Soybean Phytotoxicity from Land-Applied Biosolids

Abstract

In 1999, marginal and entire leaf necrosis, interveinal chlorosis, leaf puckering and crinkling, and stunted overall growth were observed in field-grown soybeans (Glycine max L.) in Essex County, Virginia. The soybeans had been fertilized with biosolids treated by the Zimpro™ wet air oxidation process at the Passaic Valley Sewerage Commission (PVSC) Newark, NJ wastewater treatment plant. Our objectives were (1) to replicate the symptoms in the greenhouse using soils collected from fields where phytotoxicity symptoms were observed and (2) to determine whether the causative agent was a constituent in the primary sludge or was produced by the Zimpro™ treatment process. We replicated the field phytotoxicity symptoms in the greenhouse in soybeans grown in Zimpro™ biosolids-amended field soils from Essex County and in soils amended with Zimpro™-processed biosolids obtained from PVSC. Soybeans grown in soil amended with untreated primary sludge from PVSC had phytotoxic symptoms that were generally atypical of those observed in the field but may have masked symptoms more similar to those of the field-grown soybeans. We were thus unable to determine whether the toxicant was present in the primary sludge or was a product of the Zimpro™ process. We were also unable to identify any common inorganic element as the cause of the reduction in the growth and development of the field-grown soybeans. We hypothesize that the toxicant may have been a persistent organic compound whose slow degradation in the soil was able to cause phytotoxicity several years after biosolids land application.

Full text

Soybean Phytotoxicity from Land-Applied Biosolids
KATHRYN C. HAERING*, W. LEE DANIELS and GREGORY K. EVANYLO
Department of Crop and Soil Environmental Sciences, Virginia Tech, Blacksburg, VA 24061-0404
INTRODUCTION
DURING the summer of 1999, phytotoxicity symp-
toms in soybeans (Glycine max L.) that had been
fertilized with biosolids from the Passaic Valley Sewer-
age Commission (PVSC) wastewater treatment plant in
Newark, NJ were reported in numerous fields in Essex
County, VA by a Virginia Cooperative Extension agent
(Keith Balderson, personal communication). These
symptoms included marginal necrosis, interveinal
chlorosis, leaf puckering and crinkling, stunted plant
growth and, in full expression, entire leaf necrosis.
Corn and wheat were not affected, but soybean grown
in the rotation with corn and wheat exhibited symptoms
as late as the second or third year following the
biosolids application, from which we inferred that the
suspected toxicant was not easily degraded or trans-
ported from the root zone and/or was active at very low
concentrations. The phytotoxicity symptoms appeared
to be more severe under drought-stressed conditions.
Analyses of the biosolids and routine soil and plant tis-
sue testing failed to identify a common deficiency/tox-
icity factor. Paired sampling of adjacent affected and
non-affected buffer areas showed higher nutrient status
but no difference in other potential causes (trace ele-
ment concentration, soluble salts/electrical conduc-
tance, pH) in soils under the symptomatic soybeans.
Soybean tissue analysis did not identify any potential
nutrient deficiency or trace element toxicity.
The PVSC wastewater treatment plant receives influ-
ent from approximately 260 km2of northern New Jer-
sey (PVSC, 2006). The wastewater influent is 15% in-
dustrial by volume. Industries served by the plant
include electroplaters, metal finishers, pharmaceutical
and organic chemical manufacturers, textile dyers, hos-
pitals, electronic products manufacturers, and news-
print recycling mills. Most of the wastewater from in-
dustries in the plant’s service area requires
pre-treatment because of high concentrations of heavy
metals.
After settling and gravity thickening, primary sludge
at the PVSC treatment plant undergoes secondary treat-
ment using the Zimpro™wet air oxidation process
*Author to whom correspondence should be addressed.
E-mail: khaering@vt.edu
1Journal of Residuals Science & Technology, Vol. 5, No. 1—January 2008
1544-8053/08/01 001-12
© 2008 DEStech Publications, Inc.
ABSTRACT: In 1999, marginal and entire leaf necrosis, interveinal chlorosis, leaf puck-
ering and crinkling, and stunted overall growth were observed in field-grown soybeans
(Glycine max L.) in Essex County, Virginia. The soybeans had been fertilized with
biosolids treated by the Zimpro™wet air oxidation process at the Passaic Valley Sewer-
age Commission (PVSC) Newark, NJ wastewater treatment plant. Our objectives were
(1) to replicate the symptoms in the greenhouse using soils collected from fields where
phytotoxicity symptoms were observed and (2) to determine whether the causative
agent was a constituent in the primary sludge or was produced by the Zimpro™treat-
ment process. We replicated the field phytotoxicity symptoms in the greenhouse in soy-
beans grown in Zimpro™biosolids-amended field soils from Essex County and in soils
amended with Zimpro™-processed biosolids obtained from PVSC. Soybeans grown in
soil amended with untreated primary sludge from PVSC had phytotoxic symptoms that
were generally atypical of those observed in the field but may have masked symptoms
more similar to those of the field-grown soybeans. We were thus unable to determine
whether the toxicant was present in the primarysludgeorwasaproductoftheZimpro™
process. We were also unable to identify any common inorganic element as the cause of
the reduction in the growth and development of the field-grown soybeans. We hypothe-
size that the toxicant may have been a persistent organic compound whose slow degra-
dation in the soil was able to cause phytotoxicity several years after biosolids land appli-
cation.
RESEARCH

