PHYSICOCHEMICAL REQUIREMENTS IN THE E~IRONMENT OF THE EART~ORM DAVID L. RAPI;AN*, ROY HARTENSTEIN~“, EDWARD F. NEUHAUSER~and MICHAELR. MALECKI~ *
Environmental Protection Group, U.S. Army Natick Research and Development Command, Natick, MA 01760, U.S.A. t School of Biology, Chemistry and Ecology, State University of New York, College of Environmentai Science and Forestry, Syracuse, NY 13210, U.S.A.
Summary-Survival and/or growth were used to assess optimum and potentially deleterious physicochemical conditions in the environment of the earthworm Eiseniafoetida. Maximum weight was gained between 20 and 29°C with horse manure or activated sludge as food. Maximum weight gain as a function of moisture in activated sludge occurred between 70 and 85%. A11worms died within a week at pH values < 5 or > 9; optimum pN for gain in weight centered around 7.0 Soluble salts in excess of 0.5% were lethal, though ammonium acetate caused 100% mortality at a concentration of&i%; concentrations in manures contaminated by urine or cattle shnry may be lethal, while those present in non~ontaminat~ manure, with an electrolytic conductivity of 1.5~3mnthos, support weight gain. fnorganic chemicals that are commonly used to coagulate sludges, often as a prehminary to land application, were innocuous at concen~atjons higher than those normally used at wastewater treatment plants. Anaerobically digested sludges are toxic to earthworms, and are characterized by low oxidationreduction potentials; when placed upon a soil substrate the redox potential increases slowly, and though the sludge tested in this study was nontoxic at Eh values in excess of 250mV, it provided insufficient nutriment to E.foetida to allow weight gain. With activated sludge as food, growth of E. jiietida occurred more rapidly when soil was present, independently of whether it was placed as a substrate beneath the sludge or mixed into the sludge. Growth occurred more rapidly when activated sludge was placed on substrates which ailowed drainage, though loam or asked foam appeared superior to others, suck as glass beads or sand; the growth promoting factor is related to the inorganic fraction of the soil.
demands for land disposal of wastes rise (Elliott and Stevenson, 1977), it becomes increasingly important to determine the effects of specific waste components on the soil biota and the capacity of soil to mineralize the organic components. It is especially import~t to determine tolerances of earthworms to wastes, since these worms are generally the principal tillers of soils (Satchell, 1967) and are known to increase in number and biomass in proportion to litter availability (Waters, 1955). Certain compounds, such as copper-containing fungicides, are detrimental (Rhee, 1977). as are excessive application rates of cattle slurry (Curry, 1976). Elimination of earthworms can lead to deterioration of soil structure (Westeringh, IW2f whife treatment of soil to encourage earth~vorm activity can be used to reclaim land (Dunger, 1969; Rhee, 1969) or promote its fertility (Teotia e? al., 1950). Eisenia foetida, a surface-feed& earthworm, is potentially useful for management of organic wastes (Graff, 1974: Tsukamoto and Watanabe, 1977). Its
reactions to salts, acid, base, moisture. temperature, light and distribution in soil in relation to pH are documented (Gates, I978), but in all cases the work was done without regard to the effect af the test condition on changes in weight of E. foetida. We report the growth response of E. foetida to pH, moisture,
temperature, oxidation-reduction potential, and the effects of certain salts, including inorganic salts commonly used to coagulate sludge at wastewater treatment plants before disposal on land (USEPA, 1973). METHODS AND
