J. G. Babish

Organic compounds in sludges, with emphasis on, health risks associated with land application of municipal
sudges, have been reviewed by Kover (260), Dacre (103-104), Parin et al. (349), and briefly by Connery(96).
Surveys of toxic organics in sludge

The related polyhalogenated biphenyl compounds and their levels found in sludges from various areas of the
United States are shown in Table 16. These are average values, e.g. the range in the Midwest was 240 to 1700
ppm with an average value of 765 ppm. The prevalence of polychlorinated biphenyls (PCBs) in sludge is well
documented (271,344). Furr et al. (161) found PCBs in concentrations up to 23 ppm in all sludges from
eighteen American cities. PCBs have been found in municipal sludges of Canadian cities (270). Tucker et al.
(442) found that the stability of_ PCBs isomers in sludge is directly proportional to their degree of chlorination.
PCBs are persistent in soils (320,325) and have demonstrated both acute and chronic toxicity. Acute .signs of
poisoning in humans include eye discharge and swelling of upper eyelid, acne-form eruption, and pigmentation
of the sicin (176). The demonstrated chronic toxicities of PCBs include carcino-genicity (331), teratogenicity
(316), and chromosomal alterations,(351).

Many organochlorine insecticides occur in sludges.. The insecticide DOT was banned by the Environmental
Protection Agency in 1973, aldrin/dieldrin in 1974, and chlordane in 1975. Table 17 indicates the levels of
these insecticides present in sludges. Other chlorinated compounds have been identified in sewage sludge by
Erikson and Pellizzari (141). The origins and toxic properties of these individual compounds are not well known.
Dacre (103) identified additional chlorinated compounds in wastewater; it is .almost certain that many of these
highly toxic as well as potentially carcinogenic compounds will be concentrated in municipal sludges (Table 18).
Polynuclear aromatic compounds (PAH) have been identified in sludge (282). T,he compounds include
fluoranthrene, benzo(b)fluoranthrene, benzo(k)-fluoranthrene, benzo(a)pyrene, benzo(ghi)perylene, and
indenol(l,2,3,c,d,)pyrene at concentrations of 90 to 1,600 ppb. Additionally, a number of PAH observed in
municipal sewage sludge included two proven carcinogens: benzo(a)pyrene and benz-ofluoranthrene (187,
Petroleum hydrocarbons, possibly resulting from industrial cutting oils, have been found in sludge (282). Such
petroleum hydrocarbons would be expected to have a herbicidal source, since waste oil has long been used as
a spray for control of unwanted plant growth along roadways. Phytotoxic effects in_ _plants grown on sludge-
amended soils have been ob-_ served by various investigators.. Values up to 180 ppm of the flame retardant
pentabromotoluene have been found in sewage sludge (301).

Trimethylamine in sewage sludge was converted to the potent carcinogen dimethylnitrosamine (10,11).
Dimethylnitrosamine formed in soil to which dimeth-ylamine and nitrite were added, but only when organic
matter was present (317). Two dinirroaniline herbicides continued to volatilize from soils for several weeks
following incorporation (339). The half-life of these compounds in soils is about three weeks (340).
Such chemical surveys of sewage sludges are im-Dortant, but cannot fully assist in the estimation of L hazard
associated with the organics in sludges/It is obvious from the surveys already conducted on ooth the organic
and inorganic fractions of sludge that basing land application rates or assessing "cleanness" of a sludge on Cd
or PCBs content is inadequate. However, any analytical technique employed to examine the contents of
sludges will suffer from a bias — it can only find what it is set up to detect.JThis analytical procedure involves
some pre-existing knowledge of.possible contaminants Mutagenic activity of organic extracts of sludge.

Estimating the potential hazards'from sludges requires that their toxic properties bedetermined. When tne toxic
properties are known, environmentally sound methods of disposal may be developed and employed. Cornell
University studies on ambient exposures to carcinogenic and mutagenic compounds included evaluations of
sewage sludges from thirty-four American cities (15). Table 20 relates the information obtained from the
questionnaires describing the samples. Percentage of wastewater volume from industrial sources ranged from
a high of 65% for Toledo to a low of only 1% for Lexington.

