; copper (as Cu( H03 )2
) , 1 . 0 ,
10 , 1 00 , 125 , 2 50 , 500 , 1000 ; and mercury ( as Hgsc4 ) , 0 • 001 , 0 . 01 ' 0 • 1 0 ' 1 • 0 ' 3 • 1 2 5 , 6 . 2 5 ' 1 0 ' 1 2 . 5 ' 2 5 • 0 ' 50 . 0 ,
3 5
1 0 0 . Cadmium ( as C d ( No3 )2
· 4 H20 ) , lead ( as Pb ( N03 )2
) y
and 2 , 4-D ( 2 , 4-Dichlorophenoxyacetic acid) were tested at
concentrations of 0 . 00 1 , 0 . 0 1 , 0 .• 1 , 1 . 0 , 1 0 , 3 1 . 25 , 6 2 . 50 ,
1 0 0 , 1 25 , 250 , 500 , and 1 000 mg/ l . Terbacil ( 3-tert-butyl-
5-chloro-6 methyluracil) and Zectran ( 4-d imethylamino-3 , 5-
xylyl methyl carbamate ) were t ested at concentrations o f
0 . 00 1 , 0 . 0 1 , 0 . 1 0 , 1 • 0 , 1 0 , 1 00 , and 1 000 mg/ l .
Short-Term Study . This study measured the effect of
the inhibitor on the disso lved oxygen upta�e within a 4-
hour time period , a length of time indicative of the
inhibitor ' s immediate e ffect upon the contact stabilization
unit of a sewage treatment facility. Utilizing the I I C50
or the concentration most closely approximating i t , the
percent inhibition was determined for each inhibitor for
each hour during the testing period ( i . e . , at O , 1 , 2 , 3 ,
4 hours ) . Each inhibitor series was run in triplicate and
the results were averaged .
Long-Term Study . This study extended over a 2-week
period and was indicative o f possible inhibitory effe c t s
on dissolved oxygen uptake by the bacteria and protozoans
in the activated sludge process by an inhibitor which
remains within or is continually introduced into the
sewage treatment facility.
Nine Erlenmeyer flask s , accompanied by aeration
devices , were each filled with 800 ml o f reaerated
activate d sludge and the appropriate concentration for
the inhibitor or water ( water contro l ) or ethanol ( e thanol
36
control ) equivale n t s . One flask contained only reaerated
activated sludge undergoing aeration in order to measure
endogenous dissolved oxygen uptake by the bacteria and
protozoans . To the remaining 8 flasks a gluco se solution
was added to obtain a final 0 . 0 1 % concentration . This
concentration was deemed to be a sufficient carbon and
energy source for the bacteria through experimentation on
succ e s sive d e cimal d i lutions of a 1 0% glucose solution .
Furthermore , to ensure a continually adequ�te supply,
8 ml o f the glucose solution was added daily to each flask.
Percent inhibition was calculate d by measuring changes
i n dissolved oxygen uptake at days 0 ( i . e . , after the
initial addition of the inhibitor ) , 0 . 1 1 , 1 , 3 , 6 , 9 , 1 1 ,
1 3 , and 1 4 . The 0 . 1 1 day measurement i s equivalent to
2 3 /4 hours following the addition o f the inhibitor.
The s tudy was replicated onc e . Potential for adaptation
by the microbial population to the inhibitor was derived
from these experimental proc e dure s .
• ,
37
RESULTS AND DISCUSSION
IIC 50 DETJ:.�filUNATION
Inhibitory levels resulting from various concentrations
o f the toxic substances are presented in Figures 2 through
8 . The figures represent the average o f 3 trials. , except
for the maximum concentration o f all inhibitors , which is
the average o f 6 trials , and for results obtained o f a
diluted activated sludge ( Figure 5 ) , which .is the average
o f 4 trials . All error bars show one standard deviation
on either side o f the mean.
The I I c 50 is defined as the initial inhibitory
concentration which cause s 50% inhibition o f dissolved
oxygen uptake during the initial 1 5-minute period following
the addition of the inhibitor. The rrc 50 is comparable
to the Lc50 ( Lethal Concentration for 50% o f the popula
tion ) , a concentration which changes with time as noted
by Broecker and iahn ( 1 97 7 ) . In their study on DCP
( 3 , 5-Dichlorophenol ) , the Lc50 after 20 hours was about
60 mg/l and after 9 2 hours 25 mg/ l ; the toxicity limit
after 20 hours was about 5 mg/l and after 92 hours below
2 mg/ l . They found that after a test perio d of 92 hours
the toxicity limits and LC50 were approximately 50% too
low. Thus , the definition of the rrc50 must be borne in
mind when applying laboratory results to sewage treatment
plant o peration . Table 3 lists the rrc50 de termined for
38
Table 3 . The rrc50 for six inhibitors of the activated
sludge process in a wastewater treatment plant .
a
b
I nhibitor
Mercury
Copper
Silver
Cadmium
Lead
2 , 4-D
a IIC 50 ( mg/l)
20
50
43
1 1 5
550
430
Concentrationb
h'mployed (mg/l)
25
3 1 . 25
1 00
1 2 5
500
500
from extrapolation of plotted data.
for subsequent studie s ; that 2-fold dilution of the maximum concentration applied closest to the rrc50 •
3 9
each inhibitor. I t appears that mercury toxicity occurs
at a lower concentration than the o ther inhibitors teste d .
Mowat ( 1 97 6 ) also obtained results supporting mercury as
the most toxic inhibitor.
In the determination of the cadmium I I c 50 it was
observed that the degree o f inhibition remained relatively
constant from 0 . 00 1 to 1 0 mg/l ; however a slight ( less than
1 %) stimulation o ccurred at the 1 . 0 mg/l concentration .
As shown in Figure 2 , the largest increase . in inhibition
was 1 796 and occurred between the 1 0 and 3 1 . 25 mg/l concen-
trations . Each successive doubling o f the concentration
after 3 1 . 25 mg/l was accompanied by about a 1 0% increase
in inhibition . The r r c 50 for cadmium was determined to
be 1 1 5 mg/l . Studies by Weber and Sherrard ( 1 980) indicate
that cadmium does not reduce the rate of organic removal
below 1 00 mg Cd/l . �warun ( 1 97 3 ) attributes bacterial
tolerance to cadmium to the mode of interaction . Cadmium
is known to exert its toxic effects by replacing zinc in
carboxypeptidase enzyme systems which catalyze peptide
degradation . Consequently, the indirect effects o f
cadmium on organic utili zation may n o t be observed until
large quantities of cadmium are presen t . Zwarun ( 1 97 3 )
also states that po ssibly the bacterial cell wall plays
an important role in protection from toxicity. I n addi tion ,
the uptake o f any given cation and the resulting effects
on an o rganism may be a function of the concentration o f
all other cations in the medium . Abelson and Aldous
0 0
0 O)
40
r\·r'.l J l-.11....;· I \. • . :l;01 . ,\.
