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Summary and conclusions - Factors determining the dewatering rate of
digested sludge on soils were investigated under laboratory and field
conditions.  Soil columns of Blount silt loam and Plainfield sand were
used in laboratory sludge dewatering studies.  Metal infiltration rings
were used for field studies.

Nitrate concentrations, electrical conductivity, and pH and Eh values
of effluent and soil water samples were determined.  Effluent from soil
columns and soil solution samples were collected throughout the period
in which the factors influencing the dewatering of sludge on soils were
investigated.

When sludge was first applied on soils, dewatering of the sludge was
fairly constant and the rate depended on infiltration of water into
soils and water losses by evaporation.  When the water content of sludge
was reduced to about 80 percent by weight, by dewatering on initially
moist soils, further drying of the sludge was to a large extent by eva-
poration alone.  Where antecedent soil moisture was low, infiltration
contributed to the sludge dewatering process when sludge moisture con-
tents were considerably less than 80 percent by weight.  Also on soils
where antecedent moisture contents were low and at sludge moisture
contents where further dewatering on soils was by evaporation only, the
evaporation rate was smaller than from pans.

The rate of infiltration of sludge liquid depended not only on the in-
itial soil moisture content, but also on the solids content of the sludge.
The higher the soil moisture and sludge solids contents, the lower was
the rate of infiltration.  However, antecedent moisture conditions af-
fected the rate of sludge infiltration less on sandy than on fine tex-
tured soils.

Initially the infiltration rate of sludge liquid into sand was greater
than into silt loam soils.  After a few days of successive applications
of sludge in the absence of complete drying, the rate was about the
same regardless of soil type.  It appears that after a period of time
the infiltration rate is determined by the sludge cake and not by the
soil surface.  The soils were unsaturated with respect to moisture and
their capacity to transmit moisture was always greater than the infil-
tration rate determined or controlled by the sludge cake.

When successive sludge applications were made at time intervals such
that the sludge cake was not allowed to dry, infiltration rates decreased
to very low levels.  But if the sludge cake was allowed to dry, the in-
itial infiltration capacity was more or less completely recovered.

Under laboratory conditions, evaporative losses of water from digested
sludge in pans were not detected when the moisture content of sludge
was reduced to about 8 to 10 percent of the dry weight.
                                 192

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The changes in soil pH and redox potentials were small following
various rates and frequencies of sludge applications.

Nitrate nitrogen concentrations continued to increase in the soil with
successive sludge applications both in laboratory and field studies.
Soil solution nitrate concentrations ranged between 100 and 300 ppm
where a total of 50 cm of sludge was applied in 70 days during field
studies.

From nitrate concentrations and redox potential measurements, it ap-
peared that anaerobic conditions were seldom, if ever, produced in the
soil by exceedingly high sludge loading rates.

Soil conductivity values were increased from an average value of about
1 mmhos/cm where only water applications were made to about 2.5 mmhos/cm
where a total of 50 cm of sludge was applied in 70 days.  From limited
data, it appears that soluble salts will be leached to deeper soil depths
in a humic region.   During exceptionally dry summers, the salt buildup
in the surface of a soil like Blount silt loam, with continuous periodic
sludge applications, will exceed that which was found with the total
50 cm application and may cause a plant moisture stress severe enough
to reduce crop yields.
                                  193

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                             SECTION VI
                      MICROBIOLOGICAL STUDIES
Influence of Soil Moisture on Fecal Coliform Survival

Introduction - A laboratory study of the possible bacterial contamina-
tion of the environment resulting from land application of liquid sludge
is the subject of a master's thesis prepared by James Schwing,  Depart-
ment of Civil Engineering, University of Illinois.  Specifically, the
purpose of the study was to evaluate the effect of the moisture content
of Plainfield sand on survival of fecal coliforms.

The study was preceded by a review of literature on survival of patho-
genic organisms in waste treatment processes including anaerobic diges-
tion and on their survival and movement in soils.  Conventional treatment
processes achieve significant reductions in the number of intestinal
bacteria and appreciable numbers of the organisms are transported to
the anaerobic digestion process in sludge.  The environment of  the di-
gester is unfavorable to pathogenic organisms; however, complete removal
cannot be anticipated and land disposal systems must be operated with
consideration to the effect of the organisms in the environment.  Bacteria
can be effectively removed from water leaching through soil - particularly
after a build-up of solids occurs in the interstices of the soil.  In
addition, intestinal bacteria may die off with time in the unfavorable
soil environment.  Some of the factors which influence the rate of die
off include temperature, the level of organic material in the soil, pH,
and the moisture content of the soil.

Based on the review of literature, the most critical period for passage
of pathogens through the Plainfield sand would be immediately after ini-
tiation of a land disposal project.  After sludge solids have accumulated
in the soil voids and microbiological activity has increased in the soil,
removal of pathogens by straining would become more effective and the
extent of the bacterial front would retreat to a position closer to the
surface of the soil.  Long-term changes in the scil structure due to
sludge applications would be expected to change the response of patho-
gens to the soil environment.  The increase of organic material in the
soil might make the environment more favorable for competitory and pre-
datory soil microorganisms because of the greater retention of moisture
and availability of food.
                                  194

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Materials and method - The soil used in these experiments was condi-
tioned by adding a total of 7.6 cm of digested sludge to a 0.6 m deep
Plainfield sand column over a period of nine days.  The sludge was
added 2.54 cm at a time every three days, and following drainage the
sludge was worked into the upper 15 to 23 cm depth of soil.  Following
this conditioning phase the upper 23 cm depth of soil in the column
was removed and mixed with an additional 1.3 cm depth of digested
sludge and sufficient rain water to adjust the final moisture content
to 5 percent.  Additional rain water was added to other samples to
give moisture levels of 10, 15, and 20 percent.  These samples at the
four moisture conditions, along with a sample of sludge not mixed into
soil, were then monitored for a period of time to observe the rate of
disappearance of the fecal coliforms originating in the sludge.  Mois-
ture contents x/ere maintained at the initial levels by sealing the
samples in containers with "Saran Wrap" covers and by adjusting the
moisture content periodically as needed.  Samples from the atmosphere
overlying the sludge were collected with a syringe and analyzed with a
gas chromatograph to assure that the oxygen content remained near nor-
mal .

Disc.ussji.on of results - Results of the study of survival of fecal coli-
forms in sludge-soil mixture at 5 percent moisture content are shown in
Figure 66.  The points shown as squares represent the actual values ob-
tained while the circles represent the 95 percent confidence interval.
An initial sharp 100-fold increase in the fecal coliform population will
be noted.  This might be explained by the fact that metabolic interme-
diates formed in the anaerobic digestion process become available to the
fecal coliforms as a food source under aerobic conditions.

Figure 67 shows the survival of fecal coliforms in sludge conditioned
soil at 10 percent moisture.  Again an initial period of growth is evi-
dent.  The fecal coliform density was increased by almost 10 fold.  The
fecal coliform survival in sludge conditioned soil at 15 percent mois-
ture is shown in Figure 68.  At this moisture concentration, a 10 fold
increase in fecal coliform density is again realized.

Fecal coliform survival at 20 percent moisture in sludge conditioned
soil is shown in Figure 69.  No initial growth is exhibited by the data
for this curve.  This is probably due to the fact that at this high
moisture concentration the saturation capacity of the soil was exceeded.
Essentially anaerobic conditions were maintained as evidenced by the
appearance of black sulfide precipitates typically evident with anaerobic
conditions.  For this reason the organic intermediates of anaerobic
digestion were not readily available to the fecal coliforms as a food
source.

Data for fecal coliform survival in sludge are presented in Figure 70.
Again, as in sludge conditioned soil at 20 percent moisture, no initial
phase of growth was realized.  The reason for this was again probably
                                 195

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 E
JCD

 C
  v»
 >
4-J
"en
 c
 0
Q
 o
O
 ctf
 o
 O)
 O
                      10    15     20     25    30    35
                          Time  (days)
                                                             40
Figure 66.
           Fecal coliform survival in sludge-conditioned soil adjus-

           ted to 5 percent moisture content.  Circles represent

           95 percent confidence limits.
                             196

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 c

 >
+J

 c
 0
O

 E
 o
O
 05
 O
 CD
IL


 D)
 O
-J
                     10     15    20    25     30    35     40
                          Time (days)
 Figure 67.
Fecal coliform survival in sludge-conditioned soil adjusted
to 10 percent moisture content.  Circles represent 95 per-
cent confidence limits.
                             197

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 O
 c
 c
 CD
Q
 o
O
 cd
 o
 0)
IL

 O)
 O
                     10     15     20    25    30    35    40
                        Time   (days)
  Figure 68.  Fecal coliform survival in sludge-conditioned soil adjus-

            ted to 15 percent moisture content.  Circles represent

            95 percent confidence limits.
                           198

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                    10
15    20    25     30     35    40
                        Time  (days)
Figure 69.  Fecal coliform survival in sludge-conditioned soil  adjusted
           to 20 percent moisture content.  Circles represent  95 per-
           cent confidence limits.
                           199

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                   10     15     20    25     30    35
                      Time  (days)
Figure 70.  Fecal  coliform survival in sludge.  Circles represent
          95 percent confidence limits.
                         200

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the maintenance of anaerobic conditions, with the resulting inavaila-
bility of the intermediates of anaerobic digestion to the fecal coli-
forms as a food source.

Table 50 shows the average percentage die off of fecal coliforms after
30 days.  The data indicate that at 5 percent moisture condition the
fecal coliforms were best able to survive.  This is surprising as it
might be expected.that a higher rate of die off would occur at lower
moisture concentrations due to the inavailability of moisture.  Perhaps
the "aeration porosity limit" as suggested by Bhaumik and Clark (18)
for the condition when a favorable balance between moisture concentra-
tion and aeration exists is near 5 percent moisture for the conditions
of this study.
Table 50.  Survival of fecal coliforms in soil and sludge.
                                              Die Off of Average
Moisture Content                          Fecal Coliforms in 30 Days
   (Percent)                                      (Percent)
5
10
15
20
Sludge
72.5
99.9
99.6
96.6
99.9
Summary - Following the initial period of growth, die off of fecal coli-
forms in sludge and in soil containing sludge followed first order kine-
tics.  That is, a constant fraction of remaining organisms died during
each time interval.  The only exception £0 this was for the 5 percent
moisture condition where the die off rate decreased with time.

Studies of the survival of fecal coliforms in Plainfield sand which had
received sludge indicated that the organisms were ab.Te to survive for ex-
tended periods of time at moisture concentrations from 5 to 20 percent.
The rate of die off may be significant from the standpoint of pollution
of,surface water when precipitation causes runoff soon after the sludge
is applied and especially if it causes soil erosion from sludge-treated
land.  Travel of the fecal coliforms through the soil with leachate was
not investigated as a part of this work but, based on reports in the
literature, would not be expected to be as -severe a problem.  Many of
                                201

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the questions regarding the transport of fecal coliform off of the
soil surface and through soil will be answered from the results ob-
tained from the NEARC lysimeter studies.

At 5 percent moisture a sizeable increase in fecal coliform numbers
occurred after sludge addition and the subsequent rate of die off was
slowest.  This is of particular interest as 5 percent is probably more
representative of the average moisture concentration of the surface
layers of the Plainfield sand in the field than the other moisture
levels studied.  However, in the field rapid changes in soil surface
temperatures and exposure to sunlight may exert an over-riding influence
on fecal coliform longevity.

Porcine Enterovirus Survival and Anaerobic Sludge Digestion

Introduction - Although, to our knowledge, a disease outbreak has never
been known to have resulted from the application of anaerobically di-
gested sludge to land, we really know very little about the virologic
aspects of municipal sludges.  In light of recent proposals and programs
for sludge utilization as a fertilizer on crop lands, additional infor-
mation is needed on possible virus content and their survival during
the anaerobic digestion period.

The following study was undertaken in an attempt to gain some insight
into the fate of viruses which cause disease in man when they are sub-
jected to the environmental conditions normally maintained in a heated
anaerobic digester.  However, to overcome a number of technical problems,
an animal enterovirus was used in the study which possesses many char-
acteristics common to human enferoviruses.

Materials and methods - The virug employed in this study was a charac-
terized swine enterovirus designated ECPO-1 (20)(143).  The agent was
propagated in primary porcine kidney cell cultures prepared in 114 gram
prescription bottles employing a medium of 0.5 percent lactalbumin
hydrolysate and 10 percent fetal calf serum in Hank's Balanced Salt
Solution (HESS).

When viral induced cellular degeneration was nearly complete and in-
volved approximately 90 percent of the cells, the culture fluid was
collected and pooled.  A representative sample of the harvest was      v
taken at that time and the titer of the virus determined.  The remain-
der was frozen in 85 ml quantities at -65°C.  Just prior to use, suf-
ficient virus stock for the seeding of laboratory scale digesters was
thawed and adjusted to yield 100 ml volumes containing 10^ plaque
forming units  (PFU) per ml.

The laboratory digesters consisted of six tightly sealed, one liter
stainless steel vessels similar in design and operation to those de-
scribed by Vatthauer et_ suL.  (159).  The initial charge of sludge and
                                 202

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inoculum consisted of 900 ml of digester draw-off from the Champaign-
Urbana wastewater treatment plant.  When in operation each unit was
maintained in a 34.5°C water bath and continuously agitated by means
of a magnetically driven glass stirring rod.  All digesters were moni-
tored daily using gas production and composition, digestion of dry
matter, and pl-l of draw-off.  Gas collections were made in metalized
plastic bags on each digester and volumes measured by x^ater displace-
ment.  Gas composition was determined on an F & M Model 720 gas chro-
matograph (F & M Scientific Corp., Avondale, Penn.) equipped with a
gas sampling valve and thermal conductivity detector.  CH4 and C02
were separated using a three-meter long column of 80-100 mesh porapak Q
(Waters Associates, Inc., Farmingham, Mass.).  Dry matter and organic
matter determinations were by standard drying and ashing techniques.
The hydrogen ion determinations were made with a Corning model 8 pH
meter with a standard combination electrode.

Once in operation, 60 ml of digested sludge were removed from each
digester daily through a small stoppered port in the top of each unit
and replaced with 60 ml of fresh sewage.  On appropriate days the draw-
off from each unit was used for viral and other monitoring purposes.

When stabilization of the digesters occurred as determined by gas pro-
duction, two units were seeded with 100 ml of the virus suspension.
Two additional units for comparison remained uninoculated with virus.

All piglets were obtained by hysterectomy and maintained in isolators
of stainless steel and flexible plastic design.  Equipment, methods of
procurement and rearing were as previously described by Meyer et a^. (107)
All piglets were free of detectable microbes, and at least 10 days old
when first exposed to sludge.

Fecal samples were collected from each pig twice a day (AM and PM) on
the 3rd and 4th days post-challenge and pooled.  Twenty percent fecal
suspensions were prepared for inoculation using complete tissue culture
medium with antibiotics as diluent and filtered through a microsyringe
filter holder containing a millipore HA filter.  All cultures once in-
oculated were examined daily for viral cytopathic effect and held for'
one week before discarded as negative.  In those cases where viruses
were recovered from infected piglets, the agents were identified as
ECPO-1 by neutralization with specific antiserum.

Three separate trials, approximately six weeks apart were carried out
employing piglets of different genetic backgrounds.  In the 1st trial,
25 ml samples of sludge were collected from each of the two virus seeded
digesters and pooled.  Such samples were obtained after 30 minutes,
1 day, 4 days, 7 days, and 12 days.  The pooled samples of each time
interval were mixed well, antibiotics (penicillin and dihydrostreptomycin)
added and 10 ml volumes force fed to two piglets.
                                   203

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In the 2nd trial the general procedures were the Same as above except
that samples were withdrawn from the digesters after 1 hour,  1 day,
2 days, 3 days, and 4 days; and that the volume fed to the piglets was
increased to 20 ml, 10 ml being fed orally and the remaining  10 ml ad-
ded to a small quantity of sterile milk.

In the 3rd trial the procedure was the same as in the 2nd trial except
the samples were collected after 1 hour, 4 days, 5 days, and  6 days.

Results - Generally a period of approximately four days was required
for the digesters to stabilize.  Data collected during the individual
trials indicated comparable and uniform rates for CH^ production and
the other parameters monitored for units with or without virus except
that units shortly after tne addition of virus showed marked  but tem-
porary increases in C02 production compared to control digesters.

Virus could not be detected or demonstrated by pig infection  after the
4th day of exposure to the environment of the anaerobic digesters. All
pigs challenged with virus material exposed in digesters 72 hours or
less became infected and 2 to 6 became infected with material with-
drawn on the 4th day.  As shown in Table 51, none of the piglets were
infected with material which had been in the digesters 5 or more days.
Table 51.  Survival of swine enterovirus ECPO-1 in anaerobic sludge
           digesters.


                      Time Virus Exposed in Digester
Trial
          11      23      45      6      7     12
         hr*  day   days   days   days   days   days   days   days

   I      +    +                    +                    00
          +    +                    0                    00
  II      +    +      +      +      0
          +    +      +      -f      +

 III      +                         000

          +                         000
   Trial I  30 min
+  Infected piglet
0  Non-infected piglet
                                       ?04

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While emphasis was on recovery of virus from piglets exposed to sludge,
it should be noted that in trial III-, six of the eight piglets exposed
to sludge at that time contracted a bacterial infection and succumbed
to the bacterial pathogen Salmonella typhimurium.
                                                                     \
Discussion - Raw sewage contains a wide variety of viruses and a signi-
ficant number of human origin.  Little or no information, however, ex-
ists on their isolation from digested sludge and the few attempts re-
corded in the literature were unsuccessful.

Because viruses may be readily absorbed onto the surface of sludge
particles resulting in rather stable virus-sludge complexes, they are
difficult to isolate.  As a result microbiologists have been reluctant
to say that the failure to isolate viruses was due to their inactiva-
tion by the digestive process per se^.

The decision to use germ-free swine as an indicator animal for detec-
ting infectious levels of virus stems from a number of factors.  First
a characterized swine enterovirus was available which possessed bio-
physical properties similar to agents commonly encountered in sewage.
Second, ECPO-1 had various desirable attributes such as its eas of cul-
tivation, identification and a minimal disease potential for man.
Third, early preliminary studies indicated that available sludge was
inherently toxic to our cell culture system and that direct isolation
by cell culture inoculations would be of limited value.

It should be recognized that in the operation of the digesters a dilu-
tion of the virus occurred with the daily draw-off and the addition of
60 ml of fresh sewage.  With a turnover rate of approximately 1/15 of
the total volume per day, one could anticipate the removal of 1/2 of
the original virus inoculum after 7 1/2 days of operation.

To help compensate for the loss of virus, as a normal consequence of
the daily feeding of the digesters, we arbitrarily started out with
what we thought was a high concentration and one not likely to be en-
countered in a conventional sludge (105 PFU) .  In addition, we increased
the amount of sludge fed to each pig form 10 ml to 20 ml during trials
II and III.

Considerably more work will naturally be required before the virologic
aspects of digested sludge will be completely known.  In the case of
the Salmonella typhimurium infection that occurred in trial III, it
appears that all facets relative to the presence of bacterial pathogens
may also need additional consideration.  Even so, it would appear, at
least in this limited study and circumstances provided in these experi-
ments, that viruses with characteristics, similar to ECPO-1, whether
complexed with sludge or not, would not appear to constitute a serious
infectious hazard after five days in digesters of the type employed.
                                 205

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Conclusions - Although additional work on a wide variety of viruses
may be required to ascertain the effectiveness of anaerobic digestion
as a means to inactivate viruses, preliminary results indicate a reduc-
tion and loss of infectivity could be expected upon a suitable five
days exposure to the environmental conditions provided by anaerobic
digestion of municipal sewage sludge.  Considering that sewage solids
are held in heated anaerobic digesters for periods of time several
times greater than needed to inactivate the swine virus used in this
study, it appears that the use of digested sludge as a fertilizer pre-
sents very little risk with regard to spreading diseases caused by
viruses.

Hygienic Aspects of Liquid Digested Sludge

Fecal coliform die off studies - For routine analyses of water, soil
and digested sludge samples, the coliform group of bacteria has been
taken as an indicator of the degree of microbial pollution.  Since
non-fecal coliforms are known to be part of the normal soil flora,
only the fecal coliforms have been considered for use in the studies
discussed here.                                \

The determination of fecal coliforms is rapidly and easily performed
by the membrane filter technique with incubation at 44.5°C in the
M-FC medium (54).  This method (referred to as MFC) reveals the pre-
sence in the liquid digested sludge of a large population of fecal
coliforms which gradually decreases upon removal of the sludge from
the digester.  A reduction from 4 x 10^ to 7 x 103 and 2 x 10^ fecal
coliforms per ml was observed after 19 and 32 days, as can be seen
from the data presented in Table 52.  A similar die off can be obser-
ved for the fecal coliform organisms contained in sludge supernatant.
In contrast to the gradual decrease or die off of fecal coliform or-
ganisms originally present in digested sludge, when laboratory grown
populations of Escheric.hia coli  (neotype, ATCC 11775) are added to
non-treated or autoclaved digested sludge they die off very rapidly,
as evident by the data presented in Table 53.  In view of this dif-
ference of behavior, the question is raised as to whether the organisms
found in the digested sludge, by  the MFC technique are truly of the
fecal coliform group.  Short of  serological tagging, the IMViC test
and the elevated temperature MPN-EC  (most probable number - EC medium)
method, as a confirmatory test from positive presumptive tubes, are
the only other two ways to identify fecal coliforms.  Both techniques
have indicated the presence of fecal coliforms in the liquid digested
sludge.  From the data presented in Table 54 it can be seen that agree-
ment in fecal coliform counts was obtained with the MFC and MPN-EC
methods.  Furthermore, on the basis of the IMViC test, Fuller and
Litsky  (49) have shown that digested sludge harbors a population of
fecal coliforms, which is on the order of 105 cells per milliliter.
                                 206

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Table 52.  Number of fecal coliforms per ml of impounded liquid
           digested sludge.
Sludge
Sample 0
Total sludge 4 x 10*
Sludge supernatant 3 x 103
Days
19
7 x 103
2 x 101

32
2 x 102
0
Table 53.  Bactericidal properties of digested sludge toward labor-
           atory-grown Escherichia coli as determined by the membrane
           filter-high temperature method.
Fecal coliform, cells/ml
Incuba-
tion, hr
0
24
Digested
sludge
25 x 102
20 x 102
Digested sludge
supplemented
with E. coli
25 x 106
41 x 102
Autoclaved
digested sludge
plus E. coli
26 x 106
<10
Table 54.  Comparison between the membrane filter-high temperature
           (MFC) and the MPN-EC medium techniques for counting fecal
           coliforms.
                     Fecal coliform, cells/ml
  Source of
digested sludge
   MPN-EC medium
95% confidence limit
   MFC
Chicago, Calumet Plant

Urbana-Champaign

Urbana-Champaign
4.9 x 103 - 4.2 x 102
1.5 x 105 - 1.2 x 104
3.4 x 105 - 3.7 x 102
1.6 x 103
1.6 x 105
8.0 x 103
                                 207

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The bacteriolytic effect on the E_, col±_ of stock culture may have been
a function of the bacterial strain.  However, the phenomenon assumed a
higher significance when it was discovered that digested sludge-adapted
fecal coliforms could be transformed into susceptible strains.  Speci-
fically, eight distinctive fecal coliform strains, isolated from diges-
ted sludge, were maintained in lactose broth.  Following one transfer
in the broth, six strains survived reintroduction into autoclaved di-
gested sludge, but two were killed.  After several transfers covering
a two-month period, all isolates no longer adapted to the digested
sludge environment and thus behaved like E_. coli ATCC 11775.  The above
results prompted further study of the bactericidal properties.

