A Study of Carabidae of Arable Land with Special Reference to Effects of Soil Applied Pesticides

A study of Carabidae of arable land with special reference to effects of soil applied pesticides

BRIAN R. CRITCHLEY, B.Sc.(Wales) Dip.Agric.Sci.(Cantab0

A thesis submitted in part fulfilment of the requirements for the Degree of Doctor of Philosophy in the University of London.

Imperial College Field Station Sunninghill Ascot, Berkshire. May, 1968 2.

ABSTRACT

Carabidae in arable fields were studied at the Imperial College Field Station from November, 1964 to July, 1967 inclusive. Sampling was by pitfall trapping using modified plastic, plant pots. Within-trap predation was reduced by separating large from small species with a zinc gauze grid. Eighty-five species of Carabidae were recorded but only 15-25 species were caught in sufficient numbers (>1% of total catch per annum) to be considered as potentially valuable predators. Absolute numbers of Bembidion lampros were estimated using the mark and recapture method and were equivalent to two individuals per square foot of land in June during the summer peak of activity. Natural mortality factors were studied in carabid larvae with special emphasis on insect parasitism. The residual contact and fumigant toxicities of thionazin in sandy loam soil to adult Carabidae were studied in different environmental conditions and with individuals of different species, size, sex, age, and physiological condition. The minimal effective dosage (the smallest amount to give >95% kill as an end point) at 15°C as determined by time/mortality studies with Bembidion lampros and Pterostichus vulgaris was 1-4 p.p.m. (2-8 lb.a.i./6" acre). The relative toxicities of four organophosphorus pesticides to B.lampros, P.vulgaris. and Agonum dorsale were phorate>thionazin>disulfoton>menazon. Menazon was non toxic at normal field dosages. 3.

The effects on Carabidae of thionazin, ethylene dibromide, ehloropicrin and Dazomet applied to the soil for the control of Heterodera rostochiensis in potatoes were studied in field experiments during 1965-67. Thionazin broadcast at 10 and 40 lb/acre greatly reduced numbers of Carabidae in the summer as did Dazomet and ethylene dibromide at 400 and 800 lb/acre respectively. The ecological significance of these results is considered. The persistence of thionazin residues in soil, water, and in adult Carabidae were analysed by gas chromatography. Downward leaching of thionazin by water in sandy loam soil was demonstrated in field and laboratory experiments, and thionazin was identified in the bodies of dead Carabidae. 4.

CONTENTS

Eau

ABSTRACT 2 GENERAL INTRODUCTION .4 5

Part I

Population Studies on Carabidae in Agricultural Land 8

Part II

Factors Affecting the Toxicity and Persistence of Pesticides in Soil to Carabidae * 43

Fart III

Field Studies bn the Action of Soil Applied Pesticides on Carabidae of Arable Land 119

Part IV

Persistence of Thionazin Residues in Soil, Water and Carabids 188

GENERAL DISCUSSION 235

ACKNOWLEDGEMENTS * 238 REFERENCES 239 APPENDIX 257 5. GENERAL INTRODUCTION

Since the Second World War synthetic organic pesticides have become increasingly important in the control of pests affecting health and food production throughout the world. The benefits from their use cannot be doubted but since Rachel Carson's (1963) publication of Silent Spring there has been much concern about their possible harmful effects from misuse. In the United States the production of all pesticides for 1960 was in the region of 900 million pounds (Hayes, 1964) and it is estimated that this amount will double by 1970• In Great Britain pest problems are not on the same scale but nevertheless about 150 pesticides are in use today (Ministry of Agriculture, Fisheries & Food, 1965) and most are used in agriculture. Carabids, because of their generalized predatory habits are not considered as important natural enemies as are specific ones such as coccinellids and some parasites. Many instances can, however, be quoted of carabids as beneficial predators and papers by Wishart, Doane & Maybee (1956), Hughes (1959), Skuhravy (1959), Wright, Hughes & Worrall (1960), Hughes & Mitchell (1960), Dobson (1961), and Coaker & Williams (1963) are especially significant because they relate to Carabidae in arable fields. There is still much to be learned of their feeding habits and of their effects as predators in arable fields; also required is a knowledge 6.

of the effects on them of pesticides - and especially of the organophosphorus group which are now replacing many of the organo-chlorine pesticides formerly used as soil insecticides. Carabids are comparatively long lived as adults and have life cycles of one or more years Some species are omnivorous and some are primarily phytophagous and may be pests e.g. of strawberry fields. The great majority are predators and hence are potentially beneficial. Thus, the possibility that these insects may be seriously affected by pesticides must not be overlooked especially as, if killed, they are likely to take longer to recover than the pests against which the treatments are applied. This thesis is divided into four parts. Each is complete in itself but the four together are meant to give an overall picture of the relative abundance of Carabidae in certain arable fields and of the possible effects on them of soil applied pesticides, especially the organo-phosphate thionazin. Part I describes a study of Carabidae in arable fields not treated with pesticides. The relative abundance of the more common species was determined by pitfall trapping. Absolute numbers, movement and dispersal were assessed for one species i.e. Bembidion lampros. Some natural mortality factors affecting carabid larvae were also studied. Part II describes laboratory evaulations of the susceptibilities of adult Carabidae to various pesticides, particularly thionazin 7.

(0,0-diethyl 0-2-pyrazinyl phosphorothioate). The toxicity and persistence of the pesticides were determined in different controlled conditions and for species of different size, age, physiological condition, and behaviour. Part III describes the results of three years field experiments to study the immediate and longer term side effects of soil applied pesticides used for the Control of the potato cyst nematode, Heterodera rostochiensis. Spring applications of granular thionazin applied in-the-furrow at two rates were compared with broadcast treatments also at two rates. The immediate and longer term effects of three soil fumigants, ethylene dibromide chioropicrin and Dazomet (3,5 dimethyl tetrahydro 1, 3, 5, 2H-thiadiazine-2-thione) applied in autumn were also compared. Finally, in Part IV the persistence of thionazin in a sandy loam soil was determined in laboratory and field experiments using gas chromatography. The leaching of thionazin in soil was determined and residues in drainage water were analysed. Residues of thionazin in adult Carabidae exposed to treated soils were analysed by gas and thin layer chromatography to determine amounts of pesticide picked-up and possible hazards to organisms higher up in food chains. 8. PART

Population Studies on Carabidae in Agricultural Land

CONTENTS

?taw.

INTRODUCTION .4. • 4 • • 66666•• • • 9

MATERIALS AND METHODS • 10 1. Pitfall Traps 10 2. Sites 10

RESULTS 13 1. Carabidae Recorded 13 2. Relative Abundance of Common Species 13 3. Comparison of Carabidae from Different Sites • 16 4. Absolute Population Estimates of Bembidion. lampros • 24 5. Foraging Range and Rate of Dispersal of B.lampros 27 6. Choice of Habitat 29 7. Effects of Predators and Parasites on Numbers of Carabid Larvae 30

DISCUSSION 35 SUMMARY AND CONCLUSIONS 41 9.

INTRODUCTION

Before studying possible side effects of pesticides on Carabidae in a particular area it is necessary to determine the species* their abundance and distribution within and around the crop habitat. The common "key" species must be defined and their edblogical abundance determined. The ability of species to disperse to or from areas affected by pesticides should also be assessedo

Most adults and the larvae of a few species of Carabidae are very active on the soil surface and one of the best ways of sampling them is by pitfall traps. However, owing to their predatory habits within - trap predation must be minimised and hence an improvement was made on the standard jam jar method. Adult and larva Carabidae were trapped continuously throughout the year in either bare soil or in a crop in three arable fields at Silwood Park. An absolute population estimate of Bembidion lampros* the most commonly occurring carabid species in arable fields at Silwood Park* was made during peak activity in the summer using a mark and recapture method. Natural mortality factors* especially insect parasitism of carabid larvae, were also investigated. 10.

MATERIALS & METHODS

1. Pitfall Traps

Surface active carabid adults and larvae were trapped live in the field in pitfall traps. These consisted of plastic plant pots 3i in. deep, 31 in. wide and mottled grey in colour. Holes at the bottom of the traps were covered with fine mesh nylon gauze and permitted the exit of rainwater but not of trapped carabids. A removable zinc gauze platform with 2 mm. diameter holes rested on the inwardly sloping sides of the pot about 1 in. from the bottom. The top of the trap was level with the surface of the soil and immediately below the trap was a 3-6 in. deep pit to allow rapid drainage. One inch above the trap was a transparent (or coloured) 4 in. wide plastic cover supported by a curved piece of zinc coated wire (Fig.1). Soil within 12 in. of the traps was kept free from weeds and organic debris. The traps were emptied at least once a week. 2. Sites Carabids were collected from three arable fields at Silwood Park: Four Acre Field, Church Field and Hill Bottom. The beetles were sampled in pesticide treated and untreated potato plots in experimental areas of Four Acre Field and Church Field, in an untreated area of Four Acre Field kept fallow and cultivated at regular intervals, and on one edge of the field bordering a mixed sycanore.beech^sweet chestnut wood. In Hill Bottom carabids were trapped in g 1-acre pesticide-free Brussels sprouts crop and in an adjacent 11.

plastic lid r 1 wire support

plastic pot

zinc gauze platform

nylon gauze

drainage pit

Fig. I Longitudinal section of pitfall trap in situ. 12.

1-acre area kept fallow by tilling. The soil at all three sites was a light sandy loam. Weeds were those common to the soil type e.g. Stellaria media, Chenopodium album, Sperggla arvensisi Senecio vulgaris, Capsella bursapastoris, Polygonum aviculare, P.convulvdlus. 13.

RESULTS

1. Carabidae Recorded Table 1 lists the adult Carabidae trapped in and immediately around arable fields at Silwood Park from November, 1964 to August, 1967 (nomenclature from Moore, 1957a).. The list includes five species i.e. Bembidion andreae, B.aeneum, Harpalus rubripes, H.melleti and Amara convexiuscula not previously recorded from Silwood Park (Greenslade, 1961). Furthermore, H.melleti and A.convexiuscula do not appear to have been recorded from Berkshire (Moore, 1957b). The list is not, however, comprehensive for it does not include 25 other species recorded by Greenslade (1961) from uncultivated areas. Table 1 indicates the relative abundance of the species based on annual numbers caught in traps from an area kept fallow in Four Acre Field. "Rare" species are defined as those which individually formed <0.1%, "common"species 0.1-1.0% and "very common" species >1,0% of the annual catch.

2. Relative Abundance of "Common" and VVery Common" Species Of the 85 species recorded in Table 1, 14-15 were sufficiently common to be considered as potentially valuable predators. These included Nebria brevicollis, Notiophilus biguttatus, Loricera pilicornis, Asaphidion flavipes, Bembidion lamprosI B.tetracolum, B.femoratum, B.andreae, B. quadrimaculatum, Trechus quadristriatus, Harpalus aeneus, H.rufipes, Pterostichus vulgaris, P.madidus and Agonum dorsaleo

14. Table 1 List of Carabidae occurring at Silwood Park, November 1964 - August 1967 R = Rare; C = Common; V.C. = Very common; * = Very rare Species Species

Carabus nemoralis Mull Ri otiat3ii4 gear R C.violaceus Linn& Hertitipes (beg) VC Cychrus caraboides Stenolophus teutonus R v.rostratus (Linn.) (Schrank) Leistus spinibarbis(Fab) R Acupalpus meridianus(Linn4 R L.rufomarginatus(Duft.) A.dubius Schilsk. R L.ferrugineus (Linn.) Bradycellus ruficollis Nebria brevicollis(Fab) VC (Steph.) R otiophilus aquaticus B.verbasci (Duft.) R (Linn.) B.harpalinus (Serv.) C N.palustris Duft. Anisodactylus binotatial C N.substriatus Waterh. (Fab.)-- N.rufipes Curt. Amara plebeja (Gy11.) R/C N.biguttatus (Fab.) VC A.similata (Gyll.) R Loricera pilicornis(Fab) VC A.ovata (Fab.) R Clivina collaris(Herbst.) C A•communis (Panz.) R Asaphidion flavipes A.lunicollis Schi8dte R (Linn.) VC A.aenea (Deg.) R Bembidion lampros VC A.eurynota (Panz.) R (Herbst) A.familiaris (Duft.) R B.lampros A.anthobia Villa C v.properans Steph. c. A.tibialis (Payk.) R Bobruxellense Wermael. R A.bifrons (Gyll.) C B.tetracolum Say VC A.praetermissa (Sahib.) R B.femoratum Sturm VC A.apricaria (Payk.) R B.quadrimaculatum VC A.fulva (Deg.) R (Linn) A.consularis (Duft.) R B.andreae (Fab.) VC A.aulica (Pamir) R B.obtusum Serv. R A.convexiuscula (Marsh.) R B.aeneum Germ. R Stomis pumicatus (Panz.) R B.guttula (Fab.) R Pterostichus cupreus R Bslunalatum (Fourc..) R (Linn.) Trechus quadristriatus VC P.oaerulescens (Linn.) R (Schrank) Povernalis (Panz.) R T.obtusus Erichson R P.niger (Schal.) C Patrobus atrorufus R P.vulgaris (Linn.) VC (Strum) P.nigrita (Fab.) R Panagaeus bipustulatus R P.strenuus (Panz.) R (Fab.) P.madidus (Fab.) C Badister bipustulatus R Abax parallelepipedus R (Fab.) (Pilleet Mitt.) Harpalus aeneus (Fab.) VC Platyderus ruficollis H.latus (Linn.) R (Marsh.) R H.rubripes (Duft.) R Dalathus fuscipes (Goeze) C H,tardus (Pant.) C C.melanocephalus (Linn.) R 15.

Table 1 cont.

Species

C.piceus (Marsh.) R Laemostenus terricola (Herbst.) R Synuchus nivalis (Panz4) R Odontonyx,rotundatus (Payk.) R Agonum mulleri (Herbst.) R A,dorsale (Pont.) VC Dromius linearis (01.) R D.quadrimaculatus (Linn.) R D.melanocepbalus Dej. R Metabletus foveatus (Fourc.) C Demetrias atricapillus (Linn.) R

16, They constituted 94,.95% of the total catch in 1965 and 1966 and their seasonal abundance is indicated in Fig.2. Bembidion lampros was the most common species and comprised 50% of the total catch; they probably include individuals of B.lampros v. properans Which is regarded by Moore (1957a) as a distinct species. Greenslade (1961) estimated that populations of B.lampros at Silwood Park contained only 5-10% v. properans and relegated the latter to the rank of a variety of B.lampros. A similar policy has been adopted in this thesis. 3. Comparison of Carabidae from Different Sites Carabid species common to both cultivated and woodland soils were compared with species active in cultivated soils only. The "common" and "very common" species (Fig.2) were present in both habitats but a few others were restricted. Thus, Cychrus rostratus Leistus spinibarbis, Laerrugineus, L.rufomarginatus, Notiophilus rufipes, Abax parallelepipedus and Calathus piceus were usually restricted to the woodland border and rarely migrated into the field. All species caught in the cultivated field but not on the border can fly and include Harpalus latus, Bembidion bruxellense, B.guttula, B.aeneum, Stenolophus teutonus, Amara communist A.fulva, A.aulica, Pterostichus cupreus, Dromius linearis and D.melanocephalus. These species are probably migrants in transit because none is common in arable fields at Silwood Park. Apart from differences in species composition, 17. 40 34%

20 Nebria brevicollis

Notiophilus bi guttatus

Loricera a- 20 pilicornis I- 15 0 ER 40 ERS P

MB 20 Asaphidion NU •• flavipes 1111111111

600

400 Bembidion lampros

200

FMAMJJ A SONDJF MAMJJ ASOND 1965 1966 male d. female U male 0 female Fig. 2 Monthly catches of adult Carabidae in Four Acre Field in bare soil. 160 18 . 8.8%

120

80 Bembidion tetracolum

40 Cl) 0_ < cc Bembidion I— 20 femoratum in

Cc w 0_ o U) cr u j 120 CO 2 D Z 80

Bembidion andreae

Bembidion guadri— maculatum

J FMAMJJ ASONDJFMAMJ JASOND 1965 1966

Fig. 2 (continued) 9.

100 6.6 cio

Trechus sua dri — stria tus

Ha rpalus aenus

cc I— 20 Ha rpalu s I- rufipes

cc Lu a. Cr) CC W 140r CO 6.7% 2

Pterostichus 60 vulga ris

20 !1,gonum do rsale

0 JFMAMJJASONDJFMAMJJASOND 1965 1966

Fig. 2 (continued) 20.

differences in activity were also observed between the two sites. Thus, carabids in the arable field were more active (i.e. more were trapped) from April to June and less active in August and September, than the same species on the edge of the field (Fig.3 A-C). The main peaks of adult activity in the spring and autumn Are seemingly associated with breeding periods as shown by subsequent peaks in larval activity (Fig.3 B and C), Relatively fewer larvae were trapped because they are mainly subterranean (Sharova, 1957). Adult Carabidae are usually more active over bare ground than in dense vegetation but the winter-active larvae appear to be more active in dense vegetation (Figs.4 and 5). Perhaps vegetation protects the larvae from severe weather and from predators and also provides more food in winter. N.brevicollis larvae are primarily surface active and hence pitfall trap catches in Four Acre Field and in Hill Bottom (Figs.4 and 5) probably give a correct indication of the seasonal abundance of this species. The relative abundance of adult and larval Carabidae from bare soil in Four Acre Field and Hill Bottom, from 1 March, 1966 to 31 Nay, 1966 were also compared (Fig.6). The results indicate that carabid activity in the two sites was similar. There were more individuals and species (31 adult and 5 species of larva) in Hill Bottom than in Four Acre Field (24 adult and 2 species of larva) perhaps because they

Migrated from an adjacent Brussels sprouts crop, whereas 2 1 .