As seen in Table 21, only one sample, Dallas Central, failed to demonstrate a dose-related increase in
revertants in any of the five tester strains with or without metabolic activation (S-9). The other thirty-, three
sludge samples exhibited a  positive mutagenic, response with at least one strain; twelve of these thirty-three
samples were positive with two or more strains. Seventy-six percent (25/33) of the positive samples required
metabolic activation to demonstrate mutagenicity, 18% (6/33) were mutagenic with and without the S-9 fraction,
and 6% (2/33) were mutagenic only in the absence of any metabolic activation. The extraction blank run with
these samples was negative (data not shown).

Of the five tester strains, TA100 was most responsive (30/33) and TA1535 least responsive (0/33). This is
interesting since TA100 was derived from TA1535 and differs only in that it has the R factor, pKM 101 (6). As
previously mentioned, this plasmid codes for an error-prone DNA repair enzyme (335) which increases the
sensitivity of TA100 to mutagens. It also results in strain TA100 responding to both basepair substitution and
frameshift mutagens (335). There fore, in the absence of any response of strain TA1535, , it must be inferred
that the mutagens contained in these samples induced primarily frameshift mutagons. Strains TA98 and
TA1538 also are identical except for the pKM 101 plasmid in strain TA98. Although it is generally recognized
that the sensitivity to most mutagens is increased by the plasmid, strain TA1538
responded positively to eight of the thirty-four samples while TA98 was mutated by only five of the thirty
samples. Concordant positive responses of the two strains occurred only twice. Therefore, for general
screening of environmental samples, the exclusion of TA1538 seems unadvisable.

The requirement for metabolic activation of 76% of the positive responses indicates the presence of relatively
inert, nonpolar compounds illiciting the mutagenic response. Although such compounds can be degraded by
soil microorganisms, many factors such as soil pH, redox potential, and sunlight influence the rate of
metabolism of these compounds and hence the hazard associated with the application of these compounds
to'open lands (282). Additionally, the components of sludge  can alter the capacity of soil. microorganisms to
degrade these refractory organic..compounds..Metals commonly found in sludges (Zn, Cd, Cr, Pb) have been
shown to inhibit soil bacterial, rand fungal activity (14); studies have demonstrated that adding municipal
sludges to field soil in vivo inhibited microbial carbon transformations (370, 444,445).
The distributions for all parameters of mutageniciry (revertants/mg extract, slope of dose-response curve, and
LSD) were computed. rOver all, strains and r samples, the median response of revertants/mg extract was 36.
The cutoff values for the upper and lower quanrile were 65 and 20 revertants/mg, respectively.  The  lowest  
response  was  2  revertants/mg     • (Kalamazoo, TA1537 with activation); foe highest re.sponse     was 264
revertants/mg (Philadelphia, Northeast, TA100 with activation). These results are in good agreement with those
of Weaver et al. (474) and Hopke et al. (215> In these studies, crude acetone extracts of sewage sludge
obtained from five Illinois municipal sanitary districts induced 27 to 58 revertants/mg extracts; approximately 155
p.1 of unaltered sludge resulted in a doubling of the number of revertant colonies with TA98 after activation
with extract S-9 from the rat.

The median slope for the dos'e-response curves for all sludge extracts was 19.8 revertants/log dose, with
values for the upper and lower quantile of 33.1 and 12.2, respectively. Extracts of Galveston airport sludge
produced the shallowest slope (1.9 revertants/log dose, TA100 with activation) and Buffalo sludge had the
steepest slope (119.3 ,revertants/log dose, TAiOO with activation). .Although the cities with the highest and
lowest mutagenicitv on a revertant/mg extract basis were not the same cities _ with the steepest and shallowest
slopes, a significant positive association 'gristed between revertants/'mg extract and slope of the dose-
response curve (p < 0.01, r = 0.489, n = 54). This indicates the frequency with which the initial portion of the
dose-response curve was described, since a high response in terms of revertants/mg extract and a shallow
slope are only possible when the initial portion of the dose-response curve is omitted.
LSD values were in the microgram range; the median Value over all strains and samples was 2.6 jig of extract.
This indicates that only 2.6 fxg of extract would be necessary to produce a statistically signifi
cant (p < 0.05) increase in revertants. The upper and lower quantiles. were 16.09 and 1.0 p.g, respectively.
The lowest LSD was 0.01 pLg (Phoenix, 91st Ave, TA100 with activation) and the highest LSD was 230 (xg
(Kalamazoo, TA1537 with activation). With activation, strains TA98 and TA100 produced the lowest LSD
values, while those for TA1537 and TA1538 were approximately ten times higher. In general, LSD values were
lower with S-9 than without.        There was a significant negative association be tween revertants/mg extract
and LSD (p < 0.05, r = -0.312, n = 54), indicating that samples with high mutagenicity on a revertant/mg basis
tended to have low LSD values. A better correlation could be expected for pure chemicafs, since nonmutagenic
compounds of the complex mixtures may attenuate the response through toxicity to the test organism. —-
The association between several characteristics of the sludges and each of the three parameters of
mutagenicity was examined. The independent variables (percentage industrialization, wastewater treatment
scheme, and chemical additives) were tested individually against potency of response (revertants/mg), slope,
and LSD. No significant associa-tions were found. The lack of association between
chlorination of wastewater and mutagenicity of sludges is interesting, since chlorination of waste-water has
been shown to produce mutagens (92,385). It appears that the mutagens produced by chlorination affect the
effluent water more than the resulting sludge.

