Composition of the earthworm Eisenia foetida (savigny) and assimilation of 15 elements from sludge during growth

Comp. Biochem. Physiol., Vol. 66C, pp. 187 to 192

0306-4492/80/0701-0187502.00/0

© Pergamon Press Ltd 1980. Printed in Great Britain

COMPOSITION OF THE EARTHWORM EISENIA FOETIDA (SAVIGNY) A N D ASSIMILATION OF 15 ELEMENTS FROM SLUDGE D U R I N G GROWTH* RoY HARTENSTEIN, ALBERT L. LEAFt,~, EDWARD F. NEUHAUSER a n d DONALD H. BICKELHAUPT State University of New York College of Environmental Science and Forestry, Syracuse, NY 13210, U.S.A.

(Received 17 October 1979) Abstract--1. Concentrations of ash and 11 minerals were analyzed in non-voided Eisenia foetida in relation to body weight. Significant decrementai regressions were found with increasing body weight for ash, Ca, Mg, P, Fe, A1, Mn, and Zn. Significant incremental regressions were found for K and Cu. Nitrogen concentrations were constant and independent of body weight. 2. Similar analyses on earthworms whose gut was voided revealed a significantly higher content of N, P, Ca and Mg than in the non-voided specimens and significantly lower levels of the other minerals. Water concentration was constant at 18%, independent of body weight. 3. During growth for 4 weeks on activated sludge, biomass increased about twofold; N, P and K remained approximately constant at 10, 1 and 1%; and Ca, Mg and Na decreased from levels of 0.8, 0.3 and 0.44% to 0.7, 0.15 and 0.38% respectively. 4. With respect to potentially phytotoxic elements, no significant changes in concentration of Cd, Cr, or Ni were found between samples of E. foetida from stock culture and samples taken two weeks and four weeks after placement into fresh activated sludge. Significant amounts of Cu and Zn accumulated after two and four weeks, and a significant accumulation of Pb was found after four weeks, but not two weeks, in sludge.

INTRODUCTION

Accelerating the mineralization of organic wastes in fields t h r o u g h use of e a r t h w o r m s has been suggested by several workers (e.g. Teotia et al., 1950; Barley, 1961; Atlavinyt6 et al., 1968). Use of the e a r t h w o r m Eisenia foetida (Savigny) in the m a n a g e m e n t of biodegradable organic wastes has also been suggested (Graft, 1974; T s u k a m o t o & W a t a n a b e , 1977; Sabine, 1978; Hartenstein et al., 1979). Little is known, however, a b o u t the elemental changes that occur in this e a r t h w o r m as it ages a n d / o r passes organic material t h r o u g h its gut. The objective of this study was to obtain some data a n d information in this regard.

MATERIALS AND METHODS

Materials E.foetida was obtained from stock cultures in peat moss sprinkled with dolomite and placed on soil with horse manure and activated sludge as food. Horse manure was obtained fresh, uncontaminated by urine, as needed. Activated sludge, with about 1% solids, was obtained from the Meadowbrook-Limestone Wastewater Treatment Plant, Onondaga County, NY. The sludge was concentrated to 11% solids by centrifugation for 10min at 5000rpm. All experiments on living E. foetida were performed at 25°C. Composition of non-voided E. foetida Ash content and the concentration of 11 elements (N, P, K, Na, Ca, Mg, Mn, Fe, Zn, AI and Cu) were determined on non-voided specimens of E. foetida in the live wt range * This research was supported by the National Science Foundation. c.B.P.66/2c---E

187

0.254-1.806 g, corresponding to approx ages 4-14 weeks. Single worms were analyzed to obtain the 5 last points in the range, couples to obtain the 5 penultimate points, 3 specimens to obtain the next series of 5, and 4 or 5 Worms to obtain the first 5 points. All of the earthworms had been grown in culture dishes which contained a substrate of silt loam. The specimens used for obtaining the first 10 points were allowed to feed on either horse manure or sludge, both of which were placed upon the soil while the remaining specimens were provided with sludge only. Earthworm samples of approx 0.1-0.2g were analyzed for N by the macro-Kjeldahl procedure. The remaining elements were determined on approx 0.5 g samples, dryashed at 480°C for 20 hr and ash concentrations were determined gravimetrically. The ash residue was taken up in weak HCI solution (Wilde et al., 1972). Phosphorus was determined by the molybdovanadate procedure with a spectrophotometer. Potassium, Ca, Mg, Na, Fe, Mn, AI, Zn, Cu, Ni, Pb, Cd and Cr were determined with an atomic-absorption spectrophotometer with appropriate hollow-cathode lamps.