80 --o— edge of field --iiIii— in -side field A

O. 80 CO 1.-

N 0. 40 co iii .0 E z= 20

80 —.6.— adults 1 edge of --a— larvae field 13

ap tr er p s r be Num

80 —4—. adults } in—side ---o•—• larvae field C

ap tr er p s ber Num

DJ FMAMJJASONDJFMAMJJ ASONDJ 1965 1966 Fig. 3 Comparison of pitfall trap catches on the edge of and 15 feet within Four Acre Field. Dec. 1964 to Jan. 1967. A. Catch of adult and larva Carabidae. B. Trappings on edge of field. C. Trappings in-side field. 22 0

our Acre Field border between field & wood bare soil

s 1 soil cultivated 15 trap

er p rs be Num

Nov. Dec. Jan. Feb. Mar. Apr. 1965 1966 Fig. 4 Activity of Nebria brevicollis larvae in Four Acre Field, 1965/66

Hill Bottom 504- bare soil IBrussels sprouts

s ap

30 16 tr

er p 20 bers m Nu 10

1966 Fig. 5 Activity of Nebria brevicollis larvae in Hill Bottom, 1966

N.B. 24 Jan.-2I Feb. traps removed from bare soil. 23.

Total Carabidae (adults & larvae) 50 ..1111 Hill Bottom

•••,&••••• Four Acre Field

March April May It 1968 Fig. 6 Relative abundance of Carabidae from bare soil in Four Acre Field & Hill Bottom. 24.

there was no crop in Four Acre Field (Appendix 1). Nine- teen adult Carabidae and two species of larva were, however, common to both sites; also Bembidion spp. formed 88% and 83% of the adults caught in Hill Bottom and Four Acre Field respectively (Table 2). Table 2 Abundance of Bembidion sap. trapped in Hill

Bottom and Four, Acre Field, 1 March, 1966 to 31 May, 1966

Numbers (% of Total Catch) SPECIES HILL BOTTOM FOUR ACRE FIELD

Bembidion lampros 66.7 61.8 B.quadrimaculatum 12,8 1.6 B.andreae 6.0 10.3 B.femoratum 1.2 4.1 B.tetracolum 1.4 5.3 TOTAL 88.1 83.1

B.quadrimaculatum, B.andreae and B.femoratum were often seen in flight on warm sunny days and the trap catches were probably underestimates. 4. Absolute Population Estimates of Adult Bembidion lampros An absolute population estimate of B.lampros was obtained by the mark and recapture method. Over 1,000 adults were marked with Touch-In Belco emulsion paints (diluted 1:10 with acetone) applied with a pin head to the elytra. The beetles were released on 3 and 7 June, 1966 on the border between a Brussels sprouts crop and a fallow field from a point at the centre of 32 pitfall traps placed 25.

+ + + + + + + + + + t + + 4 +

++++++++++++++0

+ + + + + + + + + + 4 + + + + 0 0 0 + + + + + + + + + + 4. + + +

0 0 + + + + + + + + + + + + + + 0

+ ++++++++ +0 ++++ 0 0 + + + + + + + + + + + + 0 + + 0

+ +0++0++0++0++ X igl 0 0 0 0

+ + + 4 + + + + 4 + + +0 4 4 0 0 + + + + + + + + + 4,1 4 * 4 + 0

4 4 4 4 + 4 + + 4 + + + 4 + 0 0 0 + + + 4 4 ++ + + + + + + + + 0 0 0 + + + + + + + + + + + 4 + + +

+ + + + 4 4 + + + + 4 + + + 0

+ + + 4 + + + + + + + + + 4. +

Brussels sprouts crop bare soil

Fig. 7 Position of pitfall traps in Hill Bottom in mark and recapture experiment. Date Total Total marked Total marked unmarked B.lampros released B.lampros recaptured B.lampros captured Yellow(Y) Red(R) Y/R Yellow(Y) Red(R) Y/R

oir -T. al ..i. cr T. cr 4. cri * cri 4 el'

3.-71.66 452 569 0

4.vi.66 59 79 1 eg W

5.v1.66 52 65 1 2 q 6.vi.66 41 55 2 lT

7.vi.66 39 61 1 3 tU OTpT Ti

TOTAL (1) 191 260 2 8 U

7.vi.66 98 204 2 8 WET 8.-71.66 46 71 1 4 0

10.v1.66 77 123 7 0 2 2 uo 1 11.vi.66 7 6 ol

13.vi.66 194 155 8 6 5 4 UT eti

16 10 8 6 s

TOTAL (2) 324 355 o J TTTH GRAND 13M

TOTAL 515 615 18 18 8 6 1 911. P f t u p

27. in four concentric circles of radius 18', 36', 54' and 72' (Fig.7). Half the traps were in the Brussels sprouts crop and half in the bare soil. The results of daily pitfall trap catches are summarized in Table 3. An absolute population estimate was calculated from the data in Table 3 using Bailey's (1952) modified Lincoln Index i.e. p a(n + 1) r 1 where P = total population, a = number of marked individuals released, n = total unmarked individuals captured and r = number of recaptures. The approximate estimate of the variance was calculated from: var.P = a2(n + 1)(n - + 1)2(r 2) Hence the absolute population estimate of B.lampros during early June was 29, 540 ±3, 989 in an area of 16, 290 sq.ft. i.e. two individuals per sq.ft. of ground. Birth- and death- rates were calculated from equations derived from Bailey (1952) but did not differ indicating that the population was stable during the period of release and recapture. It is not expected that additions due to recruitment or losses due to death appreciably influenced the population estimate. 5. Foraging Range and Rate of Dispersal of B.lampros The distances marked individuals had covered after release were recorded (Table 4). Table 4 Recovery of yellow marked B.lampros in traps set at different distances from .a central release point on the border between a Brussels sprouts crop and a fallow field

DATE DAYS AFTER PITFALL TRAP CATCHES TOTAL RELEASE Distance from Central TRAPPED Point of Release 181 36' 54' 72' 4.vi.66 1 1 1 5.vi.66 2 2 1 3 6.vi.66 3 1 1 2 7.vi.66 4 2 1 1 4 8.vi.66 5 3 2 5 10.vi.66 7 3 1 2 1 7 11.vi.66 8 13.vi.66 10 9 2 1 2 14 TOTAL 10 21 7 5 3 36

Seven days after release one was caught in a trap in the fallow field 721 S.E. from the point of release. One was also trapped approximately 2001 to the S.E. The rate of dispersal was about 181 in 24 hours which is probably an underestimate of the amount of movement in the field since this is probably at random. Similar results were obtained with more marked individuals released on 7 June, 1966 (Table 5). Table 5 Recovery of red marked B.lampros in traps set at different distances from a central release point on the border between a Brussels sprouts crop and a fallow field

DATE DAYS AFTER PITFALL TRAP CATCHES TOTAL RELEASE Distance from Central TRAPPED Point of Release 18' 36' 54' 721 8.vi.66 1 2 10.vi.66 3 2 1 1 4 11.vi.66 4 1 1 13.vi.66 6 7 9 TOTAL 6 9 4 1 0 14

29. Table 6 also shows that the beetles moved in all directions but furthest towards the S.E. probably because activity was greater on the bare soil o± the fallow field than in the Brussels sprouts crop. Table 6 Dispersal of yellow marked B.lampros as indicated by pitfall trap catches around a central point of release

PITFALL TRAP CATCHES TOTAL DATE N NE E SE S SW W NW CATCHES 4.vi.66 1 1 5.vi.66 1 1 1 3 6.vi.66 1 1 2 7.vi.66 1 2 1 4 8.vi.66 2 2 1 5 10.vi.66 2 3 3 1 1 1 11 11.vi.66 1 1 13.vi.66 1 1 6 9 3 2 1 23 TOTAL 3 5 9 16 6 2 5 4 50

6. Choice of Habitat The numbers of marked and unmarked B.lampros trapped in the Brussels sprouts crop and in the fallow field were compared (Table 7). Table 7 Total numbers of marked and unmarked B.lampros caught in the Brussels sprouts crop and the fallow field, 3.vi.66 to 13.vi.66

PITFALL TRAP CATCHES (12 traps) Date Brussel Sprouts Fallow Field (1966) Marked Unmarked Marked Unmarked 3.vi-13.vi. 10 97 19 422 30. The results show that although fewer B.lampros were trapped in the Brussels sprouts crop than in the fallow field the ratio of marked to unmarked individuals in the Brussels sprouts crop was twice as great as in the fallow field. This suggests that the beetles Ilpreferredn the Brussels sprouts crop. 7. Effects of predatory aftajoarasites on numbers oE. carabid larvae Larvae trapped in the field were reared singly in the laboratory in 2x1 inch glass tubes containing sterilized moist sand (approximately 8% water by weight). The tubes were corked to prevent loss of water and kept at 15-23°C. The larvae were fed once a week with chopped up blowfly maggots and any food remaining after 24 hours was removed to prevent development of moulds. Whenever deaths occurred attempts were made to determine the cause. Table 8 summarizes the results of two years work on 8 species of carabid larvae from two arable fields at Silwood Park. The table indicates that less than 50% of the larvae pupated and that one of the important causes of death was insect parasitism. The commonest parasites were Phaenoser- phus viator (Hal.) and P.pallipes (Latr.) (Proctotrypidae, Hymenoptera:Parasitica). P.viator is gregarious and occurred mostly in winter-active carabid larvae e.g.

Nebria brevicollis, Pterostichus vulgaris, P.madidus, while P.pallipes was solitary and usually parasitized

Causes of mortality of carabid larvae collected from Four Acre Field and Hill Bottom

SPECIES DATES OF Nay % MORTALITY CAPTURE & exam. Other REARING P.viator P.pallipes factors Pupation Four Acre Field: Nebria brevicollis 27.1.73.v.66 294 23 2 43 31 Notiophilus spp. 14.v-19.ix.66 47 23 53 23 Loricera pilicornis 30.*0-,26.ix.66 32 63 37 Agonum dorsale 12.vii-3.x.66 11 9 64 27 ••• Carabus violaceus 31.viii-10.x.66 25 .111. 100

N.brevicollis 28,x1.66-8.v.67 346 11 5 52 32 Pterostichus 17.x.66- 91 2 90 7 vulgaris 14.iii.67 Calathus fuscipes 17.x,66-14.iii.67 16 25 69 6 Pterostichus 7.xi.66-15.v.67 10 10 •••• 90 •••• madidus Hill Bottom: Nebria brevicollis 28.11-18417-.66 182 51 45 Nebria brevicollis 21.xi.66-7.iii.67 144 6 3 55 37 Pterostichus 14.xi.66-7.ii.67 4 IMO 75 25 vulgaris Calathus fuscipes 28.xi.66-7.iii.67 9 11 89 MIN 32. summer-active larvae such as Nbtiophilus spp. Neither parasite was specific to one hdst larva species. Carabid larvae from Four Acre Field were more heavily parasitized than larvae from Hill Bottom, perhaps because there were more larvae (and hence parasites) in Four Acre Field. Other mortality factors included the following known and possible causes: injury, starvation (or lack of adequate food), parasitic nematodes (Mermithidae), fungi, protozoa, and bacteria. Injury and starvation were factors which could have been induced or enhanced by trapping, incorrect feeding and rearing conditions. Only Nebria brevicollis larvae were trapped in sufficient numbers to give reliable estimates of the causes of death (Fig.8). No first or second instar larvae trapped in the field completed development to the pupa in the laboratory yet larvae reared from eggs in the laboratory developed completely to the adult; the cause of death is unknown. Mortalities of third instar N.brevicollis larvae from P.viator and P.pallipes are recorded in Appendix 2. As many as 22 first instar larvae of P.viator were found in a single N.brevicollis larva, although more were found in larvae of other carabid species. Thus, 37 first instar P.viator larvae were dissected from a third instar Carabus violaceus larva captured on 17.x.66 and 57 from a third instar Pterostichus madidus larva captured on 14.xi.66. However, more than 9-10 P.viator rarely emerged from a single N.brevicollis larva. Fig. 8Relativeabundance ofNebriabrevicollis vs Q. E t 30 Numbers pe r 15 traps a 20 60 40 50 I0 70- 1966 toMay,1967. B. larvae inFourAcre FieldfromOctober, A. OCT NOVDECJANFEBMARAPRMAY OCT NOVDECJANFEB MARAPRMAY Border of field andwood. Within ploughed field. 1111st instar 03rd instar 02nd instar 33• I

'Om rn I Nebria brevicollis 1.• A 34.

The only other significant causes of mortality in third instar N.brevicollis larvae were probably those due to injury (either before or after trapping) and to nematode parasites. Injured larvae died before or during pupation. Two immature specimens of Hexamermis sp. (Nematoda) were found in host larvae trapped in Hill Bottom on 4.iv.66 and 11.iv.66 respectively. Both were tightly coiled in the haemocoel of the host's thorax and were 89 mm. and 102 mm. long. Many Mesodiplogaster iheritieri (Maupas)(Diplogaster- idae) were sometimes found in dead or dying larvae but probably invaded the host larvae through wounds. Ciliate protozoa were occasionally found in association with Diplogasteridae and it is possible that they are symbionts or parasites of the nematodes. 35. DISCUSSION

Pitfall traps were used extensively in this study to sample the carabid fauna from various arable fields, to determine the temporal and spatial distribution of the more abundant (and hence probably more important) species, to obtain an absolute population estimate by the method of mark and recapture and to determine dispersal and foraging range of a given species. It is generally agreed, however, that the method has serious limitations for the direct estimation of populations. Briggs (1961), for example, was unable to correlate numbers caught in pitfall traps with those obtained from soil samples and concluded that the former method was suitable only as an indication of activity. Van der Drift (1951) noted the ability of diurnal Notiophilus spp. to evade capture by pitfall traps. Greens- lade (1964d) found that catch from pitfall traps tended to vary according to the amount of ground cover and concluded that pitfall traps should not be used to compare the numbers of one species in different habitats. Mitchell (1963b) found within-trap predation a serious problem. In spite of these limitations pitfall traps are still the best method available of studying carabid populations objectively. Although some very elaborate pitfall traps have been described (Williams, 1958; Doane, 1961; Rivard,

1962) a simple but effective pitfall trap was designed which improved the efficiency of the standard jam jar method

36. of trapping. Thus, the use of a grid to separate the small from the large carabid species considerably reduced within- trap predation. Grids of varying sizes can be used to separate the species trapped into various size groups and hence could simplify later identification of the species. The traps enabled the insects to be caught live (i.e. without serious losses from predation). Other advantages of the traps were that they were easy to construct, to transport, to keep clean and to replace. Used with caution these traps were therefore able to give a considerable amount of useful and valid information. Hence, carabid species from different sites were only compared at similar time intervals and from areas of comparable habitat, either bare ground or monoculture. However, some limitations could not be overcome. These were that species which were capable of flight e.g. Bembidion spp.(B.andreae, Baemoratum, Bsquadrimaculatum, etc.), Bradycellus spp.„ Amara spp.,

Dromius sPP•: or which were active burrowers e.g. Clivina, collaris,were probably not caught as frequently as other species which were truly epigaeic. Similarly, many species of larvae with the notable exception of Nebria brevicollis, Notiophilus spp., Loricera pilicornis are mainly subterranean in habit and hence pitfall traps are inappropriate for sampling them. It is therefore stressed that no matter how efficient the method of sampling, pitfall trapping can only give an indication of the relative abundance of the species in a given habitat. Pitfall trapping is also considerably 37. influenced by weather and hence, for comparative purposes, trap records are only of use if done continuously and over a period of time e.g. several days. Pitfall trapping for three years showed that certain species are characteristic of the arable fields at Silwood Park. These included about equal numbers of species capable of flight or completely flightless (Fig.2). The flightless Bembidion lampros was easily the most abundant species and occurred predominantly in May and June. A mark and recapture experiment indicated that the adult of this species was very abundant on bare ground (e.g. 2 per sq.ft.) and that in spite of its small size (mean length about 3 mm.) it foraged 200 ft. or more. In view of its well known predatory habits (Wright, 1956; Hughes, 1959; Hughes and Mitchell, 1960; Wright, Hughes and Worrall, 1960) the use of pesticides which harm it might have serious side effects. Natural mortality factors were studied in some species. Adult Carabidae have few known arthropod predators (with the exception of spiders (Bristowel 1941))and probably occupy a position at the top of food chains. Vertebrates are probably the major predators on the adults e.g. Amphibia (frogs and toads), small mammals i.e. mice and shrews (Lavrov, 1943; Crowcroft, 1954 in Murdoch, 1966) and birds, A few insects parasitize the adult Carabidae and Thompson (1943) lists only 6 tachinids, 2 proctotrupids and one braconid. Rivard (1964) in a study of the period of egg 38. production in carabids, dissected over 5,500 individuals of some 40 species but found 19 only parasitized ones, mostly Pterostichus melanarius (=P.vulgaris). The most susceptible stages of the carabid life cycle appear to be the eggs or larvae. Eggs are generally laid at random in or on the surface of the soil while the comparatively soft bodied larvae are vulnerable to attack by parasites. The two most important insect parasites at Silwood Park were Phaenoserphus viator and P.pallipes. Eastham (1929) has given a detailed account of the biology of P.viator as a parasite of Pterostichus niger and Nixon (1938) has reviewed the genus in his Revision of the British Proctotrupinae. There is little information on the parasites apart from brief observations on breeding (Elliott and Morley, 1907-11; Thompson, 1943; Richards, 1946; Davies, 1955; Basden, 1959; Briggs, 1965) and no information on the extent to which parasitism occurs. Dissections of living and dead N.brevicollis larvae revealed that first instar larvae of P.viator first appeared in late November and early December. Prepupae and pupae emerged from the host larvae from January onwards and after a short pupa period (about 1 week) adults emerged and mating occurred within 24 hours. Adult parasites could be kept alive for several weeks or months on a sugar solution and presumably survive in nature on the nectar from flowers. Parasitism or attempts at parasitism never occurred when live host larvae were enclosed with adult female parasites. 39: Adult parasites are surprisingly good burrowers in moist sand and it is likely that parasitism occurs in the soil in the comparative confinement of the host's burrow. Twenty three per cent of third instar N.brevicollis larvae examined in 1966 were parasitized by P.viator and 11% in 1967 (Table 8). Possibly parasitism exceeds 23% in some years e.g. Greenslade (1964b), recorded that numbers of N.brevicollis adults decreased from 2,300 in 1959 to 200 in 1960 (which he tentatively related to a climatic factor). Although other carabid larvae were parasitized by P.viator insufficient were examined to give adequate estimates of parasitism.

