v°/EPA
                                 United States
                                 Environmental Protection
                                 Agency
                                 Municipal Environmental Research \ 4
                                 Laboratory                    '""•
                                 Cincinnati OH 45268
                                 Research and Development
                                 EPA-600/S2-81-024 Mar. 1981
Project Summary
                                 Helminth  and  Heavy  Metals
                                 Transmission  From
                                 Anaerobically  Digested
                                 Sewage  Sludge
                                 Paul R. Fitzgerald
                                  This summary discusses the findings
                                 of a study designed to determine the
                                 transmission to an animal host of the
                                 ova of the nematode worm Ascaris sp.
                                 that have survived through a modern
                                 sewage treatment process and are pre-
                                 sent in the sludge. Four large experi-
                                 ments and three smaller ones involv-
                                 ing 178 specific pathogen-free (SPF)
                                 pigs were used. Natural transmission
                                 of Acaris sp. from soil treated with
                                 liquid  anaerobically  digested sewage
                                 sludge that had been stored for several
                                 years occurred in a few pigs in each of
                                 four experiments. Also, natural trans-
                                 mission from Nu-Earth, a dried, stored
                                 sewage sludge, also occurred in pigs
                                 that were exposed to this material by
                                 contact in the pens.  In general, ova in
                                 anaerobically digested sludge or in Nu-
                                 Earth  remained unembryonated until
                                 after they were exposed to the air. With-
                                 in 6 weeks after exposure to air, the
                                 ova began to embryonate, and there-
                                 after,  a small percentage of the ova
                                 that embryonated became  infective
                                 for pigs.
                                  The occurrence of heavy  metals in
                                 the tissues of swine held in pens treat-
                                 ed with anaerobically digested sludge
                                 or Nu-Earth, which originated from a
                                 large municipality, was also studied.
                                 Chemical analyses of kidneys, livers,
                                 hearts, diaphragm muscles, and bones
                                 were conducted to determine the quan-
                                 tities of the heavy metals cadmium.
                                 zinc, copper, iron, lead, chromium, and
                                 nickel that were present in the tissues
                                 following exposure of the pigs to differ-
                                 ent amounts of the sewage products
                                 in or on the soil. Only cadmium accu-
                                 mulated to a significant degree in some
                                 tissues of swine exposed to sludge con-
                                 taining heavy metals. No physiological
                                 or pathological changes associated with
                                 exposure to the sludge material were
                                 detected. Examination of visceral fat
                                 from control and experimental pigs in-
                                 dicated that there was no unusual accu-
                                 mulation of organic compounds includ-
                                 ing polychlorinated biphenyls (PCB's)
                                 and the insecticides Heptachlor and
                                 Dieldrin.
                                   This Project Summary was develop-
                                 ed by EPA's Municipal Environmental
                                 Research Laboratory, Cincinnati, OH,
                                 to  announce  key  findings of the
                                 research project that is fully docu-
                                 mented in a separate report of the
                                 same title (see Project Report ordering
                                 information at back).

                                 Introduction
                                   Improvements in sewage disposal
                                 systems within the last 50 years have
                                 contributed to improved use of biological
                                 wastes to supplement the texture and
                                 fertility of soils. In using aerobically or
                                 anaerobically digested sewage sludges
                                 for soil conditioning or fertilizer, one of
                                 the important  unanswered questions,
                                 however, concerns possible adverse