Composition of voided E. foetida Sixty E.foetida, ranging from 10 to 2000 mg live wt, were dried at 105°C to obtain dry wt. 30 g of E.foetida, ranging from c. 100 to 400 mg live wt (18% solids), were taken from stock culture and placed into 900 g activated sludge (11% solids), knowing from preliminary studies that the sludge would appear to be fully converted into castings within 4 weeks. A control sample was withheld from the setups, which consisted of placement of E. foetida directly into sludge in 18 x 23 cm trays. The floor of the trays, made of bolting cloth, was suspended on corner posts over an air space beneath to maximize aeration. About half of the earthworms were removed from the sludge after 2 weeks and the remainder after 4 weeks. The earthworms were rinsed in distilled water, placed overnight into empty 100 x 20 mm petri dishes for voidance, killed by freezing at - 10°C, dried at 105°C, and analyzed as above.

RoY HARTENSTEINet al.

188 RESULTS AND DISCUSSION

Moisture content Independently of size or live weight, the concentration of water in E.foetida was 81.8~ ± 7.7 SD. All values of concentration in the following text are based on dry wt. Composition of non-voided E. foctida Ash concentration in relation to size of E. foetida over the range of about 50-400 mg (dry wt) is given in Fig. 1. A regression toward a significant decrease in ash with increase in age is seen, notwithstanding removal of the worms from two sources of food, the younger worms having had access to horse manure and sludge, the older worms sludge only. Linear regressions and statistics for ash and the 11 elements analyzed are given in Table 1. The decrease in ash may be attributed mainly to the significant decreases that occurred for Ca, M~, P, Fe and Al (Figs 1 and 2), though significant decreases occurred for Mn and Zn also (Fig. 2). In contrast, significant incremental regressions were found for K among the macroelements and Cu among the microelements (Figs 1 and 2). Nitrogen alone, among the 11 specific elements tested, neither increased nor decreased significantly with age (Fig. 1). It comprised a X% _+ SE of 9.2 ± 0.15 and exhibited a smaller coefficient of variation than was obtained for any other element (Table 2). As with the data on ash, no difference in trend was seen for any of the elements tested in the younger worms, which had been reared on sludge and manure, versus the older ones, which had been reared on sludge only.

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Biomass of populations of E. foetida, consisting of individuals ranging in age from about 3 to 6 weeks increased about two-fold during the 4-week feeding period on sludge, whose composition is given in Table 3. During the first 2 weeks most of the sludge appeared to be converted into castings. Data on changes in concentrations of ash, N, P, K, Ca, Mg and certain other elements in E. foetida upon feeding on sludge are given in Table 4. Ash concentration decreased significantly both after the second and fourth weeks, signifying a corresponding increase in organic matter, as was noted above for non-voided worms in relation to increase in weight or age (Fig. 1). Of the elements shown in Table 4, only Mn decreased significantly after both 2 and 4 weeks, and only A1 increased significantly on both of these sampling dates. Sodium was unique in not exhibiting significanee both 2 weeks and 4 weeks after initiating the experiment. All the remaining substances exhibited nonsignificant differences, a significant increase, or a significant decrease on either the second or fourth week of sampling. Despite the significant changes for N, P and K, the concentrations of these elements appeared to fluctuate with time within a narrow range. Phosphorus did not decrease and K did not increase (Table 4), which is in contrast to what appeared in analyses of the non-voided earthworms (Fig. 1). A constant concentration of about 1~o of each of these elements was found instead. Similarly, a constant amount of N was found, approximately 10~.

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189

Elements in Eisenia Table I. Ash and elemental composition of E. foetida in relation to dry weight, X, in rag, over the range c. 50-400 mg (dry wt) t*

Regression equation Ash, %

=

-0.0266X + 15.98

N, % = -0.001X + 9.44 P, % = -0.0039X + 0.56

r

(a)

-0.80

5.65 (0.0o05) 0.89 3.76 (0.002) 3.18 (0.005) 9.72 (0.ooo5) 6.9 (0.0005)

-0.207 -0.631

K, % -- 0.00578X + 0.71

0.600

Ca, % = -o.oo17x + 0.97

-0.917

Mg, %

-0.88

=

,0.0067X + 0.34

Na, ppm = - 3 . 5 4 X + 8550

-0.330

SD

SE

CV?