The biology of P.pallipes was more difficult to assess for it parasitizes both summer- and winter-active larvae. In 1966 23% of Notiophilus spp. larvae examined were killed by this parasite but otherwise few were parasitized. Davies (1959) bred P.vexator Nixon from Notiophilus biguttatus and N.rufipes, one parasite being found in each host. Possibly Davies's P.vexator could have been P.pallipes for Nixon (1938) states that "P.vexator may later be shown to be nothing but an unusual form of P.pallipes". Very few nematode parasites of Carabidae have previously been recorded. There is a single record of, a mermithid, Mermis albicans Sieb in Amara similata Gyll in Europe (Assmussi 1858 from Rivard, 1964) and one of Hexamermis sp. in Bembidion nitidum Kby in Canada (Rivard, 1964). The presence of Hexamermis sp. in Nebria brevicollis larvae is therefore 40. noteworthy but these parasites cannot be important in affecting carabid numbers. 414* SUMMARY AND CONCLUSIONS

1. Carabidae were studied for three years from November, 1964 to August, 1967 in three arable fields at Silwood Park.

2. Adults and larvae were caught live in pitfall traps consisting of modified plastic plant pots. Large species were separated from smaller ones by a removable zinc gauze platform which considerably decreased within-trap predation.

3. Eighty-five species of adult Carabidae were recorded of which about 15 were sufficiently common (i.e.> 1% of the total catch per annum) to be potentially valuable predators.

4. Bembidion lampros was the most abundant species and accounted for nearly 50% of the total in 1965 and 1966.

5. An absolute population estimate for B.lampros during the first two weeks in June 1966 indicated that 29,340 3,989 were present in an area of 16,290 sq.ft. This was equivalent to two per square foot of land.

6. The rate of dispersal of this species was about 18 feet in 24 hours but one beetle was caught nearly 200 ft. away 4 weeks after being released.

7. B.lampros was more active in an uncropped (bare soil) area than in a Brussels sprouts crop but the results suggested a "preference" for the cropped area.

8. Natural mortality factors were studied in 8 species of carabid larvae especially Nebria brevicollis which was abundant from November to April inclusive. Phaenoserphus 42. viator (Hal.), a gregarious parasite, occurred mostly in winter-active larvae while P.pallipes (Latr.), a solitary parasite, occurred mostly in summer-active larvae. The level of parasitism varied from year to year and from one field to another and was sometimes as high as 25%. Parasit- ism and other mortality factors could cause population changes in the adults of a species in some years. This could cause important changes in numbers that might complicate the effects of changes caused by pesticide treatments. 43. PART II Factors Affecting the Toxicity and Persistence of Pesticides in Soil to Carabidae

CONTENTS

page

INTRODUCTION is•••••••••1 •.. MATERIALS AND METHODS 47 1. Soil Properties 47 (a)Mechanical analysis 47 (b)Soil moisture 48 (o) Soil pH 50 2. Insecticides and Formulations 50 (a)Uses of thionazin 50 (b)Insecticidal rates and soil properties 53 (c)Preparation of treated soils 53 3. Carabids for Bioassay Tests 57 Maintenance of cultures • 57 4. Laboratory Experiments on Contact and Fumigant Action of Thionazin 6o (a)Residual contact action 60 (b)Fumigant action 61 5. Method of Assessing Toxic Effects 62 6. Statistical Treatment of Results 63 RESULTS 64 Preliminary experiments on susceptibility of adult carabids to thionazin treated soil 64 The critical dosage of thionazin in sandy loam soil to various species of carabids .... 68 Toxicity of thionazin in soil as affected by environmental factors 71 Soil moisture 71 Soil compaction 76 Temperature and light 77 Soil pH 79 44.

Toxicity of thionazin in soil as affected by biological factors 81 Species susceptibility 81 Age 87 Starvation 89 Toxicity of thionazin as affected by chemical properties 91 Fumigant effects 91

Persistence of thionazin in sandy loam soil 94 Comparative tests of contact toxicity of other soil applied systemic pesticides to carabids 98 1. Toxicity of Ground Phorate Granules in Soil. 98 2. Toxicity of Ground Disulfoton Granules in Soil 101 3. Toxicity of Ground Menazon Granules in Soil 104 Comparative toxicity of thionazin$ phorate, disulfoton and menazon to carabids 105

DISCUSSION 106 SUMMARY AND CONCLUSIONS 116 45. INTRODUCTION

The factors determining the susceptibility of an organism or group of organisms to a soil pesticide are numerous and complex and require carefully controlled, replicated experiments for their elucidation. Gough (1942) and Fleming (1942) (in Gough, 1945) have stressed the need to control conditions other than those to be studied. For studying effects of different insecticides in soil conditions it is necessary, for example, to control temperature and soil moisture and also the species, condition and age of the test insect. In the present work Carabidae could not be reared successfully and laboratory experiments had therefore to bc, done with unstandardised individuals trapped in the field. The main aim of these experiments was to determine conditions which would affect carabid susceptibility in relation to field applications of systemic organophosphorus pesticides as exemplified especially by thionazin. The factors studied which could influence the contact and fumigant toxicity of these pesticides included water, temperature, light, soil compaction, pH, size of organism, sex, age, starvation, behaviour and activity. Effects due to fumigant toxicity alone were also assessed. Persistence of the pesticide in soil was studied to determine possible long term effects. Comparative studies with three other systemic organophosphorus pesticides namely phorate, disulfoton and menazon were also done. It is stressed, however, that 46. owing to the artificially controlled and simplified environmental and biological factors, results in the final analysis can only be related to field conditions in relative and not absolute terms. 47. MATERIALS AND METHODS

1. Soil Properties.. Soil for laboratory bioassay experiments was obtained from sites representative of field plots to be treated with soil pesticides but known to contain no pesticide residues. The soil was removed by trowel from the surface 3-4 inches and was sieved to remove large stones (>10 mm.) before air- drying. After air-drying for several days it was passed through a No.10 mesh sieve to remove all small stones (2-10 mm.) and stored in an air-tight galvanised bin. (a) Mechanical analysis A mechanical analysis of the soil was done by the pipette method as described by Piper (1947). Organic carbon was determined by,Walkley and Black's Rapid Titration Method (Piper, 1947). This method is said to give a recovery of 81.2% of organic carbon with a standard deviation of 1.5%. To obtain a value for organic matter the correction factor 1.232 was first applied and then the value was multiplied by the conventional factor 1.724 for mineral soils as suggested by Waksman and Hutching (1935). Table 9 summarizes the results of this analysis. The table indicates that the soil was mainly of a sandy nature but sufficient clay, silt and organic matter were present for it to be classified as a "light sandy loam". These results are in close agreement with those of Call (1955) and Scopes (1965), for Ashurst field soil. 48. Table 9 Mechanical analysis of Ashurst field soil

Constituent Percentage Coarse sand (Particle size 2.0-0.2 mm.) 39.5 Fine sand ( n n 0.2-0.02 mm.) 35.9 Silt ( it " 0.02-0.002 mm.) 7.1 Clay ( n " <0.002 mm) 13.9 Organic carbon, Walkley & Black values 2.5 (Organic matter (calculated)) 5.3 Moisture content of air-dried soil 2.1

TOTAL 101.0

(b) Soil moisture Soil moisture is usually presented in the literature as a percentage of the field capacity. This is defined as the moisture content at which drainage has become so low that downward movement of water almost ceases. From this definition it would appear that field capacity cannot be measured very accurately and may not always be reproducible for a given soil. The sticky point, on the other hand, appears to be a more precise method of expressing soil moisture for it is the point at which all the pore spaces in the soil are completely saturated with water. It is defined by Hardy, 1923 (cited in Piper, 1947) as the moisture

content, expressed as a percentage of the, oven-dry soil, at which kneaded moist soil just ceases to adhere to smooth objects. The sticky point was determined as described in Agriculture Handbook No.60 (1954) of the United States Department of Agriculture and from a determination of the 49. moisture content at field capacity it was possible to derive a table of moisture contents for Ashurst field soil as expressed in terms of either field capacity or sticky point (Table 10). Table 10 Range of moisture contents for Ashurst field soil

SOIL MOISTURE CONTENT % sticky % by weight of % Field capacity 1 point Air-dry soil Oven-dry soil

5 1.5 3.6 16.0 10 3.0 5.1 22.7 15 4.5 6.6 29.3 20 6.0 8.1 36.0 25 7.5 9.6 42.7 30 9.0 11.1 49.3 35 10.5 12.6 56.0 40 12.0 14.1 62.7 45 13.5 15.6 69.3 5o 15.0 17.1 76.o 55 16.5 18.6 82.7 60 18.0 20.1 89.3 65 19.5 21.6 96.0 70 21.0 23.1 102.7 75 22.5 24.6 109.3 8o 24.o 26.1 116.0 85 25.5 27.6 122.7 90 27.0 29.1 129.3 95 28.5 30.6 136.o 100 30.0 32.1 142.7

1. Moisture content (% oven-dry soil) at field capacity = 22.5 (mean of two determinations) 50.

2. Insecticides & Formulations, The relative toxicities of the following systemic organophosphorus pesticides were tested for their contact and fumigant effects against adult carabids in laboratory experiments. (N.B. Names of pesticides beginning with lower case letters are common names approved by the British Standards Institute Anon (1965)). (1)Thionazin ("Nemafos", "Zinophos", "Cynem"). 0,0-diethyl 0-2 pyrazinyl phosphorothioate. Manufactured by Cyanamid of Great Britain Ltd. 5% active ingredient on Attapulgite clay. (2)Phorate ("Thimet"). 00-diethyl-S-ethyl phosphorodithioate. Manufactured by Cyanamid of Great Britain Ltd; 10% active ingredient on Fullers Earth granules. (3)Disulfoton ("Di-syston"). 00-diethyl S-2-(ethylthio) ethyl phosphorodithioate. Manufactured by Baywood Chemicals; 5% active ingredient on Fullers Earth granules. (4)Menazon ("Saphicol"). S-(4,6-diamino-1,3,5 triazin-2-yl) methyl 00-dimethyl phosphorothiolothionate. Manufactured by Plant Protection Ltd.; 5% active ingredient on Fullers Earth granules.

(a) Uses of thionazin Thionazin has both contact and systemic properties and was discovered by Cyanamid in 1956. It is now used extensively as a nematicide for the control of eelworms in tulips, Table 11 Uses of thionazin as a nematicide

Reference Pest Crop Dosage Results Rate &/or Pest Crop Formulation Control Growth P G G Pt Baker & Sasser Ditylenchus alfalfa 41 8 & 16 lb/ (1959) dipsaci acre. Gr. X Cohn & Mina Tylenchus citrus 10,40 Kg/ha X (1965) semipenetrans trees Gr X Collins & Feldman Radopholus orange 32 lb/ac.- (1965) similis trees D Cooper & Sasser Belonolaimus peanuts 4,-8,16.lb/ac. X (1960) longicaudatus Dias Netto & Meloidogyne figs 8 lb-/ac.- X Falanghe (1962) incognita Gcr.. French (1964) Ditylenchus oats 16 lb/ac.a.i. dipsaci Gr.

It 6 & 12 lb/ac. •Gr.

fi tt It 2,4,8 lb/ac.

Guile (1964) Heterodera potatoes 48 lb/ac.* rostochiensis Gr •

Lewis (1965) Heterodera tomato 24 lb/ac.E.C. rostochiensis 14 lb/ac. Gr. Table 11 cont.

Reference Pest Crop Dosage Results Rate &/or Pest Crop Formulation Control Growth P G G Pt

Miller & Perry Root-knot nursery 1000 p.p.m. X (1965) nematode plants

Motsinger & Morgan tobacco 10-20 lb/ac. X (1960) Gr.

Oliff (1966) Ditylenchus tulips 20 lb/ac. X dipsaci IV narcissus <20 lb/ac. X

Ouden & Kaai Heterodera potatoes 20g/sq.cm. xx (1963) rostochiensis Gr.

Suit & Feldman Radopholus citrus (1961) simiiis trees 8-32 lb/ac. D Zuckerman (1964) Trichodorus cranberries 8-16 lb/ac. X christiel G = Good Gr. = Granules P = Poor D = Drench Pt = Phytotoxic E.C,= Emulsifiable concentrate S = Spray * in peat soil x Time of application important 53. daffodils and chysanthemums as well as in some edible and other agricultural crops. Some of its uses as .a "nematicide" and an "insecticide" are summarized in Tables 11 and 12 respectively. The tables indicate that higher rates of thionazin are required as a rule for effective control against nematodes than for insect pests (e.g. 8-32 lb/acre as compared to 2-8 lb/acre a.i.) and that granules were more effective than emulsion or spray formulations. Thionazin has a high mammalian toxicity (oral LD50 for rat = 12 mg/Kg., Edson et al. (1964)) but, formulated as granules, is relatively safe to handle. (b)Insecticidal rates and soil properties The following procedure was adopted to convert application rates from pounds active ingredient per acre (lb a.i./ acre) to parts per million (p.p.m.) of soil and vice versa. Using a soil sampling auger 30 6-in. deep cores were removed from the treated area and air-dried. Stones were removed by sieving the air-dried soil first through a coarse sieve and then through a No.10 sieve (mesh size about 2 mm) and the soil was then weighed. The dry weight of 1 acre of soil 6 in. deep was calculated to be 2,096,000 lb/acre. 1 lb of pesticide/6 in.acre was equal to 0.48 p.p.m. and 1.0 p.p.m. was equal to 2.1 lb/acre. (c)Preparation of treated soils Soils with known concentrations of pesticide were prepared as follows. The granular pesticide (30-60 mesh) was 54. Table12 Uses of thionazin for control of insect pests D.P.E. = Delayed plant emergence; S.P.E.= emergence.Stimulated plant Reference Pest Crop': Dosage Results Rate &/or Formulation Pest Control Crop Growth Poor Good Good Phytotoxicity

Anderson, Nakakihara earWorms sweet corn 5% dust X Hall (1963)

Bacon et al. (1964) Bruchophagus seed alfalfa 1 & 2 lb/ac. (spray) X roddi Bang & Kae (1964) Chi to 2 Kg/Ha in irrigation X suppressalis rice water Bardner (1963) aphids .Crop g.a.i./100g. seed Rhopalosiphum wheat padi L. 0.40 15 days D.P.E. 0.20 X D.P.E. II It Brevicoyne kale brassicae L. 4.00 X 2.00 X Aphis fabae Scop. sugar beet 0.88 33 days 0.44 X S.P.E. - Beck (1963) Prosapia bicincta Bermudagrass lb/ac. X Bevan (1967) Agriotes spp. potatoes 4 lb/ac.

Boush & Alexander Diabrotica peanuts X X (1964) undecimpunctata 2 lb/ac. E.C. & grans. Bryden (1965) wireworms potatoes X II U 1.7 lb/ac. grans. 1.0 lb/ac. spray X Caldicott & Isherwood Erioischia edible 0.05 & 0.025 g. (1967) brassicae brassicae• a.i./plant tr II Carden (1967)a°3 2 lb/ac. X (1967)c Psila rosae celery 14 oz/ac. X Coaker & Finch (1965) Erioischia edible 0.02-0.1% a.i. brassicae braSsicae drench Davis & McEwen (1965) II radish 4 lb/gal. E.C. X Day et al. (1964) Conoderus potatoes X falli 1 & 2 lb/ac. Dean & Palmiter (1963) Magicicada apple trees 1 pt./tree septendecim (45% a.i.) Dominick (1964) Epitrix hirtipennis tobacco 4 lb/ac. (flea beetle) (grans) Finlayson et al. Hylemya brassicae Crucifers 5 lb/ac.grans. (1965) 2.5 lb/ac. E.C. Golightly (1965) Erioischia cauliflowers 0.5g/plant grans. X brassicae 0.09% a.i.per plant dips 55. Table 12 cont.

Reference Pest Crop Dosage Results Rate &/or Formulation Peet Control Crop Growth Podk X Golightly (1967) Tipula spp. barley 1 lb/ac. spray X 0.5 lb/ac, bait X X 1 lb/ac. gran. X X Graham (1967) Erioischia cauliflower 2.1 lb/ac. X brassicae X Griffiths & Bardner (1964) wireworms wheat 2.7 lb/ac.

Guthrie et al. (1963) tt .tobacco 1 lb/ac.a.i.broad- X past;21 4 or 81b/ac.in- row. Harding & Wolfenbarger leafminer Southern 2 & 4 lb/ac. (1963) peas (grans) X*

Harrison (1964) Ostrinia maize 1 lb/ac. grans. nubilalis 2 lb/ac. grans. 4 lb/ac. grans. X

Hays & Morgan (1965) Diabrotica peanuts 2 lb/ac. gran. X undecimpunctata 2 lb/ac. spray

Howitt & Cole (1962) Hylemya brassicae radish 1,2 & 4 lb/ac.

Long et al. (1961) Diatraea sugar cane 13 lb/ac. saccharalis F.