Various treatments were performed on the digested sludge to determine
the nature of its apparent toxicity toward E_. coli, as determined by
the MFC technique.  Results of these studies are presented in Table 55.
Although the digested sludges were always collected at the same waste-
water treatment plant (Calumet, Chicago) a few batches turned out to
be devoid of toxicity.  This fact rules out many factors which other-
wise would have been considered as possible causative agents for the
toxicity: the low redox potential, the lack of oxygen, the saturation
of the sludge liquid phase with carbon dioxide, methane, and possibly
the presence of sulfides.  Since toxicity was not eliminated by heat
sterilization, the bacteriolytic effect could not be attributed to a
protein, parasitic relationship, or nutritional competition with other
organisms.  The bactericidal properties were localized in the liquid
phase of digested sludge (Table 55) thus eliminating several of their
possible sources.  Most components of digested sludge occur in the
solid phase.  The liquid phase, for example, contains less than
0.05 ppm sulfide, less than 10 ppm organic carbon exclusive of meth-
ane and carbon dioxide, and only traces of heavy metals.  Because
methane and C02 saturation of the liquid phase, presence of bicarbo-
nate and ammonium ions (up to 500 ppm-N) at pH values 7.0 to 8.6, low
redox potential, and lack of oxygen are properties common to both
bacteriotoxic and non-bacteriotoxic sludge samples, they could not be
considered as the principle causative factor.

Heat-sterilized sludge and its liquid phase were assayed for antibio-
tics by the diffusion technique in nutrient agar  (Difco).  Incubation
was carried out aerobically at 27°C.  Under these conditions, no anti-
biotic activity was detectable.

Volatile fatty acids have been held responsible for the exclusion, of
—• coli- and salmonellas in the rumen of bovines by Hollowell and
Wolin (72).  However, their range of bacteriostatic and bacteriolytic
action is limited to pH values below 7.0 and to concentrations above
60 ymoles/ml; conditions which are riot prevalent in digested sludge.
Moreover, Brounlie and Grau (24) presented evidence that the elimina-
tion of salmonellas and E_. coli from bovine rumen cannot be accounted
for by volatile fatty acids alone.
                                 208

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Table 55.  Localization of bactericidal properties in digested
           sludge liquid phase*

                                                                     \
                                                  Escherichia coli
                                                  after 24 hr incu-
    Medium                                        bation, cells /ml

Saline solution, 1.0%                                  3 x 107

Digested sludge, autoclaved
Liquid phase, autoclaved

Liquid phase, nonautoclaved

Precipitate, resuspended with liquid
   phase, autoclaved                                    1CH
Precipitate, resuspended with distilled
   water, autoclaved                                 11 x
_
  Liquid phase obtained by two successive centrifugations; the first
  one at 5000 g for 20 min; the second one done at 30,000 g for 60 min
  on the supernatant liquor from the first centrifugation.  Both pre-
  cipitates were resuspended to original volume with either distilled
  water of supernatant liquor.
Langley et al . (86) upheld the view that elimination of S_. typhosa is
not caused by toxic compounds, but rather results from a nutritional
deficiency which can be satisfied by additions of tryptophane.  We
found that with E_. coli, reversal of the toxicity could be achieved by
addition of 5 g/1 tryptone (Difco) to the digested sludge.  This amount
was in excess of the usual nutritional needs.  At 2.5 g/1, tryptone did
not prevent die off of E_. coli .   The energy and growth factors brought
with tryptone could not be replaced by lactose and /or yeast extract
(Difco) .   Deficiencies in oxidizable organic material and an essential
nutrient do not create a toxic medium.  For example, the same fecal
coliform population which rapidly died off in digested sludge liquor
remained stationary in physiological saline solution.

By way of summarizing the findings up to this point it may be said that
fecal coliforms aerobically grown in a lactose broth and transferred to
the sludge, die off more rapidly if obtained from stock cultures than
if freshly isolated from the sludge.  This resistance of sludge isolates
disappears after several transfers in a lactose broth.  The bactericidal
properties are localized in the liquid phase of the sludge, but no anti-
biotics are detected by the diffusion a'gar method.  The boiling of the
sludge destroys the bactericidal properties, autoclaving does not.  The
lack of oxygen and the low oxido-reduction potential of the sludge are
                                 209

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not solely responsible for the killing.  Reversal of the bactericidal
action is obtained by the addition to the sludge of high concentrations
(5 gm/liter) of tryptone (Difco).   At the same concentrations, trypto-
phane and tyrosine reduce the death rate slightly.  Lactose or yeast
extract, or both, have no effect.   Digested sludge made from tryptone
as sole source of carbon is toxic; however, sludge made from butyrate
is not.

Effect of heavy metals on fecal coliform organisms - Differences in die
off rates of _E. coli in digested sludge as measured by the pour plate
and MFC procedures coupled with the characteristics of the toxicity
previously discussed suggested that heavy metals should be investigated
as a source of bacteriolytic behavior.  The pour plate technique gave
higher fecal coliform counts and a lower rate of die off than the MFC
method.  Shipe and Fields (143) had similar results with 12. coli cell
which had been suspended in zinc or copper sulfate solutions.  They
assumed that either toxic metals were concentrated on the membrane
surface or that some cells weakened by the metals could no longer form
colonies on the membrane.  Nearly all of the heavy metals present in
digested sludge occur in the solid phase.  Analyses of centrifuged
sludge liquor showed 0.057 to 0.10 ppm Cu, nondetectable to 0.10 ppm
Ni, and 0.075 to 0.15 ppm Zn. 'These concentrations were lower than
those which were shown to cause a reduction of 5 percent or less in
digester efficiency (Public Health Service, 1965).  Cadmium and Cr
were not detected in the liquid phase although they were present in
the solids.  Even at low concentrations, metals can be toxic to E_. coli.
For example, Malaney, et a]^. (99)  reported that as little as 0.3 ppm Cu
or 0.5 ppm Cd affects the metabolism of _E. coli.  Moreover, sublethal
concentrations of several metals can accumulate to toxic levels.

An objection to assigning the observed toxicity to heavy metals could
be raised because of precipitation of metals by carbonate, hydroxide,
and phosphate anions in the liquid phase of sludge.  To check the ef-
fect, 2 x 104 cells/ml of E_. coli ATCC 11775, as determined by the pour
plate technique with nutrient agar, were suspended in buffered and un-
buffered Cu solution.  The buffered solution was composed of
10~2M CuSO^ • 5H20 and 0.1 M phosphate at pH 7.0.  Based on the solu-
bility product of Copper phosphate (10~36.7)} the theoretical solubil-
ity of Cu in the buffered solution was 10-H-6 M.  The concentration of
Cu in the unbuffered solution was 10"^ M (0.83 ppm).  As expected, with-
in 12 hours the E_. coli cells were killed in unbuffered solution, but
survived in buffered solution.  One mechanism for metals to enter solu-
tion in digested sludge is through chelation with naturally occurring
organic compounds.  In this form they might retain toxic properties.
To test this possibility, a model system was developed with Cu and pro-
tocatechuic acid as a complexing a.gent.  It can be seen from the results
presented in Table 56 that when the Cu salt and protocatechuic acid were
present in equimoler quantities, the die off rate was approximately
                                 210

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proportional to the Cu concentration.  Neither the buffer-Cu nor the
buffer-chelate solution was toxic.  Complexation of Cu and protocate-
chuic acid apparently took place increasing the Cu toxicity.  Chela-
tion of metals is possible in digested sludge.  The digestion process
depends on a large population of viable microorganisms.  If heavy
metals in solution caused toxicity as was observed with JE. coli, di-
gestion itself would probably have been severely inhibited unless the
microbial population in the digester had acquired a high level of
tolerance toward chelated metals.  An observation of such a tolerance
was reported by Malaney _et^ al_. (99).  Within six days or less after
the addition of as much as 50 ppm Zn, 20 ppm Cu, or 16 ppm Ni, a
microbial population from sewage recovered 35 to 50% of its original
activity.  They described the toxicity as inhibitory, not lethal.
Table 56.  Toxicity of chelated copper toward Escherichia coli
Escherichia coli, cells/ml3
Incuba-
tion, hr
0
4
11
48
CuS04 • 5H20, protocatechuic
10-3c/io-3 10-4c/io-4
53 x 102 54 x 102
32 x 102 42 x 102
0 35 x 102
0 20
acid molar ratio^
10-3/0 0/10~3
77 x 102 62 x 102
119 x 102 56 x 102
79 x 102 55 x 102
137 x 102 31 x 102
a   Counts made by the pour plate technique with nutrient agar.
b   Solutions made, in 0,1 M phosphate buffer pH 7.0, heat-sterilized.
c   Protocatechuic acid added only after copper solution in phosphate
    buffer had stood at room temperature for 12 hr.
Fecal coliform survival on soils and in water - The behavior of the
sludge fecal coliforms as determined by the MFC technique has been
examined under various environmental conditions.  A gradual decrease
of the fecal coliform population was observed in the sludge cake which
develops on a soil surface amended with digested sludge, can be seen
from the results presented earlier in Table 5.  These results are in
agreement with those already obtained from various studies done on the
behavior of fecal coliforms and _E. coli in digested sludge, water and
soil samples (40)(86)(156).
                                211

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Routine analyses for fecal coliform densities have been performed on
drainage and runoff water samples originating from the Northeast Agronomy
Research Center lysimeters.  From an analysis of the data collected the
sandy soils (Plainfield) have performed as expected, i.e. no fecal coli-
forms were detected in tile drainage waters.  However, drainage waterx
samples from many Blount and Elliott plots were sometimes higher in
fecal coliform counts than expected.

Samples collected-in the spring were generally low in fecal coliforms.
That situation was probably a reflection of the fact that only four
sludge applications were made during the spring sampling period.  As
expected, surface runoff samples were generally contaminated with fecal
coliform throughout most of the sampling period.  The relatively high
contamination in tile drainage water can only be explained by contami-
nation through cracks or animal holes in the soil.  It was also noted
that as the soil temperature decreased, fecal coliform contamination
increased.  Increased longevity of fecal coliforms in cool soil and ac-
cumulating sludge residues probably accounted for the increase.

Runoff water from the check plots was almost as consistently contami-
nated as water from the plots with maximum treatment.  No specific
explanation for this pehnomenon can be given.  Warm-blooded animals all
excrete fecal organisms.  Gophers, ground squirrels, deer, and birds
frequent the plot area and may be responsible for the phenomenon.

The reader who would like to find general considerations on the hygienic
aspects of sludge disposal on land is referred to other publications
such as those by Gordon (56) and Hanks (62).

Microbiological purification of polluted waters by percolation through
artificial filters or soils is known to be an effective method of water
treatment.  Insofar as inferences can be made from traditions and ex-
periences, one may expect the percolated waters from a biofilter four
to five feet thick to be free of pathogens.  In the present case, the
challenge is at the soil-atmosphere, interface, where digested sludge
will cover acres and be accessible to runoff waters, insects, birds,
and animals.  The danger of infection from these fields will, to a
great extent, be controlled by the persistence of pathogens at the soil
surface.
                                 212

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                            SECTION VII
                        GREENHOUSE STUDIES
The Effect of Heavy Metals on Nutrient Uptake and Growth of Corn

Introduction - This study on the effect of Pb, Cu, Cr, Zn, and Ni on
nutrient uptake and growth of corn was prompted by their presence in
relatively large concentrations in digested sewage sludge.

Rohde (130) found toxic levels of Zn and Cu on soils treated with
sewage for many years near Paris, France.  Lunt (96) found Zn and
Cu toxicity symptoms in a few vegetable crops following additions of
acidic sludges.  Build-up seems likely in the upper soil horizons of
most of the heavy metals added by sludge, since studies on heavy
metal retention in soils have shown that £hey tend to accumulate in
upper horizons rather than leaching (85).  Sludge has also been known
to acidify soiJs.  This further enhances the possibility of available
toxic amounts of heavy metals in soils, since the heavy metals in
question become more soluble as pH decreases (51).

Although Pb, Cr and Ni have not been studied in connection with sludge
fertilization of soils, other studies have indicated a number of po-
tential problems due to excess levels in soil.  Lead has not been
found to be toxic, at least, to deep-rooted crops (79) (80).  It, along
with the other four heavy metals investigated here, does reduce Fe up-
take by plants, however.  Chromium has been found to be toxic in some
serpentine soils, sand cultures, and nutrient solutions (144).  Chro-
mium and Cu also interfere with P uptake.  Nickel can be even more
toxic than either Cu or Zn at similar concentrations (38)(41) (60)(66)
(123)(127).

Experimen.ta1 p ro c ed u r e - Lead, Zn, Cu, Cr, and NLwere applied to Plain-
field fine loamy sand as chemical salts at rates corresponding to quan-
tities added from 15 cm per year sludge applications for 5, 10, 15, and
20 years.  That is, rates of the added metals were equivalent to the
quantities that would be applied in 75, 150, 225, and 300 cm of sludge.
                                  213

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Concentration levels of heavy metals in sludge assumed here are given
in Table 57.  It is realized that this technique over-emphasized the
actual situation that would occur under field conditions.  However,
the intent was to simulate the highest possible concentrations of
metals that might occur from the respective sludge rates.  Thus, metal
additions were made on the assumptions that all metals in the sludge
would be released from organic form, and that the metal salts would
be retained in the upper soil horizon.  The authors are aware that
under actual conditions the organic material would only partially de-
compose and that salts would tend to leach from the upper horizon.
Table 57.  Heavy metal concentrations in sludge from the Calumet
           sewage plant.
Element
Copper
Zinc
Lead
Chromium
Nickel
kg/ha-cm
2.42
9.14
9.14
5.32
0.18
Each heavy metal was tested for its individual effect on corn growth.
No interactions between the heavy metals were studied.  Each treatment
was replicated three times in a randomized complete block design.

Some treatments were tested on an Elliott silt loam soil type for Pb,
Zn, Cu, and Cr.  These treatments were replicated in the same manner as
above.

The heavy metals were applied as the following chemical salts:  lead
acetate, zinc sulfate, cupric sulfate, chromic acetate, and nickel
sulfate.

Each greenhouse pot contained 3000 grams of soil to which 200 mg of
nitrogen was added as ammonium sulfate, 200 mg of phosphorus as mono-
calcium phosphate, and 200 mg of potassium as potassium chloride. 'The
"pots" were number 10 cans which were lined with polyethylene bags to
prevent rust from contaminating the soil.  Ten kernels of corn were
planted in each pot, and the stand thinned to eight plants after ger-
mination and emergence.  The corn plants were allowed to grow for four
weeks before harvesting.  They were cut off just above the soil.  Oven-
dry weight was used to determine stover yield.
                                 214

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 Zinc, Fe,  Mn, P, and Cu were determined in the plant tissue by emis-
 sion spectrograph.   Lead,  Cr and Ni were determined in the plant
 tissue by  atomic absorption.

 Regression analysis was used to determine significant effects on corn
 growth and its chemical content.  When a decrease or increase in yield
 or chemical content is declared significant, it will be so at the
 95 percent confidence level or higher.   All data given in tables and
 figures are mean values.

 Yield of elements was calculated by multiplying concentration levels
 in corn stover by oven-dry yield of corn stover.
•?-*,
"Results and discussion - The various Pb treatments affected corn growth
 in an erratic manner on the Plainfield sand (Figure 71).   Regression
 analysis of the yield data failed to show any significant effect on
 corn yield by adding Pb at rates up to 1224 ppm (twenty years accumula-
 tion) to either the sand or the silt loam.  However, the trend with the
 Elliott silt loam soil appears to be decreasing yield with increasing Pb
 rate.  Keatori's (80) data on barley growth showed no general trend as
 rates of Pb were increased.  His highest treatment was 2785 ppm of Pb
 as lead carbonate.   At most rates, yields of barley tops were slightly
 higher than the control.  Scharrer and Schropp (134) found very little
 effect on  plant growth when Pb was adaed to soils as lead acetate.
 They also  reported  some growth stimulation from Pb had no toxic effect
 on deep-rooted crops.  Jones and Hatch (79) reported that Pb had no
 toxic effect 011 deep-rooted crops.  Yet, they found some damage when
 some shallow-rooted vegetable crops were planted in Pb-treated soils.

 Lead was not present in the aerial portions of the corn except in
 trace amounts (Table 58).   This is in fair agreement with Keaton's (80)
 work, although he was able to show detectable concentrations of about
 2-3 ppm in barley tops.  His concentrations may be nothing more than
 an artifact due to incomplete washing, since the concentrations were
 so small and constant with all rates of lead.  He found very high con-
 centrations in root samples though.  Jones and Hatch (79) rarely found
 concentrations of lead over 10 ppm in the vegetable and legume crops
 they analyzed.  They did not find, however, concentrations in roots of
 their plants as great as Keaton had reported.  Lead concentrations in
 root samples were not determined in our experiment.

 A significant linear decrease in Fe content in corn stover occurred as
 Pb rates increased on the Plainfield fine loamy sand (Table 58) ."  There
 was no effect on iron content in the stover grown on the Elliott silt
 loam treated with Pb.  A highly significant linear decrease in P con-
 tent of the corn tissue occurred for both soils as lead rates increased.
 None of the other elements reported in Table 58 were significantly af-
 fected by  Pb,
                                 215

-------
Yield
gm/pot
                  PLAINFIEID
                       306
612
918
1224
                             Pb(ppm)
 Figure 71.  Yield (oven-dry weight) of corn at four weeks in the
           presence of Pb admixed with Elliott silt loam and Plain-
           field sand.
                         216

-------
Table 58.   Heavy metal and P content.of  corn  stover  as  influenced by
           Pb,  Cu,  Cr, Zn, and  Ni additions to  soils.
Treatments
Element
ppm
Concentrations in
Zn
ppm
Fe
ppm
Mn
ppm
corn stover
P
%
Cu
ppm
Plainfield
Pb
Pb
Pb
Pb
Pb
Cu
Cu
Cu
Cu
Cu

Cr
Cr
Cr
Cr
Cr
Zn
Zn
Zn
Zn
Zn

Ni
Ni
Ni
Ni
Ni


Pb
Pb
Pb
0
306
612
918
1224
0
81
162
243
324

0
178
356
534
712
0
306
612
918
1224

0
6
12
18
24


0
306
1224
110
141
139
91
104
110
70
87
108
132

110
93
77
66
80
110
4969
7535
9152
11776

110
147
174
219
199


59
59
57
129
149
123
83
93
129
58
60
88
81

129
110
87
73
94
129
94
95
140
96

129
142
189
148
153
Elliott

86
83
84
239
205
186
144
206
239
274
389
520
600

239
436
377
394
538
239
317
208
254
302

239
269
267
279
293
silt

79
74
59
.62
.48
.39
.26
.26
,62
.28
.48
.72
.69

.62
.35
.27
.28
.31
.62
.50
.76
1.23
1.25

.62
.77
1.02
1.02
1.02
loam

.30
.27
.18
13
15
14
11
19
13
28
76
122
260

13
12
13
8
11
13
12
13
12
12

13
16
19
21
20


6
7
9
Element varied
ppm
Pb
trace
trace
trace
trace
trace





Cr
4
26
24
40
32





Ni
trace
9
23
48
64

Pb_
trace
trace
trace
                                 217

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Table 58 (cont).   Heavy metal and P content of corn stover as influen-
          ced by Pb, Cu, Cr, Zn, and-Ni additions to soils.
  Treatments
Element    ppm
Concentrations in corn stover
Zn
ppm
Fe
ppm
Mn
ppm
P.
%
Cu
ppm
Element varied
     ppm
                       Elliott silt loam (cont)
Cu
Cu
Cu

Cr
Cr
Cr
Zn
Zn
Zn
0
81
324

0
534
712
0
306
1224
59
66
84

59
46
59
59
1000
5170
86
77
67

86
72
62
86
76
81
79
66
77

79
39
50
79
64
72
.30
.21
.16

.30
.17
.38
.30
.23
.20
6
10
18

6
8
9
6
5
8



Cr
2
14
14



Germination was reduced by high Pb addition to the Plainfield but not
the Elliott soil (Table 59).

Yield of elements was generally reduced as rate of Pb increased (Table 60)
Some of these reductions were usually due to both reduced yield and con-
centration levels in the corn.  Yield of Cu was affected relatively less
than the other elements.  Yield of P was reduced relatively more than
the other elements.

Rates of Cu corresponding to 15- and 20-year additions of sludge
(243 and 324 ppm Cu) were extremely detrimental to germination of corn
in the sandy soil (Table 59).  Germination was not affected by Cu added
to the silt loam soil.