._) '-1'"
�g 1; i o , � • <-..1 -{r(J
•
i I
.... () N 0 'J tD N \{) : .. o :\...[
I j �'
·:,) N I
,, -
• n
,., -
• ... ,
c
...., !".)
r :
C'J
4 1
( 1 9 5 0 ) were able t o demonstrate a great reduction in the
toxicity of Cd2+ ions by increasing the conc entration of
magnesium, since the toxicity of these ions at low concen
trations ( Lester et al . , 1 979 ) is a consequence of their
interference in the normal metabolic role of magnesium .
Synergism or antagonism may also occur with a mixture o f
metal s . A s tudy by Sterritt and Lester ( 1 980 ) indicated
that very low concentrations of Cd2+ potentiated the
l e thal a ction of c u2+ .
Determination o f the Copper r r c50 indicated that at
low concentrations a tenfold increase in concentration
( from 1 . 0 to 1 0 mg/ l ) resulted in a 1 6% increase in
inhibition , whereas the same increase in percent inhibition
occurred after only a doubling of the concentration at a
higher c oncentration ( from 6 2 . 5 to 1 2 5 mg/l ) . Further
doubling of the concentration from 1 25 to 250 mg/l , from
2 50 to 500 mg/ l , and from 500 to 1 000 mg/l produced only a
6.l. 2 , 2 , and 6 % change , respectively, in inhibition .
it appears tha t , for copper, once a c ertain threshold
Thus ,
inhibitory level is attained , inhibition increase s up to
a point , 1 25 mg Cu/l in this cas e , and then increases
relatively little . This reaction pattern was observed by
Barth et a l . ( 1 965b) for copper, chromium , nicke l , and
zinc . They concluded that a small dose of the heavy me tal
gave a significant reduction in treatment efficiency, but
that substantially larger doses did not further decrease
the efficiency greatly. This is po ssibly related to the
42
mode of interaction between the bacteria and the heavy
m e tal , such as copper, such that after saturation o f
interactive s i t e s , the change in percent inhibition is
relatively small and approaches constancy. Overall ,
Figure 3 indicates that there is a gradual increase in
inhibition with a leveling-off at the higher conc�ntrations .
I t was observed during the lead rrc 50 determination
that the percent inhibition did not fluctuate extensively
over the concentration range from 0 . 00 1 to . 1 0 mg/ l . At
the 3 1 . 25 mg/l concentration , actual stimulation o f
dissolved oxygen uptake o ccurred in 2 out o f 3 trials ,
while the third trial measured only a 1 % inhibitio n . The
standard deviation indicated a possible high value o f 0 . 6%
inhibit ion but the significance o f this value would
require further study . I t is probably a result o f the
inherent sludge variability, since a different sludge was
used for each of three trials . ( Tomlinson � a l . ( 1 96 6 )
found that there was variation in response o f different
batches of activated sludge to an inhibitor . ) A steady
increase in inhibition by lead from 3 1 . 2 5 to 250 mg/l
was not e d , with the exception that inhibition de creased
�lightly ( 3i�) from 1 00 to 1 2 5 mg/l . Once again , this
observed e ff�ct could have resulted from differences in
sludge sample s . A marked increase in percent inhibition
with a 3 1 % change from the 250 to 500 mg/l concentration
occurred as indicated in Figure 4 . Similarly , a 22% jump
in percent inhibition o ccurred from 500 to 1 000 mg/l .
0
1.0 1 0 :� 1.2 � 62.!::> k)O 12t> 2';:;J SCO :coo Cu CQr,!CFNT RJ.\TIO'J (.'-".C/L)
Figure 3 . The effe c t of Copper o n d i s s o lved oxygen uptake in activated sludg e .
44
�� I I
''j I I I I I
._.j I I I i
- ... ...__, ---!...--.---1., ___ ....... __ __.l. _____ , !...., ---- -.1---. 0 u () u 0 0
0 cO l_O "1" . r,; l N' �J' . . ,
__ I :J ( j ;cJ
L() - (\j 0 0 l () rJ \.0
-_() ("\]
' I ,_ ,
') -.. �
o O . -
z 0 � 0::: I-
I � l z - -. 0 O u
jl 8 - 0 () \\.'. I
45
This c o n trasts with the inhibitory effe ct observed at
s imilar high concentrations o f cadmium and coppe r . . I n
both cadmium and copper, al though inhibition coritinued to
increase at upper l e ve l s , the change s in perc e n t inhibition
were more gradual . Perhaps this is due to the fact that
lower concentrations o f bo th cadmium and copper proved
more inhibitory than similar c o n c e ntrations o f l e a d , and ,
thus , the effects o f lead inhibition were only beginning
to become pronounced at the higher c o n c e n t�ation s . This
sugg e s t i o n is suppor t e d by the fact that the rrc 50 for
lead , 5 50 mg/l , was much hisher than tho s e determined for
cadmium , 1 1 5 mg/l , and copper , 50 mg/l .
The inhibitory effe c t s o f lead were also t e s t e d in
an a c t i va t e d sludge having reduced suspended s o l i d s . A
1 : 4 d i lution o f 1 00� a c t i vated sludge result e d in a 75%
d e crease i n suspended solids per uni t volume . The result-
ing trend , shown in Figure 5 , i n d i c a t e s that the averaee
percent inhibition in the 25� activated sludge was greater
than tho se obtained at similar c o n c e n trations t e s t e d with
1 009� a c t i va t e d sludge ( Figure 4 ) . This trend may i n d i c a t e
that suspended s o l i d s t i e up some heavy m e t a l a n d d e c rease
the po ssibility o f its interaction with the microorganisms .
Tomlinson e t al . ( 1 9 6 6 ) found that the c o n c e n tration o f
t o t al s o l i d s affe c t s the a c t i v i t i e s o f the compounds beine
t e s t e d . In activated s ludge , metals can be adsorbed by
organic mat t e r ; it is important therefore to recogn i ze
that a plant may be able to withstand relatively high
0 0
4-6
I I >---�---1
I 0 () 0
() 0
0 d
-
0
(+-1 0
.p <.> (!)
. -Tl � · Q.) <!.)
b!l (:) ·-c ,.!..:; ;:j 8 rl
(/)
. "' l.(\ (i)
+> ;;,) ciJ H > � "rl �:;-:;
.. -: () f:r-1 C'J
47
concentrations of added metals without serious loss of
activity. In one study ( Gadd and Griffiths , 1 978) , i t
was found that protozoa were unaffected by copper concen
trations up to 5 mg/l and the reduction in overall effic ien
cy was only 4% even at concentrations of 25 mg/ l . Thus ,
the suspended solids content of activated s ludge appears
to be an important parameter if a s ludge i s to survive or
acclimate to the effects o f heavy metal inhibi tors .
For mercury ( Figure 6 ) , less than 1 %. �nhibition
occurred at concentrations below 0 . 1 mg/l , an average o f
3 . 4% s timulation occurred a t this leve l , and , above i t ,
progressive increases i n inhibition were note d . One
exception to the latter generali zation was a 1 7� decrease
in inhibition between 1 00 and 1 25 mg/l . As described
previously for lead , this effe c t probably resulted from
the inherent variabili ty in suspended solids and other
factors in the different s ludges . No stabilization in
inhibitory effects was indicated at higher concentration s ;
this progressive inhibition occurred a t concentrations
lower than those for any other inhibitor tested . This
feature is reflected in the rrc50 for mercury, 20 mg/l ,
which was the · lowest inhibitor concentration to show a
50% reduction in the dissolved oxygen uptake by t�e
microbial population . Higher concentrations of m�rcury
were not teste d . Neufeld ( 1 97 6 ) found that virtually
any mercury level ( 1 , 1 0 , 30 , 1 0 0 , 300 , 1 000 mg/ l ) wi ll
result in some degree of deflocculation, an indication of
:oJ[ ,
_ 8 cT () I 1 _)
1 [:: 60 ·
' l 1 C) l z.
