The Earthworm Inoculation Unit technique: An integrated system for cultivation and soil-inoculation of earthworms

PergamoIl PII:

Soil Bid. Biochem. Vol. 29. No. 3/4. pp. 251-257. 1997 Q 1997 Else&r Science Ltd. All rights reserved Printed in Great Britain soo3B-B717(%)ooo5~3 0038-0717/97 517.00 + 0.00

THE EARTHWORM INOCULATION UNIT TECHNIQUE: AN INTEGRATED SYSTEM FOR CULTIVATION AND SOIL-INOCULATION OF EARTHWORMS KEVIN

R. BUTT,* JAMES FREDERICKSON

and RICHARD

M. MORRIS

Biosystems Research Group, Faculty of Technology, The Open University, Milton Keynes MK7 6AA, U.K. (Accepted 3 February 1996)

Summary-The introduction of earthworms into degraded or newly restored land is known to promote soil improvement. Obtaining the most appropriate species in the large numbers required can be costly and time consuming using traditional techniques. Research and development of a novel approach, the Earthworm Inoculation Unit (EIU) technique, may help to overcome this. This technique combines cultivation of selected earthworms in small soil-based units, with an effective method of direct soil introduction. Successful cultivation of deep burrowing species, e.g. Lumbricus terrestris L. and Aporrectodea longa (Ude), and shallow working species, e.g. Allolobophora chlorotica (Savigny), has been achieved by optimizing environmental factors. Accelerated rates of reproduction compared with field data have been recorded. At soil-inoculation, each EIU was found to contain all three earthworm life stages, adults, cocoons and hatchlings, promoting maximum opportunity for successful colonisation. Results from field trials suggest, that for A. longa, the EIU technique can enhance survivorship in compacted clay soils compared with a more conventional inoculation method. Earthworm inoculation, where appropriate, should become an integral component of sustainable land restoration practice. In hostile soils, often associated with reclaimed land, the EIU technique may provide a means of ensuring- long_ term survival for earthworm populations. 0 1997 Elsevier Science

INTRODUCTION The presence

of earthworm

species in soils has been

demonstrated to have a positive effect on numerous soil properties (e.g. Stewart and Scullion, 1988). Action to increase earthworm numbers, in soils where they are either absent or present at low density, may be one way of assisting the rehabilitation of degraded or derelict lands (e.g. Brun et al., 1987). This increase may take several forms including stimulation through selected soil practises or the deliberate introduction of specific earthworms. Examples of the success of introduced earthworm populations have been reported for a variety of soil types (Curry, 1988). Selection of the most appropriate species for earthworm introduction trials is required and these earthworms should be in a healthy physical and physiological condition in order to promote long term population development and survival. It is possible to introduce earthworms after collection from locations where they are present in large numbers, for example by gathering behind a plough, but *Present address: Department Management, University of Preston PRl 2HE, U.K.

of Environmental Central Lancashire,

this procedure may be associated with high financial costs and such earthworms may not give rise to sus-

tainable populations (e.g. Scullion, 1992). Current techniques for introducing earthworms into soils are usually limited to surface inoculation (broadcasting) or turf addition. Both methods are attractive for particular reasons, but each appears more suited to the provision of particular types of earthworm and neither is ideal (Table 1). This paper evaluates the development of the Earthworm Inoculation Unit (EIU) technique, a method which attempts to combine the benefits of earthworm culture with positive aspects of both broadcast and turf-cutting earthworm inoculation. The EIU technique has two phases, cultivation and soil-inoculation. Cultivation involves production of individual units containing soil, feed and earthworms. Within these units a small starter culture of mature individuals, maintained under optimal conditions for a pre-determined period, reproduce at an accelerated rate compared to field earthworms. Soil-inoculation of the intact units into the field completes the process. A number of modifications to the technique which were made on the basis of experience in successive field trials are described. 251

252

Kevin R. Butt PI ul. Table

I. Merits

Technique Turf Cutting 1982)

of exlstrng earthworm

moculatmn

techmques

Advantages

Disadvantages

Protecuw mvzro-enwronment Cocoon\ transferred

Densities usually low Little control over species/numbers Mainly surface dwelling worms Cutting machines/labour required Damage to collection site

and relaymg (e.g Stockdrll.