Copper was very detrimental to growth of corn at the rates used in this
experiment.  On the Plainfield sand, both linear and quadratic regres-
sion terms for growth were highly significant (Figure 72).  Much of the
growth depression occurred with Cu rates no higher than 162 ppm.  On
the Elliott silt loam, there was a slight increase in corn yield at
81 ppm Cu.  This was not a significant increase, however.  The overall
effect of Cu was to significantly decre.ase growth of corn on the silt
loam.
                                218

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Yield
gm/pot
                         81
162
243
324
                              Cu(ppm)
   Figure 72.  Yield (oven-dry weight)  of corn at four weeks in the
            presence of Cu admixed with Elliott silt loam and Plain-
            field sand.
                         219

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Table 59.  Germination of corn 10 days after planting as influenced
           by Pb, Cu, Cr, Zn, and Ni additions to soils.
Treatment
Element
Pb
Pb
Pb
Pb
Pb
Cu
Cu
Cu
Cu
Cu
Cr
Cr ,
Cr
Cr
Cr
Zn
Zn
Zn
Zn
Zn
Ni
Ni
Ni
Ni
Ni
ppm
0
306
612
918
1224
0
81
162
243
324
0
178
356
534
712
0
306
612
918
1224
0
6
12
18
24
Plainfield
93
93
83
90
73
93
100
77
47
30
93
80
60
97
53
93
100
93
90
87
93
87
93
93
97
Germination, %
sand Elliott silt loam
93
97
—
—
97
93
93
—
—
100
93
—
—
100
93
93
90
—
—
90
	
—
—
—
—
                                 220

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Table 60.  Yield of Zn, Fe,  Mn, P, and Cu as influenced by Pb, Cu,
           Cr, Zn, and Ni.
Treatment
Element
ppm
Zn
Fe
Yield, mg
Mn

P

Cu
Plainfield sand
Pb
Pb
Pb
Pb
Pb
Cu
Cu
Cu
Cu
Cu
Cr
Cr
Cr
Cr
Cr
Zn
Zn
Zn
Zn
Zn
Ni
Ni
Ni
Ni
Ni

Pb
Pb
Pb
0
306
612
918
1224
0
81
162
243
324
0
178
356
534
712
0
306
612
918
1224
0
6
12
18
24

0
306
1224
.822
1.090
.916
.703
.646
,822
- .262
.083
.040
.024
.822
.652
.418
.437
.371
.822
17.000
3.630
2.150
2.660
.822
.970
.896
.884
.837

.488
.459
.385
.964
1.150
.811
.641
.598
.964
.217
.057
.033
.015
.964
.771
.473
.483
.435
.964
.322
.046
.033
.027
.964
.937
.973
.597
.644
Elliott
.711
.645
.567
1.78
1.59
1.23
1.11
1.28
1.78
1.03
0.37
0.19
0.11
1.78
3.06
2.05
2.61
2.49
1.78
1.09
0.10
0.0&
0.07
1.78
1.77
1.38
1.13
1.23
silt loam
.653
.575
.398
46.3
37.2
25.7
20.1
16.1
46.3
10.5
4.58
2.68
1.27
46.3
24.5
14.7
18.5
14.4
46.3
17.1
3.67
2.89
2.82
46.3
50.8
52.5
41.1
42.9

24.8
21.0
12.2
.0972
.1160
.0923
.0850
.1180
.0972
.1050
.0726
.0454
.0480
.0972
.0841
.0706
.0529
.0510
.0972
.0411
.0063
.0028
.0027
.0972
.1050
.0979
.0847
.0842

.0496
.0544
.0608
                                221

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Table 60 (cont).  Yield of Zn, Fe, Mn, P, and Cu as influenced by Pb,
          Cu, Cr, Zn, and Ni.
Treatment
Element

Cu
Cu
Cu
Cr
Cr
Cr
Zn
Zn
Zn
ppm

0
81
324
0
534
712
0
306
1224
Zn

.488
.612
.389
.488
.111
.058
.488
6.590
22.200
Yield, mg
Fe
Elliott silt
.711
.715
.310
.711
.173
.060
.711
.501
.348
Mn
loam (cont)
.653
.612
.356
.653
.094
.049
.653
.422
.309
P

24.8
19.5
7.41
24.8
4.08
3.70
24.8
15.2
8.6
Cu

.0496
.0928
.0833
.0496
.0192
.0088
.0496
.3330
.0344
Concentration levels of Cu in the aerial portions of the corn were not
very high considering the concentrations added to the Elliott silt loam
(Table 58).  This is in agreement with results reported by Reuther (127).
Copper concentration levels in corn grown on the Plainfield sand in-
creased significantly as the rate of added Cu was increased.

At the 5-year rate of Cu (81 ppm), corn grown on the sand had a severe
interveinal chlorosis symptomatic of "an iron deficiency.  Iron content
of plants was significantly reduced (Table 58).  On the silt loam soil
this chlorosis occurred at the 20-year rate of Cu although it took
longer to develop and was not as severe as the chlorosis which developed
at the 81-ppm treatment on the sandy soil.  Iron content, however, was
not significantly affected by Cu treatment at the 95 percent confidence
level.  Manganese content of corn on the sandy soil was increased as Cu
additions were increased.  This may have been due to pH changes in the
soils since the soils treated with Cu sulfate were more acid (0.5 - 0.7
pH units less) than the control.  No similar increase in Mn content oc-
curred for corn grown on the silt loam.  Zinc content of corn grown on
the sandy soil was depressed to 70 ppm at the 81-ppm Cu treatment and
then increased steadily thereafter as plants were more adversely affec-
ted by a Cu toxicity.

Copper additions reduced corn growth so much that yield of all elements
was greatly reduced as Cu rates were increased in the sandy soil
(Table 60).  Yield of elements was reduced on the silt loam soil, ex-
cept for Cr, as Cu was added.  The reduction in yield of P was much
greater than the reduction in stover yield on the silt loam soil.

                                 222

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           Chromium was more toxic to corn grown on the silt loam soil than when
           grown on the. sandy soil (Figure 73).  This was unexpected since the
           silt loam soil is more highly buffered than the sandy soil.  Growth
           reduction, although less than on the silt loam soil occurred on the
           sandy soil.  The linear regression term was significant for the sand
           even though the effect due to treatment was somewhat erratic.  On the
           silt loam soil a highly significant decrease in corn growth occurred
           as Cr additions were increased.  Severe stunting and purpling of the
           leaves occurred at the highest rate of Cr.

           Germination of corn in the sandy soil was affected by Cr, but this,
           too, did not seem to follow rates of application very closely (Table 59).

           Chromium significantly affected Fe content.  On both soils, Cr signi-
           ficantly decreased the Fe content of corn (Table 58).  At 714 ppm of
           Cr, P concentration of corn on the sandy soil was reduced to half that
           of the control, and this reduction was highly significant.  Total P
           uptake by the corn was significantly reduced on both soils (Table 60).
           Yield of Mn on sandy soil was the only element whose yield was not re-
           duced by increasing Cr application rates.

           Precipitation of phosphates by Cr in the soil is not likely since Cr
           phosphate, salts are soluble in dilute acid solutions and water.  There-
           fore, it would seem most likely that this reduction in P content in the
           plant due to hjgh Cr additions to the soil is a physiological phenomenon.
           Zinc content was also significantly reduced in corn grown on the sandy
           soil (Table 58),   This kind of reduction did not occur on the silt loam
           soil.

           Manganese content of corn and yield of Mn was significantly increased
           as amounts of Cr applied to the sandy soil were increased (Tables 58
           and 60).  This would seem to be directly related to Cr additions to the
           soil since pH of the soil did not change with treatment as it did with
           the Cu treatments.  On the silt loam soil, however, the reverse occur-
           red.  Manganese content in the corn stover decreased significantly as
           the rate of Cr applied to the silt loam increased.

           Concentrations of Cr in the corn grown on the two soils leads to another
X          paradox.  Content of the above-ground portion of corn was less on the
           silt loam soil where more damage to growth occurred, than on the sandy
           soil (Table 58).

           It would seem that perhaps a change in the valence of chromium occurred
           in the silt loam soil which did not occur in the sandy soil.  Perhaps
           this change in valence could have occurred through a biological oxida-
           tion of the chromic ion to the chromate or dichromate ion.  Microbial
           activity has been known to oxidize the manganous ion, so the possibil-
           ity of chromic ion oxidation may be quite good.  A purely chemical oxi-
           dation in the soil is remote since the chromic ion is only oxidized by
                                           223

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Yield
gm/pot
                0
178
356
534
712
                              Cr(ppm)
   Figure 73.  Yield  (oven-dry weight) of corn at four weeks in the
             presence of Cr admixed with Elliott silt loam and Plain-
             field  sand.
                        224

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rather strong oxidizers.  Since the silt loam had a good supply of
organic matter and the sandy soil hardly any, this would mean the mi-
crobe population should be at a minimum in the sandy soil but high in
the silt loam soil.  Microbes which could possibly oxidize the chromic
ion are facultative autotrophs.  Thus, the silt loam soil being more
abundant in organic matter content than the sandy soil can harbor a
larger population of these microbes.

Soane and Saunder '(144) suggested that the degree of Cr toxicity could
be influenced by Ca and P levels in soil.  Since our two soils differed
considerably in their Ca and P content, this, too, may explain the
differences in Cr toxicity between the two soils.

Oxidation state of Cr is very important because its toxicity is greatly
influenced by the valence.  Hewitt (66) reported that chromate was more
toxic than chromic, although dt is doubtful that in slightly acid soils
chromate will exist for long periods of time before it is converted to
dichromate.  This conversion, however, does not lead to a further change
in valence.  Soane and Saunder (144) used dichromate in their sand-
culture study.  They found 50 ppm of Cr as dichromate caused the same
severe symptoms that the highest rate of Cr caused in this study on the
silt loam soil.  Comparing the effect of chromic ion concentrations on
growth of corn on the sandy soil in this experiment with the effect of
dichromate ion concentrations on growth of corn on the sand-culture
experiment of Soane and Sanuders would seem to substantiate Hewitt's
finding.

Zinc was very toxic on the sandy soil even at the 5-year rate (306 ppm)
(Figure 74).   The plants were stunted and the lower leaves were bright
red.  The two highest rates of Zn caused growth to terminate shortly
after germination.  All the plants turned to a brilliant red.  On the
silt loam soil where only the 5-year and 20-year rates of Zn were ap-
plied, there was a slight growth depression at the first Zn rate and a
yield of only one-half that of the control at the highest rate.

Zinc increased to very high concentration levels in the corn for both
soils treated with Zn (Table 58).  Where 306 ppm of Zn were added, con-
centration levels in corn grown on the sandy and silt loam soils were
4969 and 1000 ppm of Zn, respectively.  Phosphorus deficiency does not
seem to be responsible for the red color which developed in the corn
on the Zn--treated sandy soil.  Phosphorus content of the plants increa-
sed as the rates of Zn increased on the sandy soil (Table 58).   The
high concentration level of P was undoubtedly due to the lack of plant
growth that prevented a dilution effect.  Phosphorus concentration
levels in the corn stover were significantly reduced as Zn application
rates were increased on the silt loam soil.

Germination was not appreciably affected by the Zn treatments (Table 59).
                                   225

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Yield
gm/pot
                       306
612
918
1224
                           Z n (ppm)
    Figure 74.  Yield (oven-dry weight) fo corn at four weeks in the
             presence of Zn admixed with Elliott silt loam and Plain-
             field sand.
                         226

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Nickel was applied only to the Plainfield sand.  Germination was not
decreased by the Ni treatments (Table 59).  Corn stover yield was sig-
nificantly decreased as the application rate of Ni was increased
(Figure 75).  Added Ni caused a highly significant increase in Ni con-
centration levels in plants (Table 58).  All chemical elements con-
sidered tended to increase in plant tissue as Ni application rates were
increased.  Yield of all elements, except Zn, tended to decrease as ap-
plication rates of Ni were increased (Table 60).

Nickel induced symptoms were very similar to those symptoms associated
with a Ca deficiency.  The corn growing on the Ni-treated soils started
to have very gummy, whip-like terminal leaves after two and one-half
weeks of growth.  These leaves failed to part completely; the tips were
glued together.  Leaf tips which appeared to be glued together died
after three or four days.  Tips of older separated leaves were riecrotic
and eventually died.  Hewitt (66) reported similar symptoms in potatoes
grown in sand culture studies with various concentration levels of ad-
ded Ni.  The older leaves of potatoes withered and the growing tips
died in the latter stages of toxicity, but this was preceded by a chlo-
rosis which Hewitt considered similar to Mn deficiency symptoms.
Soane and Saunder (144) also noted that toxic levels of Ni caused an
interveinaL chlorosis.  Chlorosis occurred only three days after emer-
gence when corn seedlings growing in a sand culture where 30 ppm Ni
were added.  Yet the Ca content of the corn increased as the applica-
tion rate of Ni was increased.   Crooke (38) observed an increase in Ca
content when 2.5 ppni Ni was present in a sand culture.  Perhaps Ni can
create a greater need for Ca in the plant.  This would explain why the
apparent Ca deficiency symptoms became worse despite increased Ca con-
tent in che com stover as application rates of Ni were increased.

Effects of Digested Sewage Sludge Added to Soil on Growth and Compo-
sition of Soybean:  Part I
Introduetion -- The experiment described here was carried out to deter-
mine the effects of heated anaerobically digested sludge mixed in large
quantities with soil which was subsequently planted to soybeans.  In
addition, an attempt was made to determine how different levels of
elements occurring :in sludge, added as salts to simulate a readily avail-
able form of the element, might interact with freshly-applied sludge to
affect plant growth.  The experiment, carried out under greenhouse con-
ditions, wan planned in a factorial manner with three levels of salt-
simulated sludge and of digested sludge.  The increments between treat-
ments increased by a factor of two, giving the highest combined treat-
ment a waight equrivalen*: of 144 t/ha of sludge solids.  The design
allosw comparison of the availability of the elements in sludge to be
compared with those added in salt form.
                                227

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Yield
gm/pot
                                12
                           N i  (ppm)
   Figure 75.  Yield (oven-dry weight) of corn at four weeks in the
            presence of Ni admixed with Plainfield sand.
                       228

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           Methods - Anaerobically digested sewage sludge was obtained from the
           Calumet Waste Treatment Plant of the Metropolitan Sanitary District of
           Greater Chicago.   The sludge represents sewerage from both industrial
           and domestic sources.  This sludge was analyzed (Table 61) for the
           elements subsequently used to prepare the salt-simulated sludge.  The
           salts were chosen to combine as many of the elements involved in the
           simulated sludge  and still maintain a highly soluble form.  For several
           of the metals it  was necessary to add acetate salts.  It was felt that
           the acetate would 'be rapidly utilized by the soil bacteria and not af-
           fect plants subsequently grown in the soil.  Sludge and simulated sludge
           were added incrementally to four kilograms of Elliott silt loam soils
           until the total amount for each treatment had been added.   The treat-
           ments amounted to 36, 72 and 144 t/ha of solids or equivalent in the
           case of simulated sludge.  The dry soil was crushed and four kilograms
           placed in a 20-cm diameter by 20-cm high plastic pot and distilled
           water was added from above and below to bring moisture content to
           20 percent by weight.  Soybean seeds QGlycine max (L.) Merr., var CorsoyJ
           were germinated in sand and when 2-to 3-cm high were transplanted to the
           pots.

           Moisture content  of the soil was maintained by daily watering, taking
           care to moisten the soil throughout its depth, and the amount of water
           added was recorded.  After 27 days, moisture content of soil was in-
           creased to 37.5 percent.  Surface area of the fourth leaf from the top
           on all plants was determined on the 35th day and leaf surface area of
           all leaves on the largest plant in each pot was determined on the 45th
           day.  Height of each plant was measured on day 12 and each week there-
           after.  Six plants from each pot were harvested to 2.5 cm above the soil
          ' surface after 22  days and during the rapid growth phase and three plants
           were harvested after 37 days, the date of initiation of blooming.  The
           plants were washed with distilled water, dried at 60°C and ground in a
           Wiley Mill to pass 40 mesh.

           Tissue was analyzed for Ca, Mg, Fe, Mn, Zn, Cu, Cd, and Ni by atomic
           absorption analysis after ashing at 500°C with care being taken to
           raise the temperature gradually and dissolving the ash in hydrochloric
           acid.  Phosphorus was determined by the vanadomolybdate yellow method
           and N by the Kjeldahl method.  Sodium and K were analyzed by flame
X          emission spectroscopy.  Also, the above elements, with the exception
           of N, were determined in a 0.1 N^ HC1 extract of an alloquot of the soil
           taken before planting.  A ratio of 0.5 gm soil to 10 ml of acid was usevd.

           Incidence of weeds was determined by counting the number of plants
           emerging in the pots after 13 days.  Germination of soybeans in the
           soil of each treatment was assessed by placing 100 seeds in Petri dishes
           containing the particular treatment and moistening the soil.  The num-
           ber of seeds germinating was counted from day 2 through 13.   This ger-
           mination experiment was not replicated.
                                           229

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Table 61.  Elemental composition of salt-simulated sludge and salts
           used in its formulation and composition of sludge from
           Calumet Sewage Treatment Works.  The Calumet sludge con-
           tained 3.13 percent solids of which 43.7 percent were
           volatilized by heating to 500°C.

Element
N

P
K
Ca
Mg
Na
Fe
Mn
Zn
Cu
S
B
Mo
Cr
Pb
Cd
Ni
Simulated Sludge
Salt Used g salt/1
(NH4)2SO .309
NH.C1 ,634
4
(NH.)H PO. 3.0006
424
NH.Mo_00. .0006
4 7 24
NH4OH . 244
KH0PO. .574
t. 4
NH4H2P04 3.066
KH2P04 .574
Ca(C2H302)2 5.275
Mg(C H 0 ) 1.610

FeC,H_0 -3H 0 5.354
b j / /
Mn(C H 0?)? .045
Zn(C H 0 ) .822
fii ( C* Ff 0 ^ 1 *^9
(NH )2SO .309
H3B03 .009
NH.Mo 0 , .0006
4 7 24
PT ( C VI 0 "^ T 71
Pb(C2H302)2 .070
Cd(C H 0 ) .033
Ni(C H 0 )0 .017
Calumet Sludge
mg element/1
800

850
165
1200
275

1000
10
170
42
75
1.5
.05
36
38
14
4
mg/1
900

626
205
1243
366
126
1230
16
148
33
n.d.
n.d.
n.d.
23
56
4
3
                                 230

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Results and discussion, general statement - All except the highest com-
bined treatment - equal to 288 t of solids/ha - supported plant growth
to the first harvest at 22 days.  However, plants died in a number of
treatments during the period between this harvest and the second harvest
at 37 days.  Plants affected were in the highest treatment of salt-sim-
ulated sludge and combinations of the second and highest levels of sim-
ulated sludge and sludge.  Reference to the section of Table 65 dealing
with the second cutting indicates those treatments which would not sup-
port growth.  Death of plants was preceded by a series of early symptoms
progressing from interveinal chlorosis, drying and rolling of the primary
leaves, through interveinal chlorosis and wrinkling of the second through
fourth trifoliate leaves.  Higher leaves appeared normal, especially in
the lower treatments.

The experiment was abandoned after 58 days because of the loss of
treatments and poor vigor in some of the surviving lower treatments.
Although toxicity due to the high levels of metals was an obvious and
appealing answer to the cause of loss of plants, we suspected salt ef-
fects.  Saturation extracts of the soils were prepared and conductivi-
ties were determined.  The conductivities (Table 62") indicate that salt
accumulation would be a considerable problem for growth of soybeans in
such a sludge-amended medium where percolating water would not remove
salts.  The salt-simulated sludge also created intolerable conditions
for soybean growth.  Sodium content of the paste extract (Table 63)
increases through each kind of treatment probably because the Na ion
is easily displaced from exchange positions on soil colloids by the more
abundant and strongly adsorbed two and higher valent ions in the sludges.
The nature of soybean growth after the soil in each treatment had been
leached to a conductivity of less than 1.0 mmho/cm or slightly more
than the 0.47 mmho/cm of the control soil is the subject of part two of
this report.  Data for pH of the soils are gathered into Table 64.  Ad-
dition of salts to the soil depressed pH by about one unit and analysis
of the data indicates main effects for both sludge and salt-simulated
sludge are present.  The range in pH involved in these treatments should
not markedly influence nutrient uptake or be physiologically detrimental,
in fact, the absorbtion of metals should be reduced substantially by
the slightly alkaline conditions.

Means of elemental contents of tissue for the first and second harvests
are given in Tables 65 through 76 for N, P, K, Ca, Mg, Fe, Mn, Zn, Cu,
Na, Ni, and Cd, respectively.  The data were treated by analysis of
variance to determine the presence of treatment effects.  To determine
mutual relationships among concentrations of elements, the data were
analyzed by correlation analysis.
                                231

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Table 62.  Effect of digested sludge and salt-simulated sludge on
           conductivity of saturation paste extract of soil.   Data
           in ramhos/cm.
DIGESTED SALT- SIMULATED
SLUDGE SLUDGE
t/ha 0 36 72 144
0 0.47 6.70 13.30 14.37
36 1.58 11.47 13.03 13.83
72 8.67 12.63 11.47 11.13
144 11.57 11.20 11.87 25.10


Average
Effect
8.71
9.98
10.98
14.93
Average
Effect 5.57 10.50 12.42 16.11
                                   232

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Table 63.   Effect of digested sludge and salt-simulated sludge on Na
           content of saturation paste extract of soil.  Data in parts
           per million of extract.
DIGESTED
SLUDGE
t/ha 0
0 4
36 9
72 21
144 45
Average
Effect 20
SALT- SIMULATED
SLUDGE
36
34
47
51
75
52
72
69
67
77
89
75
Average
144 Effect
128 59
118 60
135 71
167 94
137
                                  233

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Table 64.  Effect of digested sludge and salt-simulated sludge on pH
           of soil in which soybeans were grown.
DIGESTED SALT- SIMULATED
SLUDGE SLUDGE
t/ha 0 36
0 6.6 6.6
36 7.0 7.0
72 7.8 7.5
144 7.9 7.5
Average
Effect 7.3 7.2
72
6.4
6.8
7.1
7.2
6.9
Average
144 Effect
5.7 6.3
5.8 6.7
6.7 7.3
6.4 7.2
6.2
Least significant differences

   Interaction 0.6 (19:1)
   Average effect
     bait-simulated sludge 0.4 (99:1)
     Digested sludge 0.3  (99:1)
                                   234

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Table 65.  Effect of digested sludge and salt-simulated sludge on I? con-
           tent in soybean.  Data are in percent of oven-dry (60°C) tissue.
           Analysis of variance performed on 4 by 3 treatments for 22-day
           cutting and 4 by 2 treatments for 37-day cutting.  Average ef-
           fects for these treatments are in parentheses.

1stCutting,
DIGESTED
SLUDGE
t/ha
0
36
72
144
Average
Effect
SALT- SHUJLATED
SLUDGE
0
5.74
4.64
8.27
8.94
(6.86)
36
6.67
7.75
8.36
8.00
(7.64)
72
7.19
8.26
8.51
8.49
(8.11)
144
7.59
8.34
8.54
8.16
Average
Effect
5.76(6.48)
7.25(6.88)
8.37(8.31)
(8.47)

Least significant difference (P)

   Interaction 0.74 (99:1)
   Average effect
      Salt-simulated sludge 0.37 (99:1)
      Digested sludge 0.28 (99:1)

2nd Cutting, 37 days
DIGESTED
SLUDGE
t/ha
0
36
72
144
Average
Effect
SALT-S1MUIATED
SLUDGE
0
3.68
4.39
5.51
6.40
(5.00)
36
3.76
5.37
5.34
7.08
(5.39)
72
4.22
6.09
5.16
Average
144 Effect
3.89(3.72)
5.28(4.88)
(5.42)
(6.74) .
—
Least significant difference (P)
   Interaction 0.37 (99:1)
   Average effect
      Salt-simulated sludge 0.25(99:1)
      Digested sludge 0.25 (99:1)
                                    235

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Table 66.  Effect of digested sludge and salt-simulated sludge on P con-
           tent in soybean.  Data are in percent of oven-dry (60°C) tissue.
           Analysis of variance performed on 4 by 3 treatments for 22-day
           cutting and 4 by 2 treatments for 37-day cutting.  Average ef-
           fects for these treatments are in parentheses.