�. 40 1 . L..1.1 \...) cc
t 2or' r ./
l I --r_., ...-Qr J-- I --- -- i .
I l . 2 C I
•
0001 C.i.0 1 O.i 1.0 3.1 2 5 6.25 10 i2.5 25 50 ICO Hs COf'\CENTRAl !ON (MG/1 )
Figure 6 . The erfe c t of Mercury <>n d i s solved oxyeen uptake in n c t i v�tod sludge .
� (X)
49 i t s inhibitory effect on the bacterial populatio n .
In genera l , s i lver showed minimal ( less than 5%)
inhibition at and · b e low the 1 . 0 mg/l leve l . Inhibition
increased by a factor of 8 between 1 . 0 and 1 0 mg/l ( i . e . ,
a change o f 32%) , increased by nearly 26% over the range
from 1 0 to 1 00 mg/l , increased by 1 1 � with only a 25 mg/l
increase in concentration from 1 00 to 1 25 mg/l , and
exhibited the last marked increas e , a change o f nearly
24�, from 1 25 to 250 mg/ l . As shown in Figure 7 at 250
mg/ l , percent inhibition stabili zed at about 98%.
The inhibitory effects of the herbicide 2 , 4-D were
much less pronounced than tho se of the heavy metals tested
in this study during the IIC 50 determinations , with the
possible exception o f lead . Both 2 , 4-D and lead were
de termined to have r r c50 1 s in the range o f 500 mg/l .
Figure 8 shows that less than 2% inhibition occurred at
concentrations below 6 2 . 5 mg 2 , 4-D/1. At 62 . 5 mg/ l , the
inhibitory effects increased; a doubling in percent
inhibition o c curred at each succes sive doubling of the
concentratio n . There was little d i fference in inhibition
be tween the values measured for 1 00 and 1 25 mg/l .
The immediate inhibitory effects of Terbacil were
found to be relatively s table and small ( i . e . , less than
2%) at and below the 1 00 mg/l concentration . Only 1 7%
inhibition o f dissolved oxygen uptake o ccurred at the
1 000 mg/l level ( Figure 9 ) . Similarly, the inhibitory
effects of Zectran were small ( Figure 1 0 ) . Les s than 5�
0 0 0 0)
50
\ ·--�
'
i �r I I
.....
.. 1 .... 0
·o
0 (\j I
' '\ '- .I Q 8 �0 () LO N :..() � 0 0
0
r-· <( cc:: 1-7 U."i u z . 0 O U
�·
5 1
� \ I + I I ! i I I l +
I
I l
� - �
I I
.n N - o 0
0
0
('; ....:�· .-., �.;: '-.../
_, 2'. c=s f-: <.( er: 1-z i.jj u :-<::
- -: 0 O U
0 0
0 I
tj-�
N
.,-1
.. N
0 0 0 CD
52
�\
C> () 0 l....O tj· !'J t- i u'"'.: 1 in1 : !' ;! 1 , . :·y . . -·, , ' • -'- 1i:.l · 1 \I ' . , . ·1 ···1 -:c· .. -··· . ....... ... � �
\
I
I ·t
I
I I ·1 I ! j I
J
I I I I
r-r� i i
I l I . ... (.)
! n -1 0 0
I 8
- ()
_J u <( co 0::: l.ll
_ f-
8
[j , .. , - r,.""' l 'J ,.J
0 \\ I
'O (j)
+> ..... ·� . , ..
'0 Q) :>
rl 0 r::i (.:
4; 0
-..-·· () Q)
(;-l <,-, 0
0 0
0 co
53
�L
\ I \
\ \ \
\ t I
'1 I I
L l i I ! I I ._.,--1.
0
�· ..... �) �..0 , . � � ) , .. 0 ...,
-j rfJ
'C c;
-P ('j .... . �
0 ·rl .p - 0 ()
m i:::
·rl
C> ,J
� .p ,... , ..... � 0 ;:::; Q) ... -'......) ;...� "' " 0
'O Q)
, .... � � rl \..? 0
- 0 �- (;� \.._/ {.') -,· .,..j Q rrJ L.... � <{ 0 n::: f- � ... z (!j IJJ f I r-. u .p 7. () -: 0 0 O U N z � <( ;.: n:: .p f- () u <:..> w :,..t N 4--i C'
0 Q) d ..G E�
,::,.
Cl> n �· � 0 ·-n ... ;,... (j .,..,-� �· 0
('\ I ,_ I
54 inhibition occurred below the 1 0 mg/l concentration , at
which ( i . e . , 1 0 mg/l ) a slight ( less than 1 %) stimulation
o f dissolved oxygen uptake occurre d . However, at 1 00 mg/1 1
a 9% inhibi tion was measured and a 1 6% inhibition o ccurred
at the 1 000 mg/ l . S ince the overall inhibitory effects
of Terbacil and Zectran on activated sludge were found to
be negligible or moderate ( less than 1 7%) at all concen
trations tested during the IIC 50 de terminations , these
substances were e liminated from subsequent .analys e s .
The IIC50 , o r that 2-fold d i lution o f the maximum
concentration applied closest to the Irc 50 , was utilized
in subsequent analys e s , which were aimed at determining
the short- and long-term effects of the inhibitor upon the
activated s ludge process . According to Tomlinson e t al.
( 1 9 6 6 ) , the immediate o r short-term effects of inhibitors
are not always a goo d BUide to their influence when applied
continuously to activated sludge plants operating under
steady conditions because other orGanis.ms in activated
s ludge may develop the abi lity to decompo se the inhibitor.
Also , the long-term effects of inhibitors o ften differ
from their immediate effects since activated sludge may
possess the ability to become adapted o r resistant to the
inhibitor. Howeve r , the long-term effects may appear to
be generally much more severe than would be expected from
their short-term effe c t s , possibly because the adsorptive
or complexing capacity of the organic matter in the sludge
is limit e d and eve�tually may b ecome exhausted ( Tomlinson
5 5
et a l . , 1 966 ) . In additior. , Jackson e t al . ( 1 970) indicate
that there is still great uncertainty about the long-term
e ffects o f low inhibitor concentrations under natural
c onditions . They recall that in some cases 1 /3 o f the
48-hour LC 50 may be too high for safety in receiving
s treams due to inhibitor effects on the fish pop�lation .
Therefore application o f laboratory results to natural
ecosystems must be approached with caution and the results
o f these studies on activated sludge may b� only a partial
indication o f the inhibitory effects of the toxic substances .