(PVSC, 2006; USFilter Corporation, 2005). The pro-
cess involves mixing liquid primary sludge with air in a
reaction vessel at 220°C (425°F) and 4.485 MPa (650
psi) which decreases BOD, COD, and insoluble volatile
solids during this low pressure oxidation (PVSC, 1996;
PVSC, 2006). The material is subsequently dewatered
via a belt filter press, and the resulting Class A product
meets the alternative pollutant limits of Title 40 of the
Code of Federal Regulations (CFR) Part 503 (Stan-
dards for the Use or Disposal of Sewage Sludge, or the
Part 503 Rule) (PVSC, 1996).
Few researchers have specifically addressed the ef-
fects of Zimpro™-processed biosolids on plant growth.
Neel et al. (1978) observed no adverse effects in a
warm-season turfgrass sod grown in 10 cm of com-
posted Zimpro™-processed biosolids from Fort Laud-
erdale/Hollywood, FL. The composted biosolids pro-
duced better turf sod than a local soil and other
composted organic waste products. Wei et al. (1985)
found that Zimpro™-processed biosolids from
Oshkosh, WI applied at rates up to 112 Mg/ha for a sin-
gle application, or at 134 Mg/ha in six annual applica-
tions, lowered soil bulk density; increased hydraulic
conductivity, aggregate stability, and large pore vol-
ume; and did not adversely affect the growth of
sudangrass (Sorghum bicolor sudanensis) and corn
(Zea mays L.). Vail and Dewey (1985) applied
Zimpro™-processed biosolids from Port Elizabeth,
South Africa to turf and pastureland in a study designed
to develop metal application rate guidelines. The
biosolids contained relatively high levels of Zn (4554
mg/kg) by today’s standards and Pb (1193 mg/kg) that
exceeds the Part 503 Ceiling Concentration Limit, but a
one-time application at rates up to 224 Mg/ha produced
high-quality forage and turf whose plant tissue metal
concentrations were well below reportedly phytotoxic
levels.
Our objectives for this study were (1) to determine
whether residual phytotoxicity existed in soils that ex-
hibited yield suppression and/or various toxicity symp-
toms in the field, and (2) to determine whether the
phytotoxicity exhibited by the Zimpro™-processed
PVSC biosolids was due to a constituent in the primary
sludge or was a result of the Zimpro™wet air oxidation
treatment process. We subjected the soybean plants
used in the bioassay to both adequate moisture and
drought-stressed conditions in order to determine
whether the toxicity was exacerbated by moisture
stress.
MATERIALS AND METHODS
Experiment 1
Two coastal plain soil series, Kempsville sandy loam
(Fine-loamy, siliceous, subactive, thermic Typic
Hapludults; Field A) and Suffolk sandy loam
(Fine-loamy, siliceous, semiactive, thermic Typic
Hapludults; Field B) were collected in spring 2000
from fields in Essex County, VA that had been
amended with Zimpro™biosolids in March 1997 (Ta-
ble 1) and where phytotoxicity symptoms were ob-
served in 1999. According to the biosolids applicator’s
records, Field A sustained “total plant loss,” and the
soybeans in Field B were “severely affected.” A Suf-
folk sandy loam collected from an unamended buffer
strip adjacent to the treated area and which had the same
surface texture as the Zimpro™-amended Kempsville
2K. HAERING, W. DANIELS and G. EVANYLO
Table 1. Treatments for Greenhouse Experiments 1 and 2.
Experiment 1
Treatments
Field-applied Zimpro -processed
Biosolids Rate (Mg/ha) Additional Amendments
Control 0 Lime, P, and K applied to greenhouse pots
Field A 23.1 K applied to greenhouse pots
Field B 21.3 Lime P, and K applied to greenhouse pots
Reclamation site 78.4 33.6 Mg/ha Blue Plains wastewater treatment plant (Washington, DC)
lime-stabilized biosolids applied in field
Experiment 2
Treatment Description
Control P and K were applied according to routine soil test recommendations. Lime was applied to adjust soil pH to 6.2.
Pre-Zimpro™22.4 Mg/ha PVSC primary sludge before Zimpro™processing
Post-Zimpro™22.4 Mg/ha PVSC biosolids after Zimpro™processing

and Suffolk soils was used as a control. We also in-
cluded a treatment consisting of soil from a reclaimed
sand and gravel mine in Aylett, VA (Reclamation Site)
that had been amended with a reclamation (higher than
agronomic) rate of Zimpro™biosolids and lime-stabi-
lized biosolids (Table 1) in fall 1998 (Daniels et al.,
2002). No phytotoxic symptoms had been reported in
the plants used to revegetate the mined land, but soy-
beans were not grown at this site.
Each soil was air dried, and sieved to < 2 mm.
Subsamples of each soil were analyzed for pH, soluble
salts, and Mehlich-1 extractable P, K, Ca, Mg, Zn, and
Mn by the methods of Mullins and Heckendorn (2005)
(Table 2). Total N was determined by EPA method
351.3 (U.S. EPA, 1979); NO3-N by method SM
4500-NO3F (AWWA, 1998); NH4-N by EPA method
350.2 (U.S. EPA, 1979), and total organic C by EPA
method 415.1 (U.S. EPA, 1979). Particle size analysis
was performed by the pipette method using oven-dry
samples (Method 3A1: UDSA-NRCS, 1996). Moisture
content at field capacity was determined by pres-
sure-plate extraction at 33 kpa (1/3 bar) (Method 4B1a,
USDA-NRCS, 1996).
Phosphorus and K as solutions of reagent grade
KH2PO4and/or KCl were added to each soil at rates rec-
ommended by Virginia Tech Soil Testing Laboratory
for soybean production (Donohue and Heckendorn,
1994). Soils having a pH of less than 5.9 were limed to
6.2 with Ca(OH)2according to Virginia Tech Soil Test-
ing recommendations prior to potting the soils
(Donohue and Heckendorn, 1994). Plastic-lined pots
were used to prevent leaching of growth-inhibiting con-
stituent(s) and to accurately control soil moisture dur-
ing irrigation. Six kg of each soil was added to each per
pot, except for the Reclamation Site soil, whose higher
organic matter content allowed only 5 kg per pot. Each
soil treatment consisted of eight pots.
Water was applied to each pot to achieve 80% field
capacity to permit equilibration of inorganic soil
amendments in preparation for seeding. Five soybean
seeds (Glycine max L. var. FFR-493) treated with
Captan 50™fungicide were planted to a depth of 1.5 cm
in the surface soil of each pot. Soil moisture was main-
tained using mist irrigation until one week after germi-
nated seeds emerged from the soil. Emergence and
plant development data were collected until the first
true leaves appeared. A week after emergence, the soy-
beans were thinned to 3 plants/pot, at which time we be-
gan watering the pots to 80% field capacity. Treatment
observations were made several times per week
throughout the course of the experiment. We thinned
the soybean plants to 2 per pot and 1 per pot at two and
three, respectively, weeks after emergence.
Treatments included two moisture regimes: 80%
field capacity (WET) and 40% field capacity (DRY) ×
three soils (Suffolk without biosolids, Suffolk with
Zimpro™-processed biosolids, and Kempsville with
Zimpro™-processed biosolids). The Reclamation Site
soil was only used in the WET (80% field capacity
moisture) regime. Each of the 7 treatment combinations
was replicated four times. The DRY moisture regime
treatment was established at 30 days after emergence by
gradually decreasing moisture content to 50% field ca-
pacity for one month before reducing it to 40%.
Soybean Phytotoxicity from Land-Applied Biosolids 3
Table 2. Properties of Soils Used in Experiments 1 and 2 before Liming and Fertilization.
Control (Suffolk sl) Field A (Kempsville sl) Field B (Suffolk sl) Reclamation Site
pH 4.80 6.20 5.40 7.60
Total Organic C (%) 1.40 1.68 1.34 2.38
Total N (%) 0.08 0.09 0.08 0.29
NH4-N (mg/kg) 11.0 12 14 4
NO3-N (mg/kg) 23 20 16 19
P* (mg/kg) 17 112 42 89
K (mg/kg) 97 66 71 30
Ca (mg/kg) 228 774 330 4,988
Mg (mg/kg) 40 42 50 99
Mn (mg/kg) 19 9 12 12
Zn (mg/kg) 2 9 7 21
Fe (mg/kg) 19 44 12 15
Cu (mg/kg) 0.43 0.74 0.36 0.47
B (mg/kg) 0.11 0.12 0.09 0.21
Sand (%) 70 76 74 87
Silt (%) 26 20 24 9
Clay (%) 4 4 2 4
*P,K,Ca,Mg,Mn,Zn,Fe,Cu,BareexpressedasextractablebyMehlich#1method.