1st Cutting, 22 days
DIGESTED
SLUDGE

t/ha
0
36
72
144
Average
Effect
SALT- SIMULATED
SLUDGE

0
.42
.50
.48
.44

(.46)

36
.62
.52
.48
.48

(-52)

72
.70
.48
.37
.44

(.50)

144
.50
.44
.59
_—

.51
Average
Effect
.56(.58)
.48(.50)
.48(.44)
(.45)


Least significant difference (P)

   Interaction .13 (19:1)
   Average effect
      Salt-simulated sludge  none
      Digested sludge .05 (19:1)

2nd Cutting, 37 days
DIGESTED
SLUDGE
t/ha
0
36
72
144
Average
Effect
SALT- SIMULATED
SLUDGE
0
.43
.51
.58
.52
(.51)
36
.62
.49
.35
.34
(.45)
72
.56
.47
.52
Average
144 Effect
.54(.52)
.49(.50)
(.46)
(.43)
—
Least significant difference  (P)

   Interaction  .08  (99:1)
   Average effect
      Salt-simulated sludge   .03  (19:1)
      Digested  sludge  .04  (99:1)
                                   236

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Table 67.  Effect of digested sludge and salt-simulated sludge on K con-
           tent in soybean.  Data arc in percent of oven-dry (60°C) tissue.
           Analysis of variance performed on 4 by 3 treatments< for 22-day
           cutting and A by 2 treatments for 37-day cutting.  Average ef-
           fects for these treatments are in parentheses.

1st Cutting, 22 days
DIGESTED
SLUDGE
t/ha
0
36
72
144
Average
Effect
SALT-SIMULATED
SLUDGE
0
1.90
2.26
1.90
1.85
(1.98)
36
2.45
2.11
1.92
2.07
(2.14)
72
2.50
2.08
1.95
2.26
(2.20)
144
2.42
2.34
2.51
2.39
Average
Effect
2.32(2.28)
2.20(2.15)
2.07(1.92)
(2.06)

Least significant difference

   Interaction
   Average effect
      Salt-simulated sludge
      Digested sludge

2nd Cutting, 37 days
DIGESTED
SLUDGE
t/ha
0
36
72
144
Average
Effect
SALT- SIMULATED
SLUDGE
0
.98
.84
.71
.80
(.83)
36
.89
.72
.74
.96
(.83)
72
.80
.80
Average
144 Effect
.89(.93)
(.78)
(.72)
(.88)

Least significant difference

   Interaction
   Average effect
      Salt-simulated sludge
      Digested sludge
                                   237

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Table 68.  Effect of digested sludge and salt-simulated sludge on Ca con-
           tent in soybean.  Data are in percent of oven-dry (60°C) tissue.
           Analysis of variance performed on 4 by 3 treatments for 22-day
           cutting and 4 by 2 treatments for 37-day cutting.  Average ef-
           fects for these treatments are in parentheses.

[ st Cutting,  22_days

DIGESTED
SLUDGE
t/ha
0
36
72
144
Average
Effect
SALT- SIMULATED
SLUDGE
0
1.85
2.37
1.96
1.26
(1.86)
36
2.30
2.0?
1.38
1.00
(1.69)
72
1.91
1.24
1.03
1.00
(1.30)
144
1.61
1.26
.59
1.15
Average
Effect
1.93(2.04)
1.73(1.88)
1.24(1.45)
(1.09)

Least significant difference (P)

   Interaction 0.28 (99:1)
   Average effect
      Salt-simulated sludge 0.14(99:1)
      Digested sludge 0.1] (99:1)

•2nd Gutting, 37 days
DIGESTED
SLUDGE
t/ha
0
36
72
144
Average
Effect
SALT-SIMULATED
SLUDGE
0
1.51
3.52
3.63
3.24
(2.98)
36
2.49
4.63
3.51
3.07
(3.42)
72 144
4.08
4 . 08
Average
Effect
2.69(2.00)
(4.08)
(3.57)
(3.16)

Least significant difference  (P)

   Interaction 0.46  (99:1)
   Average effect
      Salt-simulated sludge 0.23 (99:1)
      Digested sludge 0.23 (99:1)
                                  238

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Table 69.  Effect of digested sludge and salt-simulated sludge on Mg con-
           tent in soybean.  Data are in percent oven-dry (60°C) tissue.
           Analyses of variance performed on 4 by 3 treatments for 22-day
           cutting and 4 by 2 treatments for 37-day cutting.  Average ef-
           fects for these treatments are in parentheses.

1st Cutting, 22 days
DIGESTED
SLUDGE

t/ha
0
36
72
144
Average
Effect
SALT-SIMULATED
SLUDGE

0
.89
.78
.76
.69

(.78)

36
.73
.71
.63
.60

(.67)

72
.64
.52
.48
.50

(.53)

144
.60
.50
.36
— —

.49
Average
Effect
.71(.75)
.63(.67)
.56(.62)
(.60)


Least significant difference (P)

   Interaction 0.05 (19:1)
   Average effect
      Salt-simulated sludge 0.03 (99:1)
      Digested sludge 0.02 (99:1)

2nd Cutting, 37 days
DIGESTED
SLUDGE
t/ha
0
36
72
144
Average
Effect


0
.62
1.01
1.04
1.00
(.92)
SALT- SIMULATED
SLUDGE
36 72
.90 .98
1.17
.99
.95
(1.00) .98


Average
144 Effect
.83(.76)
(1.09)
(1.02)
(.98) v

Least significant difference (P)

   Interaction 0.15 (99:1)
   Average effect
      Salt-simulated sludge 0.06 (19:1)
      Digested sludge 0.08 (99:1)
                                  239

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Table 70.  Effect of digested sludge and salt-simulated sludge on Fe con-
           tent in soybean.   Data are in parts per pillion of oven-dry (60°C)
           tissue.  Analysis of variance performed on 4 by 3 treatments for
           22-day cutting and 4 by 2 for treatments for 37-day cutting,
           Average effects for these treatments are in parentheses.    *

1st Cutting, 22 day
	 v
DIGESTED
SLUDGE

t/ha
0
36
72
144
Average
Effects
SALT- SIMULATED
SLUDGE

0
75
85
90
79

(82)

36
72
86
68
85

(78)

72
77
75
51
58

(65)

144
64
61
59
— —

61
Average
Effects
72(74)
77(82)
67(70)
(74)


Least significant difference (P)

   Interaction 16 (99:1)
   Average effect
      Salt-simulated sludge 11 (99:1)
      Digested sludge  none

2nd Cutting, 37 day
DIGESTED
SLUDGE

t/ha
0
36
72
144
Average
Effect


0
51
59
40
49

(50)
SALT- SIMULATED
SLUDGE

36 72
63 57
29
32
54

(44) 57

Average
144 Effects
57(57)
(44)
(36)
(52)

—
Least significant difference
             None
                                   240

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Table 71.  Effect of digested sludge and salt-simulated sludge on Mn con-
           tent in soybean.  Data are in parts per million of oven-dry (60°C)
           tissue.  Analysis of variance performed on 4 by 3 treatments for
           22-day cutting and 4 by 2 treatments for 37-day cutting.  Average
           effects for these treatments are in parentheses.

1st Cutting, 22 days
DIGESTED
SLUDGE

t/ha
0
36
72
144
Average
Effect
SALT-SIMULATED
SLUDGE

0
40
44
80
100

(66)

36
64
74
78
82

(74)

72
63
59
52
51

(56)

144
81
47
27
— —

52
Average
Effect
62(55)
56(59)
59(70)
(78)


Least significant difference (P)

   Interaction  18 (99:1)
   Average effect
      Salt-simulated sludge  9 (99:1)
      Digested sludge  7 (99:1)

2nd Cutting, 37 days
DIGESTED
SLUDGE
t/ha
0
36
72
1-44
Average
Effect

0
28
89
212
364
(173)
SALT- SIMULATED
SLUDGE
36 72
33 163
241
158
208 • --
(160) 163

Average
144 Effect
75(31)
(165)
( 186)
(286)

Least significant difference (P)

   Interaction  54 (99:1)
   Average effect
      Salt-simulated sludge  none
      Digested sludge  27 (99:1)
                                   241

-------
Table 72.  Effect of digested sludge and salt-simulated sludge on-Zn con-
           tent in soybean.   Data are in parts pec million of oven-dry (60°C)
           tissue.  Analysis of variance performed on 4 by 3 treatments for
           22-day cutting and 4 by 2 treatments for 37-day cutting.   Average
           effects for these treatments are in parentheses.

1st Cutting, 22 days
DIGESTED
SLUDGE
' T/ha
0
36
72
144
Average
Effect
SALT- SIMULATED
SLUDGE
o -
351
,966
771
761
(712)
36
919
892
633
637
(770)
72
1079
501
403
478
(615)
144
832
465
353
550
Average
Effect
795(783)
706(783)
540(602)
(625)

Least significant difference (P)
   Interaction  185 (99:1)
   Average effect
      Salt-simulated sludge  93 (99:1)
      Digested sludge  70 (99:1)

2nd Cutting, 37 days
DIGESTED
SLUDGE
t/ha
0
36
72
144
Average
Effect


0
172
291
367
353
(296)
SALT-SIMULATED
SLUDGE
36 72
228 420
352
188
216
(246) 420


Average
144 Effect
273(200)
(322)
(278)
(284)

Least significant difference (P)

   Interaction  135 (99:1)
   Average effect
      Salt-simulated sludge
      Digested sludge  68 (99:1)
                                   242

-------
Table 73.  Effect of digested sludge and salt-simulated sludge on Cu con-
           tent in soybean.  Data are in parts per million of oven-dry  (60°C)
           tissue.  Analysis of variance performed on 4 by 3 treatments for
           22-day cutting and 4 by 2 treatments for 37-day cutting.  Average
           effects for these treatments are in parentheses.

1st Cutting, 22 days
DIGESTED
SLUDGE

t/ha
0
36
72
144
Average
Effect
SALT-SIMULATED
SLUDGE

0
9.3
14
17
22

(16)

36
15
20
17
19

(18)

72
22
20
18
17

(19)

144
21
18
24
— —

21
Average
Effect
17(15)
18(18)
19(17)
(19)


Least significant difference (P)

   Interaction  6 (99:1)
   Average effect
      Salt-simulated sludge  2 (19:1)
      Digested sludge  none

2nd Cutting, 37 days
DIGESTED
SLUDGE
t/ha
0
36
72
144
Average
Effect

0
76
73
21
27
(49)
SALT- SIMULATED
SLUDGE
36 72
27 25
20
30
46
(31)

Average
144 Effect
43(51)
(46)
(26)
(36)

Least significant difference (P)

   Interaction  39 (99:1)
   Average effect
      Salt-simulated sludge  none
      Digested sludge  14 (19:1)
                                  243

-------
Table 74.  Effect of digested sludge and salt-simulated sludge on Na con-
           tent in soybean.  Data are _in parts per million of oven-dry (60°C)
           tissue.  Analysis of variance performed on 4 by 3 treatments for
           22-day cutting and 4 by 2 treatments for 37-day cutting.  Average
           effects for these treatments are in parentheses.            \

1st Cutting, 22 days
DIGESTED
SLUDGE
t/ha
0
36
72
144
Average
Effect
SALT-SIMULATED
SLUDGE
0
364
850
995
1192
(850)
36
372
519
680
1001
(643)
72
452
577
802
1209
(760)
144 .,
433
848
870
717
Average
Effect
405(396)
698(649)
837(826)
(1134)

Least significant difference (P)
   Interaction none
   Average effect
      Salt-simulated sludge  139 (99:1)
      Digested sludge  105 (99:1)

2nd Cutting, 37 days
DIGESTED
SLUDGE
t/ha
0
36
72
144
Average
Effect

0
134
2t)8
763
1215
(580)
SALT-SIMULATED
SLUDGE
36 72
146 198
414
1554
2574 ~
(1172) 198

Average
144 Effect
159(140)
(311)
(1158)
(1894)
(1894)
Least significant difference  (P)
   Interaction  529  (99:1)
   Average effect
      Salt-simulated sludge   264  (99:1)
      Digested sludge  264 (99:1)
                                     244

-------
Table 75.  Effect of digested sludge and salt-simulated sludge on Ni
           content in soybean.  Data are in parts per million of oven-
           
DIGESTED
SLUDGE
t/ha 0
0 5.3
36 5.3
72 '"""4.0
144 4.0
Average
Effect (4.6)
SALT-SIMULATED
SLUDGE
36 72
8.0 4.0
10.7
4.0
4.0
(6.7) 4.0
t* , '
Average
144 Effect
5.8(6.7)
(8.0)
(4.0)
(4.0)
—
Least significant difference

             None
                                  245

-------
Table 76.  Effect of digested sludge and salt-simulated sludge on Cd con-
           tent in soybean.  Data are in parts per million of oven-dry (60°C)
           tissue.  Analysis of variance performed on 4 by 3 treatments for
           22-day cutting and 4 by 2 treatments for 37-day cutting.  Average
           effects for these treatments are in parentheses.

1st Cutting, 22 days
DIGESTED
SLUDGE
t/ha
0
36
72
144
Average
Effect
SALT- SIMULATED
SLUDGE
0
1.0
3.0
3.4
2.7
(2.5)
36
5.3
5.1
2.8
2.9
(4.0)
72
6.7
3.2
2.5
2.3
(3.7)
144
8.6
3.2
1.6
4.5
Average
Effect
5.4(4.3)
3.6(3.8)
2.6(2.9)
(2.6)

Least significant difference (P)

   Interaction 1.1 (99:1)
   Average effect
      Salt-Simulated sludge  0.6 (99:1)
      Digested sludge  0.4 (99:1)

2nd Cutting, 37 days
DIGESTED
SLUDGE
t/ha
0
36
72
144
Average
Effect
SALT-SIJ1ULATED
SLUDGE
0
1.0
1.1
2.0
2.2
(1.6)
36
1.9
4.4
3.2
4.0
(3.4)
72
8.1
8.1
Average
144 Effect
3.7(2.8)
(2.8)
'(2.6)
. (3.1)

Least significant difference   (P)

   Interaction  1.1  (99:1)
   Average effect
      Salt-simulated sludge  0.6  (99:1)
      Digested sludge  0.6  (99:1)
                                     246

-------
Treatment effects for elemental content of the first cutting (22 days)
and miscellaneous data - The results of analysis of variance for main
effects and interaction for treatments are gathered into Table 84.  In-
spection of this table indicates that only the main effect for sludge on
Fe content and the. interaction of the treatments for Na are not signi-
ficant among the eleven elements analyzed.  Reference to Tables 68, 69,
72, and 76 indicates contents of Ca, Mg, Zn, and Cd, especially with
digested sludge treatment, decrease in content with increasing applica-
tion of sludge or "simulated sludge.  Ash content (Table 77), reflecting
these decreases, also undergoes a marked decline.  Na increases with
digested sludge treatment.  Production of dry matter or yield (Table 78)
and leaf surface area of all leaves at 35 days and of the fourth top
leaf at 45 days (Tables 79 and 80) are significantly related to digested
sludge treatment wherein increasing amounts of sludge applied depresses
yield.

Growth rate (Table 81) from the 12th through 21st days shows signifi-
cant negative treatment, effects for both sludge and simulated-sludge
treatments.  Interaction effects also occur.  Increasing treatments of
each kind depresses the incidence of weeds (Table 82) which were pri-
marily grasses occurring in the soil.  Similarly, the test of germi-
nation of soybean seeds (Table 83) shows significant negative effects
of digested sludge and interaction effects.  This interaction suggests
that inhibition is more closely associated with some toxicity than
with strictly osmotic effects.

Compared with values for soybean tissue reported by Walker (164) for
soybeans sampled across Illinois, N, Zn and perhaps K and Na contents
are very high.  Phosphorus contents are within the upper regions of
ranges reported,  iron contents are smaller than the lower limit of
the range reported by Walker and Mn values fall within the lower region.
Magnesium, P, K, and Ca, in the lower treatments, are in the range of
compositions cited by Ohlrogge  (115).  Contents of both N and P are
almost two times the values given by Hanway and Weber (63) for field
grown plants in Iowa.  Potassium is in the range given by Hanway and
Weber.  Compared with the data reported by Harper  (64), K, Cu and, par-
ticularly, Fe are lower at this growth stage and Zn is appreciately
"higher.

Correlations among elements and ash in the first cutting are plotted in
Figure 76.  A considerable number of significant correlations exist
among the elements.  The large number of negative correlations associa-
ted with Ca - note particularly the correlation coefficient of -.71 as-
sociated with N and Ca - and Mg (for N-Mg the r value is -.65) are
noteworthy.  The positive correlations among Zn, Cd, Fe, and Mn and the
other elements are also of interest, especially the very strong inter-
action (r = .70) between Zn and Cd.
                                 247

-------
Table 77.  Effect of digested sludge and salt-simulated sludge on ash
           content in soybean.  Data are in percent of oven-dry (60°C)
           tissue.  Analysis of variance performed on 4 by 3 treatments
           for 22-day cutting and 4 by 2 treatments for 37-day cutting.
           Average effects for these treatments are in parentheses.    v

1st Cutting, 22 days
DIGESTED
SLUDGE
t/ha
0
36
72
144
Average
Effect
SALT-SIMULATED
SLUDGE
0
9.61
11.16
9.37
8.16
(9.58)
36
11.62
9.05
7.66
7.50
(8.96)
72
9.73
6.33
5.39
6.36
(6.95)
144
7.99
6.38
6.07
6.81
Average
Effect
9.74(10.32)
8.23(8.85)
7.12(7.47)
(7.34)

Least significant difference (P)
   Interaction 1.21 (99:1)
   Average effect
      Salt-simulated sludge  0.61 (99:1)
      Digested sludge  1.21 (99:1)

2nd Cutting, 37 days
DIGESTED
SLUDGE
t/ha
0
36
72
144
Average
Effect
SALT-SIMULATED
SLUDGE

7
12
14
13
(11
0
.00
.64
.00
.09
,69)

10
16
13
12
(13
36
.93
.62
.38
.67
.40)
72 144
14 . 66
12.54
13.60
Average
Effect
10.86(8.
13.93(14
(13.69)
(12.88)


96)
.63)

Least significant difference
   Interaction  1.16  (99:1)
   Average effect
       Salt-simulated sludge  0.58  (99:1)
       Digested sludge  0.58  (99:1)
                                 248

-------
Table 78.  Effect of digested sludge and salt-simulated sludge on dry
           matter production in soybean.  Data are in grams per plant
           reported on oven-dry (60°C) basis.  Analysis of variance per-
           formed on 4 by 3 treatments for 22-day cutting and 4 by 2
           treatments for 37-day cutting.   Average effects for these
           treatments are in parentheses.
1st Cutting, 22 days

DIGESTED
SLUDGE

t/ha
0
36
72
144
Average
Effect
SALT-SIMULATED
SLUDGE

0
1.26
.95
.97
.93

(1.03)

36
1.09
.96
.87
.89

(.95)

72
.97
.88
.99
.83

(.92)

144
1.08
.73
.55
—

.79
Average
Effect
1.10(1.04)
.88(0.93)
.84(0.94)
(.88)


Least significant difference (P)

   Interaction  None
   Average effect
      Salt-simulated sludge  None
      Digested sludge  .09 (19:1)
2nd Cutting. 37 days
DIGESTED
SLUDGE
t/ha
0
36
72
144
Average
Effect
SALT-SIMULATED
SLUDGE
0
1.37
1.37
1.06
.94
(1.18)
36
3.14
.76
.59
.67
(1.29)
72 144
1.66
.34
1.00
Average
Effect
2.06(2.25)
.82(1.06)
(.82)
(.80)

Least significant difference  (P)

   Interaction 0.82 (99:1)
   Average effect
      Salt-simulated sludge  None
      Digested sludge  0.41  (99:1)
                                  249

-------
Table 79.  Effect of digested sludge and salt-simulated sludge on leaf
           surface area of all leaves of the largest plant in each pot.
           Data are in square centimeters.  Analysis of variance per-
           formed on 4 by 2 treatments.  Average effects for these
           treatments are in parentheses.
DIGESTED
SLUDGE
t/ha 0
0 127
36 123
72 131
144 91
Average
Effect (IIS)
SALT-SIMULATED
SLUDGE
36 72
376 174
95
56
75
(150) 174


Average
144 Effect
56 183(251)
(109)
(94)
(83)
56
Least significant difference (P)

   Interaction  72 (99:1)
   Average effect
      Salt-simulated sludge  none
      Digested sludge  36 (99:1)
                                   250

-------
         Table  80.   Effect  of digested  sludge  and  salt-simulated  sludge on leaf
                     surface of  fourth top  leaf of  the  tallest  plant  in each pot.
                     Data are in square  centimeters.  Analysis  of  variance per-
                     formed  on 4 by  2 treatments.   Average  effects for  these
                     treatments  are  in parentheses.
DIGESTED
SLUDGE
t/ha 0
0 18
36 17
72 14
144 10
SALT-SIMULATED
SLUDGE
36
25
12
8
7
Average
72 144 Effect
21 9 18(22)
4 — 11 (14)
(11)
(8)
         Average
         Effect         (15)         (13)          12
         Least significant difference   (P)

            Interaction  7 (99:1)
v           Average effect
               Salt-simulated sludge  none
               Digested sludge  4  (99:1)
                                           251

-------
Table 81.  Effects of digested sludge and salt-simulated sludge on growth
           rate of soybean over tvo periods.  Data are in centimeters per
           day.  Analysis of variance 'performed on 4 by 3 treatments for
           12-to 21-day period and on 4 by 2 treatments for 21-to 41-day
           period.  Average effects for these treatments are in parentheses.