Mathias & Roberts (1967) Leptohylemyia winter wheat 161b/ac.a.i. coarctata. 5% grans. Mulla (1965) Hippelates sp. Citrus grove 2 lb/ac. 4 lb/ac. X 10 lb/ac. X Raw (1965) wireworms Spring wheat 1.5 lb/ac. spray Rogerson (1967) Erioischia edible 1.0-2.0 lb/ac. brassicae brassicae (spray) Savage & Harrison (1962) Myzus persicae tobacco 1 lb/ac. 4 ibiac.(5% grans) X Schaeffers (1963) Cyclamen mite strawberry 2 lb. E.C./ac.

Schwartz et al. (1961) Epilacha Lima beans 2.1 lb/ac. varivestis (grans)

Shanks & Howitt (1964) Scutigerella mint 5 lb/acre immaculata (sp. or grans.) (Myriapoda) Shanks & Gans (1965) tt It St. G. Light (1967) Erioischia edible 2-5 lb/ac. brassicae brassicae (grans.) X* relative to phorate. Xx better than phorate 56. Table 12 cont. Reference Pest Crop Dosage Results Rate &/or Formulation Pest Control Crop Growth Poor Good Good Phytotoxicity Steinhaur et al. (1962) alfalfa weevil, alfalfa pea aphid & 4 lb/ac. grans spittlebug 2 lb/ac. spray X Vernon (1965) Erioischia cauliflowers brassicae 0.25 g/plant X (grans) Wells & Guyer (1967) wireworms potato 2 lb/ac.in-row X (10% grans) Woodville (1967) Merodon • narcissus equestris bulbs 0.23% dip. X Wright (1965) carrot fly swedes 2.5 lb/ac. X (grans) 57. first ground to a fine powder using a mortar and pestle the whole process being done in a polthene bag to prevent dispersal and inhalation of the pesticide. The grounding improved distribution of the pesticide in the soil. Walker (1963) has shown that such a process may initially make disulfoton slightly more toxic but does not affect persistence. The highest concentrations were prepared and lower concentrations obtained by dilution with untreated soil. The air-dry soils were mixed in a Kenwood Mixmaster food mixer in a large polythene bag which prevented dispersio of dust. Mixing took about 20 minutes interrupted 3 or 4 times to scrape consolidated soil from the base of the stainless steel bowl. The treated, dry soils were then sealed in polythene bags and stored at -18°C until required for bioassays. Untreated soils were similarly mixed and stored.

3. Carabids for Bioassay Tests. Maintenance of cultures Carabids for bioassay tests were collected in pitfall traps from areas which had no known pesticide treatments. The beetles were separated according to species and sex and kept in plastic boxes of varying sizes containing damp peat. Those with injuries (e.g. missing appendages) or with ecto- parasitic mites or fungi were discarded. The healthy beetles were kept in a room at 15-23°C and were fed at least once a week with fourth instar blowfly maggots. Uneaten food was removed after 24 hours to prevent development of moulds. 58. At moat five individuals of large speCies were. put in one box measuring 9 in. x 54 in. x 3 in. More of the smaller species could be kept together without significant cannib- alism. For bioassay experiments it was important to find a suitable food which (1) was acceptable to all the test species (2) was available throughout the year and (3) could be eaten in known amounts. None of the foods previously recorded proved satisfactory for all purposes. In the laboratory large species of carabids could eat approximately their own weight of earthworms in 24 hours (Table 13). Table 13 Mean weight of earthworm eaten by Pterostichus madidus individuals in 24 hours

Rep. Mean Weight of Mean Weight of Weight Earthworm to No, 6 P.madidus Earthworm Weight Beetle (n) (mg.) Eaten Eaten 1 116.1 + 4.8 95.9 + 1403 0.83/1.00 (S.D.11.8) (S.D.34.9) 2 126.1 + 8.9 121.5 + 20.6 0.96/1.00 (S.D.21.8) (S.D.50.3) 3 148.1 + 12.8 117.7 + 37.7 0.80/1.00 (s.D.31.3) (S.D.92.3)

Food uneaten after 24 hours generally remained uneaten and was removed to prevent development of moulds. An experiment with Bembidion tetracolum showed that individuals of mean weight 7.9 + 0.3 mg. could eat 5.9 + 0.4 tag. of earthworm in 24 hours i.e. approximately 0.74 x their own weight. Scherney (1955) has shown that large species such as Carabus auratus, C.cancellatus, Pseudophonus pubescens and 59. Pterostichus vulgaris could eat 1.36-3.4 x their own weight in food daily. However, it is also known that some carabids can starve for weeks or months providing there is adequate water (van Dinther$ 1964). Earthworms were readily eaten by most species of carabids but did not entirely satisfy requirement (2). A study of possible alternatives showed that blowfly maggots from laboratory cultures of Phormia terraenovae R.D. proved excellent as a standard. Fourth instar larvae about to pupate proved most satisfactory because (1) they were easy to collect in very large numbers (2) they were of about the right size for fending whole to the larger carabid species and (3) they did not contain undigested food in their guts which when present could cause early putrifaction. The maggots were killed by freezing them at -18°C. (They could be stored indefinitely at -18°C and provided a regular supply of food throughout the year). The maggots were killed because in normal circumstances they would soon become puparia which were unsuitable even to the larger species. After removal from the deep freeze and before being fed to the carabids, the maggots were drenched first in boiling water to harden their contents and then in tepid water. The large maggots were chopped up in appropriate sized pieces for feeding to the smaller carabid species. No special provision was made for supplying water because the beetles obtained this from the peat which was 60. lightly sprinkled with distilled water once a week. Bembidion tetracolum, Harpalus aeneus, Laemostenus terricola among other species kept in this way lived in cultures for 1-2 years.

4. Laboratory Experiments on Contact and Fumigant Action of Thionazin. (a) Residual contact action Except where otherwise stated the procedure was essen- tially as follows: Treated and untreated soils were removed from the deep freeze and allowed to thaw out. 100g. 1g. samples of soil were placed in circular transparent plastic containers, 10 cm. internal diameter, 4 cm. deep and with clip-on lids. Sufficient distilled water was added to each 100g. soil sample to give a moisture content of 15% by weight (see Table10 for conversion of moisture content to per cent of "field capacity" or "sticky point"). The soil developed a good crumb structure when mixed with the water. The soil samples filled each container to a depth of about 2.0 cm. (approx. 1 in.) and permitted the beetles to burrow. In each experiment three replicate containers were used for each treatment (including the controls) and each sex, The soils were then placed in a constant temperature room or . cabinet and left over-night for the soil temperature to equilibrate with the ambient air temperature. Most experiments were done at 15°C 4- 0.5°C. This temperature was 61. chosen because it was similar to the mean summer soil temperature at a depth of 1-2 in. in Ashurst field soil. Carabids were taken at random in groups of five of each sex. They were then conditioned for 24 hours at the appropriate temperature and with 16 hours day provided by fluorescent lighting. The beetles were fed with appropriate amounts of chopped up blowfly maggots so mortalities from starvation during treatment would interfere minimally with those caused by the pesticide. Five beetles were put in each container of treated soil. Great care was taken to standard- ize as many environmental factors as possible so that valid comparisons could be made of results of different tests. (b) Fumigant action A test to determine the concentration x time product of thionazin vapour at the L.T.50 for a given species was done as follows: air of known humidity and flow rate was drawn over thionazin granules and then over the test insects which were confined in an open ended glass tube. Humidity was controlled and maintained at 80% R.H. by passing the air through a sintered glass bubbler containing a solution of potassium hydroxide (KOH) of known specific gravity (Solomon, 1951). Flow rate was determined by a soap bubble flow meter and a steady current of air was maintained through the apparatus. Glassware was thoroughly cleaned before assembly and plastic rather than rubber bungs were used to join the various sections together. Thionazin granules were 62. packed in a 5 cm. column at one end of a long tube and the insect cage was placed at the other end. The air which passed over the pesticide granules and the insects was bubbled through a 5 ml. solution of ethyl alcohol - which was kept cool in a thermos flask containing water and ice to reduce loss by evaporation - to trap any toxic vapours and was then released in a fume chamber. An identical control was set up in parallel with a'btivated charcoal replacing the thionazin granules and the experiment was done at room temperature ( 19°C f 2°C).

5 Method of Assessing Toxic Effects Time/mortality studies were done in all bioassays. Toxicit was defined in terms of rate of kill at .articular temperature and was usually measured as time (hours) for 50% kill at a given temperature. This often requires frequent examinations of the test insects on or in treated soils and involves disturbing the insects, a factor which may affect mortalities. As this procedure was adopted in all the bioassay tests it is probable that all the results were affected to a similar degree and hence this factor was standardized. In spite of this disadvantage and the fact that it is time consuming this method enables the maximum information on mortalities to be obtained from the comparatively few test insects that could be collected.

Test insects were separated into three categories when assessing toxic effects. Normal and slightly affected (A) 65.

badly affected or moribUhd (M) and dead (D). Normal carabids showed no symptoms of abnormal behaviour and almost all readily burrowed in treated and untreated soils. Slightly affected individuals exhibited agitated and unco- ordinated movements and those badly affected or moribund could not right themselves when overturned. Dead insects showed either no movement or very slight movements of legs and antennae which sometimes persisted for days and weeks.

6. Statistical Treatment of Results Badly affected or moribund individuals were included as dead insects in calculating per cent mortalities. The cummulative percentage kills were converted into probits according to Finney's (1952) Table I after correction for control mortalities (Abbott, 1925). The probits were plotted against the logarithms of time and the best fitting straight line was then drawn by eye through the points on the graph. Values of L.T.50 (i.e. the time for 50% mortality to occur in any one treatment) were used for comparing and assessing the effects of the pesticide treatments since it is at this point that the 95% fiducial limits are smallest and hence give the most precise estimate of the effect of the treatment. Probit regression lines were computed to obtain a measure of the goodness of fit of each time-response line and to find confidence limits for each

L.T.50. 64. RESULTS

Preliminary experiments on susceptibility of adult carabids to thionazin treated soil

Pilot experiments with a wide range of dosage rates (10, 50, 250 and 1,250 p.p.m.) indicated that concentrations greater than 50 p.p.m. were greatly in excess of the minimal effective dose and were of little use for experimental purposes since mortalities occurred too rapidly to be recorded accurately. Further pilot experiments were then done on a restricted range of concentrations from 0.5-32.0 p.p.m, with intermediate concentrations chosen on a logarithmic scale. The results of this experiment using Bembidion tetracolum as test insects are shown in Fig.9 and Appendix 3 and the L.T.50's for each concentration are summarized in Table 14. The slope of the regression line for 32 p.p.m. thionazin (Tablel4) was much steeper than that of lower concentrations and the L.T.50 much less than that of the critical or minimal effective dosage. The slopes of the lines at concentrations between 1 and 16 p.p.m. were seemingly parallel so that the L.T.50's are representative of relative toxicities at all time intervals. The critical dosage based on L.T.50 was approximately 0.5-1.0 p.p.m.

The criterion for the critical dosage was here assumed to be the largest concentration which took longer than 168 hours (1 week) to produce 50% kills in a given species at 15°C. After this arbitrary time degradation of the pesticide 65*

Bembidion tetracolum (dV 6.5 32 8 4 2 I ppm ppm ppm ppm ppm

6.0

5.5 KILL

OF 5.0 Fig, 9 T OBI

PR 4.5

3.5 Is, 0.0 0.4 0.8 1.2 1.6 ' 2-0 2.4 2.8 LOG TIME (HOURS)

6.5 0 / 2ppm/ 2 ppm • I P Pm • PPIr ("de V i? d'd QS A I •

5.5

16 5.0 Fig. io

EB 0 cc 0- 4.5

4.0

3.5 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 LOG TIME (HOURS) Figs. 9 & 10 Probit regression Ones for Bembidion tetracolum on sandy loam soil treated with ground thionazin granules a kept at 15°C (lines calculated) Table 14 Toxicity of thionazin in sandy loam soil to Bembidion tetracolum (d.44 at 15°C.+ 0.5°C; soil moisture approx. 20% by weight

Concentration Slope of L.T.501 95% Fiducial Toxicity relative to (p.p.m.) Regression Line (hours) limits (hours) mortalities at 1 p.p.m.

32 14.95 + 2.09 1.12 + 0.02 1.07 & 1.17 16 5.77 +'1.04 3.98 + 0.22 3.55 & 4.41 x 55.0 8 5.13 + 1.17 6.31 + 0.51 5.31 & 7.31 x 34.7

4 4.08 + 0,68 20.89 + 1.87 17.22 & 24.56 x 10.5 . 2 3.72 + 0.73 46.77 + 2.87 39.18 & 54.36 x 4.7

1 4.82 1.- o.68 218.8 + 15.1 189.2 & 248.4 OM. 0.5 1.80 + 0.34 955 + 151.6 658 & 1613 *

1 Residual contact action 67.

as well as movement of the insect away from the sites of

treatments might be expected to become limiting factors. Mortalities at 0.5 p.p.m. differed little from those in the controls which were now dying from starvation. This experiment confirmed the remarkable ability of some carabid individuals to withstand long periods of starvation. The last individual in control (non-treated) soils died on about the 60th day whereas one individual still remained alive on soil treated at 0.5 p.p.m. The proportions and mortalities of the sexes were not recorded separately in this experiment and hence the estimated critical dosage cannot be compared with data obtained in later experiments. From this and other pilot experiments the need to control all possible variables became apparent and in subsequent tests mortalities of the sexes were recorded separately, soil moisture was regulated more exactly (in most experiments soil was maintained at a moisture content of 15% by weight = 50% of sticky point = 76% of field capacity (Table10)) and test insects were preconditioned for standard times (e.g. 24 hours) to the ambient temperature of the C.T. room. Tests were done in closed vessels for in spite of probable increased fumigant effects the results were reproducible because (1) evaporation

of water from the soils was standardised at a very low level (2) relative humidity was maintained at a constant and high level (96-100%) and (3) cross canteinntton by fumigation between adjacent treatments, although most unlikely, was 68. avoided.

Tho critical dosage of thionazin in sandy loam soil to various species of carabids.

The results of bioassay tests with Bembidion tetracolum are shown in Fig.10 Appendix 4 and L.T.50's are summarized in Table 15. After 240 hours (10 days) only one female had died on soils treated at 0.5 p.p.m. The experiment was terminated after 336 hours (14 days) no further mortalities having occurred on the soils treated at 0.5 p.p.m.

Table 15 Toxicity of thionazin in sandy loam soil to Bembidion tetracolum Wand ?? ) at 15°C

p 4,05 ° C.

Concentration Sex L.T.50 95% fiducial (p.p.m.) (hours) limits (hours)

2.0 14.79 + 0.44 13.92 & 15.66 2.0 17.38 + 0.60 16.20 & 18.56 1.0 48.98 + 1.92 45.23 & 52.73 1.0 102.3 •4- 4.94 92.6 & 112.0

The results indicated clear differences in susceptibilities of male and female individuals possibly because of differences in weight; surface area relationships and were especially marked at the concentration of 1.0 p.p.m. (Fig.10) which proved to be near the critical dosage in this experiment. Critical dosages at 15°C were also determined for Bembidion lampros and Pterostichus vulgaris which were the most

69. abundant small and large species respectively in arable fields at Ashurst Lodge (Part.III). Time/mortality data for these experiments are presented in Appendices 5(A and B) and 6 (A and B) and L.T.50's with 95% fiducial limits are summarized in Table 16. Table16 Toxicity of thionazin in sandy loam soil to Bembidion lampros and Pterostichus vulgaris as determined by L.T.50 values

Species Sex Concn. L.T.50 95% fiducial (p.p.m•) (hours) limits (hours)

B.lampros 16 1.48 0.051 1.38 & 1.58 16 1.62 0.056 1.51 & 1.73. ote 8 3.24 0.112 3.02 & 3.46 8 3.80 0.227 3.36 & 4.24 4 12.30 0.566 11.19 & 13.41 4 13.18 0.637 11.93 & 14.43 2 52.48 4.587 43.49 & 61.47 2 44.67 7.050 37.62 & 51.72 1 446.7 76.03 297.7 & 595.7 1 316.2 35.64 246.4 & 386.1 P.vulgaris 8 34.67 1.75 31.23 & 38.11 99 8 66.07 3.95 58.33 & 73.81 4 81.28 4.11 73.22 & 89.34 99 4 501.2 108.4 288.8 & 713.6

Differences in susceptibilities of male and female Bembidion lampros were not so marked as for Pterostichus vulgaris and this was attributed to differences in weight or size (Table 22). Unlike P.vulgaris the mean weights of 70.

male and female B.lampros were not significantly different (P =

Toxicity of thionazin in soil as affected by environmental factors Soil moisture: Newly emerged adults of Bembidion tetracolum were used in bioassay experiments to assess the influence of water on the toxicity of thionazin granules in soil (other factors being kept constant as described in "Methods") and the results are recorded and analysed in Appendix 7(A and B). Table 17 summarizes the L.T.50 values for each regression

line and the values of the L.T.500 s (hours) have been plotted against soil moisture (% field capacity) in Fig.13. Control 72. 2.40 Best bid i on tetracolum

2-00 (4312)

r. U) cc 1.60 0

o 1.20 Fig. 11 -J 0 LO t_ 0.80

0.40

0.00 0.0 0.3 0.6 0.9 1.2 LOG CONCENTRATION (P PM)

2.80

2.40 Bembidion lampros • MALE ()FEMALE

2.00 cc O I1.60 0 Fig. 12

0 1.20 Lr) 1- 0-80

.0.40

0.00 0.0 0.3 0.6 0.9 1.2 LOG CONCENTRATION (PPM) Figs. II & 12 Relationship between LT5O (log hours) a log concentration (ppm) for adults of Bembidion tetracolum a B. lampros exposed to sandy loam soil treated with ground thionazin granules.