We sampled the lower symptomatic trifoliolate
leaves from each plant of the Control and
Zimpro™-amended treatment soils at anthesis (R2, 64
days after emergence). At 66 days after emergence, we
sampled the three upper (younger) completely devel-
oped trifoliolate leaves from the Field A, Field B, and
Control treatments to test for nutrient deficiency/toxic-
ity in soybean (Sabbe et al., 2000). There were too few
leaves remaining on the Reclamation Site treatment
plants to allow sampling at this time. All plant tissue
samples were dried for 4 to 6 days at 55°C, ground in a
stainless steel Wiley mill, weighed into Pyrex beakers,
and ashed in a muffle furnace at 450°C for 16 h. Ash
was dissolved in 2 mL of concentrated HNO3on a hot
plate and then refluxed for 2 h with 10 mL of 3 MHCl.
After digestion, solutions were filtered and diluted to
25 mL with 0.1 MHCl. Samples were analyzed for Zn,
Cu, B, Mn, Fe, P, Mg, Ca and K by inductively coupled
plasma (ICP) atomic emission spectrometry using 40
mg L−1Y as an internal standard.
We harvested whole plants 145 days after emergence
by cutting plants at the base. At this point, all of the
seeds in the Reclamation Site plants had attained matu-
rity, but some of those in the Field A and Field B treat-
ments had not. Pods were separated from plants, and
both pods and whole plants were oven dried at 55°C un-
til constant weight was attained. We hand-removed as
much of the root mass as possible from each pot and vi-
sually compared root mass, nodulation, and other root-
ing characteristics. The soil from each pot was then
mixed and subsampled. Soil samples were analyzed for
pH, soluble salts, and Mehlich 1 extractable P, Ca, Mg,
K, Mn, Zn, Fe, Cu, and B by the methods of Mullins and
Heckendorn, 2005.
Experiment 2
For experiment 2, we used both primary sludge from
the PVSC plant (Pre-Zimpro™) and the product of this
same primary sludge after undergoing the Zimpro™
process at the PVSC plant (Post-Zimpro™)(Table3).
The primary sludge from the PVSC wastewater treat-
ment plant was collected after screening and settling,
but before digestion. The sludge was then dried to ap-
proximately 50% moisture at the wastewater treatment
plant without Zimpro™method processing. Chemical
and physical analyses of both the primary sludge and
Zimpro™-processed biosolids was performed by the
following methods: percent solids (SM 25408;
AWWA, 1998), total N (Method 351.3; U. S. EPA,
1979); NO3(SM 4500-NO3F; AWWA, 1998); NH4-N
(Method 350.2; U.S. EPA, 1979); total organic C
(Method 415.1; U.S. EPA, 1979); chloride (SM
4500-CL D; AWWA, 1998); and P, K, S, Ca, Mg, Na,
Fe, Al, Mn, Cu, Zn, and B (SW 846-6010B; U.S. EPA,
1995). Calcium carbonate equivalence was determined
by ASTM method C602-95a (ASTM, 2001).
All treatments were applied to the limed control soil
(Suffolk sandy loam) that was used in Experiment 1.
Plastic-lined, 4.5 liter pots were filled with 5 kg soil and
amended with the equivalent of 22.4 Mg/ha (dry weight
basis) of the two PVSC materials. Both the primary
sludge and the Zimpro™-processed biosolids were
hand-mixed into the soil. A third treatment consisted of
5 kg of unamended control soil. As in Experiment 1, all
three soil treatments received P and K as solutions of re-
agent grade KH2PO4and/or KCl at rates recommended
by Virginia Tech Soil Testing Laboratory for soybean
production (Donohue and Heckendorn, 1994). Treat-
4K. HAERING, W. DANIELS and G. EVANYLO
Table 3. Properties of Primary Sludge and
Zimpro -processed Biosolids from
PVSC Wastewater Treatment Plant.
PVSC primary
Sludge
(Pre-Zimpro )
PVSC
Zimprotm-processed
Primary Biosolids
(Post-Zimpro )
Solids (g/kg) 500.78 820.44
CCE (g/kg) <0.1 <0.1
Total Organic C (g/kg) 434.2 347.5
Total N (g/kg) 53.4 29.8
C/N ratio 81.0 117.0
NH4-N (g/kg) 8.9 10.1
NO3-N + NO2-N (g/kg) 0.02 nd*
Organic N (g/kg) 44.5 19.7
P (g/kg) 18.1 28.1
K(g/kg) 5.9 2.1
S(g/kg) 9.6 4.1
Ca (g/kg) 18.4 22.3
Mg (g/kg) 5.0 5.8
Na (g/kg) 9.4 2.4
Fe (g/kg) 8.7 16.0
Al (g/kg) 12.4 23.5
Cl (g/kg) 9.9 2.4
Mn (mg/kg) 355 544
Cu (mg/kg) 947 1,210
Zn (mg/kg) 82 1,580
B (mg/kg) 20 12
Cd (mg/kg) 6.0 11.0
Ni (mg/kg) 49 59
Pb (mg/kg) 91 125
As (mg/kg) 3.4 2.9
Hg (mg/kg) 1.6 2.7
Se (mg/kg) 2.2 1.9
Mo (mg/kg) 93 30
*nd = not detected