Short-Term and Long-Term Studies
The results obtained from short-term studies of the
inhibitors over a 4-hour period are shown in Figures 1 1 ,
1 4 , 1 7 , 2 0 , 23 , and 26 . For each separate inhibitor,
results were obtained from three replicates of the same
sludge sample , but the sludge sample differed for each
inhibitor tested. Error bars indicate one standard
deviation o n e i ther side o f the mean . Replicates of the
long-term study o f each inhibitor are shown in Figures
( 1 2 , 1 3 ) , ( 1 5 , 1 6 ) , ( 1 8 , 1 9 ) , ( 2 1 , 22) , ( 24 , 2 5 ) , and
( 27 , 28 ) . Each of the two replicates ( i . e . , Replicate 1
and Replicate 2 ) came from different sludge samples and
becaus e of the observed variability, the results were
not averaged . Each long-term study spanned a 2-week
peri o d .
The short-term s tudy o n cadmium indicated an iP-itial
inhibition o f 43% followed by an add i tional 1 7� increase
56
i n inhibition after only 1 hour in the presence of 1 25 mg
Cd/l . Further inhibition occurred gradually and tapered
off with time ; typical changes are 7% over 1 to 2 hours ,
5% over 2 to 3 hours , and about 2% from 3 to 4 hours . As
shown by Figure 1 1 , the maximum inhibition achieved after
4 hours was 75%, an increase of 32% over the 4-hour peri o d .
Both replicates o f the cadmium long-term study i ndicate
a marked increase in inhibition within the first 3 hours ,
then a gradual decrease in inhibition foll�wed by increased
inhibitory effects after 9 day s . Inhibition aeain declined
after day 1 1 but began increasing within the last 2 days
o f the study . Overall , Replicate 1 indicates there was
l e s s than a 5% recovery from the initial rrc 50 inhibitory
effects , and less than 2% recovery in replicate 2 .
Therefor e , from Figures 1 2 and 1 3 i t appears that little
adaptation by the microorganisms occurred in the presence
o f 1 1 2 mg Cd/l within 2 weeks . These results are
comparable to those obtained by Cheng et a l . ( 1 97 5 ) in a
study which revealed a rapid initial uptake o f cadmium by
sludge , followed by a long-term slower rate of uptake .
The amounts o f cadmium uptake after 1 0 minutes of me tal
s ludge contact time were 8 6 , 8 4 , and 74 %, respectively,
for initial cadmium concentrations of 2 . 0 8 , 1 0 . 3 , and
2 5 . 5 mg/l . The association o f metal with the sludge was
essentially comple te after 1 0 minutes o f contact time .
These data demonstrate that activated s ludge has the
abi lity to remove and accu.mula te metal ions f:com s o lu t i o n
�00
90
80
:z: 70 0 I--CD 60 I ::z:
!-- so .... ., ..:::.. w u 0:: I 1J 40 o._
30
20
10
57
r
·---··'--·-·---.-1....--- --l-·------....l. -----l-...-..-�"-
() 2 .:; -� T1 1.1.c.' (· Jo• .,., ,..) 1 • '" • 1 -1, JI\ .;:)
Figure 1 1 . Short-term effe c t s o f Cadmium r r c 50 on d i s solved oxygen uptake i n a c t i vated s lud:Ze .
2'. 0 f-en T z
f--z Ld u er: l.i.J Q_
7 0 f-rD -::r: ::.::: 1 --z LLI u CY-LJJ Cl .
58
i OO 90 80 70 60 50 / 40 3 0 2 0 1 0
00!1 I 3 6 9 1 1
! 0 0
T l M E (Dt-rs) Figure 1 2 . Long-term effe c t s o f Cadmi�� rrc50 on d i s solved oxygen uptake i � a c t i vated sludge l Replicate 1 ) .
9 0 ' ("" 0 r (j
7 0 6 0 50 4-0 -��.----/ 3 0 2. 0
1 0 0.Q'I U .. I 3 . I I . i3 ! 4
Figure 1 3 . Long-term effe c t s o f Cadmiun I I C s o on d i �: s o l vcd oxnv::n uptake i 11 act ivated sludge ( ae pl i c a t e 2 ) .
59
in a rapid initial phas e , followed by a second slower phase
o f uptak e . Cheng et al . conclude that under aerobic
condi t i o n s , metal uptake by the .biomass i s characteri zed
by a very rapid phase of 3 to 1 0 minutes , followed by a
long-term , slow-phase uptake . At lower metal concentration s ,
metal i s taken up by the biofloc through the for�ation of
metal-organic complexes . At higher concentrations , in
addition to sludge uptake , metal ion prec i pitation from
s o lution may occur , which may explain the gradual reduction
in inhibition observed at these leve l s . I t is o f interest
to note that Cheng et al . observed similar uptake patterns
for lead and coppe r .
I n the present investigatio n , the maximum inhibition
observed for copper in the short-term study was equivalent
to that measured for cadmium , namely , 75%. Simi larly �
there was a 40% initial inhibition for copper which is
comparable to the initial inhibition noted for cadmium .
However , the concentration employed for copper was 3 1 . 2 5
mg/l, and that for cadmium 1 25 mg/ l . According to
McDermo t t � a l . ( 1 96 3 ) reduction in treatment effi ciency
caused by 1 0 , 1 5 , and 25 mg/l of copper averaged 4% or
les s . In the copper short-term study , a marked increase
i n inhibition occurred during the first hour following
addition of the inhibi tor. After this initial 3 2% increase
in the first hour, further increases were small : 4� from
1 to 2 hours ; 3% from 2 to 3 hour s ; and 0 . 2� fro� 3 to 4
hours ( Figure 1 4 ) . In a study d o �1e " ., .. . .. . · . .
1 0 0
9 0
8 0
z 70 0 f-(1) :c z 6 0
I-z - o l;j � u er w 40 0..
30
20
1 0
0
60
I 2 Tl:vlE (tOURS)
t--r-
3
Figure 1 4 . Sho=t-term effects of Copper I I C�0 on dissolved oxygen uptake in activated s ludee .
6 1
( 1 9 59 ) , curves for the cumulative oxygen utilization by
sewage microorganisms in the presence o f 0 to 20 mg Cu/l
showed an initial lag in oxidation , which varied in duration
according to the conc entration of copper, followed by
recovery and a more or less rapid uptake of oxyge n .
During the long-term study, both replicates showed
a rapid increase ip inhibition within the first 2 3 /4 hours
after addition of the copper concentration employed .
Similar results for Replicates 1 and 2 o f t�e long-term
study are indicated in Figures 1 5 and 1 6 , respectively.
Both sets of results show alternating periods o f inhibition
and semi-recovery. Overall , there was only a 1 % difference
in initial and final percent inhibition measurements in
Replicate 1 and no measurable inhibition occurred at d ay
1 4 o f Replicate 2 . The periodicity of inhibition followed
by semi-recovery is indicative of changes in the bac t e r i � l
population probably related to the process of acclimation .