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       effects of dispensing of possible disease-
       causing materials to the environment.
         Farmers throughout the world have
       used human and animal fecal material
       as fertilizer for hundreds of years. Only
       since  the early 1900's has the use of
       such materials been recognized as the
       source of many diseases. More recently, it
       has been determined that a few bacterial,
       viral, protozoan, and helminth parasites
       are able to persist in waste products
       originating from  humans or animals.
       Although the majority of pathogenic
       organisms of viral, bacterial,  protozoan,
       or helminth origin  are destroyed by
       exposure to an external environment,
       many validated instances of serious
       outbreak of disease  have occurred
       involving these organisms. In under-
       developed countries, many parasitic
       diseases are literally a "way-of-hfe."
         During recent years, researchers
       have found that ova of ascarid round-
       worms are the most resistant organ-
       isms in wastes from human and animal
       sources. These ova are known to survive
       in soil for many months, even years, and
       thus serve as a  potential source of
       disease.
         In addition to the bacteria, viruses,
       protozoan, helminths, etc., other mate-
       rials found in sludges may cause disease
       in man and animals. Among these  are
       toxic chemicals such as heavy metals
       (e.g., Cd, Pb, Hg,  Cr, etc.)  and organic
       compounds (e.g, PCB's) of various kinds
       and combinations. The route of these
       chemicals to man or animals is through
       direct ingestion of sludge, or through
       the food chain that  starts with crops
       grown on sludge-amended soil. The
       relationship between exposure or dose
       and illness is complex and  is not  the
       subject of this investigation  However,
       the chance of illness is clearly related to
       the flow of the toxic substance up  the
       food chain. By establishing relationships
       between the initial dose or exposure
       and the absolute concentrations in  the
       intermediate and final hosts in the food
       chain, the ultimate threat of illness can
       be put in perspective.
         In the work summarized  here, one
       objective was to determine the possibility
       of transmission of one pathogen (Ascaris
       lumbricoides suum), a resistant para-
       site, from soil treated with sludge to a
       susceptible host (a pig). This parasite is
       almost  identical  in  nature and  in  its
       mode of infection  to the Ascaris lumb-
       ricoides, which infects man. Conse-
       quently, information developed on trans-
       mission bears directly on the dose-
infection relationship for pigs and is
most likely very similar to  the dose-
infection relationship in humans. A
second objective was to determine the
uptake of heavy metals and organic
compounds in the pigs exposed to
sludge. From this  information,  the
possible exposure of humans who ulti-
mately consume the pigs can be  esti-
mated.

Procedure
  A total of 178 pigs free from parasitic
infections  (specific-pathogen-free:SPF)
were used to study whether anaerobic-
ally digested  sludge, added to soil at
different concentration levels, could be
a potential source of infestation with the
nematode Ascaris lumbricoides suum.
The soil was treated with various levels
of liquid anaerobically digested sludges
(from 22 to 65 dry metric tons  per
hectare) or with dried sludge (Nu-Earth)
added to the soil to a depth of 15.3 cm.
Pigs were confined in pens (9.8 x  14.6
m) to which the sludge had been added.
Pigs living in  each of these environ-
ments, for approximately 4 months,
were compared with  pigs  living in
similar control pens, under the same
environmental conditions, but without
sludge added to the pens.

Results
  • In each of two of the experiments,
    eight young pigs were necropsied 2
    to 3 weeks after introduction  into
    the pens and lung and liver tissues
    were Baermannized to  recover
    migrating stages of the worms.
    Larvae were recovered from the
    lungs of 2 of 16 necropsied. Of 134
    pigs that survived the full experi-
    ment to  necropsy, 43  contained
    ascarid worms in the intestines,
    including four from the control
    pens. These four represent a  con-
    tamination of the control area that
    apparently occurred accidentally
    during the  course  of  the study.
    These results represent an infection
    rate of 28% in the experimental
    groups and 3% in the control groups.
    Of the 134 pigs, 11 were housed in
    pens treated with previously stored
    Nu-Earth; of these, 6 were infected.
  • An attempt was made todetermine
    the rate of transmission of the
    human ascarid worm, obtained
    from a source in Columbia, South
    America, to pigs.  Fifteen SPF  pigs
    were confined in two sludge-free
 pens. Embryonated or unembryo-
 nated ova in water were sprayed
 onto the soil or vegetation at the
 rate of 572,000 ova in each of the
 two pens. The ova were extracted
 from  the uteri of adult female
 human Ascaris lumbricoides; the
 portion of embryonated were em-
 bryonated in 1% formalin with
 continuous aeration  for 30 days.
 The pigs were allowed to remain in
 the environment for 4 months,
 During that time, all of the vegeta-
 tion was consumed and the pigs
 did what pigs do: ate soil, breathed
 dust, rolled in water and mudholes
 after rainstorms, etc.  At necropsy,
 4 months after being placed in the
 pens,  no worms were  found in
 either group of pigs.
1 Three pigs were fed anaerobically
 digested sludge in which there
 were naturally occurring ascarid
 ova as part of their  normal food
 diet. The source of the ascarid ova,
 of course, could have been humans
 or pigs. Before the feeding step, the
 liquid anaerobically digested sludge
 was aerated by bubbling air through
 it for  104 days. It was then fed to
 each of the three pigs during a 30
 day period. Forty-seven days after
 the end of the feeding period, the
 pigs were necropsied and the gastro-
 intestinal tracts were examined for
 ascarid worms. Worms found in
 the intestine of one pig  indicated
 that ova naturally present in  the
 sludge could be infective for pigs.
 The rate of embryonation of ascarid
 ova (removed from the uteri of
 gravid female worms) was deter-
 mined in the presence of untreated
 soil, soil treated with liquid anaero-
 bically digested sludge, or Nu-
 Earth. At the end of 6 weeks'
 exposure to the soil to which liquid
 sludge or Nu-Earth had been added,
 an average  71% of the ova had
 embryonated and were presumed
 to be  infective. The worms were
 determined to be viable by observa-
 tion of movement  of larval  stages
 within the ova. None of the ova
 were  fed to susceptible pigs to
 ascertain infectivity.
 Tissues taken from the pigs at  ne-
 cropsy were analyzed for heavy met-
 als  and organic compounds. Dia-
 phragm muscle, heart, liver, kidney,
 and bone were selected for analysis
 for heavy metals, and visceral fat