E~~~t~d~ was obtained from a culture kept in rimed peat moss on silt loam, pH 6.5, with horse manure and activated sludge as food. Buss Bed-ding a culture medium for earthworms, consisting of minerals and newsprint (44% ash), was purchased from Buss Manufacturing Co., Lanark, IL. Anaerobic sludge with about lo”/, solids and activated sludge with about I--2% solids were obtained respectively from the centrifuge of the Metropolitan, and aerobic digester of the Meadowbroo~-Limestone, wastewater treatment plants in Unondaga County, NY; activated sludge was also obtained from drying beds at the latter plant. Horse manure was obtained fresh as needed, uncontaminated by urine. Cellulose was purchased from Sigma Chemical Co, St. Louis, MQ. The effect of temperature in horse manure OR weight change of E. foetida after 2 weeks was studied in Petri dishes (20 x 100mm) with 1 worm per dish The effect of temperature and moisture in activated sludge on gain in weight after 10 days was studied in Petri dishes containing 10 worms each. Ta test weight changes of E. ,jbetida in relation to
KAPL.A~ t’f ul
pH, activated sludge was first adjusted to integral units of pH from 2 to 9 with 12 N HCI or 10 N NaOH, centrifuged at 5000 rev min- ’ for 10 min to obtain a sediment with 11% solids, and placed into Petri dishes with 1 worm per dish for 2 weeks at 25°C. The dishes were inspected daily to determine survival. pH was measured when worms had died, or after 2 weeks, when survivors were weighed. The initial live weight of the 55 worms was !? = 156 mg & 3 SE. Weight change of E. foetida in response to salts was tested in two different experiments. In one, at IYC, aqueous solutions were added to a substrate comprised of 15% silt loam and 857; Buss Bed-ding. Dishes 4.5 x 10 cm dia were filled with this medium, which held 1.5 times its own weight of moisture, and into which 20 worms were placed for 2 weeks. The second setup, at 25”C, was designed to test the effects of sludge conditioners, CaCO,, Al&XI&, FeSO, and FeCl,. Aqueous solutions were added to activated sludge (2% solids) to achieve concentrations up to 10% (w/w). After thorough mixing and adjustment of pH to 7.Q, the sludge was centrifuged at 5000 rev min- ’ for 10min and placed into Petri dishes containing 20 g air-dried soil and 8 ml water. Each dish contained 5 earthworms which were weighed initially and after 14 days. Electrolytic conductivity was measured with a Horizon Model 1484 meter which was checked against KC1 standards (Daniels er al., 1956). To measure electrolytic conductivity in manure the electrode was placed into the manure and distilled water was added gradually: conductivity was taken as the maximum reading. The influence of soil or drainage was tested in two separate experiments. Activated sludge (40 g) from drying beds was placed upon soil or peat alone, or mixed with soil in Petri dishes containing 5 worms per dish at 25°C. The worms were weighed periodically up to 45 days. In the second experiment, fresh activated sludge with 11% solids was placed on a 1 cm
Temperature ( a C ) Fig. 1. Survival, and gain or loss in weight by E. foetida, in relation to temperature, with horse manure as food. N = 3.
thick layer of silt loam, ashed silt loam, sand, glass beads (91 mm dia), methyl cellulose, or diatomaceous earth in dishes 4.5 x 1Ocm dia. A fresh supply of sludge was added weekly for 12 weeks. Each dish was provided with 5 E. foetida which had emerged from cocoons during the preceding 2 weeks. Toxicity of anaerobic sludge as a function of oxidation-reduction potential was tested by placing worms on the surface of a layer of sludge 1 cm deep and checking worm survival after 24 h. Oxidationreduction potentials were measured with a Corning Model 130 pH meter and were corrected for the calome1 electrode (Allen, 1974). Eh was recorded 10min after inserting the electrode into the sludge.
Fig. 2. Weight gained by Efoetida, with activated sludge as food, in relation to temperature and percent moisture. N = 3. SEs are given at only one temperature to illustrated degree of variance in general.