12 to 21 Days
DIGESTED
SLUDGE
t/ha
0
36
72
144
Average
Effect
SALT- SIMULATED
SLUDGE
0
.371
.319
.275
.265
(.308)
36
.403
.262
.219
.281
(.291)
72 . 144 .
.365 .184
.155
.081
. 109
(.178) .184
Average
Effect
.33K.380)
.245(.246)
(.192)
(.218)

Least significant difference (P)

   Interaction  None
   Average effect
      Salt-simulated sludge  ,060 (99:1)
      Digested sludge ".046 (99:1)

21 to 41 Days
DIGESTED
SLUDGE
t/ha
0
36
72
144
Average
Effect

0
.258
.196
.125
.095
(.168)
SALT-SIMULATED
SLUDGE
36 72
.432 .212
.100
.133
.070
(.184) .212

144 Average
Effect
.30K.345)
(.148)
(.129)
(.082)

Least significant difference  (P)

   Interaction   .100  (99:1)
   Average effect
      Salt-simulated  sludge   None
      Digested sludge   .050  (99:1)
                                   252

-------
         Table 82.  Effect of digested sludge and salt-simulated sludge on weed
                    germination.  Data in number of plants germinating.
         DIGESTED
          SLUDGE
            SALT-SIMULATED
                SLUDGE
           t/ha
 0
36
72
144
Average
Effect
             0

            36

            72

           144
142

 40

 45

 33
70

43

42

15
18

18

22

 8
 5

 2

 2

 1
  58

  26

  28

  14
         Average
         Effect
 65
42
16
N.
                                           253

-------
Table 83.  Germination of soybean seed in soil used for unleached
           experiment.  Data are in percent of seed planted.  Only
           one trial per treatment was performed.
DIGESTED
SLUDGE
t/ha 0
0 94
36 100
72 94
144 56
AVERAGE
Effect 86
SALT-SIMULATED
SLUDGE
Average
36 72 144 Effect
86 94 91 91
92 98 55 86
91 88 41 78
87 86 8 59
86 92 49
                                  254

-------
                                N
             ASH
v\    38    P
    Cu
               Zn
               Fe
Figure 76.   Graphical representation of simple linear  correlations
            among macroelements ao
-------
Treatment effects for elemental contents of the^second cutting (37 days),
or the initiation of blooming - Except for the intermediate level of
salt-simulated sludge at the zero level of digested sludge, all treat-
ment blocks at the intermediate and highest treatment levels of salt-
simulated sludge were lost because of death during the time interval
between the first and second cuttings (compare, e.g., -cutting* in*
Table 78 above).  The data for 0 and 36 t/ha of simulated sludge across
the four treatment levels of digested sludge were treated by analysis .
of variance.  Zinc, Mg, Ca, P, Na, N, Cd, and ash show interaction and
main effects for both sludge and salt-simulated treatments.  Manganese
and K show highly significant sludge treatment and interaction effects.
Copper levels are affected by salt-simulated treatments and interaction
effects are present.  Iron is conspicuous for absence of treatments
and interaction effects of either kind.  Nitrogen and Mn, particularly
with digested sludge, and Ca increase in content with treatment, where-
as P declines.  Compared with the first cutting, Cu undergoes a two-
fold increase in content.  Yield of the second cutting shows a highly
significant main effect with sludge treatment and there is a highly
significant interaction effect.  Growth rate measured from the 21st
through 41st day (Table 81), leaf surface area of all leaves on the
35th day and of the fourth leaf on the 45th day (Table 79), and evapo-
ration on the day of harvest (data not presented here) all show signi-
ficant main effect for digested sludge treatment and interaction x^ith
the salt-simulated sludge.  Evaporation also is influenced by simulated
sludge (Table 84).

Compared with values for soybean tissue reported by Walker (164) for
soybeans in Illinois, Ca, Mn, Zn, Cu, and Na contents are very high.
Potassium contents fall within the lower range as reported by Walker.
Phosphorus, K, Ca, and Mg are in the range of contents summarized by
Ohlrogge (115) whereas N, as in the first cutting, remains high - al-
most two times the upper range found by Ohlrogge.  Nitrogen is also
above the highest contents found by Hanway and Weber (63) , and P is
two times that in field-produced plants at a similar growth stage.
With regard to N contents, it should be noted that only roots of plants
in the control and lowest simulated sludge treatment were nodulated.
Potassium is at the lowest level observed by Hanway and Weber for nod-
ulating plants without K fertility.  Potassium and Fe are about four
times lower and Cu two times lower than Harper (64) found at this
growth stage, whereas, Ca, Mg and Mn are about two times higher.

Correlations among elements and ash in the second cutting are plotted
in Figure 77.  As in the first cutting, a considerable number of sig-
nificant correlations exist among the elements.  The fewer negative
correlations associated with Ca and Mg are noteworthy with the high
positive correlation's between Ca and Mg (r = .92) and Na and N {r =  .90)
especially strong.  Copper and Cd are negatively correlated in contrast
to the positive correlation of Cd and Zn.  Modest but noteworthy cor-
relations of the first cutting not existing in the second cutting are
N and Zn, P and K, Fe and Mg, Fe and Mn, ana Cd and Na.
                                256
-------


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               ASH
       Cd
K
                                                              Mg
                 Zn
                                                 Fe
Figure 77.   Graphical representation of simple  linear correlations
            among macroelements in soybean  aerial plant parts harves-
            ted at 37 days, appearance of first blooms.  Numbers are
            correlation coefficients multiplied by 100.  Coefficients
            greater than 33 are significant at  the .05 level and coef-
            ficients greater than 40 are significant at the .01 level.
            Dashed lines are negative correlations.
                               258
-------
Effect of sodium on soybean - To assess the effect on plant growth of
the level.of Na occurring in the digested sludge utilized in the ex-
periment, two treatments of salt-simulated sludge were prepared using
Elliott silt loam.  One treatment was prepared at the level of 18 and
one at 36 t of solids/ha.  The reader will recall that Na was not added
to the salt combination (Table 61).  After the plants had grown 30 days,
Na, as NaCl, equivalent to a sludge application of 144 t of solids/ha
was added to the lower treatment and Na,  as CH3COONa, equivalent to the
same sludge application was added to the higher salt-simulated treat-
ment.  After seven days the plants in both treatments developed chlo-
rosis, browning and drying symptoms indistinguishable from those ob-
served in the factorial experiment.

Relationships among cuttings and soil extracts - Data for contents of
P, K, Ca, Mg, Fe, Mn, ~Zn, Cu, Na", Ni, Cd, and Pb in the acid extracts
of the soils are given in Tables 85 through 96, respectively.  These
data are set do'cn so that the render can observe the similar amounts
of the elements extractable with dilute acid from either salt-simulated
or digested sludge.  Inspection, of the average effects of each treat-
ment indicates that only Fe, Cd and Ni differ markedly, there being a
substantial interaction effect of unknown source for Fe.  Of course,
Na was not added in the salt mixture.  These data and similar results
laid out in part two of this report can be taken as empirical substan-
tiation of the choice of salts and method of soil preparation.

Values for correlation coefficients of the elements as determined in
the two cuttings and in the acid extracts of the soils of the respective
treatments are arranged in Table 97.  For the elements analyzed, only
Mn and P in the first cutting are not significantly correlated with the
acid-extract contents of these elements.   Among the elements that are
significantly correlated, Zn, Fe, Mn, Ca, Mg, and P are negatively cor-
related.  These would usually be considered to correlate positively
with the extract values.  Correlation of elements in the second cutting
with the same extract values results in notably fewer significant rela-
tionships, probably in response to the considerable physiological stress
experienced during the period between cuttings.  Sodium, P and Cd are
the only elements that can be correlated with the soil extract, and among
these P is negative.  Between cuttings, K, Mg, Fe, and Cu are negatively
correlated and Mn, Na and Cd are positively correlated.  Extractable Na
is especially highly correlated with plant levels, a fact that lends
support to the contention that salinity has affected plant development."
The increase in Cd is important because of concern for this potentially
toxic element to animals.

Summary and conclusions^ - Under the conditions of moisture content main-
tained, that is, at or slightly above field capacity, and the levels of
sludge applied in this experiment, considerably negative effects on
plant growth are to be expected.  The effects of suppression of growth
and toxicity are expressed primarily through osmotic influences on soil
                                  259
-------
Table 85.  Effect of digested sludge and salt-simulated sludge on P
           in 0.1 N^ HC1 extracts of soils in which soybeans were grown.
           Data in parts per million oven-dry (110°C) soil.
DIGESTED SALT- SIMULATED
SLUDGE SLUDGE
t/ha 0 36 72 144
0 60 280 550 - 1077
36 210 413 727 1137
72 377 623 927 1220
144 617 ' 863 1113 3013
AVERAGE
EFFECT 316 545 829 1612


Average
Effect
494
622
787
1402


Least significant difference

   Interaction  319 (99:1)
   Average effect
      Digested sludge  101  (99:1)
      Salt-Simulated sludge  159  (99:1)
                                 260
-------
Table 86-  Effect of digested sludge and salt-simulated sludge on
           K in 0.1 IJ HC1 extracts of soils in which soybeans were
           grown.  Data in parts per million oven-dry (110°C) soil.
DIGESTED SALT-SIMULATED
SLUDGE SLUDGE
t/ha 0 36 72
0 156 195 254
36 172 225 287
72 208 261 330
144 265 331 399
AVERAGE
EFFECT 200 253 318
Average
144 Effect
377 246
413 274
418 304
885 470
523
Least significant difference

   Interaction  76 (99:1)
   Average effect
      Salt-simulated sludge  38 (99:1)
      Digested sludge  24 (99:1)
                               261
-------
Table 87.  Effect of digested sludge and salt-simulated sludge on
           Ca in 0.1 N_ HC1 extracts of soils in which soybeans were
           grown.  Data in percent of oven-dry (110°C) soil.
DIGESTED SALT-SIMULATED
SLUDGE SLUDGE
t/ha 0 36 72
0 .37 .46 .48
36 .44 .52 .59
72 .49 .59 .68
144 .62 .69 .80
Average
Effect .48 .56 .64


Average
144 Effect
.64 .49
.70 .56
.72 .62
.85 .74
.73
Least significant difference

   Interaction   .05  (19:1)
   Average effect
      Salt-simulated sludge .04  (99:1)
      Digested sludge   .02  (99:1)
                                   262
-------
Table 88.  Effect of digested sludge and salt-simulated sludge on
           Mg in 0.1 N_ HC1 extracts of soils in which soybeans were
           grown.  Data are in parts per million of oven-dry (110°C)
           soil.
DIGESTED SALT-SIMULATED
SLUDGE SLUDGE
t/ha 0 36 72
0 780 796 744
36 848 753 826
72 918 852 920
144 978 996 1060
Average
Effect 881 849 888


Average
144 Effect
902 806
1006 858
937 907
1148 1046
998
Least significant difference
   Interaction  134 (19:1)
   Average effect
      Digested sludge  57 (99:1)
      Salt-simulated sludge  90 (99:1)
                                  263
-------
Table 89.  Effect of digested sludge and salf-simulated sludge on
           Fe in 0.1 N^ HC1 extracts of soils in which soybeans were
           grown.  Data are in parts per million of oven-dry (110°C)
           soil.
DIGESTED SALT- SIMULATED
SLUDGE SLUDGE
t/ha 0 36 72 144
0 233 340 519 1097
36 425 399 612 1596
72 557 457 633 1441
144 652 579 791 1751
Average
Effect
547
758
772
943
Average
Effect 467 444 639 1471
Least significant difference
   Interaction  207  (99:1)
   Average effect
       Salt-simulated sludge  103  (99:1)
       Digested sludge  65  (99:1)
                                   264
-------
Table 90-  Effect of digested sludge and salt-simulated sludge on
           Mn in 0.1 FJ HC1 extracts of soils in which soybeans were
           grown.  Data are in parts per million of oven-dry  (110°C)
           soil.
DIGESTED
 SLUDGE
            SALT-SIMULATED
                SLUDGE
  t/ha
            36
             72
             144
             Average
             Effect
    0

   36

   72

  144
145

194

265

200
222

199

233

255
231

228

220

288
276

284

315

330
218

226

258

268
Average
Effect
201
227
242
301
Least significant difference
   Interaction  61 (99:1)
   Average effect
      Salt-simulated sludge  30 (99:1)
      Digested sludge  19 (99:1)
                                  265
-------
Table 91.  Effect of digested sludge and salt-simulated sludge on
           Zn in 0.1 N[ HC1 extracts of soils in which soybeans were
           grown.  Data are in parts per million of oven-dry (110°C)
           soil.
DIGESTED SALT-SIMULATED
SLUDGE SLUDGE
t/ha 0 36 72
0 12 115 217
36 84 165 303
72 158 264 382
144 293 392 453
Average
Effect 137 234 339
Average
144 Effect
418 190
483 259
510 328
587 431
500
Least significant difference

   Interaction  47 (99:1)
   Average effect
      Salt-simulated sludge  24  (99:1)
      Digested sludge  15 (99:1)
                                   266
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Table 92.  Effect of digested sludge and salt-simulated sludge on
           Cu in 0.1 _N HC1 extracts of soils in which soybeans were
           grown.  Data in parts per million of oven-dry (110°C)
           soil.
DIGESTED
SLUDGE
t/ha 0
0 12
36 27
72 52
144 80
Average
Effect 43
SALT- SIMULATED
SLUDGE
Average
36 72 144 Effect
25 51 86 44
45 70 100 60
68 88 116 81
96 112 131 105
58 80 108
Least significant difference

   Interaction  11 (19:1)
   Average effect
      Digested sludge  4 (99:1)
      Salt-simulated sludge  7 (99:1)
                                 267
-------
Table 93.  Effect of digested sludge and salt-simulated sludge on
           Na in 0.1 .N HC1 extracts of soils in which soybeans
           were grown.  Data are in parts per million of oven-dry
           (110°C) soil.
DIGESTED SALT-SIMULATED
SLUDGE SLUDGE
t/ha 0 36 72
Average
144 Effect
    0

   36

   72

'  144
 79

110

160

234
 52

 96

130

215
 64

 96

143

241
 86

142

132

252
 70

111

141

236
Average
Effect
146
123
136
153
Least significant difference
   Interaction  26  (19:1)
   Average effect
      Digested sludge  11  (99:1)
      Salt-simulated sludge  18  (99:1)
                                  268
-------
•*• t-
             Table  94.  Effect  of  digested  sludge  and  salt-simulated sludge on
                        Ni  in 0.1  N^ HC1  extracts of  soils  in which soybeans were
                        grown.  Data are in parts  per  million of oven-dry (110°C)
                        soil.
DIGESTED SALT-SIMULATED
SLUDGE SLUDGE
t/ha 0 36 72
0 3.0 5.2 7.7
36 4.3 5.7 7.8
72 5.0 6.5 8.3
144 6.2 7.8 9.0
Average
Effect 4.6 6.3 8.2
Average
144 Effect
11 6.7
10 7.0
10 7.4
11 8.5
10
             Least significant difference

                Interaction  1.2  (19:1)
                Average effect
                   Salt-simulated  sludge   0.8  (99:1)
                   Digested sludge  0.5  (99:1)
                                               269
-------
Table 95.  Effect of digested sludge and salt-simulated sludge on
           Cd in 0.1 N^ HC1 extracts of soils in which soybeans were
           grown.  Data are in parts per million of oven-dry (110°C)
           soil.
DIGESTED SALT- SIMULATED
SLUDGE SLUDGE
t/ha 0 36 72
0 1.0 6.5 14
36 2.8 8.2 16
72 4.3 11 18
•144 7.9 14 19
Average
Effect 4.0 9.9 17


Average
144 Effect
16 11
26 13
27 15
27 17
26
Least significant difference
   Interaction  2.2 (99:1)
   Average effect
      Salt-simulated sludge  1.1 (99:1)
      Digested sludge  0.7 (99:1)
                                   270
-------
Table 96.  Effect of digested sludge and salt-simulated sludge  on
           Pb in 0.1 _N HC1 extracts of soil in which soybeans were
           grown.  Data are in parts per million of oven-dry  (110°C),
           soil.
DIGESTED SALT-SIMULATED
SLUDGE SLUDGE
t/ha 0 36 72
0 15 19 34
36 26 31 50
72 47 50 64
144 64 73 96
Average
Effect 38 43 61
Average
144 Effect
61 32
71 44
95 64
99 83
82
Least significant difference
   Interaction 18 (19:1)
   Average effect
      Salt-simulated sludge  12 (99:1)
      Digested sludge  7 (99:1)
                                   271
-------

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water and subsequently on elemental absorption by the plant.  The ef-
fect of Na appears strongest among the elements analyzed, although the
effects of other elements under the conditions of salt-induced physio-
logical stress are not evaluated.  Nitrogen, P and Zn appear to be
easily available from sludge sources to soybeans and occurred at lev-
els in aerial plant parts up to the time of first bloom exceeding those
found in field-cultivated plants.  Germination is inhibited by sludge
but this effect is, somewhat counteracted by simulated sludge.
                                 273
-------
Effects of Digested Sewage Sludge Ad.ded to Soil on Growth and Compo-
sition of Soybean:  Part II

Introduction - In part one of the experiment described here application
of relatively high amounts of liquid anaerobically digested sludge and
salt-simulated sludge to soil created salt conditions unfavorable for
growth of soybeans.  Results are presented here for the growth of soy-
beans in the same- treatments after the treated soils had been leached
to a conductivity nearly equivalent to that of the untreated or control
soil.  Field conditions such as those represented in this experiment
would be expected in a humid climate where soluble salts are removed
by percolating ground water.

Methods - Soils from each of the treatments used in the earlier experi-
ment were leached to a conductivity in the leachate of less than
1.0 mmhO/cm, slightly higher than the conductivity level of the leach-
ate from untreated Elliott silt loam used in the trial.  After leaching,
the soils were crushed, mixed and returned to their respective plastic
pots and 12 soybean Cciycine max (L.) Merr. var. Corsoyll seedlings
2-3 cm in height were planted in each pot.  Moisture content of the
soil was maintained, by weighing, at 37.5 percent.  The plants were
grown under greenhouse conditions during late July, August, September,
and October.  Daylength after August 10 was controlled at 14 hours.
The mean high and low temperatures for the period of the experiment
were 86.3 and 63.8°C,respectively.  After 19 days, six plants were
harvested, washed with distilled water, dried and ground in a Wiley
Mill to less than 40 mesh for analysis.  Similarly, three plants were
harvested after 30 days.  The remaining three plants were left to ma-
ture - 93 days - and seed as well as the aerial plant parts were har-
vested for analysis.  Germination in the soil of each treatment was
assessed by placing 100 seeds in Petri dishes containing the particular
treatment and moistening the soil.  The number of seeds germinating was
counted from days 3 through 5.

Tissue was analyzed for Ca, Mg, Fe, Mn, Zn, Cu, Cd, Cr, and Ni by atomic
absorption analysis after ashing at 500°C and dissolving the ash in
hydrochloric acid.  Phosphorus was determined by the vanadomolybdate
yellow method and N by the Kjeldahl method.  Sodium and K were analyzed
by flame emission spectroscopy.  Also, the above elements, with the ex-
ception of N, were determined in a 0.1 N^ HC1 extract of the soil using
a ratio of 0.5 gm soil to 10 ml acid and two-hour equilibration time.
A 1  to 1 soil-water paste was prepared for pH determination.

Results and discussion, general statement - All treatments supported
plants to maturity, although one replicate in the treatment combining
maximum rates of salt-simulated sludge-and digested sludge at the maxi-
mum  rate of 144 t of solids/ha of each and one replicate in the treat-
ment combining salt-simulated sludge at the rate of 72 t of solids/ha
                                 274
-------
with the maximum rate of digested sludge, did not produce pods.  In
subsequent analysis and correlation of the third cutting and seed data,
values for these missing data were calculated as the average of  the
remaining two replicates within the treatment.  Germination of soybean
seeds in the soil after leaching experienced none of the adverse treat-
ment effects occurring before leaching, with from 96 to 100 percent of
the seeds germinating in all treatment rates.  Soil pH  (Table 98) is
closely related to sludge treatment, with a negative simulated-sludge
interaction.  For all, except perhaps the lowest salt treatments, pH
is satisfactory for soybean nutrition.

Treatment effects of sludge and simulated sludge on soil Ifevels of
macro- and microelements - On extraction of elements from soil, the
levels of all elements measured in 0.1 N^ HC1 extracts (Tables 99
through 110 for P, K, Ca, Mg, Fe, Mn, Zn, Cu, Na, Ni, Cd, and Pb,
respectively) are very significantly (P < .01 in all cases, treatment
effects collected in Table: 138) affected by the treatments.  Also,
cump.it i :;on of I he mean effects; for e.;ich sludge type for the elements
cons 1-dered :mj;gesi .-, th.it the original levels added, the process of
leachlns' and the acid extraction employed combine to yield remarkably
similar concentrations for each incremental increase of sludge type
added.  These similarities are empirical evidence that  the attempt to
simulate the levels and availability of the elements in sludge was
successful.  Of special interest is the fact that interaction effects
between the treatments occur for all elements except Zn, Ni and Cu.
Correlation of the leached values with unleached data indicate that
levels of Zn, Mg and Cd extracted after the leaching procedure are
nearly equivalent to those extracted before leaching (for concentra-
tions prior to leaching compare Tables 85 through 96 in part one of
this section).  Copper, Fe, Mn, Ca, P, K, and Ni are from one-half to
three-quarters of the unleached levels.  In contrast, for simulated
treatments, Na levels decline to about one-third of those in the un-
leached series and Pb is about one-third the unleached values in both
sludge and simulated series.  Coefficients of determination for Zn,
Ca, P, K, Cu, and Cd in the above comparisons were above 0.80.  The
lowest coefficients xvrre Na at 0.29, a reflection of the rather com-
plete removal of readi.'y accessible sources of this ion by the leaeh-
ing procedure, and Mn at 0.32, perhaps a reflection of transformation
of this oxidation responsive ion into forms not readily available to
hydrochloric acid.  It should be noted that Na was the only element
among the analytes not added in the simulated sludge.  It occurs at
concentrations of about 125 ppm in the sludge used in the experiment.