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     was selected for analysis for the
     organic compounds (Tables 1 and
     2).
   Cadmium in the kidney and liver was
 the only heavy metal showing significant
 accumulation. Some heavy metals pre-
 sent in sludges may accumulate in the
 tissues at levels related to the  level of
 exposure, i.e., animals exposed to great-
 er concentrations of sludge had greater
 tissue concentration of some heavy
 metals. In these studies, pigs exposed to
 dried sludge (Nu-Earth) accumulated
 the greatest quantities of heavy metals.
 For example, the average accumulation
 of cadmium in the kidneys was 9.8 ppm
 dry weight. On a wet weight basis this
 would be less than 3 ppm. In humans,
 clinical evidence of damage to the
 kidney is said to begin when approxi-
 mately 200 ppm, wet weight,  have
 accumulated.
  The  results of chemical analysis for
 organic compounds  (PCB's, Dieldrin,
 Heptachlor) showed there was no differ-
 ence between experimental and control
 animals in terms of accumulation. These
 compounds were not accumulated in
 the animal tissues to levels greater than
 is normally found in the environment.

Conclusions
  Exposure of swine to different levels
of anaerobically digested sludge from a
large  municipality  has shown  that a
potential for transmission of the nema-
tode Ascaris sp. exists. The results of
this study show that some ova from this
parasite worm are able to withstand the
rigorous treatment of a modern sewage
treatment plant and are subsequently
infective to pigs. The exposure of swine
to anaerobically digested sludge was
infinitely greater than a normal human
exposure would be, yet the levels of
infestation with Ascaris sp. were low.
With ova present at an approximate
level of 4000/m2 of surface, embryona-
tion of some ova occurred, some were
ingested by pigs and eventually caused
light infestations in the gastrointestinal
tracts. Although these worms did not
cause detectable pathology in the ani-
mals they infested, a potentially serious
parasite problem could develop  in a
swme  herd continuously exposed to
anaerobically digested sludge.  This
could occur not because of the immedi-
ate damage caused by worms transmit-
ted from the sludge, but because of the
potential for buildup of numbers of ova
in the environment as sexually mature
female worms, originating from ova  in
 Table I.    Summary of chemical analyses for heavy metals in tissues of pigs exposed
           to different levels of anaerobically digested sewage sludge originating
           from the Metropolitan Sanitary District of Greater Chicago. Numbers in
           parenthesis represent deviations in numbers of animals.
                                              ppm, dry weight
 Metal & Group
Number of
 Animals  Kidney
Liver
Diaphragm
  Muscle
Heart   Bone
 Cadmium
   Control                 14
   22-25 DMT/Ha sludge    18
   33 DMT/Ha sludge       4
   65 DMT/Ha sludge       4
   Nu-Earth                4
   NBS control (bovine)
 Chromium
   Control                 12
   22-25 DMT/Ha sludge    12
   33 DMT/Ha sludge       4
   65 DMT/Ha sludge       4
   Nu-Earth               —
 Copper
   Control                 14
   22-25 DMT/Ha sludge    18
   33 DMT/Ha sludge       4
   65 DMT/Ha sludge       4
   Nu-Earth                4
   NBS control (bovine)
 Iron
   Control
   22-25 DMT/Ha sludge
   33 DMT/Ha sludge
   65 DMT/Ha sludge
   Nu-Earth
 Lead
   Control                 14
   22-25 DMT/Ha sludge    18
   33 DMT/Ha sludge       4
   65 DMT/Ha sludge       4
   Nu-Earth                4
   NBS control (bovine)
 Nickel
   Control                 12
   22-25 DMT/Ha sludge    12
   33 DMT/Ha sludge       4
   65 DMT/Ha sludge       4
   Nu-Earth               —
 Zinc
   Control                 14
   22-25 DMT/Ha sludge    18
   33 DMT/Ha sludge       4
   65 DMT/Ha sludge       4
   Nu-Earth                4
   NBS control (bovine)
            0.47
            2.30
            2.10
            3.83
            9.80
 0.23
 0.34
 0.29
 0.58
 1.50
 0.27
    0.04
    0.06
    0.07
    0.15
    0.06
                            0.12
                            0.17
                            0.14
                            0.09
 0.05
 0.05
 0.22
 0.09
 0.06
                    0.08
                    0.09
                    0.18
                    0.06
37.00 26.00
33.00 25.00
28.00 37.00
33.00 23.00
44.00 33.00
188.00
7.00
10.00
9.00
8.00
7.00