The lack of association between the percentage of industrialization and any parameter of mutagenicity also is
an unexpected finding. It has been generally assumed that sludges from domestic areas were "cleaner" than
sludges from treatment plants receiving a large percentage of wastewater from industrial sources. The results
of this study indicate that mutagenic activity of sludges is independent of the percentage of industrialization.
The potential health hazards of the mutagenic components of sewage sludge are difficult to assess. A recent
study (63) indicated that rats consuming cabbage and beets grown on sludge^amended soil produced urine
which was mutagenic to TA98 in the presence /  of a metabolic activating system. Thus, it seems possible for
the mutagenic components of sludge to be transported through the crops grown on it.
Acute toxicity of organic extracts of sludge

A further characterization of the toxicity of the organic fraction of sludge was made on the basis of lethality. The
extract of the Dallas sludge was the only sample negative in the test for mutagenicity, and was chosen as a
possible nontoxic sludge extract (15). Since the extract of the Boston (Deer Island) sludge was mutagenic with
metabolic activation to tester strains TA1537 and TA100 (106 and 86 revertants/mg extract, respectively), this
sample was selected as an example of a relatively toxic sludge extract.

Mice used in this study were male BALB/c, obtained from a colony maintained at the New York State College of
Veterinary Medicine. All animals were between three and four months of age and were fed Agway PHM 3,000
ad libitum. The light/dark cycle was maintained at eighteen/six hours, respectively, with a room temperature of
72°F and relative humidity of 55%. They were housed five per cage in polycarbonate cages with softwood
bedding chips.

All animals were fasted overnight prior, to dosing. Sludge extracts" were administered intraperitoneally at a
constant volume of approximately 9.5 ml DMSO/kg (approx. 0.2 ml/mouse). Concentrations of sludge extract
ranged from 5.2 mg/ml DMSO to 45.9 mg/ml DMSO. Control animals received DMSO only.' Observations were
made on all animals continuously for the first four hours post dosing, two times each day for the next three
days, and one time each day for the following ten days.

At the termination of the study, all surviving animals were killed by cervical dislocation and examined for gross
lesions. Additionally, all animals dying during the observation period were subjected to a routine gross
necropsy. Livers, kidneys, 'and spleens were weighed.

Since many congeners of PCBs are potent inducers of hepatic cytochrome P-450 (332) and since PCBs
content of sludges is currently used to set standards for land application, the biological response to P-450 was
measured in animals treated with sludge extracts. In this way a comparison could be made to find the more
sensitive indicator of the presence of problematic organic compounds, and response of P-450. Hepatic
cytochrome P-450 content was determined by the method of Schoene et al. (388) and expressed per g liver,
per mg 9,000 x g supernatant fraction protein, and amount per total liver. Protein was determined by the
method of Sutherland et al. (430). All determinations were made after the fourteen-day observation period
when surviving animals were killed.

The median lethal dose and its 95% confidence in terval were calculated according to the method 'of"' Weil
(477). Body weights, and the gross weights and relative organ weights were analyzed by one-way analysis of
variance; differences among means were determined using Tukey's HSD test (408). The probability of a Type I
error was set at the nominal 5% level.