Treatment effects, jirst cutting or beginning of rapid growth phase -
Data for yield of the first cutting are presented in Table 111.  Data
for tissue analyses of the elements determined in the plants at 19 days
are presented in Tables 112 through 136.   The tables are arranged in
the order N, P,  K, Ca, Mg, Fe, Mn, Zn, Cu, Na, Ni, Cd, and ash weight.
                                275
-------
Table 98.  Effects of digested sludge and salt-simulated sludge on pH
           of soil in which soybeans were grown.  Determinations made
           on a 1:1, by weight, soil-water paste.
SIMULATED DIGESTED
SLUDGE SLUDGE
t/ha 0 36 72
0 6.3 6.0 6.1
36 6.4 6.4 6.4
72 6.5 6.6 6.8
144 7.3 7.3 7.4
AVERAGE
EFFECT 6.6 6.6 6.7


Average
144 Effect
6.5 6.2
6.5 6.4
6.9 6.7
6.6 7.1

6.6
Least significant difference  (P)
   Interaction =0.2 (99:1)
   Average effect
      Salt-simulated sludge = 0.1 (99:1)
                                  276
-------
Table 99.  Effects of digested sludge and salt-simulated sludge on P  \
           contents in 0.1 .N HC1 extracts of soils  in which  soybeans
           were grown.  Data are reported in parts  per million of
           oven-dry (110°C) soil.
SIMULATED DIGESTED
SLUDGE SLUDGE
t/ha 0 36 72
0 103 200 427
36 157 387 607
72 390 647 857
144 837 947 1157
AVERAGE
EFFECT 372 545 762
144
747
563
1177
1973
1115
Average
Effect
369
428
768
1228

Least significant difference (P)

   Interaction = 168 (99:1)
   Average effect
      Digested sludge = 53 (99:1)
      Salt-simulated sludge = 84 (99:1)
                                 277
-------
Table 100.  Effects of digested sludge and salt-simulated sludge on K
           contents in 0.1 N_ HC1 extracts of soils in which soybeans
           were grown.  Data are reported in parts per million of
           oven-dry (110°C) soil.
S IMULATED D IGE S TED
SLUDGE SLUDGE
t/ha 0 36 72
0 110 117 150
36 110 173 197
72 160 213 240
144 287 333 357
'AVERAGE
EFFECT 167 209 236
Average
144 Effect
197 143
230 178
267 220
480 364
293
Least significant difference  (P)
   Interaction = 23  (99:1)
   Average effect
      Digested sludge = 7 (99:1)
      Salt-simulated sludge = 11 (99:1)
                                  278
-------
Table 101.  Effects of digested sludge and salt-simulated sludge on Ca
           contents in 0.1 N_ HC1 extracts of soils in which soybeans
           were grown.  Data are reported in percent of oven-dry  (110°C)
           soils.
SIMULATED DIGESTED
SLUDGE SLUDGE
t/ha 0 36 72
0 .30 .29 .30
36 .30 .30 .35
72 .33 .36 .42
144 .44 .50 .52
AVERAGE
EFFECT .34 .36 .40
Average
144 Effect
.38 .32
.43 .34
.49 .40
.59 .51
.47
Least significant difference
    Interaction = .03 (99:1)
    Average effect
       Digested sludge - .01 (99:1)
       Salt-simulated sludge = .01 (99:1)
                                 279
-------
Table 102. Effects of digested sludge and salt-simulated sludge on Mg
           contents in 0.1 N_ HC1 extracts of soils in which soybeans
           were grown.  Data are reported in parts per million of
           oven-dry (110°C) soil.
SIMULATED DIGESTED
SLUDGE SLUDGE
t/ha 0 36 72
0 747 660 667
36 670 643 733
72 627 693 853
144 837 930 1013
AVERAGE
EFFECT 720 732 817
144
783
880
990
1230
971
Average
Effect
714
732
791
1002

Least significant difference (P)
   Interaction = 76 (99:1)
   Average effect
      Digested sludge = 24 (99:1)
      Salt-simulated sludge = 38  (99:1)
                                 280
-------
Table 103. Effects of dipest'i-il .sludge  and  salt-simulated  sludge on F«
           contents in 0.1 N_ 11C1 extracts  of  soils  in which soybeans \
           were grown.  Data are reported  in  parts  per million of
           oven-dry (110°C) soil.
SIMULATED DIGESTED
SLUDGE SLUDGE
t/ha 0 36 72
0 188 307 348
36 240 299 480
72 333 397 594
144 543 793 989
AVERAGE
EFFECT 326 449 603


144
702
774
973
1363

953


Average
Effect
386
448
574
922


Least significant difference  (P)
   Interaction = 124  (19:1)
   Average effect
      Digested sludge = 53  (99:1)
      Salt-simulated  sludge = 84  (99:1)
                                  281
-------
Table 104.  Effects of digested sludge and salt-simulated sludge on Mn
           contents in 0.1 N^HC1 extracts of soils in which soybeans
           were grown.  Data are reported in parts per million of
           oven-dry (110°C) soil.
SIMULATED DIGESTED
SLUDGE SLUDGE
t/ha 0 36 72 144
0 127 169 214 271
36 129 224 255 256
72 168 216 250 256
144 226 226 251 348
AVERAGE
EFFECT 162 209 242 283


Average
Effect
195
216
222
262


Least significant difference (P)
   Interaction = 50 (99:1)
   Average effect
      Digested sludge = 16 (99:1)
      Salt-simulated sludge = 25 (99:1)
                                 282
-------
Table 105.  Effects of digested sludge and salt-simulated sludge on Zn
           contents in 0.1 N HC1 extracts of soils in which soybeans
           were grown.  Data are reported in parts per million of oven-
           dry (110°C) soil.
SIMULATED DIGESTED
SLUDGE * SLUDGE
t/ha 0 36 72
0 12 77 141
36 102 175 246
72 201 281 357
144 403 470 532
•AVERAGE
EFFECTS 180 251 319


Average
144 Effect
268 124
392 229
476 329
681 522

454
Least significant difference (P)
   Interaction = 25 (19:1)
   Average effect
      Digested sludge = 11 (99:1)
      Salt-simulated sludge = 17 (99:1)
                                 283
-------
Table 106. Effects of digested sludge and salt-simulated sludge on Cu
           contents in 0.1 N. HC1 extracts of soils in which soybeans
           were grown.  Data are reported in parts per million of
           oven-dry (110°C) soil.
SIMULATED DIGESTED
SLUDGE SLUDGE
t/ha 0 36
0 3.2 13
36 15 27
72 31 42
144 61 70
AVERAGE
EFFECT 27 38
72
30
44
61
85

55
Average
144 Effect
56 26
65 38
81 53
110 81

78
Least significant difference (P)
   Interaction = 9 (19:1)
   Average effect
      Digested sludge = 4  (99:1)
      Salt-simulated sludge = 6 (99:1)
                                  284
-------
Table 107 • Effects of digested sludge and salt-simulated sludge on Na
           contents in 0.1 N_ HC1 extracts of soils in which soybeans
           were grown.  Data are reported in parts per million of oven-
           dry (110°C) soil.
SIMULATED
SLUDGE
t/ha 0
0 37
36 42
72 22
144 32
AVERAGE
EFFECT 33
DIGESTED
SLUDGE
36
40
45
33
39

39
72
50
50.
42
73

54
Average
144 Effect
62 47
54 48
51 37
50 48

54
Least significant difference (p)
   Interaction = 14 (99:1)
   Average effect
      Digested sludge = 4 (99:1)
      Salt-simulated sludge = 7 (99:1)
                                 285
-------
Table 108 .  Effects of digested sludge and salt-simulated sludge on Ni
           contents in 0.1 N_ HC1 extracts of soils in which soybeans  v
           were grown.  Data are reported in parts permillion of oven-
           dry (110°C) soil.
SIMULATED
~ "SLUDGE
t/ha
0
36
72
144
AVERAGE
EFFECT
DIGESTED
SLUDGE
0 36 72
2.9 4.1 5.3
4.5 5.3 6.7
6.3 5.2 6.5
8.1 8.0 8.1
5.5 5.7 6.7

Average
144 Effect
5.9 4.6
7.2 5.9
6.7 6.2
8.6 8.2
7.1
Least significant difference (P)
   Interaction =1.8  (19:1)
   Average effect
      Digested sludge = 0.8 (99:1)
      Salt-simulated  sludge = 1.2  (99:1)
                                 286
-------
Table 109. Effects of digested sludge and salt-simulated sludge on Cd
           contents in 0.1 N_ HC1 extracts of soils in which soybeans  v
           were grown.  Data are reported in parts per million of oven-
         .  dry (110°C) soil.
SIMULATED . DIGESTED
SLUDGE SLUDGE
t/ha 0 36 72
0 .1 1.8 3.7
36 5.9 7.6 9.3
72 11 13 15
144 23 23 23
AVERAGE
EFFECT 10 11 13
Average
144 Effect
7.4 3.2
12 8.8
18 14
34 26
IS
Least significant difference (P)
   Interaction = 3.3 (99:1)
   Average effect
      Digested sludge = 1.0 (99:1)
      Salt-simulated sludge =1.7 (99:1)
                                 287
-------
Table 110.  Effects of digested sludge and salt-simulated sludge on Pb
           contents in 0.1 N_ HC1 extracts of soils in which soybeans
           were grown.  Data are reported in parts per million of oven-
           dry (110°C) soil.
SIMULATED DIGESTED
SLUDGE SLUDGE
t/ha 0 36
0 1.3 9.2
36 8.7 15
72 19 24
144 24 27
AVERAGE
EFFECT 13 19
72
16
21
29
26
23
Average
144 Effect
23 12
23 17
31 26
11 22
22
Least significant difference  (P)
   Interaction =3.7 (99:1)
   Average effect
      Digested sludge = 1.2 (99:1)
      Salt-simulated sludge = 1.8 (99:1)
                                 288
-------












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-------
Table 113. Effects of digested sludge and salt-simulated sludge on ¥
           content in soybean seed.  Data is percent of oven-dry (6Qoc)
           tissue.
SIMULATED DIGESTED
SLUDGE SLUDGE
t/ha 0 36 72 144
0 6.71 6.24 6.19 6.44
36 6.53 5.74 5.38 5.87
72 5.39 6.49 5.89 6.18
144 5.18 5.92 6.51 7.03
AVERAGE
EFFECT 5.95 6.10 5.99 6.38
Average
Effect
6.40
5.88
5.99
6.16


Least significant difference (P)
   Interaction = .96 (99:1)
   Average effect
      Salt-simulated sludge = .36 (19:1)
                                 291
-------





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-------
Table 115. Effects of digested sludge and salt-simulated sludge on P
           content in soybean seed.  Data are reported in percent of
           oven-dry  (60°C) tissue.
SIMULATED
SLUDGE
t/ha 0
0 .52
36 .66
72 .80
144 .80
AVERAGE
EFFECT .70
DIGESTED

36
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.80
.84
.75

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SLUDGE
72
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.78
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144
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Average
Effect
.69
.76
.78
.72


Least significant difference (P)
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   Average effect
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      Digested sludge =  .07 (99:1)
                                293
-------





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Table 117. Effects of digested .sludge and salt-simulated sludge on K
           content in soybean seed.  Data are reported in percent of
           oven-dry (60°C) tissue.
SIMULATED DIGESTED
SLUDGE SLUDGE
t/ha G 36 72
0 1.74 1.77 1.83
36 1.72 1.89 2.03
72 2.00 2.06 1.84
144 1.91 1.88 1.76
AVERAGE
EFFECT 1.84 1.90 1.87

„ I," - ».
*Vs ' r
144
1.77
1.99
1.72
1.70

1.80

Tt .
Average
Effect
1.78
1.91
1.90
1.81


Least significant difference (P)
   Interaction = .15 (99:1)
   Average effect
      Digested sludge = .05 (99:1)
      Salt-simulated sludge = .08 (99:1)
                                295
-------




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Table. 119- Effects of digested sludge and salt-simulated sludge on Ca
           contend in soybean seed.  Data are reported in percent of
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SIMULATED
SLUDGE
t/ha
0
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SLUDGE •
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.28
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                                297
-------





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Table 121. Effects of digested sludge and salt-simulated sludge on Mg
           content in soybean seed.  Data are reported in percent of
           oven-dry (60°C) tissue.
SIMULATED DIGESTED
SLUDGE SLUDGE
t/ha 0 36 72
0 .28 .30 .27
36 .29 .28 .28
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      Salt-simulated sludge = .01 (99:1)
                                299
-------





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Table 123. Effects of digested sludge and salt-simulated sludge on  Fe
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SIMULATED
SLUDGE
t/ha 0
0 54
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72 45
144 56
AVERAGE
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                               301
-------


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Table 125. Effects of digested sludge and salt-simulated sludge on
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SIMULATED
SLUDGE
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0 41
36 28
72 29
144 64
AVERAGE
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63 93
94 103
84 71
66 53

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                                 303
-------




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-------
Table 127. Effects of digested sludge and salt-simulated sludge on
           Zn content in soybean seed.  Data are reported in parts
           per million of oven-dry (60°C) tissue.
SIMULATED
SLUDGE
t/ha 0
0 40
36 62
72 75
144 64
AVERAGE
EFFECT 60
DIGESTED
SLUDGE
36
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62
67
74

67
72
64
64
64
82

68
144
60
67
69
80

69
Average
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58
64
69
75


Least significant difference (P)
   Interaction = 5 (99:1)
   Average effect
      Digested sludge = 2 (99:1)
      Salt-simulated sludge = 3 (99:1)
                               305
-------


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-------
Table 129. Effects of digested sludge and salt-simulated sludge on
           Cu content in soybean seed.  Data are reported in parts
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SIMULATED
SLUDGE

t/ha 0
0 11
36 8
72 8
144 11
AVERAGE
EFFECT 10
DIGESTED
SLUDGE

36 72
10 9
7 10
11 12
13 14

10 11


Average
144 Effect
11 10
12 9
13 11
11 12

12
Least significant difference (P)
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      Salt-simulated sludge = 1 (99:1)
                                307
-------



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Table 131. Effects of digested sludge and salt-simulated sludge on
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SIMULATED
SLUDGE
t/ha 0
0 37
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144 28
AVERAGE
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                                309
-------




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Table 133. Effects of digested sludge and salt-simulated sludge on
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S IMULATED
SLUDGE
t/ha 0
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72 4.2
144 4.6

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                                311
-------





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-------
Table 135.. Effects of digested sludge and  salt-simulated  sludge  on
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SIMULATED
SLUDGE
t/ha 0
0 0.4
36 2.2
72 2.9
144 3.3
AVERAGE
EFFECT 2 . 2
DIGESTED
SLUDGE
36 72 144
0.6 1.0 1.0
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                                313
-------



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weight.  Only l-'e 
-------
Table 137.  Effects of digested sludge and salt-simulated sludge on
           ash content of soybean seed.  Data are reported in per-
           cent of oven-dry (60°C) tissue.
SIMULATED
SLUDGE
t/ha
0
36
72
144
AVERAGE
EFFECT
DIGESTED
SLUDGE
0 36 72
5.54 6.03 6.20
5.87 6.55 6.68
6.35 6.78 6.14
6.59 6.20 5.72
6.09 6.39 6.18

144
6.17
6.10
5.80
5.69
5.94

Average
Effect
5.98
6.30
6.27
6.05

Least significant difference (P)
   Interaction = .40 (99:1)
   Average effect
      Digested sludge = .13 (99:1)
      Salt-simulated sludge =.20  (99:1)
                                316
-------
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ments, but P is about two times higher than their values.  As in the
first cutting, K, Fe and Cu are less than values cited by Harper (64)
for soybeans grown outdoors in sand culture.  Manganese values are
three to four times those reported by Harper.

Treatment effects, third cutting or mature plant at harvest - Analyses
of the third cutting are for the aerial plant parts without seeds and
pods.  Leaves were collected as they dropped.  Data for elemental con-
centrations and a'sh content are presented in Tables 112 through 136.
With the exceptions of Ca s Fe, Cu, Na, Ni, and Cd, digested-sludge
treatments show main effects for the elements analyzed.  The contents
of more elements are affected by salt-simulated sludge treatment with
only Ca, Mg, Na, and Ni lacking in significant treatment effects.
Interaction effects occur for all elements except Ca, Fe and Ni.  The
production of dry matter at maturity is highly significant for both
main effects and interaction, the combination of sludges reducing yield
somewhat (Figure 78) at the highest treatment levels.

Nitrogen and Mg are higher than values reported by Ohlrogge (115) as
is P, which is in the range that Webb et al. (167) cited for Mg defi-
cient plants.  Manganese values, except for the two lower treatments
of salt-simulated without digested sludge, are much higher than any
data cited by Ohlrogge.  Compared with data of Hanway and Weber (63)
for whole plant analyses at harvest, N tends to be lower, P three-times
higher and K is lower by one-third.  Nitrogen is one-half and K is
one-quarter the levels cited by Harper (64), whereas, Ca and Mg are
three-times higher, P two-times and Mn and Zn four-to ten-times higher.
Iron is slightly higher.

Treatment effects, seed - Among the data obtained, those for seed
analyses (Tables 113 through 137 and summary in Table 138) are of most
immediate Interest with regard to application of sludge on cropland
because seed is the product utilized in commerce.  Therefore, it is
particularly noteworthy that more main and interaction effects are
significant for seed than in any of the three harvests of plant tissue.
Only main effects of Mg and Na for- digested and Ni for salt-simulated
sludge and Ca and Na for interaction are not significant.  As in yield
of stems and leaves at maturity, the weight of beans is significant
for both main effects and interaction and the interaction is similar
to that in the third cutting wherein the combination substantially re-
duces yield (Table 139 and Figure 79).  Oil content  (Table 140) is
reflected in significant treatment effects for interaction and digested
sludge., sludge tending to diminish oil content probably partly because
of dilution due to protein increases.

Treatment effects, summary - Application of salt-simulated or digested
sludge results in significant differences in content of all elements
analyzed for in this experiment.  Only Ca, Na and, particularly, Ni
content of the aerial plant parts show decreasing tendencies for sig-
                                318
-------
Yield
  (g)
           0       18       36      72      144
             Salt - Simulated  Sludge
                        (t/ha)
 Figure 78.  Weight of aerial plant  portion of soybean at maturity
             without seeds and pods.  Weights are for one plant.  The
             surface is generated by the  equation Y = 1.03 -I- 0.75 X,
+ 2.19 X  + .009
- 0.18
                                            -  .092
, where KI is
The standard
             simulated salt and  X2  is  digested sludge.
             partial regression  coefficients are: Xj_, 0.63; X2 , 1.84;
             X±2, .064; X22,-1.29;  Xj_X2 , -0.45.  The multiple corre-
             lation coefficient  is  0.68.
                             319
-------
Table 135. Effects of digested sludge and salt-simulated sludge on
           seed weight.  Data are reported in grams of oven-dry
           (60°C) seed from 3 plants.
SIMULATED DIGESTED
SLUDGE SLUDGE
t/ha 0 36 72
0 5.41 10.55 12.14
36 9.72 10.10 12.57
72 10.20 8.95 13.31
144 12.78 12.60 8.59
AVERAGE
EFFECT 9.53 10.55 11.65


144
11.63
12.30
14.83
6.56

11.33


Average
Effect
9.93
11.17
11.82
10.13


Least significant difference  (P)
   Interaction =5.57  (99:1)
   Average effect
      Digested sludge =1.76  (93:1)
                                320
-------
Bean
 Wt
 (g)
        6.75
        6.00
                   18
              Salt - Simulated  Sludge
                         (t/ha)
Figure 79.  Weight  of  seeds  produced by digested- and simulated-
            sludge  applications  to Blount silt loam.  Weights are
            for seeds  collected  from three plants.  The surface is
            generated  by  the equation Y = 6.35 + 1.54 X^ + 1.42 \2
            - 0.11  Xi2 --  .068 X22 - 0.18 X]_X2, where Xi is simulated
            sludge  and X2 is digested sludge.  The standard partial
            regression coefficients are: Xi, 1.63; X2, 1.51; X^2^ -.99;
            X2*, -.63; X]_X2,  -1.11.  The multiple correlation coeffi-
            cient is 0.70.
                              321
-------
Table 140. Effects of digested sludge and salt-simulated sludge on
           oil content of soybean seed.   Data are reported in per-
           cent of moisture-free seed.
SIMULATED
SLUDGE
r/lia 0
0 2.1. (i
36 21.2
72 22.2
144 22.9
AVERAGE
EFFECT 21.8
DIGESTED
SLUDGE
16 7?
21.4 21.')
22.5 21.9
20.0 22.2
21.6 19.7

21.4 21.3


144
2J .2
21.1
20.7
19.2

20.6


Average
Effect
21.3
21.7
21.3
20.9


Least significant difference
   Interaction = 2.0 (99:1)
   Average effect
      Digested sludge = 0.6 (99:1)
                               322
-------
nifleant treatment effects as the soybean plant develops, however,
these elements do show treatment responses for elemental contents of
the seed.

Correlation?: oJ" elemental contents among cuttings and with soil -
Relationships among elements in the tissue of the first cutting are
evident in Figure 80.  The large number of significant relationships,
most of which are negative, with Na is noteworthy.  Also, the majority
of interelcmenLai ielationships with Ca and Mg are negative.  The
highest correlation coefficient, .89, occurs between Ca and Na.  Con-
siderably fewrn significant, relationships occur in the second cutting
(Figure 81) ar.;i aaiong these the sense of the relationship has changed
to negative for Cd and Ca contents.  At harvest the numbor of signi-
ficant iel it i •-• Kshipu  (.Figure 82) increased substantially over that of
the second cutting,   hoie negative relationships occur and Cu-Cd and
Cu-Zri correlations have changed to negative relationships.

Fewer significant correlations  (Figure 83) occur in the content of ele-
ments in seed than among elemental contents of the aerial plant parts
at harvest.  Nitrogen and Ca are noteworthy in that all correlations
associated with these elements are negative.  The four correlations as-
sociated with Ni contrast with only one correlation that exists among
all of the three cuttings of aerial plant parts.

Reference to Table? 141 and 142 offers an opportunity to follow the
correlation pt elemental content through the three cuttings, seed and
with soil extract.  Among the macroelements, P, K, and Mn, and Fe, to
a lessei extent, exhibit significant correlations between the first
harvest, subsequent cuttings, seed, and soil extract.  Among the micro-
elements, on'ly /'n shows a similar tendency for first cutting levels to
correlate with Jater cuttings, seed and soil.  In the seed, levels of
N, P, Mg, Fe, 
-------
    Cd
    Cu
                s'   \
              '       I
-  \  -< *       >'46
  >wx   x^v      /
 '  \   ^--xx   /
48   \     ^"^^^jv/i
     \38           Mg
          66
                Zn
         Fe
Figure  80.  Graphical  representation of simple linear correlations
           among macroelements and microelements in soybean aerial
           plant parts harvested at 21 days, the beginning of the
           rapid growth phase.  Numbers are correlation coefficients
           multiplied by .100.  Coefficients greater than 28 are sig-
           nificant at the .01 level.  Dashed lines are negative
           correlations.
                            324
-------
               Ni
                                                              50
    Cu
           Mg
          62
               Zn
Fe
Figure 81.   Graphical representation of simple linear  correlations
            among macroelements and microelements  in soybean aerial
            plant parts harvested at 32 days,  the  initiation of
            blooming.  Numbers are correlation coefficients multi-
            plied by 100.  Coefficients greater than 28 are signi-
            ficant at .05 level and coefficients greater than 37
            are  significant at the .01 level.   Dashed  lines are
            negative correlations.
                             325
-------
               Ni
    Cu
           Mg
                Zn
Fe
Figure 82.   Graphical representation of simple linear  correlations
            among macroelements and microelements  in soybean aerial
            plant parts harvested at 95 days,  the  date of harvest.
            Numbers  are correlation coefficients multiplied by 100.
            Coefficients greater than 28 are significant at .05 level
            and  coefficients greater than 37 are significant at the
            .01  level.  Dashed lines are negative  correlations.
                              326
-------
                Zn
Fe
Figure 83.  Graphical representation of  simple  linear  correlations
            among macroelements and  microelements  in soybean seeds.
            Numbers are correlation  coefficients, multiplied by 100.
            Coefficients greater than 28 are  significant at .05
            level and coefficients greater  than 37 are significant
            at the .01 level.   Dashed lines are negative correlations.
                               327
-------
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loading rate and this element is the source of adverse physiological
and pathological effects.  The highest levels observed in the seed
are below the 5 to 10 ppm of diet that Weber and Reid (168) found re-
duced b~one citric-acid levels in mice.  Assuming the concentration
effect in processing beans for meal the level of Cd remains below the
considered detrimental to livestock especially in view of the fact
that soybeans grown in sludge amended soil will be blended or mixed
with beans of low, native Cd content originating from usual agricul-
tural production.'  Of course, the important aspect of availability of
Cd in meal to absorption in the animal is not considered here,  Ad-
ditionally, the ameliorative effects of increasing amounts of Zn,
creating a similar Zn to Cd ratio, should be of benefit to any ulti-
mate animal use of the bean.  Except for K which occurs in small amounts,
digested sludge used in the study is a ready source for plant uptake of
all major elements and minor elements determined in this study.  Also,
the lack of toxicity symptoms and the positive results for yield sug-
gest that the ratios of elements taken up by soybean under the condi-
tions of this experiment are in favorable ranges for soybean growth and
development although the data suggest that application rates in excess
of 144 t/ha, especially over short time intervals, may be associated
with yield decline.
                                330
-------
                           SECTION VIII
                    SUPPLEMENTAL FIELD STUDIES
Plant Responses to Applications of Digested Sludge in Field Studies

Introduction - Yields of both corn and soybeans have been significantly
increased during the last four growing seasons by furrow irrigation
with digested sludge drawn from the heated anaerobic digester at the
Metropolitan Sanitary District of Chicago's Southwest Plant.  Sludge,
soils, and plant tissue samples were analyzed to determine nutrient
and certain nonnutrient chemical element accumulations in soils and
uptake by various grain, forage, and fiber crop plants.  Absorption
of some chemical elements by grain and forage crop plants has been in-
creased by sludge applications but concentrations in plant tissues are
considerably below levels considered to be harmful to animals consuming
the crops.