ments again included two moisture regimes: 80% field
capacity (WET) and 40% field capacity (DRY) ×three
soil treatments (Suffolk without biosolids, Suffolk with
PVSC primary biosolids, and Suffolk with PVSC
Zimpro™-processed biosolids). Each of the 7 treatment
combinations was replicated four times.
We used the same watering, seeding, germination,
observation, and soybean thinning procedures as in Ex-
periment 1. We initiated drought stress for the 40% field
capacity treatments three weeks after emergence. The
soybean plants were harvested 65 days after emer-
gence, at which time approximately half of the plants
were flowering. We harvested the whole plant because
their small size required that the entire plant be used to
provide enough material for analysis. Whole plant sam-
pling and drying, root observations, and soil sampling
and analysis were conducted as described for Experi-
ment 1.
Pest Control
Pest control for each greenhouse experiment was
provided by spraying Avid 0.15 EC™miticide
(Abamectin) and Conserve SC™insecticide (Spinosad,
including Spinosyn A and Spinosyn D).
Statistical Analyses
The WET (80% field capacity) and DRY (40% field
capacity) treatments of each experiment were statisti-
cally analyzed as separate completely randomized ex-
periments with four replications per treatment. Thus,
we did not statistically compare data between the WET
and DRY treatments of the experiments. Within each
moisture regime of each experiment, treatment varia-
tions were analyzed by a least squares analysis of vari-
ance procedure (SAS Institute, 2002). Where the over-
all experiment-wide F-test was significant (p< 0.05),
treatment means were separated by Fisher’s LSD pro-
cedure.
RESULTS AND DISCUSSION
Soil Properties
Neither of the agricultural soils from Fields A and B
exhibited any potential deficiencies in essential nutri-
ents (Table 2; Donohue and Heckendorn, 1994) or ab-
normally elevated soil levels of potentially toxic trace
elements (Kabata-Pendias and Pendias, 1984). Only
the pH of the Field B soil (5.40) was considered to pose
growth-limiting conditions for soybean. This was cor-
rected with lime for the greenhouse experiment.
Sludge and Biosolids Characteristics
The characteristics of the PVSC primary sludge and
Zimpro™-processed biosolids are presented in Table 3.
The Zimpro™process apparently caused the loss of or-
ganic C and N. None of the measured macro or trace el-
ements were present at concentrations that would be ex-
pected to cause phytotoxicity, although the USEPA 503
ceiling concentration limits for Mo (75 mg/kg) were ex-
ceeded in the primary sludge.
Experiment1—SoybeanGrowthinPreviously
Zimpro -amended Soils
Germination and Seedling Development
There were no observable differences in germination
rate and early seedling development that could be at-
tributed to treatment. Within two weeks after emer-
gence, marginal necrotic spotting appeared on the first
true leaves in the Reclamation Site and Field A treat-
ments and marginal chlorosis appeared on the older
leaves in the Field B treatment. Toxicity symptoms in-
creased in rate and intensity in the order Reclamation
Site > Field A > Field B.
Symptom Progression Before Flowering
Between 15 and 30 days after emergence, Reclama-
tion Site treatment plant leaves, which had been small,
puckered, and crinkled during the emergence period,
developed total necrosis. The earlier-described mar-
ginal necrotic spotting spread from the leaf tip to the en-
tire leaf margin, and then moved inward. This effect
progressed most rapidly in the oldest leaves (Figure 1),
which eventually senesced and dropped. These leaves
first showed dark brown necrotic spotting at the margin
near the leaf tip, which then developed into a band of
dark brown marginal necrosis which eventually extend-
ing around the entire leaf. The band then moved in-
wards, leaving dead tissue. The inside of the leaf then
became almost completely yellow, and the leaf dropped
off. All of the leaves on the Reclamation Site treatment
plants, except for the newest trifoliolates, had marginal
necrosis throughout the rest of the experiment.
The identical progression of symptoms occurred in
the Field A plants, except the severity was less than in
the Reclamation Site treatment. The Field B plants were
Soybean Phytotoxicity from Land-Applied Biosolids 5

generally smaller than the Field A and Control plants.
Many of the first true leaves and first trifoliolates on
Field B plants exhibited diffuse, marginal chlorosis but
not the marginal necrotic spotting observed in the Field
A and the Reclamation Site treatments. Only a few
leaves were slightly puckered and crinkled. Field B
plants generally had fewer trifoliolate leaves than the
Field A and Control plants.
Subsequent imposition of drought stress to the Con-
trol, Field A, and Field B treatments at 30 days after
emergence reduced the overall growth of plants in all
DRY treatments but did not exacerbate the previously
observed leaf symptoms.
Symptom Progression After Flowering
Flowers appeared on all but one of the Reclamation
Site treatment plants between 52 and 57 days after
emergence. The final Reclamation Site treatment plant
flowered at 72 days after emergence. The order of ob-
served flowering among treatments was Control = Field
A < Field B < Reclamation Site, with drought inducing
earlier flowering. We removed lower and upper
trifoliolates from all plants at 56 to 58 days after emer-
gence for tissue analysis. At this time, approximately
one-third of the older leaves in the Field A treatment in
the WET moisture regime and one-half of the older
leaves in the DRY moisture regime had some marginal
necrosis. All leaves on the Field B plants under both
moisture regimes exhibited more interveinal chlorosis
than the Control and Field A plants. Chlorosis was more
pronounced in plants in the Field B DRY treatment. The
leaves of the Field B plants became progressively
chlorotic after flowering but the plants set pods. The
proportion of symptom-affected to non-symptomatic
leaves was higher in plants in the Field A and Field B
DRY moisture regime than in the WET moisture re-
gime throughout the experiment. The Reclamation Site
plants dropped many leaves during the experiment but
rebounded from near death after flowering to produce
new leaves that exhibited severe marginal necrosis and
few or no small pods.
The symptoms on the Field A plants were similar to
those that appeared on the Reclamation Site plants and
unlike those that appeared on Field B plants. It is thus
possible that the symptoms on the Field B plants may
not have been caused by the same agent as that which
caused necrosis in the Reclamation Site and Field A
plants.
Observations at Harvest
We harvested pods at 145 days after emergence, at
which time all plants had set seed. The DRY plants were
shorter and reached the full pod stage earlier than the
WET plants. The combined leaf and stem weights and
the height of plants in the WET soil moisture regime
were greater in the Control than in any
Zimpro™-amended treatment (Table 4). The Field A
and Field B plants accumulated more biomass and grew
taller than Reclamation Site plants. There were no treat-
ment differences among the growth characteristics of
6K. HAERING, W. DANIELS and G. EVANYLO
Table 4. Effects of Experiment 1 Treatments on Soybean Weight, Height, Pod Number, and Pod Weight.
Treatment
Leaf + Stem wt. (g) Height (cm) Pod Number Pod wt. (g)
WET* DRY WET DRY WET DRY WET DRY
Control 36.02a 18.60a 96.3a 71.4a 40a 26a 13.5a 10.3a
Field A 24.95b 14.38a 84.1b 68.6a 38a 23a 15.8a 10.5a
Field B 28.50b 17.67a 83.9b 67.5a 44a 31a 13.6a 10.8a
Reclamation Site** 5.80c — 47.3c — 19b — 7.4b —
*Means followed by the same letter within columns are not significantly different (p = 0.05).
**The reclamation site soil did not undergo a DRY moisture regime.
Figure 1. Close-up of marginal necrosis, crinkling and puckering,
and overall growth stunting in soybeans growing in the Reclamation
Site treatment with adequate moisture in Experiment 1. Approxi-
mately 78 Mg/ha Zimpro -processed biosolids had been applied to
the mine soil in the field 18 months previously.