Post-dosing effects

Both treated and control animals exhibited signs of discomfort immediately after dosing. These signs were
manifest as frequent examinations of the injection site and increased activity. However, fifteen to Jhirty minutes
after dosing, the Boston sludge-treated animals began to show signs of effects on the central nervous system.
Orientation was affected to some degree in all groups as animals rotated repeatedly ii) both clockwise or
counter-clockwise directions; one animal (dosed at 32.9 mg/kg) turned over continuously for a period of four to
six minutes. Additionally,  motor coordination  and  gait were  adversely affected by the Boston sludge extract in
nearly  all the animals. Animals dosed with Dallas sludge extract showed few of the signs of toxicity of the
Boston sludge extract groups and were more nearly like control animals.        •• ..-,

.. All signs of excessive activity had diminished in all animals within the first four hours. By twenty-four
hours post-dosing, decreased activity and decreased muscle tone were observed in all sludge groups,
while control animals appeared normal. Deaths were recorded at the highest dose for both sludge extracts.
Within six days, all moribund animals at all dose levels had either recovered or died. - ^Mean   time-to-death   
for   Boston   sludge   extract ranged from 4.5 to 1.8 days, decreasing with increasing dose. The same
decrease in mean time-to-death with increasing dose was observed for Dallas sludge extract, although the
range of 3 to 1 days was some what shorter (Table 22).

' Body weights were adversely affected in only one group with sufficient survivors to allow for a statistical
comparison. Animals receiving the Dallas sludge extract at 57.3 mg/kg lost 30% of their initial weight. Food
consumption data were lost for this group; however, examination of Table 22 shows that con- •' sumption of 2.3
g feed/day/mouse was sufficient to prevent a significant reduction in mean body weights during the observation
period, high-dose groups for both Boston and Dallas sludge extracts did not consume any food before dying.
For the Boston sludge extract, the median lethal dose was 23.4 mg/kg and the 95% confidence interval
was 17.8 to 30.9 mg/kg. These figures were calculated from responses at 14.6, 22.0, 32.9, and 49.3 mg
The median lethal dose for the Dallas sludge extract was 46.5 mg/kg, with a 95% confidence interval of ,35.1'
to, 61.6 mg/kg. Responses at doses of 25.3, • 38.1, 57.3, and 86.0 were used in calculations of the LD5n and
its confidence interval. Although the Boston sludge was more toxic, as demonstrated by an LD50 approximately
one-half the Dallas sludge, both of these sludge extracts would be rconsidered extremely toxic (256). This
rating is applied to compounds with LD50 values below 50 mg/kg.

Necropsy observations

No gross lesions were observed in any animals which could be associated with the test materials. Additionally,
no treatment effects were seen in gross or relative organ weights. Hepatic cytochrome P-450 levels were not
significantly, different from control values (data not shown). These results indicate that PCBs content of these
sludges was probably low. In fact, analysis of PCBs content of these sludges indicated relatively low levels of
0.84 and 1.00 ppm for Boston and Dallas sludges, respectively.

Over all, this study demonstrates that an organic extract of municipal sewage sludge can be extremely toxic.
This toxicity did not correlate with PCBs consent or mutagenicity; although the mutagenic sample: was more
toxic the quantitative difference was not qualitatively significant.

The relative hazard of the toxic component(s) of these sludges is difficult to assess from this study. However, it
is clear that setting.standards of sludge application and classification based on analytical measurement of one
or any number of compounds is inadequate for protection of public health. An effective sludge monitoring and
classification program must consider a combination of analytical and biological assays.
Risk assessment of land application of sludges

These studies (15,16.17,87,134,135,160,162) emphasize the importance of the largely unstudied organic
fraction of sludges.  Because analytical techniques for specific organics are expensive and the organic content
may be extremely variable, this fraction has generally been ignored in estimating the hazards associated with
commercial users of sludges (457). The continued commercial use of sludge or considerations of methods of
disposal should take into account all potential risks. This can be done only with the development of monitoring
programs which assess both the organic and inorganic constituents of, municipal sewage sludges.
Moreover, the absence of information concerning  the organic constituents of municipal sewage sludges must
not be equated with low risk or safety. Under conditions .of minimal data, maximal risk must be assumed in
order to protect public health.