73. Table 17 Toxicity of thionazin in soil as affected by moisture content. Concentration of a.i. = 16 p.p.m., temperature 15°C ±- 0.5°C'

Soil Bembidion tetracolum Moisture AA (% by wt. oo of air dry soil) L.T.50 95% Fiduc. L.T.50 95% Fiduc. (hours) Limits (hours) Limits (hours) (hours)

0 * * * 1.5(16) 8.71+0.300 8.12 & 9.30 13.80+1.310 12.49 & 3.0(23) 4.07+0.094 3.89 & 4.25 6.61+0.243 6.13 & 7.09 4.5(29) 2.95+0.095 2.76 & 3.14 4.68+0.118 4.45 & 4.91 6.0(36) 2.57+0.053- 2.47 & 2.67 3663+0.142 3.35 & 3.91 7.5(43) 2.75+0.160 2.59 & 2.91 2.89+0.116 3.66 & 4.12 9.0(49) 1.95+0.049 1.85 & 2.05 3.09+0.078 2.94 & 3.24 12.0(63) 1.62+0.045 1.53 & 1.71 3.31+0.076 3.16 & 3.46 15.0(76) 2.00+0.028 1.94 & 2.06 2.63+0.067 2.50 & 2.76 18.0(89) 1.91+0.035 1.84 & 1.98 2.46+0.045 2.37 &c2.55. 24.0(116) 2.09+0.043 2.01 & 2.17 2.34+0.054 2.23 & 2.45 28.5(136) 2.09+0.111 1.87 & 2.31 2.75+0.082 2.59 & 2.91 30.0(143) 2.29+0.053 2.19 & 2.39 2.95+0.068 2.82 & 3.08

Figures in brackets denote moisture content as a percentage of field capacity. 74. mortalities did not occur on any of the damp soils and are not included in the tables but some occurred on the air dry soils and an explanation for this is given later. The results showed that there was a rapid increase in the toxicity of the pesticide with a relatively small increase in soil moisture (the relationship being concave curvilinear with an increase in moisture from 5 to 50-60% of field capacity) but toxicity did not increase with increasing moisture content above 60% and appeared to decrease very slightly above the 100% level. Two factors besides water may have influenced or contributed to the toxicities. These were (1) texture of soil in the absence of water and (2) soil compactness. On the soils which were kept dry 100% kills occurred on the treated soil within 9 hours but control mortalities were highly significant and were attributed to the fine texture of the soil possibly clogging up the spiracles, restricting movement and probably increasing desiccation through effects on the waterproofing mechanism as well as because the air was dryer. The fine texture of the soil resulted from the mixing it underwent in the Kenwood food mixer but the addition of water normally restored a good crumb structure by aggregating the finer soil particles. Mortalities on untreated soils of a coarser nature still occurred but at a latter stage when they were of little significance and were probably mainly due to natural desiccation rather than to enhanced desiccation through the 75.

Bembidion tetracolum RS) • male • female 8 HOU (

6 L T 50

0 0 20 40 60 80 100 120 140 SOIL MOISTURE I% FIELD CAPACITY / Fig. 13 Effect of soil moisture on the mortality of male a female Bembidion tetracolum exposed to soils treated with ground thionazin granules at a dosage of 16 p.p.m. and kept at I5°C (drawn by eye).

6.5 • • • Bembidion tetracolum

6-0

_I 5.5 uncompacted soil

U. 0 5•• I- 03 compacted 0 soil CC 4.5

4.0

3.5 0.2 0.3 0-4 0.5 LOG TIME (HOURS) Fig. 14 Effect of soil compaction on the mortality of Dembidion Ietracoluni exposed to sandy loam soil treated with ground thionazin granules a kept at 15°C (lines calculated). 76. physical action of the soil particles. Soil compaction was also affected by water and this probably influenced the burrowing activities of the carabids and hence the rate of pick up of pesticide. Soil compaction can be regulated by compressing a fixed weight of soil into a fixed volume. However, without compression, soils at both extremes of moisture content i.e. air-dry and water logged were found to be more compact than those at intermediate levels, the former because they lacked any crumb structure and the latter because they lacked any air spaces. Soil compaction: The effect of soil compaction was studied in greater detail using soils at 50% of the "sticky point". Soils in three replicate containers were compacted to a depth of 10.0 mm. (mean depth 9.9 mm. from 9 measurements) and left uncompacted in another three containers (mean depth 19.9 mm., 9 measurements). The results of this experiment are shown in Fig.14and Appendix 8 and L.T.50's are summarized in Table 18. Table 0 Toxicity of thionazin in compacted and uncompacted soil to Bembidion tetracolum females as determined by L.T.501 s (hours). Concentration a.i. = 16 p.p. m., temperature 15°C 0.5°C, soil moisture 15% ly_yeight (50% of sticky point).

Soil L.T.50 (hours) 95% fiducial limits (hours) Uncompacted 2.40 + 0.039 2.32 & 2.48 Compacted 2.88 7 0.033 2.82 & 2,04 77. There was therefore a greater initial kill of susceptible individuals on the uncompacted soils probably because they were able to burrow and came into greater contact with the pesticide. Differences in mortalities were more marked in susceptible than in resistant individuals as indicated by

convergence of the regression lines as per cent mortalities increased (Fig.14). Temperature and light: Absolute toxicity cannot be expressed in terms of rate of kill unless experiments using different concentrations are continued to the end point. In this section, however, toxicity is expressed in terms of rate of kill. As might be expected temperature markedly affected the toxicity of thionazin in soil as shown by the results of bioassay tests with Nebria brevicollis tabulated in Appendix 9 (A and B). Table 19 summarizes the L.T.50's at the various treatments and Fig.15 shows that the relationship of the L.T.50's (log hours) to temperature was concave curvilinear. Thionazin exhibited a positive temperature coefficient mortalities occurring 2.2 x faster at 10°C, 8.1 x faster at 15°C and 11.8 x faster at 22°C than at 7°C as determined by male Nebria brevicollis individuals at L.T.50 hours. The L.T.50 (hours) for female individuals of this species at 7°C was in excess of 168 hours and hence 16 p.p.m. was about the critical dosage at this temperature. Light, because of its effects on activity, also had a significant effect on mortalities as can be seen from 78.

Nebria brevicollis

0 male • female

7 10 15 20 22 TEMPERATURE (°C ) Fig. 15 Effect of temperature on the mortality of Nebria brevicollis exposed to sandy loam soil treated with ground thionazin granules at 16 ppm.

6.5 Nebria brevicollis crdl

6.0

continuous continuous —I 5.5 dark light

ur O 5.0 1- 0 cc a- 4.5

4.0

3.5 PS 1.6 1.7 1.8 1.9 2.0 2.1 2.2 LOG TIME (HOURS) Fig. 16 Effect of light on the mortality of male Nebria brevicollis exposed to sandy loam soil treated with ground thionazin granules at 16 ppm & kept at 7°C. (lines calculated). 79. Fig.l6. Nebria brevicollis is strictly nocturnal (Williams, 1959) and the rate of kill at L.T.50 (hours) was 1.6 x faster (at 7°C) in the dark than in continuous light.

Table 19 Toxicity of thionazin in sandy loam soil to Nebria brevicollis as affected by temperature and light (concentration of a.i. = 16 p.p.m., soil moisture 76% of field capacity)

Temp. Light Sex L.T.50 95% fiducial (°C) (Hours/24 hours) (hours) limits (hours)

70+0.5 24/24 89.13+3.49 82.30 95.96 7+0.5 24/24 177.8+9.22 158.6 197.0 7 +0.5 0/24 56.23+2.72 50.91 61.55 10+0.5 12/24 39.81+1.47 36.94 42.68 10+0.5 1 2/24 39.81+1.74 36.40 43.22 15+0.5 16/24 lo.96+o.28 10.42 11.50 15+0.5 16/24 42?- 13.49+0.31 12.88 14.10 22+0.5 24/24 eV 7.5944).3o 7.01 8.17 22+0.5 24/24 lo.96+o.38 10.22 11.70

* callow individuals

Soil pH: The effects of pH on the toxicity of thionazin in soil seemed relatively complex, as indi6ated by bioassay tests with Pterostichus vulgaris (Appendix 10(A and B). Table 20 summarizes the L.T.504 s for each time/mortality curve. Soils of different pH were prepared by adding powdered CaCO3 to an acid sandy loam soil (pH 4.8) from Four Acre Field and were then immediately treated with ground up thionazin 80. Table 20 Toxicity of thionazin in soil to Pterostichus vulgaris as affected by soil pH (Concentration of a.i. = 16 p.p.m., temperature 15°C .5°C)

CaCO3 pH Ageing Sex L.T.50 95% fiducial g/Kg. of soil (hours) limits(hours) soil (days)

0 4.8 1 6.46 + 0.49 5.50 & 7.42 o 4.8 1 8.32 + 0.29 7.76 & 8.88 1 5.7 1 6.76 + 0.51 5.76 & 7.76 1 ' 5.7 1 8.32 + 0.27 7.79 & 8.85 5 6.6 1 5.62 + 0.70 4.25 & 6.99 5 6.6 1 10.23+ 0.40 9.45 & 11.01 5o 6.9 1 5.62 + 0.85 3.95 & 7.29 5o 6.9 1 8.32 + 0.21 7.91 & 8.73

0 4.9 7 13.18 0.91 11.40 & 14.96 1 6.0 7 11.75 ± 0.30 11.17 & 12.33

5 7.6 7 12.02 * 0.33 11.37 & 12.67 50 7.6 7 6.61 * 0.40 5.84 & 7.38 0 4.9 14 ece 5.25 + 0.23 4.80 & 5.70 1 6.0 14 ore 8.71 1 0.34 8.04 & 9.38 5 7.6 14 &? 8.32 10.34 7.65 & 8.99 5o 7.6 14 (IV 6.92 44 0.33 6.27 & 7.57 81. granules. The addition of small quantities of CaCO3 e.g. 1-5 g./Kg. soil appreciably increased pH but larger quantities did not raise the pH above 7.6 (Table 20). The addition of CaCO3 did not have an immediate effect on the toxicity of thionazin in soil but, after 2 weeks of ageing at 15°C rates of kill on the very acid soils were significantly quicker = <0.05) than those on the slightly acid (pH 6.0) or alkaline (PH 7.6) soils. Determinations of thionazin by gas chromatography showed that the concentration of the chemically active ingredient was much higher in the acid soils (9.8 p.p.m.) than in soils treated with 1, 5 and 50 g. CaCO3/Kg. soil which contained 8.0, 6.0 and 6.9 p.p.m. of thionazin respectively. Loss of the pesticide had obviously occurred more rapidly on the alkaline soils and although this had initially resulted in quicker mortalities (see L.T.50 values at 1 week - Table 20) it was sufficiently greater after 2 weeks to cause an appreciable decrease in toxicity relative to that in the acid soils. These results on the persistance of thionazin in acid and alkaline soil are very similar to those obtained by Griffiths et al.(1967).

Toxicity of thionazin in soil as affected by biological factors: Syecies susceptibility: The relative susceptibilities of carabid species exposed to soils freshly treated with ground up granules of thionazin are recorded in Appendix 11(A and B). Table 21 82.

summarizes the L.T.501 s for each species under the stated conditions and Fig.17 attempts to correlate the L.T.50's with the mean weights of the species. Carabids chosen for these studies were those which occurred most abundantly in and around arable fields and included equal numbers of predominantly diurnal species (i.e. Trechus quadristriatus, Bembidion lampros, B.andreae, B.tetracolum, Asaphidion flavipes, Agonum dorsale and Loricera pilicornis) and predominantly nocturnal species (Nebria brevicollis, Harpalus aeneus, H.rufipes, Pterostichus madidus, P.vulgaris, Calathus fuscipes and Abax parallelepipedus). The species also included representatives of all weights and sizes ranging from about 2 mg. to >250 mg. (Table 22). Comparison of the L.T.50's showed that with but a few exceptions the smaller species of mean weight 2-10 mg. were 11-13 x more susceptible to a lethal concentration of thionazin than were the larger species (represented by Pterostichus vulgaris). The L.T.50's showed a closer relationship with surface area as indicated by mean weight 2/3 than by weight for carabid species of similar behaviour and habits (Fig.17). In addition the slope (b) of the regression line was equal to the average of the L.T.50 (log hours) per mg. weight2/3 - which can be denoted by a constant K (Table 21) - and further confirms this relationship. However, in addition to susceptibilities due to weight, size or sexual differences (the latter factor being correlated with size as shown by K

1-2 1— Af

ciaN b 0 Ap 1.0

cc • 0.8 0 0 51, k

0 0.6 0 male • female 0 ►n Af Asaphidion flavipes Ap Abax parallelepipedus I- Ha Harpalus aeneus --1 0-46- H r H. rufipes 0 00 0 H a Nb Nebria brevicollis

0.2

0.00 10 20 30 40 50 (WEIGHT—mgt4 Fig. 17 Effect of size on the mortality of Carabidae exposed to sandy loam soil treated with ground thionazin granules at 16 ppm a kept at I5°C.

TABLE 21 Toxicity of thionazin in sandy loam soil to various carabid species relative to L.T.50 of female Pterostichus vulgaris (Concentration of a.i. 16 p.p.m.t temperature 15°C 4. 0.5°C, soil moisture 157 by weight of air dry soil)

Species Sex L.T450 95% Fiducial tiv Relative (hours) Limits (hours) (L.T,50 toxicity per V2/3) at L.T.50 Trechus quadristriatus of 1.55 4- 0.06 1.42 & i.68 1.03 12.6 tt it 99 1.74 4- 0.08 1.59 & 1.89 1.03 11.2 1 Bembidion lampros do'? 1.48 + 0.051 1.38 & 1.58 1.02 13.2 11 11 99 1.62 7 0.056 1.51 & 1.73 1.03 12.0 Asaphidion flavipes ece 13.18 + 0.46 12.29 & 14.07 3.07 1.5 tt It 99 18.62 7 0.81 17.02 & 20.22 3.38 1.1 Bembidion andreae 1.66 -1 0.05 1.56 & 1.76 1.24 11.8 It tt 79 1.82 + 0.05 1.73 & 1.91 1.26 10.7 B.tetracolum2 car 2.00 0.03 It 1.94 & 2.06 1.19 9.8 11 2.63 4- 0.07 2.50 & 2.76 1.27 7.4 Agonum dorsale dog 2.00 + 0.06 1.89 & 2.11 1.16 9.8 11 Ti 19 2.46 + 0.06 2.34 & 2.58 1.16 7.9 Loricera pilicornis otr 2.24 ± 0.03 2.18 & 2.30 1.14 8.7 11 11 99 2.75 + 0.03 2.69 & 2.81 1.17 7.1 Harpalus aeneus3 crc7 2.29 + 0.07 2.14 & 2.44 1.07 8.5 11 2.63 + 0.09 2.45 & 2.81 1.07 7.4 Nebria brevicollis4 ol? 10.96 + 0.28 10.42 & 11.50 1.17 1.8 13.49 4- 0-.31 12.88 & 14.10 1.18 1.5 Calathus fuscipes cre 5.62 + 0.25 5.14 & 6.10 1.13 3.5 11 1.11 2.6 19 7.41 + 0.27 6.87 & 7.95 TABLE 21 Cont.

Species Sex L.T.50 95% Fiducial 'K' Relative (hours) Limits (hours) (L.T.50 toxicity per W2/3) at L.T.50 Harpalus rufipes de 5.01 0.14 4.74 & 5.28 1.08 3.9 22 5.25 ± 0.17 4.92 & 5.58 1.07 3.7 Pterostichus de 11.48 4- 0.48 10.55 & 12.41 1.10 1.7 madidus II I1 21? 14.79 4- 0.58 13.65 & 15.92 1.10 1.3 P.vulgaris eoe 11.48 + 0.45 10.60 & 12.36 1.09 1.7 11 19.50 ± 0.85 17.83 & 21.17 1.09 1.o Abax parallelepipedus citr 9.55.2 0.42 8.73 & 10.37 1.06 2.0 13.49 +- 0.65 12.21 & 14.77 1.06 1.5

1 = Table 16 2 = Table 23 3 = Table 23 4 = Table 19

86. TABLE 22 Mean weights of carabid species commonly occur'. ring in arable fields

Mean Weight of Adults (mg) Species Differ, in wt. No. Mean Stand. No, Mean Stand. between wd. wt. Dev. Wd. wt. Dev. sexes <10 mg. Trechus 100 1.6 0.62 100 2.4 0.52 * * * quadristriatus Bembidion 100 2.2 0.48 100 2.1 0.43 N.S. lampros Asaphidion 50 3.4 0.46 50 3.8 0.64 * * * flavipes Bembidion 100 3.6 0.60 100 4.1 0.80 * * * andreae B.tetracolum 100 7.6 0.89 100 7.9 1.06 * * 10-50 mg. Agonum dorsale 100 10.4 1.78 100 14.5 2.45 * * * Loricera 100 15.0 1.35 100 1.6.5 2.80 * * * pilicornis Harpalus aeneus 56 39.4 6.82 56 42.7 6.97 * 50-250 mg. Nebria 100 58.6 8.02 100 65.3 9.97 *** brevicollis Calathus 100 52.4 6.97 too 78.5 11.66 *** fuscipes Harpalus 100 98.1 16.47 100 119.5 22.10 *** rufipes Pterostichus 100 121.0 14.90 100 144.5 24.97 *** madidus P.vulgaris 100 149.0 16.33 100 195.2 30.26 *** > 250 mg. Abax parallel- 50 260.7 23.89 50 288.9 39.49 *** elaipedus * * * = Mean weight of sexes significantly different at P =<0.001 ** P =<0.01 * = Ir P =<0.05 N.S. = Not Significant. wd.= weighed; Stand.=Standard; wt.= weight; Dev. = Deviation; 87.

(log 10) values) striking differences in susceptibilities were noted as a result of differences in behaviour and activity. Thus Asaphidion flavipes and to a lesser extent Nebria brevicollis were less susceptible to thionazin than other species of comparable size (Fig.17) because they rarely burrowed into the soil while Harpalus aeneus and H.rufipes were more susceptible because they readily burrowed. Reasons for these behaviours are given in the discussion.