McDermo tt et al . ( 1 96 3 ) found that s lug doses of a few
hours ' duration with up to 50 mg Cu/l had only a s light
e ffect on treatment e1ficiency; on the other hand , those
slug doses greater than 50 mg/l had severe effects on the
efficiency o f an unacclimated system . However, the system
returned to normal in about 1 00 hours . The 1 00 , 2 1 0 , and
4 1 0 mg/l doses caused severe effects over the 1irs t 48
hours , in proportion somewhat to the copper d o s e ; but
even with doses to 4 1 0 mg/l , the sludge was not destroye d ,
and it recovered to normal i n about 1 00 hours . A study
z 0 !-OJ :r: z f-z w � Lu CL
:..:-:: 0 f-CD ]-• 7 . �
f--z w u 0:::: l Ll (\
100 90 -8 0 7 0 6 0 5 0 -4 0 -30 -20 lO -
00!1 I
62
3 6 9 Tl�J:E (o.:.:rs)
I
1 1 13 1.:�
Figure 1 5 . Long-term effects o ! Copp�= IIC 5 0 on dissolved oxygen uptake .:. n a c t i vate d sludge ( Replicate 1 ) .
100· 90 80 -70 60 50 -40 30 2 0 -10
'
0011 1 . , 0 6 9 Tl· · r ( J • vi -.
I:"\'·(· ,, J L._ .Jf ' .:-.
1 1
Figure 1 6 . Lo:ic;-te:::'m e ffe :":.s o 1' Cop?er I I C 0 on dis solved. oxyecn uv�ake in actfvated s ludge ( P.e?li cnte 2 ) .
..
63
by Bar.th et al . ( 1 965b) showed that treatment plants
receiving heavy metal slug doses in the range of 1 to 9
mg/l on an almost continuous basis exhibi t e d no serious
reduction in effi c iency of the aerobic or anaerobic
treatment of sewage . Both McDermott et al . ( 1 9 6 3 ) and
Barth e t al . ( 1 965b) concluded that the maximum copper
concentration that does not have a detectable effe c t on
treatment efficiency i s 1 mg/l . In addition , McDermott
e t a l . ( 1 96 3 ) found 0 . 8 mg/l as the maximum co pper
concentration necessary to obtain an effluent no t signif
icantly affected ( i . e . , turbid ) , since increased turbidity
o ccurred with increased copper concentration beyond this
level . Williamson and Nelson ( 1 981 ) found that the toxic
level for biological nitrification was 20 . 0 mg Cu/l .
Thu s , levels o f inhibitors may significantly affect one
process and not another. It is therefore pertinent to
consider parameters of the activated sludge which affec t
the interaction between heavy metal and activated �ludge
since interaction affects treatment efficiency. A study
done by Tomlinson e t a l . ( 1 96 6 ) indicated that , in c o ntrast
to organic compounds , copper and mercury affec ted
Nitrosornonas in mixtures of activated sludge and sewage
less than they d i d in pure cultures o f the same organism.
They c o ncluded that this discrepancy was very likely due
to the formation o f chemical complexes or physical inter
actions between the metal and organic matter in the sewage
or sludge . This conc lusion was sup)o rted by �territt �nd
6 4
Le ster ( 1 980 ) , who determined that the toxic effects of
metals on the activated sludge process can be alleviated
by increasing the concentration of suspended solids
( biomass ) . In addition , they found that the density of a
bacterial culture or population may also influence the
toxici t y o f heavy metals . The inhibitory effects o f copper
on the growth o f bacterial cultures can be eliminated by
the addition of more living or dead c e lls , and , therefore ,
adaptation may occur . Lester et al . ( 1 979 ) determined
that the e ffects o f slug doses o f both copper and lead were
similar in that there was an immediate and drastic reduc tion
in the t otal population within 2 hours followed by recovery
o f the resis tant bacteria beginning 1 2 to 24 hou�s after
the addition of metal . Although this initial marked
inhibition was evident in dissolved oxygen uptake in the
present study, minimal recovery had begun only in the case
of copper . These dat� may b e due to d i ffering magnitudes
in the 1 rc 50 1 s . The initial inhibition resulting from the
employed concentrations probably varie d because lead is
adsorbed by activated s ludge more e fficiently thar. are
copper and cadmium ( Sterritt and Lester, 1 980 ) .
The results obtained from the short-term study o f
lead inhibi tion are indicated i n Figure 1 7 . Initially , a
40% inhibition occurred , which is a value similar to those
observed for both cadmium and copper, although the rrc50 for each differe d . However, unlike either cadmi�n or
copper, the inhibition by lead incre&sed to a maximum o f
6 5
iOO -
90 -
80
2 70 0 I--
f Co 60 -_I
r i�-r1 · z
� so - �r' . w u 0: 8: 40
30
20
!O
, ___ __,_ ___ _..._ ___ , ____ J __ . __ __._ __
0 2 3 · 1 1Mt. G.ouRs)
.• '-r
Figure 1 7 . Short-term effects o f Lead I I C � o o n dissolved oxygen uptake in activated J
sludg A .
66
60% over the 4-hour period . I ncrease i n inhibition during
the short-term study occurred gradually; for each o f 4
successive hours i t changed 4%, 8%, 5% and 2%, respec tively.
During the lead long-term s tudy there was no great fluctua
tion in inhibition over the 2-week period . Replicate 1
showed 53% average inhibition with a 4% s tandard deviatio n .
The effe c t s of lead are relatively consistent and little
recovery o ccurred ( le s s than 2�) within 2 weeks ( Figure 1 8 ) .
Although the results from Replicate 2 ( Fig�re 19) were
generally similar to those from Replicate 1 , an increase
in inhibi tion was observed between days 1 3 and 1 4 in
Replicate 2 . A lack of fluctuation in inhibition is
probably due to the interacitve mode of lead with the
microbial populatio n . Tornabene and Edwards ( 1972) found
that lead in concentrations up to 2 . 5 grams/l had no effect
on the bacteria Micrococcus luteus or Azo tobacter spp .
They determined that when bacteria were exposed to 2 . 5 g/l
of lead , over 99% of the lead associated with the cells
was on the cell walls and outer membranes of the bac teria.
This indicated that less than 11; of the lead had penetrated
into the cytoplasm . They concluded that this phenomer.on
was the reason that extremely high lead concentrations
had no e ffect on the bacteri a . In contrast , unadapted
populations o f Vorticell� convallaria var simili s , a
sessile peritrich protozoan found �bundan tly in healthy
rivers , activated s ludge , percolating filters and slow
sand filters , were sub j ected to a range of lead concen-
� )
' 100 L 90 7 80 0 f.--: 70 Q) J: 60 �-: ,.. o r.____ I- J 7. 40. L!..l
ii 30 g: 2 0
1 0
6 7
........ _._ __ ...___ ___ ..__ __ __. __ _.__ __ ...___, . _ 0011 1 3 9 I i !3 1·'1·
TlM� (DtlVS) Figure 1 8 . Long- t e rm e f fe c t s o f Lead I I C �0 on d i s s o lved oxygen uptake ir: acti vated sludge ( Replicate 1 ) .
!GO'· 9 0
. , 80 -(j t=: 70 rn r 60 / � 50 �------� z 40 /-------------u J I 2i 30 g_: 2 0
10 L!'.L.-..1.---.L----'----_..._--.___--L---... 1-·� .. 00:� I 3 o 9 : l /.3 l ,�
T : M �·. (o.�vs) Figure 19 . Long-term e ffect2 of l�ad I I C�0 on d i s s o l v e d oxygen uptake in acti vated s ludge ( J� p l i c � t � 2 ) .
68
trations in a s tudy done by Sartory and Lloyd ( 1 976 ) .