16.00
17.00
19.00
16.00
18.00

14
18
4
4
4
166.00 488.00
155.00 541.00
144.00549.00
172.00586.00
198.00 453.00
99.00
103.00
91.00
119.00
73.00
180.00
187.00
169.00
180.00
152.00
            0.31
            0.31
            0.29
            0.32
            0.44
 0.33
 0.34
 0.36
 0.31
 0.47
 0.35
          138.00 264.00
          133.00 263.00
          117.00248.00
          128.00210.00
          140.00 286.00
                 131.00
    0.22
    0.28
    0.17
    0.20
    0.21
                            0.39
                            0.41
                            0.30
                            0.60
        123.00(9}
        /2S.001111
        119.00
        108.00
        113.00
 0.20
 0.23
 0.18
 0.38
 0.26
            75.00
            74.00
            65.00
            74.00
            77.00
0.03('
0.02"
0.04
                                             3.50''
                                             5.30"
                                             3.80
                                            44.00('
                                            44.00"
                                            42.00
0.63''
0.69(t
1.77
       98.00''
      118.00"
      122.00
the sludge, began to lay eggs which
would "seed" the environment. Sub-
sequent swine, exposed to the area,
could be exposed to much greater
numbers of ova, and a potential parasite
                 problem could develop into a serious
                 actual problem.
                   Under the conditions of this study, the
                 health of the animals did not appear to
                 be threatened by the parasites transmit-
                                                                                     I US GOVERNMENT PRINTING OFFICE 1981 -757-0 1Z/7I06

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     Table 2.    Summary of chemical analyses for organic compounds in visceral fat of
                pigs exposed to different levels of anaerobically digested sludge originating
                from the Metropolitan Sanitary District of Greater Chicago.

                                                       ppm, wet weight
Exposure
Control
22 DMT/Ha sludge
Nu-Earth
Number of
Pigs
17
9
10
Polychlorinated
Biphenyls
0.158
0.111
0.120
Heptachlor
Epoxide Dieldrin
0.063 0.051
0.005 0.006
0.002 0.003
     ted through the sludge. If humans were
     exposed to fields with the same sludge
     application densities, they would ingest
     far less sludge than did the pigs. Con-
     sequently, it is not likely that man would
     become seriously parasitized from expo-
     sure to anaerobically digested sludge
     under current sanitary conditions in the
     United States.
       The results of this study have shown
     that cadmium was the only one of seven
     heavy metals which  accumulated to a
     significant degree  in tissues  of swine
     exposed to sludge containing heavy
     metals. Of the five tissues examined for
     heavy metals, only kidney and liver
     accumulated cadmium at levels greater
     than in controls. Although the accumula-
     tion  in these organs was statistically
     greater than in controls, the accumula-
     tion was far below a  lethal level.
       The presence of polychlorinated bi-
     phenyls  and  the organic  insecticides
     Heptachlor and Dieldrin in tissues of
     experimental animals, at levels com-
     parable to controls, indicates that these
     organic compounds did not appear to be
     an immediate threat to animal health
     under the conditions of these studies.
     The facts revealed herein suggest that
          serious exposure to anaerobically di-
          gested sludge, containing organic com-
          pounds at levels described in this study,
          would cause no significant detrimental
          effect on the animals.
            The full report was submitted in
          fulfillment of Grant No. R805315-01 by
          the University of Illinois under  the
          sponsorship of the U.S. Environmental
          Protection Agency.
             Paul R. Fitzgerald is with the University of Illinois,  Urbana, IL 61801.
             Gerald Stern is the EPA Project Officer (see below).
             The complete report, entitled "Helminth and Heavy Metals Transmission from
               Anaerobically  Digested Sewage  Sludge," (Order No.  PB  81-161  846;
               Cost: $8.00, subject to change) will be available only from:
                     National Technical Information Service
                     5285 Port Royal Road
                     Springfield, VA 22161
                     Telephone: 703-487-4650
             The EPA Project Officer can be contacted at:
                     Municipal Environmental Research Laboratory
                     U.S.  Environmental Protection Agency
                     Cincinnati, OH 45268
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Postage and
Fees Paid
Environmental
Protection
Agency
EPA 335
Official Business
Penalty for Private Use 8300

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