Experimental procedure:  corn - The continuous corn experiment was con-
ducted on 6 x 22 m plots and was replicated four times.  The soil was
Blount silt loam which had been cropped to alfalfa in 1965 and 1966.

The check plots were not fertilized in 1968 but 269 kg of N and 302 kg
of P (?2^5 equivalent) per hectare were applied annually during three
following years.  All plots received a broadcast application of 224 kg
of P per hectare (K^O equivalent) during the last three years.  All
inorganic fertilizer was applied in the spring before plowing and rid-
ging the soiJ.  After the ridges and furrows were established, corn
was planted on the ridge tops 76 cm apart at a row spacing to give a
plant population at harvest of about 44,440 in 1968 and 61,700 plants
per hectare in following years.  The first application of sludge after
planting was usually made when the corn plants had reached a height of
about 15 cm.  The treatments were 0-, 0.64-^ 1.3-j and 2.5-cm applications
applied as frequently during the growing season as drying conditions
of the sludge would permit.  Preemergent herbicides were used to con-
trol weeds and the plots were never cultivated during the growing sea-
son.
                                331
-------
Results of continuous cprn_ study ~ Average corn yields for three
rates of sludge iriigation, as compared with no sludge, are given in
Table 143.  Also given are the total liquid and dry solids applied
each year.
Table 143.  Corn yield obtained with sludge treatments and sludge
            treatment levels.
            Rate of Application
Greatest Application Rate
Year

1968
1969
1970
1971
4 yr
ave.
0
cm

4.16
8.96
5.53
6.06
6.18
0.64
cm
yield ton
6.03
9.34
7.48
6.50
7.34
1.3
cm
2.5
cm
per hectare
7.16
9.42
7.62
6.92
7.78
7.02
9.44
8.63
7.88
8.24
Total liquid
cm/yr
17.14
25.4
22.86
25.4*
90.80
Total dry solids
t/ha
51.5
47.3
69.0
96.5
264.3
*  An additional 12.7 cm were applied after the growing season con-
   taining 3.38 percent solids or on a dry weight basis, 41.2 tons
   per hectare.
In Table 143 it may seetn that with regard to 1969 yields the plots
treated with one-fourth maximum applications were as.great as those
from plots receiving higher rates.  Considering the carry-over of
nutrients from sludge application made during the previous year, plus
those made during the growing season, the lack of response to higher
applications of sludge was probably due to the fact that over 33 cm
of well-distributed rainfall occurred during the months of June and
July (Table 144).  It is somewhat remarkable though, that 25.4 cm of  v
additional water supplied as sludge did not cause a decrease in
yields for the highest treatment.  One might well expect a decrease
during a season of such high amounts of rainfall, because Blount silt
loam is poorly drained.  It appears that yields were substantially in-
creased as a result of thef additional water supjilied by the sludge
Irrigation treatments in 1970, a year in which 17.8 cm of rain fell
in June and July.  While a favorable response was obtained with sludge
applications in 1971, the increase in yields was less than expected
in a year during which only 11.2 cm of rain fell in June and July.
                                 332
-------
In some similar sludge-treated plots on the same field in 1971 the
conductivities of saturated extracts of soils were found to be greater
than 5 mmhos/cm.  Thus, under the extremely dry conditions experienced
during the 1971 growing season, high soluble salt contents may have
adversely affected corn yields on sludge-treated plots.
Table 144.  Monthly rainfall during the growing season, as cm.
Year    April     May     June    July    August    September    Totals
5.38
13.00
33.18
6.73
13.97
17.93
11.00
4.19
6.43
15.67
6.81
6.98
3.43
1.78
2.54
4.95
9.35
3.22
22.07
5.46
44.91
60.73
65.99
29.84
1968    6.35
1969    9.12
1970   10.39
1971    1.52
Although most oi the water appJled by sludge irrigation is lost by eva-
poration because the suspended solids seal the surface of the soil, this
sealing apparently serves to conserve available water stored in the root
zone.  On the other hand, when rainfall is insufficient to leach soluble
salts to lower depths, the increase in osmotic pressure in soil solu-
tions with higher rates of sludge applications may off-set some of the
water conserving advantage.  Nevertheless, it is noteworthy that neither
during the relatively wet growing season of 1969 nor the dry season of
1971 did yields decrease with increased sludge applications.

Digested sludge on a dry weight basis is a low grade fertilizer, but
when frequently applied as a liquid by irrigation methods large amounts
of almost all plant nutrients are supplied.  The total amounts of plant
macronutrients added to the soil as a result of maximum irrigation of
corn with digested sludge are given in Table 145.  These amounts have
been incrementally applied as constituents of the applied digested
sludge on maximum-treated plots during four years.  The aggregate of
103.4 cm of digested sludge applied during four years is equivalent to
a total solids loading of 305.5 dry tons per hectare.


Table 145.  Total plant macronutrients in kilograms per hectare applied
            as a constituent of sludge on corn plots receiving maximum
            treatments during four years.
Total N
14,426
NH4~N P
6,462 8,445
K
1,277
Ca
9,990
Mg
2,890
S
1,131
                                 333
-------
Digested sludge nearly always contains large quantities'-of plant es-
sential tnicronutricnts.  The amounts'applied on the maximum-treated
corn plots as a constituent of sludge are presented in Table 146.
Table 146.  Total plant essential micronutrients in kilograms per
            hectare applied as a constituent of sludge on corn plots
            receiving maximum treatments during four years.
  Fe        Zn        Cu        Mn        Mo         B         Cl
14,717     2,072     538       179       0.4        18       2,262
Plants are the source of certain minor elements that are essential to
the growth of animals but not for the plant itself.  In addition to
most of the elements listed in Table 146, animals require small amounts
of the elements listed in Table 147.  The data in Table 147 indicate
that relatively large amounts of essential elements for animals have
been added on the maximum-treated plots.
Table 147.  Minor elements (kilograms per hectare) applied as a
            constituent of sludge on crop plots receiving maximum
            treatments during four years.  These elements are con-
            sidered essential for animals but not for plants.
 Na            Cr            Co     ^       Se      -    •  -Hi
605           1288          1.2           1.6           130
Plants absorb many minor elements which are not presently considered
to be essential for either plants or animals.  Like most natural pro-
ducts, digested sludge contains nonessential trace elements.  The ele-
ments listed in Table 148 are those most frequently mentioned as
having potential for producing a detrimental effect on animals which
have consumed feed containing some concentration in excess of the
critical level.  The total amounts of four nonessential minor elements
applied during a four year period as a constituent of sludge on max-
imum-treated corn plots are given in Table 148.
                                 334
-------
Table 148.  Additional total trace elements (in kilograms per hectare)
            applied as a constituent of sludge on corn plots receiving
            maximum treatments during four years.  These elements are
            not considered essential for either plants or animals.
          Pb             Hg             Cd             Sn
         459            0.16           146            18.4
Some changes in soil chemical parameters with digested sludge applica-
tions are of interest.  First, in the absence of a continuous liming
program, frequent sludge applications will result in a lowering of the
pH in the soij surface as evident from the data presented in Table 149.
After the application of 42.5 cm during a two-year period the soil was
reduced from a pH value of 5.6 to 4,9.  The depression of soil pH
values probably was caused by the large amounts of nitrogen applied as
a constituent of digested sludge.  The pH values were allowed to reach
much lower values than would be permitted under a normal soil management
program, because we x\ranted to see how soil pll would effect the absorp-
tion of trace elements by corn plants, which is discussed later on.  In
the fall of 1970, limestone was applied on the plots at rates calculated
to raise the soil pH to a value of at least 6.  As much as 11.2 tons per
hectare of limestone were applied on the maximum sludge treated plots.
When the soils were sampled in the latter part of April, a few weeks
before planting the 1971 crop, only slight increases in soil pH values
were noted as a result of lime applications.  Another item to be noted
from Table 149 is the marked increase in plant available phosphorus
(Pi) with increasingly greater sludge applications.  After two years
of digested sludge application the' concentration levels of phosphorus
(Pp.) which are slowly available to plants from precipitants in soils
were greater than could be read from the standard laboratory calibra-
tion curve, even at the lowest sludge application rate.  The apparent
decrease in plant available potassium from 1969 to 1971 may be normal
variation related to soil conditions at time of sampling, sample hand-
ling, etc., but the trend is of interest.  Available K appears to have
been reduced in all plots, even though 224 kg per hectare of (K/?0 equi-
valent) K fertilizer were applied annually over all plots.  Soil contents
of plant available K do not appear to be influenced by sludge loading
rates.

After the first three years of applying digested sludge at the annual
rates given in Table 143, it was possible to detect significant changes
in the total concentrations of some elements in the surface of the soil.
As shown in Table 150, the concentration increases in soils of total P,
Cu, Cd, and Hg with increased loading rates were significant at the
                                335
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 1-percent  level.  Concentration  increases of Cr, Pb and Zn in the soil
 surface  horizon were  found  to be significant at the 5-percent level.
 Changes  in concentration  levels  were not noted  for all elements for
 several  reasons.  Digested  sludge may contain concentrations that are
 about  the  same as those in  soils, the chemical  elements have migrated
 with percolating water to lower  depths, or the  amount of a particular
 chemical species in soils is so  large that the  amounts added as a
 constituent  of sludge cannot yet be detected above the normal varia-
 tion between soil samples.

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 provides an  estimation of the plant availability or mobility of the
 several  species of elements.  As shown in Table 151, digested sludge
 applications have increased the  mobility of P, Na, Cr, Cu, Pb, Ni, Zn,
 and Cd in  the zero to 15.2-cm depth of Blount silt loam.  In the
 30.5-to  45.7-cm depth increased  mobilities of Na, Cr, Cu, Zn and Cd
 with sludge  applications  are highly significant, although actual change
 in conceiitration are  very small  for some species.  The increased mo-
 bilities of  Mn and Pb with  sludge application are significant at the
 5-percent  level.  An  important observation that may be made from the
 data in  Table 151 is  that whereas there is little doubt that available
 phosphorus increases  with sludge applications in the plow layer, in-
 creases  in concentration levels  in deeper soil horizons are highly
 variable.

 Data are gathered into Table 152 to show the relative proportion or
 percent  of the several total chemical elements extractable with
 0.1 N^ HC1.   Except for K, Ca, Mg, Fe, and Mn, extractability increases
 for all  elements with increased  sludge applications.

 As can be seen in Table 153, sludge applications were highly correlated
 with increased contents of N, P  and Zn in corn leaf tissue.  In the
 case of  Mg,  sludge applications  were highly correlated with a decreased
 content  of the element in the plant leaves.   Concentrations of the ele-
 ments Ca, Mn, Cd, and B as found in corn leaf tissue correlated with
 sludge applications at the 5-percent level of significance.  Only Zn
 and Cd concentrations in corn grain correlated at the 1-percent level
 with sludge  application.   The content of K in corn grain significantly
 correlated with sludge application at the 5-percent level.

 To summarize, it is evident that a favorable yield response can be ex-
 pected from relatively large sludge applications in a year of normal
weather  conditions.   However, corn yields were not decreased by sludge
 applications during a very wet growing season,   Trace elements added
 as constituents of sludge have not presented a problem, even though the
 Blount silt loam is a poorly drained soil and soil pH was permitted to
decrease to a low value with respect to crop production.   Since trace
elements would be most mobile or available to plants in poorly drained,
acid soils, the concentrations of trace element  in corn tissue samples
                                 338
-------






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arc higher then would be expected where internal soil drainage is bet-
ter and soil pH Is maintained at a value of 6 or greater.   Thus, corn
plants did not accumulate, toxic levels of trace elements even under
soil conditions x-.Thich should favor such a development.

Experimental procedure:   soybeans_ - The continuous soybean experiment
was established to test, under field conditions, the availability to
crop plants of phosphorus in digested sludge.  However, because of
what we now know about the rapid build-up of available phosphorus with
sludge applications, our present interest in continuing this study is
to see how the high soil contents of available phosphorus will effect
soybean rmtrlMon,  Soybeans were chosen for this study in order to
eliminate the effects that the nitrogen in the sludge might have on a
non-leguminous pLant,

Three replications of 12 x 12 m plots were established on Blount silt
loam in the fall of 1968 for the following treatments:  (1) zero or
control, (2) maximum, (3) one-half maximum,  (4) one-fourth maximum ap-
plication rates of sludge, and (5) well water supplied at the same time
and rate as the maximum sludge application.  The plots were split and
superphosphate was applied by broadcasting on one-half of each plot at
a rate to provide 269 kiJograms per hectare of P20s equivalent each
year.  Also, all plots received a broadcast application of potassium
chloride to provide 269 kilotrams per hectare of K20 equivalent.  After
fertilizer was applied,  the Blount silt loam plots were fall plowed.
During tae following years all inorganic fertilizer was applied before
spring plowing.  The soybeans were planted on the ridges and furrow-
irrigated with sludge and water in the same manner as was used for the
corn study described earlier.

Results and discussion - Soybean yields for phosphate, sludge and water
treatments are given in Table 154.  A significant increase in yields
ia response to additional phosphorus applications had not been observed
in three years.  Evidently the soil has sufficient available P to meet
the demands of soybean plants, even though the Blount silt loam study
site was selected because this soil type generally is somewhat defi-
cient in available P.  In all three years yields have been significantly
(P > .01) increased witu increased sludge- applications.  During the
first two years, water alone significantly increased yields, but not
in 1971.  Except, for the first year9 1969, the maximum sludge treatment
produced an increased yield over an equivalent amount of applied well
water.  However, the increased soybean yields for the maximum appli-
cation of sludge were better than the equivalent water treatment only
at the 5 percent  Level of significance in 1970.  While it is probably
too soon to speculate, Jt can be seen in Table 154 that there is a
trend toward decreased soybean yield by years for all treatments.  The
failure to obtain a  favorable yield response to the 20.3 cm of irriga-
tion water applied during the. very dry season of 1971  (see Table 144)
cannot be explained.
                                 342
-------
Table 154,  Soybean yield responses to phosphorus, sludge, and
            water applications.
Year
P205
kg /ha
Rate of sludge application
0 cm
0.64 cm
1.3 cm
2.5 cm
Water2-''
_ _ _ _ _ Metric ton per hectare - - - -
1969

1970

1971

0
269
0
269
0
269
2.28
2.53
1.93
1.89
1.77
1 . 53
3.02
3.00
2.76
2.57
1.93
1.87
3.24
3.16
2.98
2.84
2.10
2.08
3.36
3.50
2.84
3.19
2.13
2.12
2.92
3.48
2.57
2.59
1.50
1.74
Total
sludge
applied
en
20.3
20.3
22.9
22.9
33. ^
33. O^7
a/  Water was applied at the same rate and time as the maximum sludge
    application,
b/  20.32 of the 33.02 cm of sludge were applied during the growing
    season.
Several chemical elements (Table 155) were significantly increased in
the surface horizon of Blount silt loam by applications of digested
sludge.  Howevers no significant increases in soil contents of total
K, Mg, Mn, Na, or Ni were detected.  Failure to observe a significant
increase in total amounts of these elements is probably due to the
fact that sludge applications have not supplied amounts of the ele-
ments in sufficient quantities to exceed amounts removed in grain and
leaching Lo lower soil depths.  Also, the amounts of these elements
added are small compared to levels that occur naturally.  Applications
of inorganic P afLected only the accumulation of total Fe in the sur-
face of the soil,  Increases in surface-soil Fe contents with increased
sludge applications were significantly greater (P>0.05) in the absence
of the additiona.1 inorganic P.  Perhaps the inorganic P increased the
mobility of the iron, resulting in a greater movement of the element
to lower sol! depths, although no differences in extractable Fe con-
tent were observed in the 30.5-to 45.7-cm depth (Table 156).  Appli-
cations of inorganic P fertilizer did not significantly affect the
total P content in the soil surface, which is to be expected because
the levels added correspond to about ten percent of the naturally oc-
curring amount.

Concentration levels of chemical elements extractable with 0.1 IN HCl
from samples of soil collected from soybean plots are presented in
Table 156.  As may be seen in Table 156, the extractability of all
                                 343
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elements except Fe was significantly increased in the soil surface
horizon  (0 tc 15.2 cm) by digested sludge applications.  But the
application oi inorganic P did not affect the extractability of any
of  the elements ia the surface horizon.  It did, however, have a sig-
nificant affect, on the extractability of several elements from subsoil
samples  (30.5 to 45.7 cm).  Applications of inorganic P fertilizer
significantly decreased the levels of extractable Mn, Ni and Zn from
subsoil  samples.  Digested sludge applications significantly increased
the levels of" extractable K, Mg, Fe, Na, and Cr from subsoil samples.
In  the absence of inorganic P fertilizer applications, the application
of digested sludge decreased the extractability of Mn from subsoil
samples.  Where inorganic P fertilizer was applied, digested sludge
applications did not affect the extractability of Mn.  Thus, for sub-
soil extractabde Mn, a highly significant (P>0.01) interaction effect
between P fertilizer and sludge applications was found.

Among samples from the surface horizon some of the concentration values
(Table 156) for O.i N_ HC1 extractable Cd are greater from surface soil
samples  than are found for total Cd as presented in Table 155.  These
discrepancies are due in part to experimental error and to the varia-
bility among samples.  Even more important though is the fact that nearly
all of the Cd added in sludge is extracted by the acid.  Comparison of
the data givt-n in Tables 155 and 156 for increasing sludge treatments
bears out this relationship.  Cadmium supplied to soils as a constituent
of digested sludge exhibits a high degree of availability for absorption
by crop plants as indicated by the fact that extractable amounts are
comparable to amounts added.

Contents (Table 157) of P, Mg, Mn, Na, Zn, and B in soybean leaves col-
lected at the early bloom stage were significantly increased by digested
sludge apyliii ations.  Increased concentrations of these six elements in
soybean 3eaves with sludge treatment were significant at the one percent
level.  Analyses for Hg contents in soybean leaves from selected plots
were made and the results are also shown in Table 157.  Although the
Hg concentration data were not statistically analyzed, they do show a
trend toward an increased relationship between leaf contents of the
element and quantities of sludge applied.  It has been observed from
other studies that the Hg content in plant tissues is decreased by di-
gested sludge applications.  The additional inorganic P fertilizer
apparently increased the content of Mg in soybean leaves.  The effect
of phosphorus fertilizer on Mg levels in soybean leaves was significant
at the 5-percent level.

Increased concentrations of K, Ca, Mn, Na, Zn, and Cd were found to be
significantly (P>0.01) increased in mature soybean grain with the ap-
plication of digested sludge.  Although significant at only the 5 per-
cent level, it can be seen in Table 158 that P and Mg concentrations
are also increased in soybean seeds with increased sludge applications.
                                     347
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Furthermore, the increases in concentrations of Mn, Mg and Ca in soy-
bean grain observed with applications of inorganic P fertilizer are
significant af the 5-percent level.

Contents of Cr, Pb and Ni in soybean leaves and grain were below de-
tectable limits for the method used.  These elements are present at
the hundreds of part per billion level or less.

In view of the fact that concentrations of both plant available and
slowly-available forms of P are increased in soils at a rapid rate
following applications of digested sludge, the split-plot soybean
study has afforded an excellent opportunity to make some observation
regarding the effect of heavy phosphorus fertilizations on the nobil-
ity and plant availability of other elements.  It appears that the
P build-up in soils may affect the mobility of Fe and Mn and may affect
the absorption by plants and/or translocation within the plant of Mn,
Mg and Ca.

Experimental procedure;  Kenaf - Kenaf (Hibiscus -cannabinus) is grown
in several tropical countries as a source of textile and cordage fiber.
Fibers of the plant have characteristics or properties that make it com-
parable to most softwoods and superior to hardwoods as a raw material
for making paper products.  It can also be used as a blend to improve
the pulping quality of less satisfactory materials used in the production
of paper.

Although very little was known about the fertility requirements or
adaptability of kanaf to northern Illinois climatic conditions,-it
seemed in 1968 to be an ideal crop for use in a digested sludge utili-
zation study.  Thus, a field study was established to determine the
yield response of several varieties of kenaf to digested sludge appli-
cation.  During the first year, 1968, only one variety was planted in
102 cm spaced rows in 4-row plots, 12-m long.  Each of the four rates
(same as for t:he corn study) of sludge application on kenaf plots was
replicated, four times.  In 1969 and 1970, the plots used for kenaf in
.1968 were split to accommodate two varieties which were planted with
a grain drill jfn 61-cra row spacing by May 15 each year.  In 1969 each
treatment for each variety was again replicated four times but in 1970  .
the treatments were replicated only twice since the east one-half of
the area was used, to establish an alfalfa study.  Digested sludge was
applied between the rows of Venaf during the growing seasons beginning
each year when the plants had reached a height of 20 to 25 cm.

Results and discus sign of the kena_f__£tud_y_ - The kenaf yields are re-
ported in Table 159 witb respect to variety, year, and treatment.  The
maximum treated plots received a total of 36.3 to 51.5 tons per hectare
of sludge on a dry weight basis each year and during the latter two
years the control or check plots were treated with 269 kilograms of N,
305 kilograms of P  (P705), and 134 kilograms of K  (K20)per hectare.
                                 350
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However, kenaf yields veie rtat significantly increased by the added
fertility.  Either fcenai" has a very low fertility requirement or the
varieties available are not well adapted to climatic conditions in
Illinois or both.  At any rate, the yields obtained from kenaf are
insufficient for it to compete with corn or soybeans for land on an
economical return basis.