Armes

The results of a bioassay test with callow (newly emerged) and mature (second year) adults of Harpalus aeneus are shown in Figs.18,19.Table 23 summarizes the L.T.50 values for the probit regression lines.

Table 23 Toxicity of thionazin in sandy loam soil to Har~alus aeneus as affected byyage of the insect. (Concentration of ail. = 16 p.p.m., temperature 15°C, soil moisture 15% by weight)

AGE SEX L.T.50 (hours) 95% fiducial limits(hours)

Callow, newly emerged d& 1.86 4. 0.06 1.74 & 1.98 2.34 0.01 2.32 & 2.36

Mature, second year dvd? 2.29 4. 0.07 2.14 & 2.44 n 2.63 ± 0.09 2.45 & 2.81

In both sexes callow individuals were more susceptible at the L.T.501 8 than were mature individuals and whereas the slopes of the regression lines of the older individuals did 88.

6.6

Harpalus aeneus

6.0

5-5 • callow mature U- 0 5,0 Fig. 18

a 4.6

4.0

3.5 0.2 0.3 0.4 0.5 0.6 LOG TIME (HOURS)

6.5

Harpalus aeneus

6.0

callow mature ur 5. Fig, 19

0 cc 4.5

4.0

I 1 3.5 I 0.2 0.3 0.4 0.5 0,6 LOG TIME (HOURS) Figs. 18 & 19 Effect of age on.,the mortality of male a female Harpalus aeneus exposed to sandy loam soil treated with ground thionazin granules at 16 ppm a kept at 15°C (lines calculated) 89.

not appear to differ significantly from parallelism, those of the callow individuals did (Appendix i2). This difference in susceptibility of male and female callow individuals is a well known phenomenon and may be related partly with differences in chitinization of the cuticle and partly with the physiological condition of the insects associated with developing sexual organs. Starvation: Bioassay experiments were done with two species Bembidion tetracolum and Agonum dorsale, on soils treated with 2 p.p.m. and 16 p.p.m. of thionazin respectively and the results are shown in Figs.20 and 21. Table 24 Effect of starvation on the mortality of Bembidion tetracolum and A onum dorsale on sand loam soil treated at 2 p.p.m. and 16 p.p.m. respectively (Temperature 15°C, soil moisture 15% by weight)

SPECIES SEX DAYS L.T.50 95% FIDUCIAL STARVATION (HOURS) LIMITS (HOURS) BEFORE TEST B.tetracolum 4 19.50+0.54 18.45 & 20.55 If 0 21.88+0.55 20.79 & 22.97 It 24 4 22.39+0.52 21.38 & 23.40 7q. 0 25.12+0.64 23.87 & 26.37 A.dorsale 4 2.04+0.07 1.91 & 2.17 It o 1.82+0.05 1.71 & 1.93 4 2.04+0.05 1.94 & 2.14 11 -27 0 2.14+0.07 2.00 & 2.28

Table 24 summarizes the L.T.50 values of the time/mortality curves and data for the regression lines are presented in

90.

Bembidion tetracolum • starved ale (S.M.) O fed c?ol (r.m) 6.0 • starved $it (S.F.) • fed $g (F.F.) LL

OF KI Fig 20

PROBIT 4.5

4.0

3.5 1.1 1.2 1.3 1.4 1.5 1.6 LOG TIME (HOURS)

Agonum dorsale • starved die (S.11) o fed d'ol(F.m) 6.0 • starved vi (S.F.) .5 fed $? (F.F.) ILL K

5'0 Fig 21 T OF OBI

PR 4.5

4.0

0.2 0.3 0'4 0.5 LOG TIME (HOURS)

Figs. 20 & 21 Effect of starvation-on.the mortality of Bembidion'tetracolum & Agonum dorsale on sandy loam soil treated with ground'thionazin granules at 2 IT:p.m and 16 p.p.m respectively and kept ,at 15°C (lines calculated). 91. Appendices13 & /4. Starved B.tetracolum of both sexes were killed more quickly than fed ones by 2 p.p.m, thionazin (P =<0.05). The effects of 16 p.p.m. on A.dorsale were indistinguishable because both starved and fed individuals were killed too quickly. Toxicity of thionazin as affected by chemical properties Fumigant effects: Three experiments were done; (1) to assess fumigant effects in both closed and open containers as used in previous tests (2) to determine the effect of temperature on fumigant toxicities and (3) to obtain an estimate of the Concentration x Time product at the L.T.50. In (1) and (2) conditions were the same as before but the insects were sus- pended 2 in. above the soils on a perforated zinc platform. In this way they were allowed room for comparatively free movement without coming into contact with the soils. Time/ mortality data for these tests are shown in Appendix 15 (A and B) and Table 25 summarizes the L.T.50 values for the stated conditions. Tests in the closed containers at 22°C showed that the first mortalities occurred after 9 hours in the males and 13 hours in the females as compared to 5 and 7 hours respectively by residual contact action (Appendix 9a). Fumigant effects were 2.3 x greater in closed than open containers but control mortalities occurred in the open containers after 36 hours (Appendix 15a) probably as a result of desiccation. Control mortalities were not important in the closed containers and results were 92. reproducible as shown by L.T.50's in Group Replicates 1 & 2 (Table 25) and by the slopes of the regression lines (Appendix 15b). In terms of L.T.50's for male individuals in closed containers, fumigant effects were 19.5 times greater at 22°C than at 7°C. Table 25 Effect of temperature on the fumigant toxicity of thionazin in soil in closed and open containers as determined by bioassay with Nebria brevicollis (Concentration of a.i. 16 p.p.m,, soil moisture 15% by weight, hours light per 24 hours, zero)

TYPE OF TEMP. SEX L.T.50 95% FIDUCIAL CONTAINER (°C) (HOURS) LIMITS (HOURS)

Closed 7 245.5+10.7 224.5 & 266.5 9 (Gp.Rep.1) 22 12.59+0.35 11.91 & 13.27

tl 22 17.78+0086 16.10 & 19.46 " (Gp.Rep.2) 22 12.30+0.31 11.69 & 12.91

Open 22 28.18+7.52 13.44 & 42.92

(Gp.Rep. = Group replicate)

In (3) air of known humidity and flow rate was drawn over thionazin granules and then over the test insects and was then bubbled in 5 ml. of ethyl alcohol solution to trap any toxic vapours. The toxic effect of the thionazin as vapour in the air stream was noted by recording the rate at which mortalities occurred among the test insects. In a pilot experiment with Trechus quadristriatus (20 insects per treatment) 100% mortalities occurred within 24 hours in the 93. treated tube while no mortalities were recorded in the control„ In the same circumstances Bembidion laMpros (4) responded as follows: (1)After one hour - primary symptoffis of poisoning were noted in several individuals (i.e. unco-ordinated and jerky movements). (2)Time : two hours - 50% of treated insects seriously affected i.e. L.T.50. These were unable to right themselves after falling over on to their dorsal surfaces. (3)Time : three hours - all individuals showing symptoms of poisonings (4)Time : four hours : all beetles seriously affected and probably beyond recovery. The presence of thionazin in the 5 ml. of ethyl alcohol solution was determined by ultra- violet spectrophotometry. Comparison with a series of standard solutions of thionazin indicated a concentration of 6 µg/ml. The concentration m time product at the L.T.50 was therefore calculated as follows: Rate of flow of soap film mg 100 ml. in 80 secs. = 4,537 ml./hour. From assay, concentration of thionazin = 6 µg/ml./4 hours. Therefore total concentration of thionazin = 5 x 6 = 30 pg. hours i.e. 30 pg.of thionazin were present in 4 x 4.537 litres of air and the C.T. product at L.T.50

was therefore 30 x 2 or 3.3 pg.hrs./1. 4.537 x 4 94. Persistence of thionazin in sandy loam soil Persistence was determined by comparing L.T.50's of male and female Bembidion tetracolum on soils aged at 15°C with L.T.50's on soils freshly treated with known concentrations of the pesticide i.e. the "standards". The results of these studies with determinations of the L.T.50's (log hours) from the calculated probit regression lines are shown in Appendix 16a (I and II) and 16b (I and II) respectively and the L.T.50's (in hours) with 95% fiducial limits are summarized in Tables 26 and 27 for the standard and the aged soils repectively. The relationship of the L.T.50's (in log hours) to log concentration of the standards are shown in Fig.22 and from these regression lines it was possible to read off the concentration of the pesticide in the aged soils for the appropriate L.T.50 values in Appendix 16b (II). Table 26 Determination of L.T.50 values for male and female Bembidion tetracolum on soils treated with known concentrations of thionazin i.e. the "standards" CONC. SEX L.T.50 95% FIDUCIAL LIMITS (p.p.m.) (hours) 7 cfril 5.62 + 0.17 5.29 & 5.95 7 72 8.32 + 0.19 7.95 & 8.69 6 47 10.72 + 0.35 10.04 & 11.40 5 81)." 7.59 + 0.18 7.25 & 7.93 5 72 12.30 0.34 11.64 & 12.96 4 99 15.85 + 0.69 14.49 & 17.21 3 17.38 + 0.64 16.13 & 18.63 3 ad" 24.00 + 0.72 22.59 & 25.41 95.

Table 27 Persistence df thionazi,h in soil aged in closed and open containers, at. 15°C as determined by L.T.50 values bf male and female Bembidion tetracolum

TIME TYPE OF SEX L.T.50 95% FIDUCIAL (weeks) CONTAINER (hours) LIMITS (HOURS)

0 Closed cre 3.16 + 0.08 3.00 & 3.32 0 Open JO3.24 + 0.05 3.15 & 3.33 0 Closed 9? 5.62 + 0.16 5.32 & 5.92 O Open 7? 5.75 +.0.13 5.49 & 6.01 1 Closed db7I 8.71 + 0.20 8.32 & 9.10 1 Open edi 6.92 + 0.19 6.55 & 7.29 1 Closed V? 13.80 + 0.54 12.74 & 14.86 1 Open V? 12.59 + 0.38 11.85 & 13.33 2 Closed dY 15.14 + 0.38 14.39 & 15.89 2 Open oVI 14.13 + 0.42 13.30 & 14.96 2 Closed ?? 21.88 + 0.65 20.60 & 23.16 2 Open ?? 21.38 + 0.39 20.61 & 22.15 3 Closed ?c?' 22.39 + 0.52 21.38 & 23.40 3 Open d84 22.39 + 0.31 21.78 & 23.00 3 Closed 22 31.62 + 0.66 30.34 & 32.90 3 Open ql 30.90 + 0.50 29.93 & 31.87 4 Closed d6/1 38.02 + 0.61 36.82 & 39.22 4 Open dol% 31.62 + 0.87 29.11 & 33.33 4 Closed 45? 51.29 + 1.42 48.53 & 54.05 4 Open n 48.98 + 1.24 46.55 & 51.41

Estimates of the residues based on mortalities of both male and female individuals in either closed or open containers were very similar as shown in Table 28.

96 .

I.2 ) 1.0 PPM ON (

TI 0.8 Fig. 22 NTRA 0.6 — NCE

CO 0.4 OG L

0.2

0.0 0.4 0.6 0.8 1.0 I.2 I.4 I.6 I.8 L T 50 (log hours) I.2

M) PP 0.8 N ( TIO 0.6 Fig. 23 TRA NCEN 0.4 CO G O L 0.2

0.0 0 I 2 TIME (WEEKS) Fig. 22 Mortality of male a female Bembidion tetracolum (as shown by LT5O values) when exposed to sandy loam soil freshly treated with ground thionazin granules Fig. 23 Persistence of thionazin in sandy loam soil as shown by LT5O values of B.letracolum on soil aged at I5°C.

07e Tabl.e 28 persistencl4ofm hignaiin residues in soil as determined by bioassay tests with Bembidion tetracolum TIME TYPE OF CONCENTRATION (weeks) CONTAINER (p.p:in.). based ont- oP4

0 Closed 10.7 10.0 0 Open 10.5 9.8 1 Closed 4.8 4.7 1 Open 5.8 5.0 2 Closed 3.2 3.2 2 Open 3.3 3.2 3 Closed 24.3 2.3 3 Open 2.3 2.4 4 Closed 1.6 1.6 4 Open 1.8 1.6

Two sources of error are evident in this experiment. The first is that estimates of L.T.50's on the standards were not obtained at each time interval for direct comparison with the L.T.50's on the aged soils and hence no account was taken of possible differences in susceptibilities of the test organisms with time. The second is that the L.T.50's on the standards did not adequately cover the range of those obtained on the aged soils and hence estimates of the "unknown" concentrations in the aged soils had to be 'obtained by extrapolation of the standard log concentration/ L.T.50 regression lines. In both cases these were due to a chronic shortage of test organisms which severely limited the number of tests that could be done. The results, nevertheless, give an indication of the persistence of thionazin in terms of biologically active ingredient and suggest a

98.

very rapid breakdown, log concentration being linearly related to time (Fig.23). The relationship between normal concentration and time of bi"eakdown is concave curvilinear,

which seems to be typical of the degradation of all pesticides in soil. Extended studies on the pei-sistence of thionazin in soil at 15°C indicated that at a concentration of 8 p.p.m. in light sandy loam (which is equivalent to a dosage rate of approximately 16 lb a.i./acre) thionazin would not be a serious threat to most carabid species 21 weeks after the time of application (Table 29 Appendix 17 and Fig.24)4,

Table 29 Persistence of thionazin in sandy loam soil over a period of 21 weeks as determined by bioassay with Bembidion tetracolum

TIME SEX L.T.50 95% FIDUCIAL (weeks) (hours) LIMITS (hours) 4 4 27.54 + 1.14 25.31 & 29.77 13 4 53.70 + 1.24 51.28 & 56.12 21 eq. 269.2 + 13.0 243.7 & 294.7

Comparative tests of contact toxicity of other soil applied

systemic pesticides to carabids (1) Toxicity of ground phorate granules in soil Phorate treated soils were prepared as for thionazin and conditions were as before. The toxicity of phorate was compared on three species i.e. Trechus quadristriatus, Nebria brevicollis and Pterostichus vulgaris which represented 99.

6.5.-

13 /1 6.0 weeks weeks

5.5

—J J

18 5.0 I— ca 0 cc a.

4.5

4.0

3.5 I I I I 1 J I 0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 LOG TIME (HOURS)

Fig. 24 Persistence of thionazin in sandy loam soil as shown by time/mortality studies with Bembidion tetracolum exposed to soils treated at 16 ppm a aged for 0, 4,13, & 21 weeks respectively at 15°C (lines calculated). 100. broadly the size range of the species commonly occurring in arable fields. Results of time/mortality tests are recorded in Appendix la (A and B) and the L.T.50 (hours) from the calculated probit regression lines are summarized in Table 30 .

Table 30 Toxicity of phorate in sandy loam soil at 15°C + 0.5°C to several carabid species based on L.T.50's

SPECIES SEX CONC. L.T.50 95% FIDUCIAL (p.p.m.) (hours) LIMITS(hours)

Trechus quadristriatus 32 1,02+0.03 0.97 & 1.07 16 1.10+0.03 1.05 & 1.15 I/ 4 2.63+0.07 2.50 & 2.76 4.90+0.08 075 & 505

32 2.51+0.06 2.40 & 2.62 16 3.31+0.05 3.22 & 3.40 4 5.89+0.15 5.60 & 6.18 1 17.78+0.45 16.90 & 18.66 32 3.16+0.08 3.00 & 3.32 16 3.80+0.13 3.54 & 4.06 4 7.59+0.30 7.01 & 8.17 1 22.39+0.57 21.28 & 23.5o Pterostichus vulgaris 32 1.45+0.03 1.40 & 1.50 II 11 16 1.91+0.06 1.77 & 2.05 4 6.17+0.30 5.59 & 6.75 ti 1 26.30+2.06 21.71 & 30.89 It 32 1.86+0.04 1.78 & 1.94 16 2.69+0.09 2.52 & 2.86 it 4 10.23+0.57 9.12 & 11.34 1 79.43+12.42 55.08 & 103.78 1014 Phorate was several times more toxic than thionazin at all the concentrations tested; for instance, at 16 p.p.m. its relative toxicity to Nebria brevicollis based on L.T.50's was 3.4 x and 3.6 x greater for male and female individuals respectively and for Pterostichus vulgaris it was 6.2 x and 1.9 x more toxic to male and female individuals respectively. The critical dosage of phorate at 15°C was appreciably less than 1 p.p.m. for most species of carabids. The persistence of phorate in sandy loam soil at 15°C 0.5°C was determined by bioassay with Pterostichus vulgaris and the results of time/mortality data are recorded in Appendix 19 (A and B) and L.T.50's are summarized in Table 31. Although these results are not truly comparable with those of thionazin they suggest that the rate of loss of phorate from sandy loam soil was 1-2 times faster than for thionazin (cp. Table 30 )

(2) Toxicity of ground disulfoton granules in soil Disulfoton was not appreciably toxic to carabids at concentrations less than 32 p.p.m. (= 64 lb. a.i./acre) when tested at 15°C. Time/mortality tests with three species exposed to soils treated at 32 p.p.m. are recorded in Appendix 20 (A and B) and the L.T.50's from the calculated probit regression lines are summarized in Table 32. 102. Table 31 Persistence of phorate in sandy loam soil at 15°C as determined by bioassay with Pterostichus vulgaris

CONCN. TIME SEX L4T.50 95% FIDUCIAL (p.p.m.) (weeks) (hours) LIMITS (hoUrs)

32 01 1.45 + 0.03 1.40 & 1.50 32 1.86 + 0.04 1.78 & 1.94 16 1.91 + 0.06 1.77 & 2.05 16 2.69 + 0.09 2.52 & 2.86 4 6.17 + 0.30 5.59 & 6.75 4 10.23 + 0.57 9.12 11.34 1 26.30 + 2.06 21.71 30.89 1 79.43 + 12.42 55.08 103.78

32 4 do 2.46 + 0.13 2.20 & 2.72 32 3.24 + 0.12 3.01 & 3.47 16 as 4.07 + 0.20 3.68 & 4.46 16 4.90 ± 0.03 4.30 & 5.50 4 EN, 50.12 ± 6.57 37.24 & 63.00 4 104.7 ± 17.58 70.2 & 139.2 1 N.S. 1 N.S.