3.40

0.76

29.3

9.20 0.318

0.71 0.043

0.15 0.009

7.7 13.5

1.66

0.98

0.22

59.0

0.69

o.19

0.043

27.5

0.277

0.078

0.017

28.1

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3.44

7900

10,090

2400

127.7

168

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(0.005)

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-0.688

4.02

1415

751.9

168

48.8

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-0.618

3.33

10.9

28.8

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-0.717

Cu, ppm = O.081X + 49

4.34 (0.0OO5) 1.99 (0.05) 5.06 (0.0OO5)

0.426

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-0.767

1153

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158

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62.4

19.7

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190

Roy HARTENSTEINet al. Table 2. Comparison of ash and elemental composition of non-voided and voided E. foetida Macroelements Treatment

Ash

Non-voided

11.59 (0.76)

Voided

7.67 (0.18)

P

X % (+SE) K

Ca

Mg

Na

9.20 (0.15)

0.32 (0.009)

1.66 (0.22)

0.69 (0.043)

0.23 (0.017)

0.79 (0.24)

10.05 (0.18)

1.027 (0.02)

1.14 (0.04)

0.81 (0.02)

0.29 (0.01)

0.45 (0.03)

N

Microelements X ppm ( + SE)

Non-voided Voided

Fe

Zn

Cu

AI

Mn

1415 (168) 684 (37)

168 (10.9) 116 (1.9)

624 (4.4) 8.0 (2.5)

1154 (158) 437 (27)

71 (8) 36 (0.7)

Based on these data, E. foetida may be viewed by individuals interested in waste conversion systems, or soil scientists, as a potential organic fertilizer agent with an N P K content of about 10:1:1. Calcium and Fe were significantly higher and Mg significantly lower at the end of four weeks. Whereas Ca increased very slightly overall, from 0.81 to 0.90°/~ the change in Mg was prominent, from 0.29 to 0.15%. Relative to non-voided specimens, the values obtained for Ca in the voided worms fell within the higher region of the Ca range (Fig. I), corresponding to lower weights of the non-voided worms. In contrast, the trend for Mg in the voided worms paralleled the trend for the nonvoided and spanned the entire Mg concentration range (Fig. 1). The levels of iron present in the voided worms approximated those of the larger non-voided worms (Fig. 1). In summary, the data on elemental concentrations of voided E.foetida indicate a constant level of approximately 10% N, 1% P, 1% K, 0.8% Ca, 0.3% Mg, 0.4% Na, 0.00% AI, 0.07% Fe, and 36 ppm Mn. These values may be compared with analytical data on freeze dried earthworms, from Abe et al. (1979): 64.9% crude protein, 0.66%P, 0.73%K, 0.28%Ca, 0.14%Mg, 0.72%Na, 0.02%A1, 0.04%Fe, and 32 ppm Mn.

Changes in composition of heavy metals in E. foetida during growth on slud#e Cadmium, Cr, Ni, Zn, Pb and Cu are heavy metals that are potentially phytotoxic (CAST, 1976). No significant changes were observed between the levels of Cd, Cr or Ni present in E. foetida in the stock culture and samples of this culture taken 2 weeks and 4 weeks after placement into fresh activated sludge. The average Cd level was 5.7 ppm (Table 5), which was close to the 8 ppm in the sludge (Table 3). Cadmium may be concentrated in E. foetida in excess of ambient levels (Gish & Christensen, 1973; Van Hook, 1974; Helmke et al., 1979; Hartenstein et al., 1980). The average Cr concentration in the worms, 52 ppm, was approximately half the value of the sludge Cr concentration, 108ppm. Chromium may accumulate in earthworm tissue, as was shown by an increase in tissue level in proportion to field level (Helmke et al., 1979). Mean Ni concentration in the worms, 34ppm, was one-third that of sludge, 102 ppm, and though it did not accumulate in time, was reported as being 1.9 fold concentrated over a field level in a study by Gish & Christensen (1973) on a mixed species population of earthworms.