Near the end of the first growing season for kenaf (1968) , but before
frost occurred, samples of leaf tissue were collected from the top most
part of the 1.8-to 2.1-raeter tall plants.  The kenaf leaf samples were
analyzed for total N and some metals.  Average results are presented
in Table 160, but the data were not statistically analyzed.  Neverthe-
less, the increase in ]eaf N content and decrease in leaf Mg content
with increasingly greater sludge applications aay be real differences.
Both Zn and Mn concentration levels appear to be increased with increa-
singly greater sludge applications.  On the basis of the amount of Zn
applied as a constituent of digested sludge, one might expect an in-
creased Zn uptake by plants, but not of Mn.  The digested sludge con-
tained Zn concentrations that ranged from about 150 to 200 parts per
million, while Mn concentration levels were most often in the range of
3-5 parts per million and seldom exceeded 10 parts per million on a wet
weight basis.  The amounts of Mn supplied on the plots as a constituent
of sludge were very small compared to native amounts in the soil.  None
of the concentration levels of Cr, Pb or Ni were high enough to be de-
tected by the method used.
Table 160.  Total nitrogen and selected metal contents of kenaf leaf
            tissues after the first growing season (1968) during which
            17.8 cm of liquid digested sludge (equivalent to 51.5 tons
            of solids per hectare) were applied on the maximum treated
            plots.
Sludge appl.
  Rate                 N            Mg            Zn            Mn
                     - - Percent - - -            - - -

    0                1.87          0.53          44.0          62.5
1/4 Max              3.51          0.51          77.5         100.0
1/2 Max              3.60          0.50         130.0         310.0
  Max                3.77          0.44         123.5         312.5
                                352
-------
Experimental procedure:  alfalfa - As mentioned above, one-half of the
plots formerly planted each year to kenaf were seeded to alfalfa in
the spring of 1970.  The alfalfa was established in the absence of a
nurse crop by the use of a herbicide.  The first cutting of alfalfa was
made on July 7, 1970.  Only one additional application of sludge was
made on the alfalfa plots in 1970 prior to taking the first cutting.
From Table 159, it may be seen that a total of 20.3 cm of digested
sludge was previously applied on the maximum-treated plots when the
area was in kenaf.  Alfalfa yields are given in Table 161 for the first
cutting of alfalfa.
Table 161.  Alfalfa yield obtained during the first cutting after es-
            tablishment in 1970.  Maximum-treated plots had received
            a total of 20.3 cm of digested sludge (36.3 dry tons/ha)
            before the establishment of alfalfa and 2.54 cm (36.3 dry
            tons/ha) after establishment.
Sludge appl
Rate
0
1/4 Max
1/2 Max
Max
Yields
Tons /ha
3.78
5.11
4.93
3.72
Results and discussion of alfalfa study - The first cutting yields did
not significantly differ as a result of treatment and while alfalfa was
clipped again in August and September 1970 the yields were not recorded.
After each cutting of forage additional digested sludge was applied.
The maximum-treated plots received 15.2 cm in July, 2.5 cm in August
and 2.5 cm in September of additional sludge for a total application in
1970 of 20.2 cm.

Soil samples were collected from the alfalfa plots in April 1971 after
a total of 40.5 cm of sludge had been applied on the maximum-treated
plots, during a period of three years.  Total contents of selected chemi-
cal elements in surface and subsoil-samples are given in Table 162.
Digested sludge applications have resulted in greater total concentration
levels of several metals in the surface-soil samples, but levels in sub-
soil samples have been changed very little, if at all.  In Table 163 the
concentration levels of extractable chemical elements in both surface-
and subsurface soil samples are given.  The extractability of P, Fe, Cu
                                 353
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355
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and Zn was significantly increased at the 5 percent levels in the soil
surface by sludge applications.  Only extractable Na concentrations in
surface-soils showed a highly significant increase as a result of
sludge treatments.  Furthermore, only the extractability of Na was
significantly (P>0.05) increased in subsurface-soil samples by sludge
treatments.

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samples are given in Table 164.  The plant samples were taken from the
first cutting in 1970.  The data were not statistically analyzed, but
the trend for increased concentration levels of Zn and Mn in alfalfa
with increased sludge applications is similar to findings from analyses
of other plant leaf tissue samples.
                                356
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      (NOTE:  These references have not been verified  by  the
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                             SECTION IX
                              REFERENCES
 1.   Aleera,  M.  I.,  and 11.  Alexander.  "Nutrition and Physiology of
     Nitrobacter agjLlis,"  Ajppl.  Microbiol..  8_,  pp 80-84 (1960).

 2.   Alexander, M.  "Introduction to Soil Microbiology," John Wiley and
     Sons, N.Y. (1961).

 3.   Alexander, Martin, "Nitrification.   Soil Nitrogen," American
     Society of AgronomyMonograph, No.  10,  pp 309-346 (1965).

 4.   Anderson,  M. S., "Sewage Sludge for Soil Improvement," United States
     Department of Agriculture,  Circular 972 (1955).

 5.   Anderson,  M. S., "Fertilizing Characteristics of Sewage Sludge,"
     Sewage and Industrial Wastes, 31, No.  6, pp 678-682 (1959).

 6.   Andrews, J. F.,  "Dynamic Model of the  Anaerobic Digestion Process,"
     Proc. Amer. Soc. Civil Engi., Sanit. Engi. Div., SA1, p 95 (1969).

 7.   ASCE, "Sewage Treatment Plant Design," ASCE Manual of Engineering
     Practice,  No.  36, p 6, (1959).

 8.   Baconj, V.  W. and F.  E. Daltonv "Chicago Metropolitan Sanitary District
     Makes No Little Plans," Public Works.,  97_, p 66 (1966).

 9.   Bacon, V.  W.  "The Land Reclamation Project,"  Proposal by the
     Metropolitan Sanitary District of Greater Chicago. (1967).

10.   Bacon, V«  W.  "Sludge Disposal," Industrial Water Engineering, 4,
     No, 4, p 27 (1967).

11,   Barnes, E. E., "Fertilizing Value of Garbage Tankage and Sewage
     Sludge," Ohio Agricultural  Experimental Station Bimonthly Bulletin.
     No, 26, pp 39-44, (1941).

12.   Bear, F. E., "Chemistry of the Soil," 2nd Ed., Reinhold Publ. Co.,
     N,Y. (1964),

13.   Beckwith, R. S,   "Titration Curves of Soil Organic Matter," Nature,
     184, pp 745-746   (1959).
                                   358
-------
  14.  Bennett, A- (, , , and F« Adams, "Concentrations of NH3  (Ag) Required
       for Incipient. NH.3 Toxicity to Seedlings," Soil Sci. Soc. Amer. Proc. ,
       34, pp 259 -?6J  (1970).

  15.  Benson, N, R,} "Zinc Retention by Soils," Soil Science, 101 ,
j       pp 171-179 (1966).
I
|  16.  Berg, G., "Virus Transmission by the Water Vehicle.   II.  Virus
'       Removal by Sewage Treatment Procedures," Health Library Science, 2_,
       No. 2, p 90 (1966).

  17.  Berrow, M. L. , and J. Webber, "Trace Elements in Sewage Sludge,"
       Journ. ScJ. Fed. Agric. , 23_, p 93 (1972).

  18.  Bhaumik, H. D. and f. E. Clark, "Soil Moisture Tension and Microbial
       Activity," Proceedings Soil Sci. Soc  of Amer., 12, No. 234  (1947).

  19.  Bjerrum, J., "Metalainine Formation in Aqueous Solutions," P. Haase,
       Copenhagen (1941).

  20.  Bohl, E. H,, K. V. Singh, B. B. Hancock, and L. Kasza, "Studies on
       Five Porcine Enteroviruses," Amer . J . Vet . Res . , 21,  No. 80
       pp 99-103 (1960).

  21.  Bradley, W. H, , "Tropical Lake, Copropel, and Oil Shale," Geol ._ Soc .
       Amer. Bull,. 77, pp 1333-1338 (1966).
                                                               sSJ^f-ti
  22.  Bremner 3 J. M. and A. r. Edwards, "Determination and  Iscrfepe-Ratio
       Analysis of Different Forms of Nitrogen in Soils.  I.  Apparatus and
       Procedure for Distillation and Determination of Ammonium," Soil Sci.
       Socju-AS?!- ..?£fic.jL> .?i» PP 504-507 (1965).

  23.  Bremner, J .. M. and D., R. Keeney, "Steam Distillation  Methods for
       Determination of Ammonium, Nitrate, and Nitrite," Anal. Chim. Acta. ,
       32 , pp 48b-49.s  (19o5).

  24.  BrownUe, L. E, and F. H. Grau, "Effect of Food Intake on Growth and
       Survival of Salmoneilas and Escherichia coll in the Bovine Rumen,"
       li^Gen^ Mici ob_io.U , 46, pp 125-134 (1967).

  25.  Buck, K. , "Fertilizing with Purified Town Sewage," Soils and Ferti-
               ?_0, p 296 (J957).
  26,  Burd, k. S,, "A Study of Sludge Handling and Disposal," Fed. Water
                           > Pub« WP-28-4  (1968).
  27.  Burges, A., "Micro-organisms in the Soil," Hutchinson and Co., Ltd.,
       London, England (1958).
                                     359
-------
23.  Butler, J, 11. A., "Functional Groups of Soil Humic Acids," Ph.D.
     Thesis, University of Illinois,  (1966).

29.  Butler, J. V, , "Ionic Equilibrium, A Mathematical Approach,"
     Addison-Wesley,  Reading. Massachusetts, 547 pp (1964).

30.  Butler, R. G., G. T. Orlob and P.  H. McGauhey, "Underground Movement
     of Bacterial arid Chemical Pollutants," Jjpurn. Amer. Water Works Assoc.,
     46., No. 2, p 97  (1954).

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166.  Weast, R. C., "Handbook of Chemistry and Physics," The Chemical
      Rubber Co., Cleveland, Ohio, p A248  (1968).
                                 369
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167.  Webb, J. R. ,  A. J. Ohlrogge, and S. A. Barber, "The Effect of
      Magnesium Upon the Growth and the Phosphorus Content of Soybean
      Plants," Soil Sci. Soc. Amer. , Proc. . 18_, pp 458-462 (1954).

168.  Weber, C. W.  and B. L. Reid, "Effect of Dietary Cadmium on Mice,"
      Toxic and Appl. Pharm., 14, pp 420-425 (1969).

169.  Wirts, J. J., "Pipeline Transportation and Disposal of Digested
      Sludge," Sewage and Industrial Wastes, 28, No. 2, p 121 (1956).

170.  Wolf, H. W.,  "Housefly Breeding in Sewage Sludge." Set-rage and
      Industrial Wastes, 2_7_, No. 2, p 172 (1955).

171.  Wood, J. C.,  S. E. Moschopedis, and R. M. Elofson, "Studies in
      Humic Acid Chemistry.  I.  Molecular Weights of Humic Acids in
      Sulpholane,"  Fuel, XL pp 193-201 (1961).

172.  Yatsimirskii, K. B., and V. P. Vasil'ev, "Instability Constants
      of Complex Compounds," Consultants Bureau, New York, N.Y. (1960).

173.  Yuan, T. L.,  "Comparison of Reagents for Soil Organic Matter Ex-
      traction and  Effect of pH on Subsequent Separation of Humic and
      Fulvic Acids," Soil Sci., 98_, pp 133-141 (1964).
                                  370
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                             SECTION X


                PUBLICATIONS GENERATED BY THE PROJECT
Publications
 1.  Braids, 0. C., M. Sobhan-Ardakani and J. A. E. Molina, "Liquid Di-
     gested Sewage Sludge Gives Field Crops Necessary Nutrients," Illinois
     Research, 12, No. 3, pp 6-7 (1970).

 2.  Hinesly, T. D., "Agricultural Application of Digested Sewage Sludge.
     Municipal Sewage Effluent for Irrigation," Edited by C.  W. Wilson
     and F. E. Beckett.  Louisiana Polytechnical Institute, pp 45-48 (1968).

 3.  Hinesly, T. D., "City Waste and Soil Management," Illinois Fertili-
     zer Conference Proceedings, pp 40-42 (1969).

 4.  Hinesly, T. D. and B. Sosewitz,. "Digested Sludge Disposal on Crop
     Land," J. Water Pollution Control Federation, 4.1, pp 822-828 (1969).

 5.  Hinesly, T. D., "The Utilization and Disposal of Municipal Sewage
     Wastes," Illinois Research, 12, No. 4, pp. 6-7 (1970).

 6.  Hinesly, T. D. and R. L. Judson, "Yields of Corn and Soybeans in
     Response to Applications of Heated Anaerobically Digested Sludge,"
     Illinois Fertilizer and Chemical Assoc. Proc., pp 25-26  (1971).

 7.  Hinesly, T. D., R. L. Jones, and B. Sosewitz, "Complementary Rela-
     tionships Between the Reclamation of Surface-Mined Land  and Sludge
     Disposal," Accepted for Publication in Mining Congress Journal, (1972).

 8.  Hinesly, T. D., R. L. Jones, and E. L.  Ziegler, "Effects on Corn by
     Applications of Heated Anaerobically Digested Sludge," Compost Science,
     13_, No. 4, pp 26-30 (1972).

 9.  Lynam, B. T., B. Sosewitz, and T. D. Hinesly, "Liquid Fertilizer to
     Reclaim Land and Produce Crops," Water Research,  6^, pp 545-549 (1972).

10.  Meyer, R. C., F. C. Hinds, H.  R. Isaacson, and T. D. Hinesly, "Procine
     Enterovirus Survival and Anaerobic Sludge Digestion," Proceeds of
     Int. Symp. on Livestock Wastes, Amer.  Soc. of Agr. Engineers, pp 183-184
     (1971).

11.  Molina, J. A. E., "The Ecology of Soil Bacteria," Inter. Symp.
     Edited by Gray and Parkinson.   BioScience, 19, p 184 (1969).
                                  371
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12.  Molina, J. A. E., 0. C. Braids, T.  D.  Hinesly and  J.  B.  Cropper,
     "Aeration-Induced Changes in Liquid Digested  Sewage  Sludge,"  Soil
     Sci. Soc. Araer., Proc., 35, pp 60-63,  (1971).

13.  Molina, J. A. E., 0. C. Braids, and T. D.  Hinesly, "Observations
     on Bactericidal Properties of Digested Sewage Sludge,"  Environmental
     Science and Technology, _6, pp 448-450  (1972).

14.  Schwing, J. E., "Environmental Contamination  Resulting  from Land
     Reclamation with Anaerobically Digested Sludge," Civil  Eng. Studies,
     Sanitary Engineering Series #56, 32 pp (1970).

15.  Sobhan-Ardakani, M. and F. J. Stevenson, "A Modified  Ion Exchange
     Technique for the Determination of  Stability  Constants  of Metal-Soil
     Organic Matter Complexes," Presented at the Amer.  Soc.  Agron.  meetings,
     Tucson, Arizona, 1970 and accepted  for publication in the Soil Sci.
     Soc. Amer. Proc. in 1971 (1971).
Reports


 1.  Braids, 0. C., T. D. Hinesly and J.  A.  E.  Molina,  "Trace Element
     Uptake by Several Field Crops Grown  Under  Digested Sludge Irrigation,"
     Agron. Abstr., A.S.A. Annual Meeting, Detroit,  Michigan, p 80 (1969).

 2.  Braids, 0. C., and L. F. Welch,  "Nitrate in Drainage Water from Soil
     Receiving Sludge and Fertilizer," Amer. Soc. Agri. Eng., Winter
     meeting, December 8-11, Chicago, Illinois  (1970).

 3.'  Cropper, J. B., L. F. Welch, and T.  D.  Hinesly, "The Effect of Pb,
    • Cu, Cr, Zn and Ni on Nutrient Uptake and Growth of Corn," Special
     Project Report, (1970).

 4.  Hinesly, T. D., 0. C. Braids, R. I.  Dick,  and J. A.  E.  Molina, "Disposal
     of Digested Sludge on Farm Land," (D-7) Solid Waste Research and De-
     velopment, II.  Engineering Foundation  Research Conf.,  Beaver Dam,  Wise.
     (1968).

 5.  Meyer, R. C., F. C. Hines, H. R. Isaacson  and T. D.  Hinesly, "Porcine
     Enterovirus Survival and Anaerobic Sludge  Digestion," Int. Symp. on
     Livestock Wastes. Ohio State University, Columbus, Ohio, April 19-22,
     (1971).

 6.  Mioduszewski, W. and T. D. Hinesly,  "Digested Sludge Dewaterlng on
     Soils," Paper presented at Amer. Soc. Civil Engineering Annual Meeting,
     Chicago, IL, 14 pp (1969).
                                  372
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 7.  Molina, J. A. E., 0. C. Braids and T. D. Hinesly, "Digested Sludge
     for Agricultural Benefits and Land Reclamation," Agronomy Abstr.,
     Amer. Soc. Agron. Annual Meetings, New Orleans, p 133 (1968).

 8.  Molina, J. A. E., 0. C. Braids and T. D. Hinesly, "Bactericidal
     Properties of Digested Sewage Sludge Toward Fecal Coliforms,"
     American Society for Microbiology Abstracts, Miami, Florida, p 17
     (1968).

 9.  Braids, 0. C., M. Sobhan-Ardakani, T. D. Hinesly, and J.  A. E. Molina,
     "Disposal of Digested Sewage Sludge on Farm Land as Evaluated  by a
     Lysimeter Study," Agron. Abstr., Amer. Soc. Agron. Annual Meeting,
     New Orleans, p 132 (1968).

10.  Stevenson, F. J. and M. Sobhan-Ardakani, "Organic Matter  Reactions
     Involving Micronutrients in Soils," Invitational Paper, Conference of
     Micronutrients in Agriculture, National Fertilizer Development Center,
     T.V.A., Muscle Shoals, Alabama, April 20-22 (1971).
Theses
 1.  Cropper, J. B./'Greenhouse Studies on Nutrient Uptake and Growth of
     Corn on Sludge-Treated Plots," M.S. Thesis, University of Illinois,
     Dept. of Agronomy, Urbana, Illinois (1969).

 2.  Gossett, R. G.,  "Ammonia Volatilization from Digested Sewage Sludge
     as Related to Land Application," M.S. Thesis, University of Illinois,
     Dept. of Civil Engineering, Urbana, Illinois (1972).

 3.  Schwing, J. E.,  "Environmental Contamination Resulting from Land Re-
     clamation with Anaerobically Digested Sludge," M.S.  Thesis, University
     of Illinois, Dept. of Civil Engineering, Urbana, Illinois (1970).

 4.  Sobhan-Ardakani, M., "Stability Constants of Metal-Polyelectrolyte
     Complexes Occuring Naturally in Soil and Sewage Sludge," Ph.D.  Thesis,
     University of Illinois, Dept. of Agronomy, Urbana, Illinois (1971).


Bibliographies
 1.  Jones, R. L.,  T. D. Hinesly, and R.  J. Johnson, "Arsenic  in Agricul-
     tural Ecosystems:  A Bibliography of the Literature 1950  Through 1971'
     Draft Copy in Review, prepared May,  1973.
                                  373
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 2.  Jor.es, R.  L.,  T.  D.  Hincsly,  and R.  J.  Johnson,  ';3nriua and  Beryl-
     liuu in Agricultural Ecosystems:  A  Bibliography of  the Literature
     1950 Through 1971,"  Draft Copy in Review,  prepared August, 1973.

 3.  Jones, R.  L.,  T.  D.  Hinesly,  and R.  J.  Johnson,  "Boron  in Agricul-
     tural Ecosystems: A Bibliography of the Literature  1950 Through
     1971," Draft Copy in Review,  prepared October, 1973.

 4.  Jones, R.  L.,  T.  D.  Hinesly,  and R.  J.  Johnson,  "Cadniiuia in-Agri-
     cultural Ecosystems:  A Bibliography of the Literature  1950  Through
     1971," Draft Copy in Review,  prepared May, 1972.

 5.  Jones, R.  L.,  T.  D.  Hinesly,  and R.  J.  Johnson,  "Chromium in Agri-
     cultural Ecosystems:  A Bibliography of the Literature  1950  Through
     1971," Draft Copy in Review,  prepared July, 1973.

 6.  Jones, R.  L.,  T.  D.  Hinesly,  and R.  J.  Johnson,  "Cobalt in Agri-
     cultural Ecosystems:  A Bibliography of the Literature  1950  Through
     1971," Draft Copy in Review,  prepared October, 1973.

 7.  Jones, R.  L.,  T.  D.  Hinesly,  and R.  J.  Johnson,  "Copper in Agri-
     cultural Ecosystems:  A Bibliography of the Literature  1950  Through
     1971," Draft Copy in Review,  prepared February,  1974.

 8.  Jones, R.  L.,  T.  D.  Hinesly,  and R.  J.  Johnson,  "Iron in Agricul-
     tural Ecosystems: A Bibliography of the Literature  1950 Through
     1971," Draft Copy in Review,  prepared February,  1974.

 9.  Jones, R.  L.,  T.  D.  Hinesly,  and R.  J.  Johnson,  "Lead in Agricul-
     tural Ecosystems: A Bibliography of the Literature  1950 Through
     1971," Draft Copy in Review,  prepared September, 1973.

10.  Jones, R.  L.,  T.  D.  Hinesly,  and R.  J.  Johnson,  "Manganese in
     Agricultural Ecosystems:  A Bibliography of the  Literature 1950
     Through 1971," Draft Copy in Review, prepared January,  1974.

11.  Jones, R.  L.,  T.  D.  Hinesly,  and R.  J.  Johnson,  "Mercury in  Agri-
     cultural Ecosystems:  A Bibliography of the Literature  1950  Through
     1971," Draft Copy in Review,  prepared May, 1973.

12.  Jones, R.  L.,  T.  D.  Hinesly,  and R.  J.  Johnson,  "Molybdenum  in
     Agricultural Ecosystems:  A Bibliography of the  Literature 1950
     Through 1971," Draft Copy in Review, prepared September, 1973.

13.  Jones, R.  L., T.  D.  Hinesly,  and R.  J.  Johnson,  "Nickel in Agri-
     cultural Ecosystems:  A Bibliography of the Literature  1950  Through
     1971," Draft Copy in Review,  prepared July, 1973.
                                 374
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14.  Jones. R.  L.,  T.  D.  Hinesly, and R.  J.  Johnson,  "Selenium  in  Agri-
     cultural Ecosystems:  A Bibliography of the Literature  1950 Through
     1971," Draft  Copy in Review, prepared August,  1973.

15.  Jones, R.  L.,  T.  D.  Hinesly, and R.  J.  Johnson,  "Silver in Agricul-
     tural Ecosystems:  A Bibliography of the Literature  1950 Through
     1971," Draft  Copy in Review, prepared September,  1973.

16.  Jones, R.  L.,  T.  D.  Hinesly, and R.  J.  Johnson,  "Tin in Agricultural
     Ecosystems:  A Bibliography of the Literature  1950 Through 1971,"
     Draft Copy in Review, prepared September, 1973.

17.  Jones, R.  L.,  T.  D.  Hinesly, and R.  J.  Johnson,  "Zinc in Agricul-
     tural Ecosystems:  A Bibliography of the Literature  1950 Through
     1971," Draft  Copy in Review, prepared May, 1972.

18.  Jones, R.  L.,  T.  D.  Hinesly, and R.  J.  Johnson,  "Zirconium in Agri-
     cultural Ecosystems:  A Bibliography of the Literature  1950 Through
     1971," Draft  Copy in Review, prepared September,  1973.
     1063
                                375
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