They found that thes e protozoan populations were killed
by concentrations of, and above , 0 . 00 0 5 mg/l of the free
lead metal ion . The difference in bacterial and protozoan
response is of interest since both types of organisms are
present in activated sludge . I n addition , these authors
also found this response to be true for mercury.
An init ial 37% inhibition was observed during the
short-term study of mercury. Thi s inhibit�on increased
an additional 26% within the first hour. Changes were
slower after the 2-hour mark and increased by only 2%
between the s e cond and third hour o f t e sting. No further
change in percent inhibition was exhibi ted ( Figure 20) .
Thi s maximal inhibi tion was 72%, a s contrasted to that
reported by Ghosh and Zugger ( 1 973 ) , who reported a 96%
inhibition at a similar ( 27 mg/l ) concentratio n . During
the long-term s tudy , high initial percent inhibition
occurred within the first 2 3/4 hours : 90� in Replicate 1
and 64% in Replicate 2 . Both replicates indicated a
decrease in inhibition within 24 hours but , thereaft e r ,
the results o f Replicates 1 and 2 differed markedly. The
microbial population recovered little from the inhibitory
e ffects of mercury during Replicate 1 ( Figure 21 ) , but
mercury had an actual stimulatory effect for every day
after day one in Replicate 2 ( Figure 2 2 ) . On the last
day of Replicate 2 , a 33% stimulation of the d i s solved
oxygen uptake by the bacteria occurre d . Although Mowat
�/ � . 0 f.:.: co T z !-:z w u 0:: L�J 0...
1 0 0
9 0
8 0
7 0
6 0
5 0
4 0
3 0
2 0 ·
1 0=-
I.
I I
69
r I I I �r1t.------�
____ _J ___ _.._ ___ _,___ 0 .::> I -
Tl M E (:-'.o�.ms) Figure 20 . Short-term e ffects of Mercury I I C - 0 o n dissolved oxygen uptake in ac tivated sludg� .
70
100 0 ) __, (
7- 8 0 Q 70 1- -ro 6 0 :i: z s o f- 40 -z w 30 u 0::: LLJ 2 0 Q_
1 0 L..U-.-J..�-�-���'--�--1-��.:.-.-- ·--'--
G 9 1 1 1 5 14 0011 1 -, .J
T I M E Co.t"'s) Figure 2 1 . Long-term effects of Mercury I I C �0 on dissolved oxygen uptake in activated sludge ( Replicate 1) .
! OO r so -80-?0 6 0 (5
f- 50 QI
40 ]� z ,)0 f-·- 2 0 z U..J u 1 0 0: � 0
- 1 0 -20 -30 -..:i 0 l-.L'--'------1---- ___ ,_....__ _ ___._ __ I
0011 I 6 � 1 1 L3 I.:� T I\'! t: ( :1;.ys)
Figure 2 2 . Long-ter� effe c t s o f � c � c � r y IIC �n on dis s o 1 v e d oxy ,-s c n un-cPJc c J..::i a c t i va t e d sludge ( Re pi� c a t2 � 2 ) . '
7 1
( 1 97 6 ) found that i n 2-week assays there was a decrease i n
mercury toxic i ty a t higher levels o f suspended solid s , in
the present study the sample for Replicate 1 was taken
when suspended solids were actually lower ( 5696 mg/ l ) than
for Replicate 2 ( 6666 mg/l ) . Other studie s , however,
indicate plausible explanations for these variable
observations .
Sterritt and Les ter ( 1 980) determined that resistanc e
to mercury is probably inducible , since during the lag
phase of growth cells lost viability , but this loss was
followed by a gradual increase in viability up to the
control leve l . In contrast , a study done by Neufeld and
Hermann ( 1 97 5 ) indicated that uptake is apparently not a
function of the metal concentration , nor is i t a fun c tion
of organism viability. They found that for concentrations
ranging from 0 to 1 000 mg/ l , there was a rapid mercury
uptake into the s ludge . This uptake leveled off after
about 3 hours , at which time approximately 9 5% of the
total mercury in the system had found its way into the
biomass . In addi tion they found that eventual e quilibrium
was achieved after about 2 weeks of contac t . Ghosh and
�ugger ( 1 9 73 ) also found that conceivably a large fraction
of mercury may be removed either by adsorption or by
incorporation into the cells . They also indicate ,
howeve r , that the possibility o f partial volatilization
of the mercury cannot be eliminat e d . They s tate that
other workers have isolated microorganisms capable o f
72
volatilizing mercury; these included Pseudomonas pyocyanea,
Klebsiella ( Enterobacter) aerogenes , an unidentified species
o f Pro teus , and an unidentified Diplo coccus ( Strepto coccus ) .
A great variability in the rate o f vo latili zation was
observe d , which may account for the temporary inhibitory
effect o f mercury. According to Ghosh and Zugger ( 1 97 3 )
it is possible for a n aerobic biological process to
acclimate to a .moderately high (up to 1 0 mg/ l ) dosage of
mercury within e few hours , but the time r�quired for
acc limation increases with the mercury dosage applied to
the sys t em . Similar to the results of aeplicate 2 during
this study, Ghosh and �ugger found that the oxygen uptake
rate in the presence of mercury was actually higher than
that measured for the contro l , which may have been due to
the beginning of an endogenous phase in the contro l .
Hence , there are s e veral possible factors which could
cause the variable observed result s .
The short-term effects o f the s tudy done with s ilver
are shown in Figure 23 . Inhibition was initially 32% and
more than doubled , with an increase to 78% within the
first hour of testing . After the first hour , the initial
inhibitory e ffects o f 1 00 mg Ag/l increased by about 4�
during the s econd hour , and then stabilized at 82� during
the third and fourth hours . The initial inhibition
observed in the silver long-term s tudy was similar to
that observed in the short-term study. Within the first
2 3/4 hours , a 23% change in inhibition o ccurred in
60
70 GO
� 50 f---0] .::r-: 40 z 1---z 30 LJJ u 0:: w Cl. 20
73
! O-
,___ _ _ __,__ ___ _, ___ ___,__ . __ ___,, ___ _,_ ___ ,,..._
0 2 0
Figure 23. Sho r t - term e ff e c t s of Si lver I I C 0 o n d i s s o l ve d oxygen uptake i n a c t i vated s lud�e .
74
Replicate 1 and a 47� cha�3e occurred in Replicate 2 . Thi s
sharp rise in inhibition was followed by a sharp drop in
inhibitory effects within the first 24 hours after addition
of the inhibitor in Replicate 2 . I n contras t , a small
gradual decrease in percent inhibition was observed during
this time period o f Replicate 1 . Figure 24 indicates a
higher average percent inhibition ( 6 2%) after the third
day, and more fluctuation in inhibitory effects ( 1 standard
deviation = 1 4%) during Replicate 1 . In c�ntrast , inhibi-
tion surged upward between the first and third days , and
stabilized near 49% inhibition ( 1 standard deviation = 4%)
during Replicate 2 ( Figure 25 ) . Thes e data indicate that
little , if any, recovery occurred from the initial 1 00 mg/l
d o s e . Howeve r , Mowat ( 1 976) found that some recovery did
o ccur in a 2-week study for a concentration be low that o f
the conc entration employed i n this study. Results showed
that at 1 mg Ag/l in five days there was only about 30%
uptake o f dissolved oxygen but that in 1 4 days this
improved to 45%. Another s tudy ( Wi lliamson and Nelson ,
· 1 981 ) indicated that as little as 0 . 25 mg hg/l would
inhibit the biological nitrification pro c e s s . Henc e , i t
becomes apparent that results vary according to the
concentration and pro c e s s examined . Tomlinson e t al .