32 22.4 9.12 + 0.29 8.54 & 9.70 32 15.49 + 1.00 13.53 & 17.45 16 33.88 ± 3.20 27.62 & 40.14 16 199.5 ± 18.8 162.6 & 236.4

N.S. = Not significant from control mortality 1 = from Table 30

103i

Table 32. Toxicity of disulfoton in sandy loam soil at 15°C + 0.5°C to various species of carabids.

Concentration of a•.i. = 2 P.p.m.)

SPECIES SEX L.T.50 95% FIDUCIAL (hours) LIMITS (hours)

Pterostichus vulgaris d&' 26.30 + 4.60 17.29 & 35.31 ft 1096 ± 960 - ?V & 2978 Agonum dorsale dd' 7.41 + 0.17 7.08 & 7.74 qi 8.91 + 0.35 8.23 & 9.59 Bembidion lampros d'd' 40.74 4. 2.25 36.33 & 45.15 39.81 4. 2.66 34.61 & 45.01

The results indicated that mortalities were not comparable with those of thionazin and suggested differences in chemical properties and uptake of the pesticide. Disulfoton has a lower vapour pressure than thionazin and consequently is less volatile. Toxicities would therefore be expected to be due mainly to direct contact action. This could explain why Agonum dorsale was more susceptible to the pesticide than Bembidion lampros for in spite of its larger size it was much more active in the experimental conditions then was B.lampros. Mortalities of male Pterostichus vulgaris were, however, anomalous and must be treated with some suspicion for although 86.7% of the treated insects died within 96 hours (4 days) 100% mortalities did not occur until 768 hours (32 days) (Appendix 26A). The concentration of disulfoton in the soil must therefore have been at about 104.

the critical dosage for this species. (3) Toxicity of ground menazon granules in soil Menazon was non-toxic at all dosage rates recommended for field applications which confirms its claim as being a truly selective systemic aphicide (Price-Jones, 1961). However, in spite of this, tests were done with extremely high dosages to observe any possible deleterious or toxic effects and these experiments are recorded and analysed in Appendix 21 (A and 3) and values of L.T.50's in these conditions are shown in Table 33.

Table 33 Toxicity of menazon in sandy loam soil at 15°C +

0.5°C to Harpalus rufipes and Pterostichus vulgaris SPECIES SEX CONC. L.T.50 95% FIDUCIAL (p.p.m.) (hours) LIMITS (hours)

Harpalus rufipes ece 512 1778 + 30.3 118.5 & 237.1 7V• 512 524.8 + 26.6 472.7 & 576.9 H d'd' 256 588.8 + 37.9 514,5 & 663.1 11 II 77 256 912 + 55 805 & 1019 Pterostichus vulgaris 10,000 7.24 + 0.38 6.49 & 7.99 u 77 10,000 14.13 + 0.88 12.41 & 15.85

Even at a concentration of 512 p.p.m. (equivalent to a dosage rate of 1024 lb. a.i./acre) the insects died very slowly and only 33% of the males were dead after 168 hours (the arbitrary time limit for the critical dosage).

105.

Mortalities after this time were of interest for although food was provided, feeding appeared to be inhibited with the result that death was probably due mainly to starvation. This therefore may be one consequence of so-called sub- lethal dosages.

Comparative toxicity of thionazin, phorate, disulfoton and menazon to carabids

Table 34 attempts to summarize the relative toxicities at L.T.50 (hours) of the four pesticides studied. The table also indicates that the relative toxicity in the order most toxic to least toxic was phorate > thionazin disulfoton > menazon. Table 34 Comparative toxicity of four systemic pesticides at 15°C 0.5°C to carabids at L.T.50 (hours) * unaffected

SPECIES SEX CONC. L.T.50 (hours) (p.p.m.) P T D M Trechus quad- (37 16 1.10+ ristriatus 0.03 11 11 gg 16 1.55+ 0.06'" tt it 16 1.74+ W 0.08— Nebria brevi- St 16 3.31+ 10.96+ collis 0.05 0,28 — it tt 78 16 3.80+. 13.49+ 0.13 0.31 Pterostichus SS 16 1.91+ 11.48+ vulgaris 0.06— 0.45 tt 16 2.69+ 19.50+ 0.09 o.85 It tS 32 1.45+ 26.30+ * o. o3 4.6o It ?? 32 1.91+ 1096+ * o.o6 96o— It SS 10,00o 7.24+ o.38 tt .,? 10,000 14.13+ 0.88— 106. DISCUSSION

The factors influencing the toxicity and effectiveness of a soil pesticide can be divided into three main categories : soil and environmental factors, biological factors and chemical factors. The soil and environmental factors include organic matter, mineral content, porosity, pH, temperature and moisture (both content and rainfall effects). The important biological factors are species and size of organism, sex, age, respiration and metabolism and behaviour, while those of the chemical (i.e. pesticidal) factors are rate of reaction, formulation, vapour pressure, solubility (in water) and diffusibility (in air) and rate of conversion to toxic or non toxic metabolites. These interactions must be extremely complex and will affect whole animal communities. To appreciate fully their overall significance requires an ecological approach to this problem but an evaluation of their individual effects can only be done adequately in controlled laboratory conditions which, however, have one major limitation - that results can only be assessed in relative and not absolute terms to field conditions. Caution is therefore required in interpreting laboratory experiments for results in the final analysis can only be as good as the methods used in obtaining them. Carabids are very active insects. They are unlikely to remain in one area for any length of time, although their movements might be expected to be at random and will 107. be governed by weather, food and their physiologiCal condition. Pesticide treatments may be bro4dcast over a field or may be confined to rows or to plants. The method of application will therefore affect the length of time when an insect is exposed to a toxic chemical and will influence its susceptibility to the themical. This applies especially to carabids which are likely to come into contact with pesticide treatments for limited periods of time. It seemed likely therefore that time/mortality studies would prove as important in assessing toxic effects of pesticide treatments as dosage/mortality studies. For this reason toxicity was measured in terms of rate of mortality at a given temperature rather than by the more conventional method of dosage per unit weight or area. The lack of numbers of one species at a given time also prohibited the testing of a wide range of concentrations necessary for dosage/mortality studies, while in similar circumstances time/mortality studies yielded much more information. Of the soil and environmental factors investigated soil type was kept constant throughout since the objective of this study was to correlate the more variable factors of the locality with results observed in the field. Comparative studies on the effects of organic matter and mineral content were therefore not done. Soil moisture was found to have a pronounced effect on the biological activity of thionazin in soil (Fig.13)but it was not directly proportional to toxicity which suggests that it affected processes such as 108.

adsorption and desorption of the pesticide to soil particles and of movement through the soil pores rather than chemical properties (Barlow and Hadaway, 1956; Lichtenstein, 1958). Hence water in excess of 50% of field capacity did not increase toxicity of thionazin but, if anything, slightly decreased it. This may have been due to two main causes (1) a reduction in the burrowing activities of the carabids owing to the elimination of air-spaces in the soil and (2) a reduction in the chemical activity of the pesticide. Call (1957a, 1957b) believes that sorption of ethylene dibromide to soils at field capacity is partly due to solution in the water and partly to sorption at the water inter- faces. The same physical process may occur with thionazin although Harris (1964a, 1967) has shown no indication of this in his studies with other organo-phosphorus pesticides perhaps because of a difference in experimental approach. When soil is saturated or nearly so, loss in effectiveness of the pesticide may also occur through a reduction in vaporization (Harris and Lichtenstein, 1961) although this might be expected to be small for thionazin. Harris (1964a) concluded that inactivation of pesticides in moist soils was proportiona to organic matter and that whereas the majority of pesticides under investigation became more toxic with increased soil moisture some such as Mevinphos became less toxic (Harris, 1964b).

Relative humidity of the air above the soil surface must be closely related with soil moisture but this factor 109. could not be tested in the small test containers and required a different bioassay technique. Relative humidity has been shown to affect the persistence of residues (Kalkat et al., 1961) and Lyon and Davidson (1965) found that in high humidity there was a rapid change of pesticide into the vapour phase. Another environmental factor not investigated in this study was wind. Dustan et al. (1947) found that this could reduce greatly the effectiveness of pesticides with pronounced fumigant action. In the present study experiments in closed and open containers also suggested that wind could reduce the fumigant toxicity of thionazin in soil to carabids. The effect of temperature on the toxicity of thionazin in soil was studied in the range 22°C-7°C since the upper and lower temperatures were thought to correspond nearest to the mean maximum and mean minimum soil temperatures at Ashurst Lodge during field applications in late spring and early summer. It was appreciated, however, that temperatures can and do rise or fall much above or below these values (although mostly for short intervals) but would also inevitably affect the activity and physiology of the insects and high temperatures only occur in the day when nocturnal species are inactive. Small increases in temperature, like soil water, increased the toxicity of thionazin as indicated by rate of kill; the relationship was again curvilinear (Fig.15) which suggested that other factors acting antagonistically at high 110.

temperatures are involved. Thus at high temperatures evaporation of water from the soils increased resulting in a decrease of chemical and microbial reactions and an increase in adsorption of the pesticide to soil particles. Effects of temperature on inherent toxicity and availability of the pesticide cannot be separated from those on the insect's behaviour and physiology which affects factors like pick-up of the pesticide. Thionazint like some other organophos- phates has a positive temperature coefficient but some pesticides notably D.D.T. have a negative temperature coefficient (Sweetman, 1945; Burgess and Sweetman, 1949). The effect of light on the toxicity of thionazin in soil was studied in relation to its influence on the activity of carabids some of which are predominantly nocturnal while others are predominantly diurnal. Thus with Nebria brevicollis which is strictly nocturnal (Williams, 1959). light considerably reduced activity and significantly decreased the mortality rate (Fig.16). It is concluded that light may have an appreciable effect on mortalities in bioassay tests and should be carefully standardized in terms of the time of day when the experiment is started and also of day length (see Fig.20). Effects of soil compaction were studied in a special experiment and also in experiments on soil moisture which affects soil compaction. Fleming and Baker (1935) in a study of the use of carbon disulphide as a soil fumigant found that diffusion was most rapid in permeable soils and was retarded by compaction and by a high moisture content. However, some pesticides which are mainly involatile but are soluble in water e.g. Menazon and dimethoate move more rapidly in moist than in dry soils (Burt et al., 1965). The movement and activation of thionazin in soil are probably governed by its physical and chemical properties but its toxicity to carabids must also be influenced by the beetles' ability to burrow into the soil where they would inevitably come into greater contact with the pesticide granules and a higher concentration of vapour. Soil pH did not seem to affect carabid activity but in the long term influenced the toxicity of the pesticide. The results obtained were in close agreement with those of

Griffith et al. (1967) since its persistence is decreased by decompositoon in alkaline conditions, Experiments on species susceptibility showed that the adults of small species were as a rule more susceptible than larger species. Species susceptibility was, however, modified by the behaviour of the insects and in particular by their burrowing and other activities. Carabid adults sometimes burrow in search for food (Scherney$ 1955) but this behaviour is probably more important in avoiding detection. Burrowing occurred only in fairly moist soils and if the soil was too dry the carabids attempted to hide under stones or in cracks in the soil. Some species, however, 112. particularly Asaphidion flavipes, were never seen to burrow and they were able to escape detection through their remarkable cryptic coloration. The-evolutionary significance of this coloration is not certain but it is postulated that this developed as a result of their feeding habits which involve capturing and eating epigaeic Collembola. In order to do this the beetles remain perfectly still until their prey is only a few mm. away when they are then pounced upon. The cryptic coloration therefore serves two purposes; i.e. it camouflages the predator from its prey but perhaps more important it camouflages the predator from other larger predators (e.g. birds) when it is most vulnerable to attack i.e. when waiting for its prey. It is concluded therefore that other diurnal carabid species such as Notiophilus spp. feeding predominantly on surface living Collembola would also have similar habits and consequently would also be expected to be comparatively resistant to thionazin. Van der Drift (1951) has commented on the ability of diurnally active Notiophilus spp. to evade capture by pitfall traps and attributed this partly to their small size and ability to recover balance on the edge of traps and partly to their well developed eyes which enables them to see and avoid the traps. Nebria brevicollis is also said to feed mainly on Collembola (Davies, 1953; Penny, 1966) but since this species is nocturnal in habit (Williams, 1959) it has no need for cryptic coloration• It was also shown in the 113. the results that some species, notably Harpalus aeneus and H.rufipes, are more susceptible than other carabids of comparable size. These species are predominantly phytophagous (Davies, 1953; Briggs, 1965) which probably accounts for their differences in behaviour from the predatory species. They are active meanly at night (but not all phytoithagous

carabids are nocturnal e.g. Amara spp.) and during the day they burrow into the soil to escape detection. As a result they probably come into greater contact with the soil pesticides than other species which do not burrow and could account partially for their greater susceptibility. If burrowing fails to conceal the beetles and they are disturbed or attacked by would-be-predators they are capable of ejecting a very potent repellent fluid (probably formic acid) which in

some circumstances (e.g. when confined in an enclosed space) could cause their own deaths. This behaviour may be triggered off by the pesticide and could also partially account for their increased susceptibility. It might be expected that other species with similar behaviour such as Anisodactylus binotatus and even Carabus violaceus - which was the largest carabid species found in arable fields at Silwood Park - would also be particularly susceptible to thionazin. However, some species without such behaviour may nevertheless be susceptible e.g. Abax parallelepipedus. This species occurs predominantly on the border between arable fields and woods at Silwood Park and was rarely caught within a crop. This 114. suggests that the species may not have been influenced by pesticides as much as those common to arable fields and if the latter have developed some resistance to pesticides through the years this could explain the reason for its apparent susceptibility. One of the more important chemical factors influencing the toxicity of a pesticide to carabids appears to be vapour pressure since it is this factor which determinee or regulates its volatility at a given temperature. Thus both phorate and thionazin which were extremely toxic to carabids at low dosages have relatively high vapour pressures whereas disulfoton which had a lower vapour pressure was less toxic and menazon which has no detectable vapour pressure at 25°C and is said to be involatile (Price-Jones, 1961) is non toxic at normal dosages. Menazon, however, is known to be physiol- ogically very selective in action irrespective of its physical properties. The persistence of thionazin was studied in sandy loam soil kept at 15°C + 0,5°C. When applied in soil at a dosage rate of 8 p.p.m. (16 lb a.i./6 in. acre) its toxic effects to adult carabids were visible for a maximum period of about 21 weeks but if as might be expected thionazin disappears mare rapidly in field conditions because of leaching, evaporation from the soil surface, uptake by plants and animals, break down by microbial activity (factors discussed in greater detail in Part.IV) then this period should be reduced in the 115.

field. Long term effects cannot, however, be evaluated from laboratory experiments without a knowledge of (1) direct effects on eggs, larvae and pupae of carabids (a) indirect effects on other organisms in the food chains and (3) the ability of different carabid species to recover or to recolonize a habitat made unfavourable by pesticide treatments. An attempt has been made to answer these questions in field experiments which are described in detail in Part III. 116. SUMMARY & CONCLUSIONS

1. The persistence and toxicity of organophosphorus systemic pesticides in soils to different species of adult carabids were studied in controlled laboratory conditions.

2. The effects of environmental, biological and chemical factors on toxicity were evaluated by time/mortality studies

using one pesticide i.e. thionazin and one soil type i.e. acid sandy loam (pH 6.1-6.7). Comparative tests of toxicity were done with phorate, disulfoton and menazon.

3. Tests to determine the minimal effective or "critical dosage of thionazin in soil indicate that mortalities of carabids occurs at or below the dosage rates normally required for satisfactory control of either nematode or insect pests of agricultural crops.

4. Water appreciably increases the activity and toxicity of thionazin in soil but the relationship of toxicity to soil moisture is concave curvilinear and above 50% of the field capacity there is no increase in the mortality rate while above field capacity there is a slight reduction.

5. Addition of water to the soils affects soil compaction and porosity which are also found to influence the mortality rate through their effects on burrowing behaviour of the carabids.

6. Thionazin exhibits a positive temperature coefficient of toxicity, the rate of kill being 2.2 x faster at 10°C, 117.

8.1 x faster at 15° C and 11.8 x faster at 22°C than at 7°C as asseAsed by time/mortality studies. The effect of temperature is however, modified by light and by water which affects both the activity of the carabids and of the pesticide.

7. There is no immediate effect of soil pH on toxicity but it affects persistence, loss of residues being greater in alkaline than in acid soils.

8. A direct correlation of rate of mortality with adult size is obtained for carabid species of similar behaviour. Species susceptibility is, however, found to be modified by behaviour and activity of the insects those which burrow in soil being more susceptible than those which do not. Predatory species such as Asaphidion flavipes and Nebria

brevicollis which rarely burrow in soil are found to be more resistant to thionazin than other species of comparable size whereas Harpalus rufipes and H.aenaes which are primarily phytophagous species and readily burrow in damp soil are more susceptible.

9. Newly moulted adults are more susceptible than older ones.

10. Starvation increases susceptibility.

11. Males are more susceptible than females in species where the males are smaller. 118.

12. The fumigant action of thionazin increases with a rise in temperature: The C.T. product at L.T.50 is about 3.3 gg.hrs./1. for Bembidion lamprOs:

13. The persistence of toxicity of thionazin as 5% granules in light, sandy loam soil was determined at 15°C + 0.5°C. Loss of biological activity occurs very rapidly under these conditions and the calculated "half-life" of the pesticide in soils of pH 6.1-6.7 is 1-2 weeks. Soils treated at 8 p.p.m. with thionazin (equivalent to 16 lb. active ingredient per six inch acre) are barely toxic to adult carabids after 21 weeks at 15°C. Longer term effects cannot' however, be determined without a knowledge of the effects on eggsi larvae and pupae or on other organisms of importance in food chains.