Chemlcal;physlcal extraction broadcasting (e.g. Springett.

MATERIALS

wth 1985)

High densmeb possible Species selection possible

AND METHODS

The Eurthworm inoculation

farmyard oculation

Protectwe

micro-environment absent No cocoon transfer Mainly deep burrowing worms Worms may be injured during extraction Laborious and expensive Damage to collection site

manure site

(FYM).

was applied

to each in-

Unit

Each unit consisted of an outer plastic envelope, filled with soil, earthworms and feed. The plastic envelope was formed from a sealable laboratory bag (Reedbut Packaging, Milton Keynes, U.K.) with lower corners tucked and secured with adhesive tape, to form a cylindrical shape. when filled. This shape of unit was thought necessary for ease of insertion during the inoculation stage. Sterilized, sieved topsoil, described by Butt (1991). was placed within the plastic envelope. The soil had a moisture content of 25-30% of wet soil mass. The EIUs were sealed and provided with mounted needlesized air holes. During the cultivation period, units were housed within an insulated polythene greenhouse, and kept in darkness at a temperature of 18 + 2°C by use of sub-soil heating cables. This temperature is optimal for growth and reproduction of earthworms such as L. terrestris (e.g. Butt, 1991). A proportion of EIUs was destructively sampled prior to inoculation to determine the number of earthworms and cocoons in the units. The number and mass of earthworms found in each unit was recorded and the soil-feed mixture was then searched for cocoons, by wet-sieving through a series of sieves with mesh sizes of 6.7, 3.4. 2.0 and if necessary 1.0 mm. The number of cocoons obtained and their condition, pre- or post-hatching, was recorded. Unhatched cocoons were incubated at either 20 or 15°C to determine their viability and permit an overall hatchability to be calculated for cocoons produced within EIUs. In the field, ElUs were inoculated into the soil manually. The bottom of the plastic envelope was split allowing the contents to be lowered into a prepared hole in the soil. Care was taken to minimise disturbance to the earthworms and their micro-environment and to ensure that the depth of deposited cocoons was kept constant in relation to the soil surface. A surface dressing of feed, in the form of

Field .site Field trials were conducted at a partially restored landfill site at Calvert, Buckinghamshire (Nat. grid ref. SP692238). The surface cover at this site is 1 m of compacted clay (agricultural cap), which lies above a further 1 m of more highly compacted Oxford clay (engineering cap) covering approximately 30 m of household refuse. The agricultural cap, sown with a perennial ryegrass mixture, has a bulk density of I .4 If: 0.2 g cm-s, a pH of 7.6, an organic matter content of 3.9% and a total nitrogen content less than 0.1% (Anonymous, 1986). A survey of the cap by the authors prior to inoculation. using a variety of extraction methods (Lee, 1985) produced no earthworms. This site was chosen, in part, as a particularly hostile environment in which to test the robustness of the EIU technique. Trial I (4-I EIUs) 340 EIUs were prepared during October 1990. Each was bound by a polythene envelope 0.46 m high and with a dia. of 0.24 m when filled. Four litres of soil were used in each. Approximately 500 g of feed was applied to the soil surface of each unit. This was a mixture of paper pulp and yeast extract, with a combined C to N ratio of 40-to-l. This feed had proved successful in earthworm growth experiments (Butt et al., 1992). Six fully reproductive L. terrestris. used in each starter culture, had a mean mass of 3.7 g. These were selected from field-collected earthworms and only those with swollen chtella were used. After addition of the starter culture to each unit, the plastic envelope was sealed to prevent moisture loss and also to prevent the earthworms from escaping. At the beginning of each calendar month (November to April) the units were unsealed to allow the application of a further 500 g of feed. Soil moisture content was assessed gravimetrically on a

The Earthworm Inoculation Unit technique

number of samples, and water was applied as necessary. After 26 weeks (April 1991), the populations in a random selection of 25 EIUs were assessed as described above. A further 300 intact EIUs were transported to the field site. Soil-inoculation was achieved by drilling holes, approximately 0.2 m deep, using a standard tractor-driven soil auger, 0.25 m dia, at 5 m intervals on a grid pattern. Field sampling occurred 10 months after soil inoculation (February 1992) when a cast count around the EIU points was made. Also a total of 100 EIUs were dug out and the surrounding clay was also examined for earthworm presence by digging and formalin application.