( 1 9 6 6 ) found that the degree of acc limation o f activated
sludge is related to the concentration o f inhibitor to
which the sludge was expos e d and tha t , in this sens e ,
s ludge i s similar to s o i l . In addition , other factors ,
I
75
1 0 0 90r /BO
�70 �60 �� 50 f -;..� 40 I I l <,_) 30 er if 20
1 0 �·4�.._---'-���-4--�------1-------L�--�-�� OOll l 3 G 9 I l ::-; : l�
Tltvl::: (oAys) Figure 2 4 , Long-term effects of Si l�a� I I C s o o n dissolved oxygen uptak0 i n ac t i vated sludge ( Replicate 1 ) �
1()('11-. J I
� ... _ ..
0 f-cc - . -J_ -7 �-=
' ,-z L.t J u Ct:' :.u q_
9 0 L. 8 0 70 60
\ 50 /lr- 0 \ 3 0 2 0 i oL_-L OUll I
' ---'r· () ' I
r' \:' ': ( � . . \/ \ II • - . \_ .,: , I °::>)
I ! : • .:..i , \ ..... I r
Figure 2 5 . Long-term effects of S i l v e r IIC50 on dissolved oxygen uptakG in ac tivated sludge ( Replicate 2 ) .
76
such as the sludge age , may affect heavy metal action and
remova l . Sterri t t and Lester ( 1 981 ) found an 1 8-day
sludge age is best for efficient silver removal . As
indicated by Liu et a l . ( 1 981 ) , there are common drawbacks - -
in interpreting the results of most persistence studi e s .
Often n o allowance is made for abio tic degradation
processes such as loss from hydrolysi s , volatilization ,
or photolys i s . These factors should be considered in the
case of silver, since it was introduced as �ilver n itrate ,
a compound which is photochemically active and also was
observed to form varying amounts o f precipitate in the
biological flo e .
The short-term study on the herbicide 2 , 4-D showed
an initial 40% inhibition , which is similar to the initial
inhibition observed for the heavy metals tested . However
within the first hour the inhibition decreased by a value
of 300. As shown in Figure 26 a Si� increase in inhibition
then occurred \'Ii thin the second hour, another 1 2?� occurred
in the third hour and an additional 8% increase was
observed in the fourth hour, but the maximum initial
percent inhibition was never reached or exceeded . The
final level of inhibition during the 4-hour period was
about � the value observed for most of the heavy metals .
These data indicated that the short-term effects of 2 , 4-D
are probably less severe than those effects induced by
heavy :ne tals upon the activated s ludge process . The two
replicates on the long-term inhibitory effe c t s o f 2 , 4-�
�:. 0 1 --CT) =j: 2 f-·�/ i1J u n: ! Ll i_L
1 ocJ 9 0
80
7 0 ·
::r t
�.ol ()() ;-
I 2�Y-
I 0 -
0
- 1 0
77
0
I 2
TIME (HOURS) cl.
Figure 26. Short-term effe c t s o f 2 , 4�D I I C SO on d i ssolved oxygen uptake in activated sluags .
78
were similar . As shown in Figures 27 and 28 , results from
both replicates showed inhibition occurring during the first
2 3/4 hours , stimulation during days 1 to 3 , and a return
to inhibition during days 6 through 9 . However, inhibition
proceeded throughout the remainder of the 2 weeks during
Replicate 2 , but a return in stimulation was observ-ed from
day 1 1 through 1 4 o f Replicate 1 . The actual values of
inhibition and stimulation differed considerably as shown
in Figures 27 and 2 8 .
The variability in microbial reaction ( i . e . , dissolved
oxygen uptake ) to 2 , 4-D during the two replicates may be
a result of the condition and heterogeneity o f the popula
tion itself and its ability to cause biodegradation of
2 , 4-D. According to Altom and Stri t zke ( 1 973 ) , the absence
of a lag phase in the population would indicate rapid
degradatio n . In addition , Foster and McKercher ( 1 973 )
found that the half-life o f 2 , 4-D fo llowed a defini te
pattern related to bacterial counts . Higher bacterial
numbers resulted in a shorter chemical persistenc e .
Furthermore , Liu e t al . ( 1 981 ) found that aerobi c ,
cometabolism conditions indicate acclimation occurs .
These observations may explain the decrease or variability
in inhibitory effects of 2 , 4-D over a period o f time .
Adaptation to 2 , 4-D may be a result o f biodegradation ,
and both rates probably depend upon the sludge composi tion .
Therefo re , the microbial population present in Replicate 2 o f the long-term study may have po ssessed the ability for
•
100 90 80 70
79
60 % Q I -· rn :r. -...... ,_
50 40 30 2 0 1-z w (i I 0
ti! 0
� '·-.. ) i-· -Cci T :c.: I-z !..J..J u 0::: ld Ct
- 1 0 - 20 -30 -40 / - s o . 60 u.L-' �---'-----""-----'------- -- --.!-.. .:.._ -···
00.!• I -, ...J
Figure 27. L�ng-term effe c ts of 2 , 4-D I I C 5 0 o n dissolved oxygen uptake i n a c t i vated sludge ( Replicate 1 ) .
40f 30 2 0 1 0 • / 0 -
- I 0 -20 <) 0 l __ __._ ___ �---�--_..._ __ ,__1._
OU!l 1 -1. ,) 1 1 !j 14 i- 1 \/ 1 · (D"''r) I I _ ... \ ( �
Fi�urc 28. L�ng-te r�t e .f fc; c t s o ::.., Z. , .�-D I IC r. r: on dissol vcd o;(yg e n upta1:E: :;.:i actl�ated ·:.lndce ( Re::)lica-te 2 ) .
80
more rapid biodegradation of 2 , 4-D . Thi s assumption seems
probable since the content of suspended solids for this
s ludge sample was 5696 mg/ l , while that for Replicate 1
was 6666 mg/l , an indication that adsorption should have
increased in the latter and caused a decrease in inhibition
i f this interaction does indeed reduce inhibition . Another
po ssibil i ty is that the microbial population in Replicate 1
is actually a more rapid biodegrader o f 2 , 4-D but that the
biodegradation products may still exhibit toxic ity and do
not become bound to the suspended solid s . The persistence
of 2 , 4-D is quite variable , undoubtedly reflectiLe the
multitude of conditions which affec ts the degradation
processe s . Altom and Stri t zke ( 1 973 ) found that the
average half-life o f 2 , 4-D is 4 days , but they also
reported other studies showed persis tence varying from
7 to 98 days .