Table 3. Composition, at time zero, of activated sludge into which E. foetida (Table 4) was placed for 4 weeks (N = 5) Components X%+SE Ash Calcium Magnesium Nitrogen Phosphorus Potassium

29.3 ± 4.39 ± 0.92 ± 5.8 ± 3.3 ± 0.48 ±

Xppm±SE 0.06 0.14 0.01 0.02 0.03 0.001

Aluminum Cadmium Chromium Copper Iron Lead Manganese Nickel Sodium Zinc

7830 ± 121 8 ± 0.13 108 ± 2 1035 ± 2 4 13,278 ± 31 180 ± 3.2 178 ± 3 85 ± 1.9 1194 ± 1 1417 ± 9.3

191

Elements in Eisenia Table 4. Concentrations of ash, N, P, K, and certain other elements in E. foetida in relation to time spent in fresh activated sludge (N = 5)* Time (weeks) 0

Ash

N

7.67 (0.18) 6.44 (0.08) 5.25 (0.19) - 1.23 -1.19 -2.42

2 4 2-0 4-2 4-0

X % (+ SE) K

P

10.05 (0.18) 9.41 (0.09) 9.89 (0.17) -0.64 +0.48 -0.16

1.03 (0.02) 1.08 (0.01) 1.04 (0.02) 0.05 -0.04 -0.01

1.14 (0.04) 0.89 (0.03) 1.05 (0.04) -0.25 +0.16 -0.09

Ca

Mg

Na

0.81 0.29 0.44 (0.02) (0.01) (0.03) 0.74 0.17 0.37 (0.01) (0.001) (0.03) 0.90 0.15 0.38 (0.04) (0.001) (0.34) -0.07 -0.12 -0.07 +0.16 -0.02 +0.01 +0.09 -0.10 -0.06

Mn

X ppm (+ SE) A1 Fe

36.1 (0.7) 24.2 (0.6) 18.3 (1.5) - 11.9 -5.9 -17.8

437 (27.5) 940 (55.0) 1403 (81.4) + 503 +463 +9.6_6

684 (37) 1069 (83) 989 (43) + 385 -80 +3_0.2

* P < 0.05 is in.dicated by underscore.

The remaining heavy m e t a l s ~ C u , Zn and P b - exhibited significant changes after either 2 weeks or between the second and fourth week, while for all 3 of these metals a significant increase in concentration occurred between time zero and four weeks (Table 5). The trend toward accumulation of Cu agrees with Rhee (1977) and Helmke et al. (1979) who found increasing levels in tissue corresponding to increased levels in soil. The highest levels reported by Helmke et al. (1979) 21.7 ppm, and Rhee (1977) 63 ppm, were lower than the mean level obtained here, 102 ppm, for worms which spent 2 or 4 weeks in sludge with an initial level of 1035ppm. A trend toward accumulation of Zn also was shown (Table 5), since the mean of 176ppm for the second and four week exposure periods was significantly greater than the initial level in the worms, l l 6 p p m . All other investigators also have shown that Zn is accumulable (Gish & Christensen, 1973; Van Hook, 1974; Ireland, 1975; Helmke et al., 1979; Hartenstein et al., 1980). Similarly, a trend can be seen toward a significant accumulation of Pb (Table 5), 99 ppm being present after 4 weeks in E. foetida which fed upon sludge with 180ppm. Gish & Christensen (1973) reported levels as high as 331 pprn, and Ireland (1975) as high as 4160ppm in Dendrobaena rubida from a metal mining area. CONCLUSIONS During growth E. foetida maintains its concentrations of water, N, P and K at steady-state values of 18% (live wt), 10, 1 and 1% (dry wt). In contrast, it experiences losses in Ca, Mg and Na. The losses in Ca Table 5. Concentrations of heavy metals in E. foetida in relation to time spent in fresh activated sludge (N = 5). Time (weeks) 0 2 4

2-0 4-2 4-0

Cu

Ni

8 (2.5) 113 (3.2) 92 (4.5) +105 -21 +84

32 (6.1) 30 (4.6) 40 (2.4) --2 +10 -8

.~ ppm ( + SE)* Zn Cd 116 (1.9) 188 (5.2) 165 (3.9) +72 -23 +49

6 (0.33) 4.4 (0.21) 6.6 (0.85) --1.6 +2.0 +0.4

* P < 0.05 is indicated by underscore.

Pb

Cr

62 (6.2) 56 (9.9) 99 (3.7) --6 +43 +37

38 (8) 63 (12) 63 (6) +25 0 +25

and Na during a doubling of biomass appear to be relatively trivial, 0.8 and 0.44%-0.7 and 0.383/0 respectively (dry wt basis), while the reduction in Mg seems to be quite appreciable, from 0.3 to 0.15% (dry wt). All of the potentially phytotoxic elements analyzed in this study have been shown to be accumulable in earthworm tissues in previous siudies, though only Cu, Zn and Pb accumulated under the conditions of this study; Cd, Cr and Ni did not accumulate during the short period used here, though the earthworms increased their biomass more than twofold. Dedicated to the memory of our colleague, Professor Albert L. Leaf, 16 May 1928 to 16 September 1979. REFERENCES

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