the DRY Control, Field A, and Field B plants, indicat-
ing that moisture stress was more growth limiting than
any biosolids constituent(s). The mean number of soy-
bean pods per plant and total pod weight did not differ
among the Control, Field A, and Field B treatments in
either soil moisture regime, but the Reclamation Site
treatment (WET moisture regime only) produced lower
values for these variables.
Root Observations
The root mass of the DRY plants appeared to be
smaller than that of the WET plants. Within the WET
soil moisture regime, roots of the plants in the Control,
Field A, and Field B treatments had many nodules,
while those in the Reclamation Site plants had none.
The Reclamation Site plant roots were observed to be
smaller and less fibrous than those in all of the other
treatments. When the sizes of the root masses of the
WET regime plants were compared visually, the root
masses of the Control were larger than those of the Field
A plants, which in turn were larger than those of the
Field B plants. In the DRY regime plants, the same rela-
tive size relationship was observed.
Post Harvest Soil Analysis
At the completion of the experiment, none of the ele-
ments measured in the soil were present at a concentra-
tion that would be considered detrimental to plant
growth (Table 5, Donohue and Heckendorn, 1994). Sol-
uble salts and B in the Reclamation Site soil were ele-
vated above the values in the other treatments, but B and
salt concentrations were not high enough to have
caused yield reductions in soybean (Maas, 1984).
Plant Analysis Data
We used sufficiency ranges for soybeans grown in the
southern United States (Sabbe et al., 2000) to assess the
effects of treatment on tissue nutrient levels. Tissue
analysis of samples taken from older (lower), symp-
tomatic soybean leaves was used to compare the Recla-
mation Site treatment with other treatments because the
Reclamation Site treatment did not produce enough up-
per trifoliolates for sampling.
The upper trifoliolate leaves of the soybeans growing
in the WET moisture regime Zimpro™-amended soils
were higher in Zn and P (Table 6). Plant tissue Zn in the
Field B, but not Field A, treatment was slightly above
sufficiency levels (>80 mg/kg), from which we inferred
that Zn toxicity was not the cause of the Zimpro™re-
lated symptoms. Plant tissue grown in both the Control
and Field B soils had slightly elevated Mn levels (>100
mg/kg), likely because of high native soil Mn levels, but
all other macro- and micro-nutrient concentrations in
plants grown in the Zimpro™-amended soils were
within sufficiency ranges.
The lower trifoliolate leaves of the soybean plants
(Table 7) showed no clear trends of deficiency or toxic-
ity. Tissue Zn was higher in plants grown in
Zimpro™-amended soils and exceeded the sufficiency
range in the adequately watered soils, and tissue B was
slightly below sufficiency levels in most treatments un-
der both moisture regimes. Tissue analysis levels for
older soybean leaves, however, cannot be correlated
with average values for sufficiency, because these val-
ues were established for the most recently fully-ex-
panded leaves. Tissue Mn levels were much higher in
lower trifoliolates from both the control and Field B
Soybean Phytotoxicity from Land-Applied Biosolids 7
Table 5. Effects of Experiment 1 Treatments on pH, Soluble Salts and Mehlich I Extractable Soil Elements.
Treatment pH
Soluble
Salts
(dS/m)
Mehlich I Extractable Elements (mg/kg)
Ca Mg P K Mn Zn Fe Cu B
WET Moisture Regime*
Control 6.38c 0.31b 660c 41d 21c 41a 13b 3c 10b 1.3a 0.10b
Field A 6.25d 0.30b 737bc 46c 98b 30ab 5d 11b 38a 1.3a 0.09b
Field B 6.52b 0.67b 767b 59b 49c 22b 15a 9b 10b 1.2a 0.10b
Reclamation site 7.30a 2.13a 5,780a 111a 159a 24b 10c 34a 7c 1.1a 0.21c
P < F 0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0236 <0.0001 <0.0001 <0.0001 0.26271 <0.0001
DRY Moisture Regime
Control 6.45a 0.34b 701b 42b 24c 43a 13b 3c 10b 1.1a 0.11a
Field A 6.22a 0.25b 748ab 47b 102a 36a 5c 11a 38a 1.3a 0.10a
Field B 6.50a 1.08a 821a 64a 57b 49a 16a 10b 10b 1.2a 0.11a
P < F 0.1719 0.019 0.030 0.0005 <0.0001 0.6321 0.0002 <0.0001 <0.0001 0.4521 0.5676
*Means followed by the same letter within columns by moisture regime are not significantly different (p = 0.05).

treatments than in the Field A and the Reclamation site
treatments which indicates that Mn did not cause the
phytotoxic symptoms in the Field A and Reclamation
Site treatments.
Physiological Interpretation
Based on the occurrence of symptoms in plant parts,
the toxicant appeared to be xylem-mobile (water solu-
ble) and caused effects similar to soluble salts, boron,
and a variety of known herbicides and other bio-active
compounds (personal communication, Dr. Larry Foy,
Virginia Tech). Excessive soluble salts and B damage
were eliminated as possible causes based on soil and
plant tissue analyses. Isolation and identification of
other compound(s) was not possible without extensive
screening analyses of numerous organic and additional
inorganic compounds in the Zimpro™-processed prod-
uct.
Overall Results
In Experiment 1, a reduction of soybean yield was
only observed in the Reclamation Site treatment. The
marginal necrosis, with some increased leaf pucker-
ing, tended to appear on a higher percentage of the
leaves of soybeans growing in the DRY (moisture
stressed) Zimpro™-amended treatments, but the
yield-suppressing effect of any toxicant was not exac-
erbated by drought as evidenced by the lack of differ-
ences in soybean growth among the Control, Field A
and Field B plants. We were not able to correlate soil
or tissue analytical data with the soybean leaf symp-
toms.
8K. HAERING, W. DANIELS and G. EVANYLO
Table 7. Effects of Experiment 1 Treatments on Elemental Composition of Soybean Lower Trifoliolate Leaves.
Treatment
Element (mg/kg)
Zn Cu B Mn Fe P Mg Ca K
WET Moisture Regime*
Control 74c 7b 17a 121b 89a 1.80a 4.80b 27.3b 5.5b
Field A 94b 6b 18a 28c 97a 2.36a 4.77b 24.2bc 7.5b
Field B 111a 7b 18a 226a 91a 2.20a 6.76a 40.7a 8.3b
Reclamation site 122a 13a 74a 18c 95a 2.92a 4.26b 22.0c 14.4a
P < F 0.0002 <0.0001 0.0661 <0.0001 0.293 0.2946 0.0032 <0.0001 0.0003
DRY Moisture Regime
Control 43b 5a 16a 124a 109a 1.70c 3.68a 22.6a 9.7a
Field A 66a 5a 37a 32a 120a 2.19a 4.21a 20.5a 10.6a
Field B 64a 4a 19a 207a 108a 2.00b 5.80a 37.0a 10.5a
P < F 0.0172 0.5329 0.1753 0.0002 0.0652 0.0005 0.0041 0.0069 0.326
*Means followed by the same letter within columns by moisture regime are not significantly different (p = 0.05).
Table 6. Effects of Experiment 1 Treatments on Elemental Composition of Soybean Upper Trifoliolate Leaves.
(Upper Trifoliolates of Reclamation Treatment Plants were Not Analyzed).
Treatment
Element (mg/kg)
Zn Cu B Mn Fe P Mg Ca K
WET Moisture Regime*
Control 47c 8b 26a 104a 82a 2.86c 3.22a 12.5b 17.3b
Field A 68b 7b 30a 50b 80a 3.23b 3.13a 10.6c 16.7b
Field B 91a 11a 23a 102a 93a 3.79a 3.51a 15.3a 19.4a
P < F <0.0001 <0.0001 0.6478 0.0002 0.2556 0.0004 0.1322 0.0002 0.0248
DRY Moisture Regime
Control 48b 6a 34a 103b 103a 2.39b 2.68b 11.1b 15.8b
Field A 69a 7a 20a 45a 119a 2.85a 3.08a 9.2c 15.8b
Field B 60ab 7a 17a 104b 113a 3.04a 3.13a 15.6a 19.0a
P < F 0.0501 0.6713 0.3664 0.0006 0.2079 0.0327 0.0456 0.0006 0.0545
*Means followed by the same letter within columns by moisture regime are not significantly different (p = 0.05).