253

landfill leachate as required during the summer months. After 10 months (February 1993) a visual count of surface castings by A. longa was made around the points of EIU and broadcast inoculation, as described by Evans and Guild (1947). The position of casts relative to the point of inoculation was recorded. In a number of instances (n = 25) the soil beneath the casting was dug out and burrow depth, plus the number of earthworms present, recorded. A limited number of A. chlorotica EIUs were sampled by digging away the surface to a depth of 5 cm in a surrounding area of 1 m2.

RESULTS

Trial 1 Trial 2 (2-I ENS) 300 EIUs were prepared during January 1992, each bound by an envelope 0.3 m high, with a dia. 0.12 m, providing a volume of 2-l. This was filled with soil which had been thoroughly combined with separated cattle solids (SCS) in the ratio of 7 soil:1 SCS by volume, to give an organic matter content of approximately 3%. A further 200 g of SCS, a proven feed for earthworm reproduction (Butt et al., 1992) was applied on the soil surface and the envelope sealed after addition of the starter culture. Three different starter cultures of fully reproductive, field collected adults were used; i. Four A. longa (mean mass 2.5 g) ii. Six A. chlorotica (mean mass 0.27 g) iii. A combination of four A. longa and six A. chlorotica. 100 EIUs of each treatment were set up. Earthworms and cocoons were recovered from 10 randomly selected EIUs at 12 week intervals to assess population development and to determine the optimum time for field inoculation. The method of recovery was as described above, using the 1.0 mm sieve in order to recover A. chlorotica cocoons. The remaining EIUs were re-supplied with feed at 12 week intervals. 40 intact EIUs from each treatment were transported to the described field site for soil-inoculation in April 1992. This was achieved by drilling holes, 0.15 m deep, either manually or using a tractor-driven soil auger at predetermined locations on a grid system. Plastic netting (2 cm mesh) was pinned to the soil above the EIU to provide some initial protection from avian predation. In addition 160 mature, healthy, field-collected A. longa (mean mass 2.2 g), were also inoculated into the clay cap on the same day by the broadcast method, in batches of 4 (n = 40). Each EIU and broadcast inoculation site was supplied with a surface dressing of FYM and permanently marked. A 2 ha area, which included the inoculation grid, was irrigated with treated

After two months of feeding, the majority of the seals at the tops of the EIUs failed. In a small number of cases (< 2%) total mortality of the starter culture appeared to have occurred after 2-3 months, as there was no evidence of feeding on the the soil surface. In each of the 25 EIUs examined L. terrestris was found in all life stages; reproductive adults, cocoons and hatchlings. The mean number of mature worms recovered was 5.7 per unit with a mean individual mass of 4.9 g. (maximum 8.8 g). Mean number of cocoons produced per unit was 38.4 (maximum 73), a mean rate of 1.12 cocoons worm-’ month-’ over the cultivation period. On average 29% of cocoons in each unit had hatched at the time of sampling. Incubation of the unhatched cocoons at 20°C brought the total hatchability of cocoons produced within EIUs to 55%. Hatchlings within the units were found at a number of growth stages, with mass measurements ranging from 0.1-3.8 g. All hatchlings appeared healthy, but none was fully clitellate. The number of hatchlings found accounted for only 24% of freshly-hatched cocoons within the EIUs. Field sampling revealed a complete absence of observable casts around the inoculation points. Digging out of 100 EIUs produced only 21 L. terrestris. Of these 9 were adult and 12 juvenile. All were present within the EIU soil. A more general survey of the site located a further three adults under surface debris. These were all within 10 m of the nearest EIU. Trial 2 Table 2 shows results for reproduction and cocoon hatching in the three EIU treatments over a cultivation period of 12 weeks. In all EIUs examined the full complement of starter culture adults was present; however, 61% of A. longa and 27% of A. chlorotica had entered a senescent state, indicated by regression of the clitellum, and were no longer in reproductive condition. For both species

254

Kevin R. Butt et al.