Environmental requirements of E. foeridu
horse manure B. &&da gained weight maximally and survived best over the temperature range [email protected]
°C (Fig. I), Only about 25% of the weight gained in this range was achieved by earthworms at 5”C, a temperature which also killed 307; of the popu# lation. At 33”C, 70% of the worms died, and the remainder lost weight. In activated sludge E. f&%i& which averaged about 75 mg initially increased their weight about two-foid in the temperature range 20-28°C and intermediate moisture levels during 10 days (Fig. 2). Maximum weight gain as a function of moisture occurred in the range of 7(r85%, correspanding to I.?-30% solids (Fig. 3). In sludge with 24% solids, weight gain in the temperature range of 2%-28°C was significantly higher than at 15 or 20°C. No worms died in sludge with low moisture, while many died in some of the setups with high moisture. At 90”/“,moisture, mortality at 5, t t, 15, 20, 25, and 28°C was O$0, 0, 33, 40 and 73% respectively. At 87% moisture mortality occurred only at 28”C, where 67% of the worms died. in
Considerable difficulty was experienced in establishing and majntaining a pH wtue to either side of the original value of 6.4 in activated studge with f.7% solids. Despite additions of base or acid periodi~aily over 2 days, a drift towards the original pH occurred. Notwithstanding, the sludge was concentrated to 1If/, solids by centrifugation, and worms were immediately added. All worms died within a week at initial pH values <5 or > 9, though the pH values which were measured within a day of the observed mortality dif-
Weight PH (initial)
2 3 4 5 h 7 8 9 10
6 7 7 14 I4 14 I4 14 7
7.0 8.0 8.0 5.4 5.9 6.4 6.6 7.0 8.7
“_change (X% it =I$ All dead All dead All dead -c 146 + 22 -+I87 * 19 i-234 i: f2 +202 _+20 +220 f 8 All dead
* Denotes complete mortality if fewer than 14 days are shown, though the 6 or 7 days indicated are days when mortality was first observed, whereas death may have occurred much earlier; 1007; survival if 14 days are shown. tt’alue obtained after 2 weeks or within a day after mortality. 1 P < 0.05.
fered from the initial value (Table 1). A significant
gain in weight occurred among worms exposed to initial values of pH 6-9 relative to worms at pH 5, though no finat value exceeded pH 7.0. Weight changes in E. j&&da in the presence of increasing concentrations of salts added to a substrate of Buss Bed-ding and soil are shown in Table 2. With the exception of NH,-acetate and the relatively insoluble salts CaCO, and CaSO,, E. foetida lost weight or died on exposure to a salt concentration of 0.5%. Greater susceptibility was shown toward
Fig. 3. Weight gained by E..&&&, with activated sludge as fond, in relation to moisture and temperature. Values derived from Fig. 2. SEs are given for onty one solids concentration to ikstrate degree of variance in general.
DAVIDL. KAPLAXer ul.
Table 2. Percent change in weight of E. fberida after 2 weeks at 15°C in relation to con~ntration of salt added to a substrate of 85% Buss Bed-ding, 15%soil. N = 3
Wght change (X + SEX,)
+8.8 i 5.10 +9.4 _+ 3.85 - 16.4 i_ 1.42 all dead all dead +9.3 & 5.41 +9.2 5 4.04 all dead all dead f2.8 + 2.60 + 20.9 i 4.7 I - 10.1 * 8.80 all dead + 14.0 j 1.34 +7.0 + 1.63 -19.7 f 1.36 ail dead +6.3 + 0.90 + 18.5 + 0.71 -21.8 all dead + 14.9 f 2.13 all dead ali dead all dead +5.7 * 1.46 +2.8 2 1.03 f5.9 * 2.30 +3.2 * 0.97 +lS.l + 0.86 +2.5 + 5.26 + 13.86 5 4.30 +11.6 f 3.54
1000 5000 1o.ooo
0 loo0 5000
IO,000 0 1000
5000 K acetate
10.000 0 1000 5000 10.000 0 1000
5ooo NH, acetate
1000 5000 CaCOj
10,ooo 0 10,000 25,000 SO,000 0
10,000 25,000 50,000