Experiment 2—Effects of PVSC Primary Sludge
and PVSC Zimpro -processed Biosolids on
Soybean Growth
Germination and Seedling Development
There were no observable effects of treatment on ger-
mination and emergence rate, but plants in the Control
treatment were uniformly taller than those in both the
Pre- and Post-Zimpro™amended soils one week after
emergence. Many of the first true leaves in the Pre- and
Post-Zimpro™treatments exhibited crinkling symp-
toms.
Marginal necrosis similar to that observed in the Rec-
lamation Site and Field A plants in Experiment 1 was
visible on almost all of the first true leaves of the
Post-Zimpro™plants approximately three weeks after
emergence. The symptom progression was similar to
that observed in Experiment 1, i.e., necrotic spotting at
the leaf tip which spread to form a band of dark necrotic
tissue around the entire leaflet. Interveinal chlorosis
and leaf puckering also occurred. Younger leaves on the
Pre-Zimpro™plants exhibited puckering but not necro-
sis. The size of the leaves decreased according to treat-
ment in the order Control > Post-Zimpro™>
Pre-Zimpro™. Marginal necrosis of the older
trifoliolates was more extensive on Post-Zimpro™than
Pre-Zimpro™plants, but the Pre-Zimpro™older
trifoliolates additionally exhibited white spotting, and
more extensive interveinal chlorosis, puckering, and
crinkling. Drought exacerbated the crinkling/pucker-
ing effect caused by both the Pre-Zimpro™and
Post-Zimpro™treatments.
Observations at Harvest
Both Pre- and Post-Zimpro™plants flowered earlier
than the Control plants, which did not begin to flower
until harvest (65 days after emergence). The
Post-Zimpro™plants in both moisture regimes were
larger than the Pre-Zimpro™plants at harvest, despite
exhibiting marginal necrosis on approximately 50% to
75% of the leaves. The Post-Zimpro™plants grown un-
der the DRY moisture regime resembled the Reclama-
tion Site plants from Experiment 1 in expression and
extent of symptoms.
Only above-ground biomass and height were mea-
sured because the plants were harvested at the time of
flowering (Table 8). There were no differences in plant
biomass or height in the DRY treatment, but the bio-
mass and height of Pre-Zimpro™and Post-Zimpro™
plants were lower than Control plants in the WET mois-
ture regime. This indicated to us that the addition of
both PVSC Zimpro™and the primary sludge from
which the Zimpro™biosolids were produced retarded
soybean growth up to at least early flowering.
Growth was reduced in the order Control >
Post-Zimpro™> Pre-Zimpro™indicating that either
(1) the plant growth limiting factor(s) was present in the
primary sludge, (2) Zimpro™processing did not exac-
erbate the detrimental effect of the toxicant, and/or (3)
the symptoms and reduced plant growth were caused by
different factors in the two PVSC materials. The
Pre-Zimpro™sludge caused some symptoms in soy-
bean seedlings (e.g., white spotting) that were morpho-
logically different than symptoms in seedlings grown in
the Post-Zimpro™treatment or in soybeans observed in
the field.
Root Observations
Plant root mass was longer and more branched under
WET than DRY soil moisture conditions in all treat-
ments. In both the WET and DRY moisture regimes, the
root mass of the Control plants was larger than the root
mass of the Post-Zimpro™and Pre-Zimpro™plants. As
with the biomass response, the root mass of the Control
treatment under both moisture regimes was greater than
either the Pre- and Post-Zimpro™treatments.
Nodulation was only observed on plants in the WET re-
gime of the Control treatment.
Soil Properties
Both primary sludge and Zimpro™-processed
biosolids additions reduced soil pH and increased con-
centrations of soluble salts and nearly every macro- and
micro-nutrient (Table 9). The addition of the
Pre-Zimpro™primary sludge resulted in a lower pH
than that generated by the Post-Zimpro™biosolids,
possibly because the primary sludge contained oxidiz-
able acidity-producing organic N and S compounds
and/or had higher soluble salt concentrations that
Soybean Phytotoxicity from Land-Applied Biosolids 9
Table 8. Effect of PVSC Primary Sludge (Pre-Zimpro )
and Zimpro –processed Biosolids (Post-Zimpro )on
Soybean Biomass and Height in Experiment 2.
Treatment
Biomass (g) Height (cm)
WET* DRY WET DRY
Control 11.99a 1.70a 66.5a 26.6a
Pre-Zimpro™2.80b 1.01a 32.2c 21.5a
Post-Zimpro™5.62c 1.25a 46.2b 24.4a
*Means followed by the same letter within columns are not significantlydiffer-
ent (p = 0.05).