Table 2. Number of earthworms and cc~coons in 2-1 EIUs (n = 10) after 12 weeks at a temperature of 18 f 2°C (SE is given for combined reproductive output) Earthworm Species

No. of adults in starter culture

Mean mass (g)

Mean reproductive output COWOIlS

Hatchlings

se.

Total earthworms per EIU at inoculation

A. longa

(monoculture) A. chlorotico (monoculture)

4

2.50

24.6

4.4

54.0

33.0

6

0.33

37.x

37 8

+8.0

81.6

A. longa + A. chlorotica

4

2.10

I2.h

4.4

f2.5

21.0

0.30

28.8

13.9

+2.4

48.7

) IO 6

) 69.7

reproductive rate (cocoons worm-’ month-‘) was significantly greater in monoculture (r-tests: A. longa, P < 0.05; A. chlorotica, P < 0.001). Of 372 A. longa and 667 A. chlorotica cocoons incubated at 15°C successful hatching of 242 (65%) and 413 (62%) occurred, respectively. All cocoons produced a single hatchling. Results from sampling at successive 12 week intervals are shown in Fig. 1. For each species in each treatment the potential number of earthworms for inoculation decreased after the first 12 week period. No cocoons were found on sampling after did 38 weeks and only in A. chkwotica monoculture cocoon production resume after that time. as some of the hatchlings became mature. None of the A. longa hatchlings reached maturity during the monitored periods and all of the identifiable starter culture adults were no longer clitellate after 25 weeks. From field investigation of the A. longa monoculture and mixed culture EIUs, 228 clearly defined

2 w

90

casts were located. some points

Casts were found over 2 m from

of inoculation

but the mean

was 0.6 m (Fig. 2). Mean burrow was

0.18 m (maximum

longa were recovered were excavated,

occasions.

tive condition

(mean

found

the

around

broadcast

0.28 m).

Two

method.

mass sites

A.

mature

on three occasions

and a single mature

each of the other

distance

depth below casts when casts specimen

on

All were in reproduc2.7 g). No casting

of inoculation

Extensive

digging

was

using

the

and formalin

80

i

60

r

20

Time (weeks) Fig. I. Mean number of cocoons and hatchhngs (*SE) in 2-l ElUs at 12 + 1 week intervals after addition of mature earthworm starter cultures ‘(n = 4 for A. longa; n = 6 for A. chlorotica). (A. longa monoculture 0, A. longa in mixed culture ? 1? , A. chlorotica monoculture 0, A. chlorotica in mixed culture ? .?Cocoon component of each treatment ?? ).

0

0.25

0.50 Distance

0.75

1.00

1.2s

of cast position

1.50

I.75

2.00

2.25

from EIUs (m)

Fig. 2. Movements of A. longa from EIIJ positions, revealed by surface cast counts, 10 months after field inoculation (n = 228. x = 0.6 m).

The Earthworm Inoculation Unit technique

application in this area did not produce a single earthworm. Digging in areas around A. chlorotica EIUs (n = 5) produced a mean number of eight earthworms per unit, of which five were clitellate. All of these earthworms were found within a radius of 0.3 m from the point of inoculation and most remained within the EIU soil. Due to the nature of the clay it is possible that some of the smaller hatchlings may not have been located.

DISCUSSION

Trial I

The use of 4-l EIUs was based on the premise that the larger volume of soil would permit growth in earthworm numbers over a lengthy period and the production, within the EIU, of a second generearthworms. ation of reproductively active Phenological data of Butt et al. (1992) suggested that after approximately 26 weeks this second generation should have been reproducing themselves. The carrying capacity of L. terrestris for this system would have fallen in the range 60-100 g live mass (Butt et al., 1994) permitting the presence of some 12-20 recently developed reproductive adults. Data from Hartenstein and Amico (1983) suggest a maximum carrying capacity towards the upper end of this range. The failure of population development as described was in part due to the loss of recruits through the failure of the seals. Results suggested that 81% of hatchlings produced had escaped from the EIUs. Subsequent tests indicated that the adults in the starter cultures, due to their larger masses fell back if they ventured up the inner edges of the moisture-laden polythene envelope. The use of paper pulp and yeast extract as a feed material, whilst known to promote hatchling growth, did not result in maximum reproduction. However, the recorded rate of 1.12 cocoons worm-’ month-’ did fall within the range of values for earthworms of similar origin obtained at higher and lower temperatures, fed with SCS. The necessity to provide optimal nutritional and temperature requirements for different stages in the life cycle of this species were again made obvious (Butt et al., 1992). The results from field sampling for inoculated L. terrestris were extremely poor. Their survival after 10 months represented much less than 1% of approximately 1700 adults, 800 juveniles and 8200 cocoons which had been inoculated in the EIUs. This can possibly be explained by a number of environmental factors and associated observations. On the day following inoculation, site workers reported that flocks of birds (Corvidae) from the SBB 2913-4-C