1.5.-3 mmhos, allowed E. ~~etida to develop from hat~hling to adult in about h w-eeks (Hartenstein et al., 1979). These fmdings are related to toxic effects observed by worm-growers who have inadvertently fed manure contaminated by urine to E.foerida. Such manures may have electrolytic conductivity values in 15 mmhos, in contrast to excess of about 1.5-3 mmhos found in uncontaminated manure. Our findings may also relate to the toxicity of high concentrations of cattle slurry found by Curry (19761 to be responsible for decinlatlng earthworms in field pasture. Response to sludge coagulants No deleterious effect on gain in weight was evident at concentrations of sludge coagulant up to 0.1x, which is above levels normally encountered at wastewater treatment plants (Fig. 4). At higher concentrations limestone had no de~imental effect. but less weight was gained with FeSO,+, FeC13, and Al,(SO& at increasing concentrations above O.tJs;).A concentration of loo/ alum or FeCI, caused 100% mortality. It appears that land application of sludges which were treated with inorganic coagulants described here will not be deleterious to earthworms. at least not to E. fbetidu, assuming the sludge is otherwise amenable to ingestion. To what extent this will hold true with repeated applications, however, remains to be determined. Redox potehnl
N&acetate, where death occurred at a concentration of 0.1%. Electrolytic conductivity in fresh activated sludge (l-2% solids) and in activated sludges obtained from drying beds ranged from about 0.9 to 1.5 mmhos. E. foetida gained weight when it consumed these sludges. Horse manure without urine, with a value of about
of anaerobic sludge
Placement of E. fbetida into sludge freshly removed from an anaerobic digester (Mitchell et al., 1977) or in freshly-passed human excreta results in 100% mortality within a few hours. When anaerobic sludge ages in the presence of air, Eh increases at rates inversely related to sludge depth (Fig. 5). Bioassay of samples on days when redox was measured showed that the worms could survive on anaerobic sludge only if the sludge had an Eh greater than 25OmV. Despite survival for several weeks, however, the worms lost weight. Possibly the sludge failed as a food because it had become humified (Terry rt al., 1979). Substrate deficient in microbial biomass may be ingested to a greater extent than activated sludge but will nonethe-
E ti I +600
Fig. 4. Effect of sludge coaguiants on growth of E.ji.i.efidn. N = 4.
Environmental requirements of E. foetida
i days )
Fig. 5. Oxidation reduction potential of anaerobic sludge with time in relation to depth of sludge cake placed upon soil. Experiment was started on July 25; (A) 9 cm, (m) 6 cm, (0) 3 cm.
, sludge on soil
sludge on neat
OlII 0 P
Fig. 6. EtTect of soil vs peat moss on growth of E. foetida SEs. less lead results).
Influence of’soil or drainage Significant increases in growth occurred when soil was present, regardless of whether it was mixed into the sludge or placed as a substrate beneath the sludge (Fig. 6). After 45 days earthworms reared with soil present weighed about 1.5 times more than worms without soil. More weight was gained when sludge was placed upon a substrate, except where methyl cellulose was used, which itself became soggy and did not allow free circulation of air and drainage of the sludge (Fig. 7). Worms grown with soil and sand as substrates were significantly heavier than worms grown in sludge alone, though no significance was found between soil and sand as substrates. Although weight gains in experiments which allowed drainage were not significantly different from
with aged activated sludge as food. Bars are
those in experiments which did not, sexual maturity was achieved sooner with certain substrates than with others (Table 3). With loam or ashed loam as substrate E. foetida became sexually mature somewhat earlier than when placed in sludge alone or in sludge underlain by one of the other substrates. Table 3. Age, in weeks, at which 50% of populations of E. foetidu became clitellate (C,,) in relation to substrate present beneath activated sludge as food Substrate None Sand Glass beads Methyl cellulose Diatomaceous earth Loam Ashed loam
C5,, (weeks) 1.2 6.1 6.1 6.5 6.3 5.6 5.1
DAVID L. KAPLAN et al.
k & 600 5 400
Fig. 7. Influence of substrates placed below activated sludge as food, on growth and development of E. foetida. N = 3. Bars are SEs. The curve for glass beads coincided with the curve for sand and was omitted for clarity.