would lower pH measured in water by displacing po-
tential acidity from the solid matrix. Chloride concen-
trations in the Pre-Zimpro™primary sludge were much
higher than in the Post-Zimpro™biosolids, but the sol-
uble salt concentration in the Pre-Zimpro™-amended
soil would not have been expected to reduce soybean
growth (Maas, 1984). A combination of increased Mn
concentration, reduced soil pH, and, perhaps, reducing
conditions may have promoted Mn toxicity
(Kabata-Pendias and Pendias, 1984).
Plant Analysis Data
Manganese and Zn concentrations in plant tissue
grown on the Pre- and Post-Zimpro™treatments were
higher than in the Control, presumably due to the soil
enrichment of Mn and Zn from primary sludge or
biosolids application coupled with lower soil pH (Table
10). Tissue Zn concentration under the WET moisture
regime appeared to be inversely related to soil pH.
Tissue Mn concentrations (745 and 861 mg/kg) of
whole plants grown in the Pre-Zimpro treatment was
greater than published phytotoxicity threshold values
of 400–500 mg/kg for leaf tissue by Kabata-Pendias
and Pendias (1984) and 720 mg/kg for whole plant by
Fageria (2001). Soybeans grown in this treatment may
have suffered Mn toxicity, symptoms which include
stunting and crinkled young leaves, Fe chlorosis, ne-
crotic spotting, and uneven chlorophyll distribution in
older leaves (Kabata-Pendias and Pendias, 1984). Man-
ganese uptake by plants is positively correlated with
soil organic matter concentration and negatively corre-
lated with soil pH and redox. Symptoms of Mn toxicity
10 K. HAERING, W. DANIELS and G. EVANYLO
Table 9. Effect of PVSC Primary Sludge (Pre-Zimpro )andZimpro –processed Biosolids (Post-Zimpro )
on pH, Soluble Salts, and Mehlich I Extractable Soil Nutrients in Experiment 2.
Treatment pH
Soluble
Salts
(dS/m)
Mehlich I Extractable Elements (mg/kg)
Ca Mg P K Mn Zn Fe Cu B
WET Moisture Regime*
Control 6.20a 0.30c 702bc 41c 24b 61c 19c 3c 10b 0.9b 0.13a
Pre-Zimpro™5.20c 2.26a 767a 68a 43a 121a 46a 4b 14a 1.6a 0.15a
Post-Zimpro™5.62b 1.12b 743a 55b 38a 99b 33b 5a 13a 1.1b 0.15a
P < F <0.0001 <0.0001 0.0202 <0.0001 0.0003 <0.0001 <0.0001 <0.0001 <0.0001 0.0073 0.2187
DRY Moisture Regime
Control 6.50a 0.70c 749a 46c 30c 107b 20c 3c 10b 0.9b 0.14b
Pre-Zimpro™5.25c 2.17a 779a 69a 39b 129a 49a 4b 14a 1.4a 0.16a
Post-Zimpro™5.45b 1.37b 762a 56b 48a 118ab 39b 6a 14a 1.1ab 0.14b
P < F <0.0001 0.0007 0.396 0.0001 0.0019 0.0688 <0.0001 0.0012 0.0007 0.03 0.0016
*Means followed by the same letter within columns by moisture regime are not significantly different (p = 0.05).
Table 10. Effect of PVSC Primary Sludge (Pre-Zimpro )andZimpro -processed Biosolids (Post-Zimpro )
on Soybean Whole Plant Tissue Elemental Composition at Flowering in Experiment 2.
Treatment
Element (mg/kg)
Zn Cu B Mn Fe P Mg Ca K
WET Moisture Regime*
Control 34b 10a 27a 119b 85a 2.79a 3.35b 15.8c 23.9a
Pre-Zimpro™91a 22a 31a 861a 104a 1.98a 4.09a 26.3a 22.5a
Post-Zimpro™64a 9a 25a 289b 90a 2.48a 3.96a 20.4b 23.2a
P < F 0.0989 0.6106 0.1289 0.01 0.4844 0.1115 0.0292 0.0007 0.1736
DRY Moisture Regime
Control 48a 4a 30a 110c 82a 1.37a 3.33a 20.1b 23.9a
Pre-Zimpro™43a 3a 33a 745a 69a 1.19a 3.64a 27.6a 22.5a
Post-Zimpro™47a 5a 38a 460b 74a 1.73a 3.17a 18.7b 23.2a
P < F 0.0281 0.1414 0.5877 0.0014 0.2814 0.3453 0.1573 0.0195 0.687
*Means followed by the same letter within columns by moisture regime are not significantly different (p = 0.05).

are generally observed in younger leaves, but there is
some evidence that Mn can be translocated to older
plant tissue when present at high soil levels
(Kabata-Pendias and Pendias, 1984). Drought-stressed
plants in the Post-Zimpro™treatment also accumulated
high concentrations of Mn (460 mg/kg).
Whole plant Zn and Cu levels in the WET moisture
regime of the Pre-Zimpro™treatment were also high,
but the variability between plants in this treatment was
so great that the difference was not statistically signifi-
cant. These differential metal levels may also have been
the result of the acidic soil pH levels discussed earlier.
Overall Results
Amendment of soil with the Pre-Zimpro™sludge re-
sulted in soybean plants that were smaller than soy-
beans grown in soil amended with resultant
Post-Zimpro™biosolids. The soybeans grown in the
Pre-Zimpro™-amended soil exhibited symptoms such
as white spotting, crinkling and puckering, and
chlorosis, but did not exhibit the distinctive continuous
marginal necrosis typical of the leaves of soybeans
growninZimpro™-amended soil. Soil testing and plant
analysis indicated that yield and morphology of plants
grown in soil amended with the primary Pre-Zimpro™
sludge may have been caused by Mn toxicity. Soil and
plant tissue analyses did not reveal any elemental toxic-
ity or soluble salt injury that might have directly caused
the plant symptoms in either the Pre-Zimpro™or
Post-Zimpro™treatments.
CONCLUSIONS
We were able to successfully replicate the 1999
field-observed phytotoxicity symptoms in soybeans
grown in soils amended with Zimpro™-processed
PVSC biosolids in the greenhouse. We observed the
identical progression of symptoms (i.e., leaf pucker-
ing/crinkling, marginal necrosis and necrotic spotting,
interveinal chlorosis, stunted growth and, finally, full
necrosis) that was reported for soybeans in
Zimpro™-amended soils in Essex County, VA. These
symptoms appear to be specifically and consistently as-
sociated with Zimpro™-processed biosolids from the
PVSC treatment plant. The symptoms were most evi-
dent in plants growing in the treatment which had re-
ceived the highest rate of Zimpro™-processed biosolids
(Reclamation Site). Moisture stress reduced plant size,
increased the number of leaves with phytotoxic symp-
toms, and exacerbated symptoms in some treatments,
but did not cause any symptoms that were not also ob-
served in soybeans which received adequate moisture.
Poor plant growth and phytotoxicity symptoms were
observed in soybeans growing in soils amended with
both the Pre-Zimpro™sludge and the Post-Zimpro™
biosolids from the PVSC wastewater treatment plant.
The appearance of the symptoms and the extent of plant
growth reduction indicated that either (1) the toxic
agents in the two materials were different or (2) the det-
rimental effects of the Pre-Zimpro™sludge masked the
symptoms observed in the field and in the first green-
house experiment. The plant tissue analysis and symp-
toms expressed by the plants grown in the
Pre-Zimpro™-amended soils were indicative of Mn
toxicity. The composition of the primary sludge (high
organic C content, high soluble salt content) may have
induced the low soil pH and redox conditions that favor
solubilization of Mn. The soil treated with
Pre-Zimpro™sludge had a lower pH, higher electrical
conductivity, and higher concentration of Mehlich I
extractable Mn than those treated with the Post-
Zimpro™biosolids although the concentration of total
Mn was lower in the sludge than in the resulting
biosolids.
It is not clear from our work whether or not the toxic
agent was a product of the Zimpro™process. The agent
may have been present in the raw sludge and, subse-
quently concentrated and/or altered by the Zimpro™
process. Some symptoms in the Essex County field soy-
beans were similar to the Mn toxicity-like symptoms
induced by the Pre-Zimpro™sludge in the greenhouse
experiment. In the field soils, however, the causative
agent persisted for several years after application of
Zimpro™-processed biosolids. Such persistence, cou-
pled with the lower than toxic concentrations of Mn in
plant tissue and soil samples, likely eliminate Mn toxic-
ity as the cause of the phytotoxic symptoms in the Essex
County field soybeans.
The toxicant may be an organic compound which ex-
hibits species-specific plant growth regulator activity
because soybean, but not corn or wheat, were affected,
and may be slowly mineralized or released/desorbed
into solution for plant uptake. Similar phytotoxicities
have been documented following the application of
other waste by-products onto land. Several pyridine
carboxylic acid herbicides persisted at concentrations
that caused phytotoxicity in sensitive crops grown in
soils amended with compost produced from turfgrass
that had been treated with clopyralid or with compost
Soybean Phytotoxicity from Land-Applied Biosolids 11