Recovery o f mixed microbial populations to the effects
of inhibitors is a complex phenomenon . Many factors
influence the probability of i t s o ccurrence . Adaptation
within species , selection o f mutants within s pe c i e s , and
selection between species may be involved in the process
( Lester e t al . , 1 979 ) . l1urthermore , resistanc e to heavy · .
metal toxicity may be expressed in two ways . Non-specific
resistance may arise from differences in physio logical
states o f the oreanism , and inheritable specific
resistance transfer factors ( RTF' s ) for particular heavy
metals are known to occur. Impermeability or detoxif'ica-
81
tion by chelation are both non-specific resistance
mechanism s , and the formation o f a complex o r chelate with
bacterial extracellular polymers is an important aspe c t o f
me tal removal from wastewater ( Sterri t t and Le ster, 1 980) .
I n addi tion , micro organisms may si�ply acc limate o r adapt
by modifying chemicals that they canho t utilize �s sources
of energy or nutri e n t s . This proc e s s i s come tabolism and
i t is enhanced by an increase in the density o f organisms
( Jacobson et al . , 1 980) . In addition to tne de�sity o f
the organi sms themselve s , an increase i n suspended solids
seems to indicate a general trend toward reduced toxicity.
However, Mowat ( 1 97 6 ) found that this toxi city reduc tion
was not evident i n many instances , e s pe c ially when there
was a time lapse between experiment s . in her s tudy, all
the metals assayed , vi z . , Ag , Cd , Cu, Hg , showed decreases
in toxicity at higher levels of suspended solids after
· 2 week� i ncubati o n . As shown in the resul t s of this
study, some fluctuation or peri odicity occurred during the
short-term and long-term studi e s . Interpretat i o n o f the
results from these experiments i s compl icated by the
mult iplic i ty o f variables inevi tably associated with a
complex system such as activated sludge . · Fac tors such
as flow rate , number and concen tration of substr�t e s ,
presence o f other toxic ions and compo s i t i o n o f the
bacterial flora affect interpre tations ( Le ster et al . ,
1 979 ) . Furthermore , the variation in the effect o f a
given substance with concentration rnigl� t be expec"tea to
-
82
provide information about the mechanism of inhibition.
However, as indicated by Tomlinson et al . ( 1 96 6 ) , it is
not possible to distinguish between various types of non
competitive mechanisms because differences between the
shapes of the curves are , in general , fairly small relative
to the accuracy of the data. Application of experimental
results to the activated sludge sewage treatment pro cess
i s de sirable , but this aim must be approached with an
awareness o f the complexity o f the system • .
83
CO.NCLUSIOJ�S
1 . The inhibitory effects on dissolved oxygen uptake
in activated sludge o f Terbacil and Zectran at concentra
tions from 0 . 00 1 to 1 000 mg/l were negligible or moderate
( less than 1 7%) .
2 . The initial inhibitory concentration for 50�
inhibition ( rrc 50 ) o f dissolved oxygen uptake during the
initial 1 5-minute period after the addition o f each
inhibitor in mg/l was for Hg ( 20 ) , for Cu ( 50 ) , for Ag
( 4 3 ) , for Cd ( 1 1 5 ) , for Pb ( 5 O O ) , an a for 2 , 4-D ( 5 O o ) •
3 . · I n general , the short-term effects of the
i nhibitors Hg , Cu , Ag , Cd , Pb , and 2 , 4-D at concentrations
near their respective I r c 50 fo llowed a similar pattern .
Initially there was a marked inhibition of dissolved
oxygen uptake followed by small increases in inhibition
to a maximum after 3 or 4 hours in the presence of the
inhibito r .
4 . Variability in the percent inhibition of
dissolved oxygen uptake by the microbial population in
activate d sludge caused by the inhibi tor is due in part
to the inherent variability o f different sludge samples .
5 . A periodicity of inhibition fo llowed by semi
recovery was exhibited <luring the long-term s tudies o f
the inhibitors tested .
6 . I n general , little i f any adaptation to the
inhibitors was exhibited by the heavy me tals cadrniun ,
-
84
copper, lead and s ilver d�ring a 2-week peri o d .
7 . Stimulation o ccurred during some phases of the
long-term studies o f both mercury and 2 , 4-D and was
probably due to noncompe titive resistance factors .
8 . The inhibitory effects o f the heavy metals Hg ,
Cu, Ag , C d , and Pb and o f the herbicide 2 , 4-D are.
significant to the proc e s s o f d i s so lved oxygen uptake in
activated sludge .
/
·.
-
8 5
LITERATUR.S CITED
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Altom , J .D . and J . F . Stritzke . 1 973 . Degradation of Dicamba, Picloram, and four phenoxy herbicides in soils . Weed S c i . 21 : 556-560.
American Public Health As sociation . 1 976 . Standard methods for the examination o f water and was tewater , 1 4th e d . American Public Health Asso ciation , Inc . , \'/ashing ton , D . C .
Barth , E . F . , J . N . English, B . V . Salot to , B . N . Jackson and M .B . Ettinger. 1 965a. Fie ld survey of four municipal wastewater treatment plants receiving metallic waste s . J . \'/at . Pollut . Control Fed . 3 7 : 1 1 0 1 - 1 1 21 .
Barth, E . � . , M . B . Ettinger, B . V . Salotto and G . � . McDermo t t . 1 96 5b . Summary report on the effe c t s o f heavy metals o� the biological treatment processes . J. Wat . Pollut . Control Fed . 3 7 : 86-9 6 .
Berth , P . 1 978 . Recent developments i n the fi eld o f inorganic builders . Jour. Ame r . O i l Che m . Soc . 5 5 : 52-58.
Blok, J . 1 97 4 . Respirometric measurements on activated s ludge . Water Res . 8 : 1 1 - 1 8 .
Bragg , P . D . and D . J . Rainnie . 1 974 . The effect o f silver ions on the re spiratory chain of Escherichia co l i . Can . J . Microbiol . 20 : 883-889 .
Bro ecker, B . and R . Zahn . 1 97 7 . The performance of activated sludge plants compared with the results of various bacterial toxicity tests--a study with 3 , 5-Dichlorophenol . Water R e s . 1 1 : 1 6 5-1 7 2 .
Brown , H .C . , C . P . Hensley, G . L . McKinney and J . L . Robinson . 1 973 . Efficiency o f heavy metals removal in municipal sewage treatment plant s . �nviro n . Le tters . 5 : 1 03 - 1 1 4 .
Brown , M . J . and J . N . Lester. 1 979 . Metal removal in activated sludge : the ro le of bacterial extre. c e l l u2.·:.::r· polymers . Water n.c s . 1 3 : 81 7-83 7 .
86
Bunch , R . L . and C . W . Chambers . 1 96 7 . A biodegradability test for organic compounds . J . Wat . Pollu t . Control Fe d . 39 : 1 81 - 1 87 .
Carpenter , D . F. , N . G . McCormick , J . B . Cornell and A . M . Kaplan . 1 978. Microbial transformation of 1 4clabeled 2 , 4 , 6-Trinitrotoluene in an activated sludge system . Appl . Environ . Microbi o l . 3 5 : 949-9 5 4 .
Carrondo , M . J . T . , J . N . Le ster and R . Perry. 1 981 . Type A zeolite in the activated sludge process-I I . Heavy metal remova l . J . Wat . Pol lut . Contro I" �"'e d . 53 : 344-35 1 .
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