It is apparent that the inclusion of materials allowing excess water to move out of the activated sludge is beneficial to earthworm weight gain. Interestingly, both soil and ashed soil stimulated an earlier maturation (formation of clitellum) of the earthworms. The soil factors which promoted this pattern of development are as yet unidentified, but since an ashed loam was about as effective as loam itself, the beneficial factor has an inorganic composition. Possibly the reduction in earthworm populations observed by Curry (1976) was due, in part, to poor drainage of the excessive applications of cattle slurry. Acknowledgement-This work was supported by a grant from the National Science Foundation’s program Research Applied to National Needs (RANN). REFERENCES ALLEN S. E. (Ed.) (1974) Chemical Analysis of Ecological Material. Wiley, New York. CURRY J. P. (1976) Some effects of animal manures on earthworms in grassland. Pedobiologia 16, 425-438. DANIELS F., MATHEWS J. H., WILLIAMS J. W., BENDER P. and ALBERTY R. A. (1956) Experimental Physical Chemistry. 5th edn, McGraw-Hill, New York. DUNGER VON W. (1969) Fragen der naturlichen und experimentellen Besiedlung Kulturfeindlicher Boden durch Lumbriciden. Pedobiologia 9, 14&151. ELLIOTT L. F. and STEVENSON F. J. (Eds) (1977) Soils for Management of Organic Wastes and Waste Waters. American Society of Agronomy, Madison, WI. GATES C. E. (1978) Contributions to a revision of the earthworm family lumbricidae. XXII. The genus Eisenia in North America. Megadrilogica 3, 131-147.
GRAFF 0. (1974) Gewinnung von Biomasse aus Abfallstoffen durch Kultur des Kompostregenwurms Eisenia foetida (Savigny 1826). Landbauforsch Volkenrode 24, 137-142. HARTENSTEIN R., NEUHAUSER E. F. and KAPLAN D. (1979) Reproductive potential of the earthworm Eiseniafoetida. Oecologia 43, 329-340. MITCHELL M. J., MULLIGAN R. M., HARTENSTEIN R. and NEUHAUSER E. F. (1977) Conversion of sludges into “topsoils” by earthworms. Compost Science 18, 28-32. RHEE J. A. VAN (1969) Development of earthworm populations in polder soils. Pedobiologia 9, 133-140. RHEE J. A. VAN (1977) Effects of soil pollution on earthworms. Pedobiologia 17, 201-208. SATCHELL J. E. (1967) Lumbricidae. In Soil Biology (A. Burges and F. Raw, Eds), pp. 259-322. Academic Press. London. TEOTIA S. P., DULEY F. L. and MCCALLA T. M. (1950) Effect of stubble mulching on number and activity of earthworms. Nebraska Agricultural Station Research Bulletin 165, 3-20. TERRY R. E., NELSON D. W. and SOMMERS L. E. (1979) Carbon cycling during sewage sludge decomposition in soils. Soil Science Society of America Proceedings 43, 494-499. TSUKAMOTO J. and WATANABE H. (1977) Influence of temperature on hatching and growth of Eisenia foetida (Olinochaeta. Lumbricidae). Pedobioloaia 17. 338-342. UsEPA (1973) Physical-Chemical Wastewater Treatment Plant Design. Technology Transfer EPA 625/4-73-002a. United States Government Printing Office. WESTERINGH W. VANUE (1972) Deterioration of soil structure in worm free orchard soils. Pedobiologia 12, 6-l 5. WATERS R. A. S. (1955) Numbers and weights of earthworms under a highly productive pasture. New Zealand Journal of Science and Technology 36, 516-525.