produced from manure excreted by livestock that had
ingested picloram-treated tall grass hay (Bezdicek et
al., 2001; Houck and Burkhart, 2001; Rynk, 2002). De-
spite the mineralization and subsequent reduction in ac-
tivity in these herbicides during the biological degrada-
tion that occurred in the compost windrows, these
compounds remained bioactive at concentrations of
less than 10 ppb. These herbicides mimic plant growth
regulators and cause symptoms similar to those de-
scribed in the field soybeans in Virginia, such as crin-
kling, puckering, chlorosis, and necrosis.
Further analysis of a wide range of organic chemical
constituents would be required to determine the exact
cause of the phytotoxicity that might initially be present
in the wastewater treatment plant sludge or created dur-
ing the wet air oxidation processing of the biosolids. An
exhaustive review of industrial inputs and analyses of
wastewater sludge and finished product, which was be-
yond the scope of this investigation, would be needed.
ACKNOWLEDGEMENTS
This project was funded by Synagro Inc. in coopera-
tion with PVSC. We would like to thank Rufus Chaney
(USDA-ARS Beltsville) for his help with all phases of
this project. We are also grateful for assistance pro-
vided by Ursula Kukier, Cheryl Szeles, Steve Nagle,
Ron Alls, and W.T. Price with field, greenhouse and
laboratory work. Larry Foy and Glenn Buss’s input on
soybean growth, physiology, and deficiency/toxicity
symptoms was invaluable, as was Jody Booze-Daniels’
help with greenhouse procedures. We would also like
to thank John Gatsch of PVSC and Brian Cauthorne,
Sharon Hogan, and Lisa Williams of Synagro for their
help in obtaining much-needed materials and informa-
tion.
REFERENCES
ASTM, 2001. C602-95a: Standard Specification for Agricultural Liming
Materials. Book of Standards Volume 04.01. Amer. Soc. Testing and
Materials, West Conshohocken, PA.
AWWA. 1998. Standard methods for the examination of water and
wastewater. 20th edition. Co-published by Amer. Water Works Assn.,
Amer. Public Health Assn., and Water. Environ. Fed.
Bezdicek, D., M. Fauci, D. Caldwell, R. Finch, and J. Lang. 2001. Persistent
herbicides in compost. BioCycle 42(7): 25–30.
Daniels, W. Lee, Steve Nagle, G. Richard Whittecar, and Greg Evanylo.
2002. Effects of biosolids application on ground water nitrate-N levels in
sand and gravel mine reclamation in Virginia. p. 645–674. In Proc. of the
2002 Natl. Meeting of the Am. Soc. of Mining and Reclamation,
Lexington, KY. 9–13 June 2002. ASMR, Lexington, KY.
Donohue, S.J., and S.E. Heckendorn. 1994. Soil test recommendations for
Virginia. Virginia Coop. Ext. Publ. 834.
Fageria, N.K. 2001. Adequate and toxic levels of copper and manganese in
upland rice, common bean, corn, soybean, and wheat grown on an
Oxisol. Commun. Soil Sci. Plan Anal. 32 (9&10):1659–1676.
Houck, N.J. and E.P. Burkhart. 2001. Penn State research uncovers
clopyralid in compost. BioCycle, July. p. 32–33.
Kabata-Pendias, Alina, and Henryk Pendias. 1984. Trace elements in soil
and plants. CRC Press, Boca Raton, FL.
Maas, E.V. 1984. Salt tolerance of plants. p. 57–75. In B.R. Christie (ed.)
Handbook of plant science in agriculture. CRC Press. Inc., Boca Raton,
FL.
Mullins, G.L. and S.L. Heckendorn. 2005. Laboratory procedures: Virginia
Tech Soil Testing Laboratory. Virginia Cooperative Extension Publica-
tion 452-881.
Neel, P.L., E.O. Burt, P. Busey, and G.H. Snyder. 1978. Sod production in
shallow beds of waste materials. J. Amer. Soc. Hort. Sci. 103(4):
549–553.
PVSC, 1996. New Jersey sludge management plan, NJPDES Permit
#NJ0021016. Passaic Valley Sewerage Commissioners, Newark, NJ.
PVSC, 2006. Passaic Valley Sewerage Commissioners (web site). Avail-
able on-line at http://www.pvsc.com.
Rynk, R. 2003. Prevalence and fate of clopyralid in compost. BioCycle
43(2): 57–60.
Sabbe, W.E., G.M. Lessman, and P.F. Bell. 2000. Field crops: soybean. IN
C. Ray Campbell (ed.) Reference sufficiency ranges for plant analysis in
the Southern regions of the United States. Southern Coop. Series Bull.
#394. Available on-line at http://www.agr.state.nc.us/agronomi/saaesd/
s394.htm.
SAS Institute. 2002. SAS/STAT user’s guide. Version 8.2 for Windows.
SAS Inst., Cary, NC.
U.S. EPA, 1979. Methods for Chemical Analysis of Water and Wastes
(EPA/600/4-79/020) Natl. Tech. Info. Svc., Springfield, VA.
U.S. EPA, 1995. Test Methods for Evaluating Solid Waste. Third edition,
third update. (SW-846). Natl. Tech. Info. Svc., Springfield, VA.
USFilter Corporation, 2005. Zimpro™wet oxidation (web site). Avail-
able on-line at http://www.usfilter.com/water/Business+Centers/
Zimpro_Products/Zimpro_Products/Zimpro_Wet_Air_Oxidation.htm.
USDA-NRCS. 1996. Soil survey laboratory methods manual. Soil Survey
Investigations Report No. 42, Version 3.0. U.S. Dept. of Agric., Nat. Res.
Cons. Service, Natl. Soil Survey Ctr. U.S. Govt. Printing Ofc., Washing-
ton, DC.
Vail, J.W., and D.G. Dewey. 1985. Controlling metal uptake from
heat-treated sludge applied to grasslands. Water Sci. Tech.
17:599–610.
Wei, Q.F., B. Lowery, and A.E. Peterson. 1985. Effect of sludge application
on physical properties of a silty clay loam soil. J. Environ. Qual.
14:178–180.
12 K. HAERING, W. DANIELS and G. EVANYLO