255

nearby working landfill area had descended on the inoculation plots and were Seen to be eating worms. These avian predators would have been able to detect the regular grid pattern from the air and may have proceeded to prey selectively on this new resource as described by MacArthur and Pianka (1966). Rainfall was also found to collect within the EIUs which, in the heavy clay, often acted as wells and waterlogging may have driven out remaining earthworms. The need for L. terrestris to burrow deeply in order to escape adverse soil conditions such as drought, may also have affected their survival and been hampered by the compact nature of the clay capping. Burrowing activity of L. terrestris has been shown to cease as the bulk density of the soil approaches 1.6 g cme3 (Rushton, 1986). In contrast A. longa , which tends not to burrow as deeply as L. terrestris, is capable of forming burrows in soil compacted to at least 1.49 g cmm3 (Kretzschmar, 1991) and may achieve this partially by means of geophagy (Lee, 1985). Due to this inability to burrow successfully over-surface migrations of L. terrestris similar to those reported by Mather and Christensen (1992), may have occurred. This was indicated by the adults found beneath debris, resulting in further earthworm loss from the inoculation area. L. terrestris may not therefore have been ideally suited to this particular field site but was chosen primarily because the sponsors desired soil inoculation of this deep burrowing species.

Triaf 2 2-l EIUs proved to have several advantages over 4-l units. Due to the smaller volume, fewer earthworms were required as a starter culture and a target of three life stages, rather than reaching the carrying capacity of the unit could more easily be achieved. A feed-only-once approach meant that there were no problems associated with sealing and re-sealing of the units. The smaller volume also meant ease of handling. Use of A. longa and A. chlorotica was thought more appropriate for the heavy clay agricultural cap due to the behaviour of these species under adverse soil conditions (e.g. Lee, 1985). The decision to inoculate after 12 weeks was supported by observations of units that were maintained and sampled over longer periods (Fig. 1). Fewer cocoons and hatchlings were recovered from 2-l EIUs sampled at subsequent 12 week intervals over a 12 month period, even though extra SCS was applied to the surface after each 12 weeks. The size of the unit and the number of earthworms in the starter cultures appear to have been ideally suited to the 12 week cultivation period, as predicted experiments. unpublished preliminary by Incorporation of SCS into the soil for this trial was

256

Kevin R. Butt

necessary for the feeding requirements of A. chlorotica. However further incorporation of this nature was not practical for those EIUs kept longer than 12 weeks and may account in part for the decline in A. chlorotica numbers. The presence of both A. longs and A. chlorotictr in a single EIU (mixed culture) led to a significant decrease in reproductive performance for both species when compared with single species units (monoculture; Table 2). However if the total production for two mixed culture units is compared with one of monoculture for each species then the former produced more cocoons and hatchlings. These results suggest that the presence of the two species together can enhance their individual performance in a given volume. For example, it is possible that by covering the surface layer of SCS with casts, A. longa makes more of this material available to A. chlorotica. As these two species are often associated in natural soil situations (e.g Lee, 1985) this type of (mutually) beneficial behaviour might not be unexpected. Direct interactions between soil dwelling earthworm species have not been examined in detail and future research of this nature may prove fruitful. The netting above the 2-1 EIUs appeared to deter the attention of predatory birds. Observations, from a distance on the two days immediately following 21 EIU inoculation indicated some interest by birds in the site. but no detectable predation. The success of the EIU technique compared with the broadcast method for A. longa survival has in part been attributed to the protective micro-environment. This would appear to allow the EIU inoculum a better chance of initial adaptation to the new environment, although the reservoir of both hatchlings and cocoons may have produced some of those individuals detected through cast counting. Cultivation of earthworms on a large scale always has potential problems, not least of which is contamination of the whole system. However the EIU system with individual discrete units reduces the likelihood of such an event occurring. Cocoons of the three species examined in these trials all hatched successfully, suggesting that polythene bound units were suitable, for a short period at least, for this type of production and could be used in future phenological studies. Further development of the EIU technique should ultimately remove the reliance on field collection of earthworms for soil inoculation, as current trials are producing the required starter cultures from a stock source of bred individuals. The EIU technique has passed through a number of iterative stages during its development, only some of which have been described in this paper, but can be adapted to suit particular species. The future of this technique depends on its use in trials under a variety of conditions utilising different

er al.

species in a range of combinations. This may be a move towards developments in vermiculture technology envisaged by Curry and Cotton (1983) as necessary for mass rearing of soil-dwelling earthworms for introduction projects. Acknowledgements--Mr Ian Stewart for assistance EIU sampling and soil inoculation. Part of this work sponsored by Shanks and McEwan (Southern) Ltd. technique is the subject of British patent GB 2 240456

with was This B.

REFERENCES

Anonymous (1986) The Analysis of Agricultural Materials (A manual of the analytical techniques used by the Agricultural Development and Advisory Service). Her Majesty’s Stationery Office, London. Brun J. J., Cluzeau D., Trehen P. and Bouche M. B. (1987) Biostimulation: perspectives et limites de I’amtlioration biologique des sob par stimulation ou introduction d’esptces lombriciennes. Revue d’Ecologie el de Biologie du Sol 24, 6877701. Butt K. R. (1991) The effects of temperature on the intensive production of Lumbricus terresfris L. (Oligochaeta: Lumbricidae). Pedobiologia 35, 251-264. Butt K. R.. Frederickson J. and Morris R. M. (1992) The Intensive production of Lumbricus terresrris L. for soil amelioration. Soil Biology and Biochemistry 24, 13211325. Butt K. R., Frederickson J. and Morris R. M. (1994) Effect of earthworm density on the growth and reproduction of Lumbricus terrestris L. (Oligochaeta: Lumbricidae) in culture. Pedobiologia 38, 254261. Curry J. P. (1988) The ecology of earthworms in reclaimed soils and their influence on soil fertility. In Earthworms in Waste and Environmental Managemenr (C. A. Edwards and E. F. Neuhauser, Eds), pp. 251-261. SPB Academic Publishing, The Hague. Curry J. P. and Cotton D. C. F. (1983) Earthworms and land reclamation. in Earthworm Ecology; From Darwin to Vermiculture (J. E. Satchell, Ed.), pp. 215-228. Chapman and Hall, London. Evans A. C. and Guild W. J. McL. (1947) Studies on the relationships between earthworms and soil fertility. I. Biological studies in the field. Annals of Applied Biology 34, 307-330. Hartenstein R. and Amico L. (1983) Production and carrying capacity for the earthworm Lumbricus rerresrris in culture. Soil Biology and Biochemisfry 15, 51-54. Kretzschmar A. (1991) Burrowing ability of the earthworm Aporreciodea longa limited by soil compaction and water potential. Biology and Fertility of Soils 11, 48-5 I.

Lee K. E. (1985) Earthworms: Their Ecology and Relationships with Soils and Land Use. Academic Press, Sydney. MacArthur R. H. and Pianka E. R. (1966) On the optimal use of a patchy environment. American Naturalist 100, 606-609. Mather J. G. and Christensen 0. (1992) Surface migration of earthworms in grassland. Pedobioiogia 36, 51-57. Rushton S. P. (1986) The effects of soil compaction on Lumbricus terrestris and its possible implications for populations on land reclaimed from open-cast coal mining. Pedobiologia 29, 85-90. Scullion J. (1992) Earthworms and the rehabilitation of disturbed land, University of Wales. Review of Science and Technology 9, 5-30. Springett J. A. (1985) Effect of introducing Ailolobophora longa Ude on root distribution and some soil properties

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Management (C. A. Edwards and E. F. Neuhauser, Eds), pp. 263-272. SPB Academic Publishing, The Hague. Stockdill S. hi. J. (1982) Effects of introduced earthworms on the productivity of New Zealand pastures. Pedobiologia 9, 93-98.