14. Comparative tests with phorate, disulfoton and menazon indicate that the order of potency of these pesticides is phorate > thionazin > disulfoton > menazon. Menazon is non toxic at normal field dosages.

119. PART III Field Studies on the Action of Soil Applied Pesticides on Carabidae of Arable Land CONTENTS

INTRODUCTION 121 MATERIALS AND METHODS 4. 123 1. Soil Treatments 123 2. Crops 123 3. Sites and Design 123 4. Soil Characteristics 124 5. Pitfall Traps 124 6. Pretreatment Cultivations 124 7. Method of Treatment 125 8. Method of Sampling Carabids 127 9. Times of Applications and Sampling 127 (i) Thionazin soil treatments 127 (ii)Soil fumigant treatments 127 RESULTS 129 1. Carabidae in Arable Fields 129 2. Species Distribution within Sites 134 3. Seasonal abundance (Spring & Summer) 139 4.Soil Fauna other than Carabidae 142 5. Carabidae in Treated Potato Plots 142 Effect of pesticide treatments on numbers 142 6. Effect of Pesticide Treatments on Individual Species 149 7. Effect of Thionazin Soil Treatments on Crop Growth 153 8. Effect of Thionazin Treatments on the Sex Ratio of Trapped Carabids 155 9. Direct Effects of Thionazin Soil Treatments on the Mortality of Carabidae 155 10.Long Term Effects of Thionazin Soil Treatments 158 (i) Seasonal abundance (Autumn) 158 (ii)Residual effects on total relative numbers 160

120.

page 11. Effect of Fumigant Soil Treatments on Surface Active Carabids • 166

(a)Direct effects 6 166 (b)Longer term effects 66 166

DISCUSSION 446 173

SUMMARY AND CONCLUSIONS 6iii;•••••••• 183 121. INTRODUCTION

Carabids, like many other soil invertebrates, are more or less ubiquitous in arable fields and are likely to come into contact with any pesticide which is applied either to a crop or directly to the soil. The literature on the effects of pesticides on soil fauna is now extensive and most of it concerns work done on the effects of chlorinated hydrocarbons such as aldrin, dieldrin, BHC and DDT on earthworms, nematodes, dipterous and coleopterous larvae, phalangids, Collembola and Acarina (Fleming and Hawley, 1950; Gould and Hamstead, 1951; Sheals, 1953, 1955; Weber, 1953; Edwards and Dennis, 1960; Heath, 1960; Menhinick, 1962; van der Drift, 1963; Bauer, 1964; Fox et al., 1964; Davis, 1965; Edwards, 1965a & b; Edwards et al., 1967). In comparison little attention seems to have been paid to the carabids (Grigor leva, 1952; Wright, Hughes and Worrall, 1960; Wright, 1962; Skuhravy and Zeleny, 1965; Coaker, 1966; Mowat and Coaker, 1967 and Dempster, 1967) and again virtually all of this work has been concerned with the very persistent organochlorine pesticides. In the cases where pest outbreaks occurred following the use of soil pesticide treatments these were often attributed to the suppression of predatory com- ponents of the habitat and in particular to carabid adults and larvae. As yet little work has been done on the side effects of organophosphorus pesticides in soil but papers by Raw (1965), Way and Scopes (1965) and Edwards and Thompson (1967) are of note for they concern the use of such chemicals 122. including systemic pesticides which will undoubtedly be used in greater amounts in the future. Opportunity was taken of utilizing thionazin (and other pesticide treated plots) in an Agricultural Research Council (A.R.C.) sponsored long term field experiment init- iated by the late Professor B.G. Peters and his colleagues for the control of potato cyst nematode, Heterodera rostochiensis. The experiments were done at two sites, both situated at the Imperial College Field Station, and field work was started in the autumn of 1964. The effects of the pesticide treatments on the carabids were determined by comparing the number and species trapped in the treated plots with those in the controls. Sampling was done by pitfall trapping which had two main aims; namely to establish indicator species for the prevailing conditions during the growth of a crop and secondly to assess the immediate and the possible long term effects of the pesticide treatments on the total beetle population. Observations were made on the growth of the crop, on the lack or abundance of weeds and of other soil invertebrates to ascertain whether sampling was representative of the soil treatments and also to obtain an overall picture of the extent of heterogeneity of the. environment. 123. MATERIALS AND METHODS

1. Soil Treatments ; pesticides, dosages and formulations. The following four pesticides at three dosage rates (zero, low and high) were investigated:

DOSAGE LEGEND PESTICIDE FORMULATION (lb/6"Ac.) a•i• Low High

Al Thionazin, broadcast Granules(5% a.i.) 10 40 A2 it in-row tt 2 8 B Ethylene dibromide Liquid 200 800 C Chloropicrin io 200 800 D Dazomet Dust (85% w*i.) 100 400

2. Crops: Potatoes (var.Majestic), tick beans, and barley. 3. Sites and Design: a. Four Acre Field: 28 plots, each 6 yds by 12 yds separated by 3 foot wide pathways. b. Church Field : 24 plots, each 6 yds by 12 yds separated by 3 foot wide pathways. In 1967, four plots were added to include thionazin broadcast at both low and high dosage rates each new treatment being replicated once.

The experiments were designed in consultation with Professor Quenouille (Murdie, 1968) for evaluation by regression analysis and as such had single plot replicates. The design was not appropriate for a comparatively short term investigation on the side effects of the pesticides in question 124. but the plots were nevertheless utilized to avoid unnecessary duplication and to economise on labour, space and expenses. After the first year of treatment, half the plots were again planted with potatoes and pitfall trapping was restricted to these areas. 4. Soil Characteristics The soil at both the sites consisted of a light sandy loam containing few stones being on clay subsoil overlying Eocene sands of the Bagshot and Bracklesham beds. Slight differences in topography and soil pH existed between the two sites. Thus, in Four Acre Field there was a slight slope in an E-W direction and during periods of rain the soil rapidly became water-logged owing to the impervious nature of the subsoil. As a result of leaching and continual cultivations, the soil at this site was very acid e.g. pH 4,5-4'9. In Church Field, on the other hand, the site was fairly level, drainage was better and the soil was not so acid (pH 5.4-5.9). 5. Pitfall Traps These consisted of the modified plant pots which are fully described in Part I. 6. Pretreatment Cultivations Prior to the commencement of the experiments the soil in both Four Acre Field and Church Field was ploughed once and harrowed several times in the autumn and spring of 1964 and '65 respectively. After the plots were established 125. similar cultivations were done whenever necessary around the plots to prevent excessive growth of weeds. All the plots were rotovated td a depth of six inches and before the potatoes were planted, I.C.I.No.1 fertilizer (N:P:K = 1:1:12) was applied broadcast to the soil at the rate of 10 cwt/acre (based on N.A.A.S. advice). No other pretreatment applications were deemed necessary primarily to reduce the number of possible variables but in 1967, soil analyses revealed the very acidic nature of the soils as well as a magnesium ion deficiency (B.Pain, 1967) which were thought to have been the causes for the poor potato yields in the previous years. Carbonate of lime and Kieserite (M004) were therefore applied to all the plots, the carbonate of lime at the rate of 30 cwt/acre in Church Field and 80 cwt/ acre in Four Acre Field and the Kieserite at 10 cwt/acre at both sites. 7. Method of Treatment Thionazin was applied as 5% granules in the in-row treatments using a combined dipper-hopper made of brass (Call, 1968). In the broadcast treatments the plots were divided into yard squares and doses were applied by means of a flour sifter. After the in-row treatments the potatoes were planted by hand in 6 rows per plot, 24 potatoes per row and the ridges split while in the broadcast treatments, the thionazin was applied after the potatoes were planted and the soil again raked flat. 126. N.B. In 1967, thionazin was not available as 5% granules and 10% granules were Used instead. This necessitated a correction being made to the volume of granules applied per yard to account for the higher concentration of thionazin. However, after completing the in-row treatments it was discovered that the density of 10% a.i. granules was approximately 25 times greater than that of the 5% a.i. granules since Fuller's Earth had been used as the carrier instead of the usual Attapulgite clay. This resulted in thionazin being applied at a higher rate than was intended. The actual rates applied were therefore not 8 and 2 lb/acre a.i. for the high and low dosages respectively but 20 and 5 lb/ acre a.i. This density factor was taken into account when applying the broadcast treatments. The soil fumigants ethylene dibromide (E.D.B.) and chloropicrin were applied to the soil using a Shell soil fumigant injector, model H1. For this purpose the plots were divided crosswise and lengthwise into one foot square sections to form a checker-board pattern and injections were applied at the intersections to a depth of nine inches in the soil. After each injection the soil was pressed down over the hole produced by the injector nozzle to prevent rapid loss of fumigant from the soil. Dazomet was applied broadcast in a similar manner to the thionazin granules and after application was immediately rotovated to a depth of nine inches. 127. 8. Method of Sampling Carabids The carabid beetle fauna active on the soil surface of each plot was sampled by means of the pitfall traps. Two traps were placed in each plot about 2-3 days after the treatments wete applied so that they came mid way along the outside of the two centre roWs of potatoes. This gave a distance of six feet between the traps and to the nearest edge of the plot. The traps were provided with plastic covers to prevent undue predation from birds and to exclude rain and dirt as much as possible. At first sampling was done daily then every third day and finally at weekly intervals. After recording the number and species trapped from each plot the insects were weighed according to species and then either killed for mounting and identification or released but were not returned to their respective plots. 9. Times of Applications and Sampling (i)Thionazin soil treatments In each year of treatment the potatoes were planted and the treatments applied during the last week in April and the first week in May depending on weather conditions. The crop was then harvested from mid-September to early October and the yield from the two middle rows per plot were recordede Pitfall trapping was commenced within 3 days and stopped just before the crop was lifted. (ii)Soil fumigant treatments These were applied in autumn, generally in early November and pitfall trapping was commenced soon after for 128* a period of eight weeks as well as during the following spring and summer to determine both the immediate and longer term effects of the pesticides*

129. RESULTS

1. Carabidae in Arable Fields Table35 summarises and cdmpares the relative numbers of individuals and species trapped in untreated potato plots at the two experimental sites, Four Acre Field and Church Field. The numbers of carabid individuals recorded, as in all subsequent tables, give only an indication of relative abundance for they are dependent not only on the total populations available for trapping but on the activity of the individuals and on the efficiency of trapping. Table 35 A comparison of relative numbers and species of Carabidae from Four Acre Field and Church Field during the months May to September of 1965 to 1967 inclusive

Numbers/Species Year Four Acre Church Field Field

Total Relative Numbers1 1965 328 593 1966 542 938 1967 812 308 1965-67 1682 1839

Total Species2 1965 31* 27* 1966 34 34 1967 34 35 1965-67 42 44 * Includes some undetermined Amara species 1. From 4 traps (1965) or the equivalent of 4 traps (1966, 1967) in two untreated (control) potato plots each of 72 sq.yds. 2. From 20 traps (1965) and 10 traps (1966, 1967) in Four Acre Field and 12 traps (1965), 6 traps (1966) and 14 traps (1967) in Church Field in treated and untreated plots. 130. Pitfall trapping was done for similar periods using identical traps. Thus, the differences in numbers of carabids trapped reflected differences in habitat. That this may have been so is indicated by the description of the two sites (see p.124 ) where it was shown that soil conditions in Church Field were initially much more favourable than those in Four Acre Field owing to better drainage and less acid soil pH which resulted in better plant growth. This may therefore account for the greater number of individuals caught in Church Field than in Four Acre Field during the years 1965 and 1966, Pitfall trap catches in 1967, however, were not truly comparable with those in the two previous years either within or between each site and for the following reasons. Owing to deteriorating conditions in soil pH and mineral ion deficiency remedial steps were taken to improve soil conditions (see p.125). This reduced or eliminated the soil differences which had been present between the two sites. Furthermore, whereas the plots in Church Field were thoroughly weeded by hand hoeing those in Four Acre Field were not and owing to the apparent increase in build up of the potato cyst nematode in the control plots as shown by the extremely poor growth of the potato haulm - the amount of plant cover was appreciably reduced in Church Field but not in Four Acre Field where weeds were abundant. This undoubtedly accounts for the greater numbers of carabids in Four Acre Field than in Church Field and unfortunately 131. was bound to affect the comparison of results from pesticide soil treatments at the two sites in 1967. Indices of similarity were calculated to determine whether any differences in the total species composition between the sites were due to chance or whether they were real. A suitable index for this purpose is based on the logarithmic-series distribution and has been shown to be less dependent on sample size. This index provides a quantitative measure of the degree of association between the species of the two sites or of the same site in different years but its main disadvantage is that it over-emphasizes the rare species (Southwood, 1966) as they are given the same weight as the abundant ones. For the purpose of calculating the indices of similarity between the two sites, the number of species of Carabidae in Four Acre Field and Church Field and the number common to pairs of sites were set out as shown in Table 36. Mountfordts Index of Similarity was calculated from the equation:

e al + e bI = 1 + e (a+b-j)I where a = number of species in site A, b = number of species in site B and j = number of species common to both sites. I was solved by an interative method and the results are shown in Table 37. A two-dimensional diagram can then be drawn as shown in Fig.25 to indicate relationships between groups of sites. It would appear from this diagram that there was some 132.

Table 6 Number of species of Carabidae in Four Acre Field and Church Field and number common to pairs of sites

Number of species common to pairs of sites Site Total FOUR ACRE FIELD CHURCH FIELD Number Number of 1965 1966 1967 1965 1966 1967 Species 1 2 3 4 5 6 6 35 24 26 26 22 27 5 34 21 26 25 20 4 27 21 21 21 3 34 26 28 2 34 27 1 31

Table 37 Values of I x lo4 for data of carabid species in Four Acre Field and Church Field, 1965-67 inclusive

FOUR ACRE FIELD CHURCH FIELD Site Number 1965 1966 1967 1965 1966 1967 1 2 3 4 5 6

1 1280 1062 710 520 732 2 1138 699 829 775 3 699 723 775 4 605 773 5 894 6 133.

FOUR ACRE FIELD CHURCH FIELD

1965 1966 1967 1967 1966 1965

1280

1100

894

725.7 697.2

Fig. 25 Classification of carabid species from Four Acre Field a Church Field and values of indices of similarity between sites a years. 134. association between the species at the two sites but that this was not as close as that within tWe sites over the three years. 2. Species Distribution within Sites Lists of the spring and summer active species occurring at the two sites are given in Tables (38 & 39) for Four Acre Field and Church Field respectively. Tables 38 & 39 show that there was a remarkable consistency in the order of abundance of the species from the two sites and especially in the years 1965 and 1966. In both fields, however, the species Harpalus rufipes gradually increased in abundance probably as a result of annual weeds growing unchecked during the growth of the potato crop and liberating millions of seeds at the end of the season. The most common weed species were Stellaria media, Polygonum aviculare and Chenopodium album and the latter are of special significance for it is known that both adults and larvae of Harpalus rufipes and related species feed upon the seeds of this plant (Briggs, 1965). Although some effort was made to control weed growth in Church Field in 1967 this was not possible in Four Acre Field owing to lack of labour and hence conditions were ideal for the invasion of the plots by Amara species which are also known to be phytophagous (Davies, 1953). As a result Amara plebeja increased in importance from being 27th in order of abundance in 1966 to 3rd in 1967 while Amara bifrons increased from being 13th 155. Table 38 List of surface active Carabidae trapped in Four Acre Field during the growth of a potato crop Numerals indicate order of abundance e.g. 1 = most abundant; 2 = second most abundant, etc.)

1965 1966 1967 Species (19.v-27.ix) (6.v-13.ix) (1.v-11.ix) Carabus violaceus "24 27 18 Nebria brevicollis 11 17 Notiophilus substriatus 17 28 N.biguttatus 20 11 23 Loricera pil4.cornis 13 10 8 Clivina collaris 24 17 16 Asaphidion flavipes 20 27 9 Bembidion lampros 1 1 1 B.tetracolum 20 ••• 28 B.femoratum 21 B.andreae 5 15 23 B.quadrimaculatum 10 4 7 Trechus quadristriatus 7 5 12 Badister bipustulatus 24 Harpalus aeneus 6 6 10 H.tardus 20 21 23 H.melleti IMO 28 H.rufipes 3 3 2 Acupalpus meridianus 16 21 18 Bradycellus verbasci ••• 28 B,harpalinus 27 Anisodactylus binotatus 9 9 4 Amara plebeja 27 3 A. similata 24 12 11 A.aenea 27 28 A.familiaris 27 14 A.tibialis 28 A.bifrons 12 13 5 136.

Table 38 List of cai.abidhe in Four Acre Field cont.

1965 1966 1967 Species (19.v-27.ix) (6.v-13.ix) (1.v-11.ix)

A.apricaria 24 27 A.fulva 18 21 23 Pterostichus caerulescens 24 17 23 P.niger 24 13 13 P.vulgaris 2 2 6 P.madidus 8 8 14 Abax parallelepipedus 18 27 - Calathus fuscipes 4 6 18 C.melanocephalus 24 - 18 C.piceus - • 21 - Synuchus nivalis - - 28 Agonum dorsale 15 21 16 Dromius melanocephalus 24 - - Metabletus foveatus 13 15 18 137. Table 39 List of surface active Carabidae trapped in Church Field during the growth of a potato crop (Numerals indicate order of abundance)

1965 1966 1967 Species (15.v-27.ix) (6,v-13.ix) (9.v-12.ix) Carabus violaceus 15 9 21 Nebria brevicollis 5 16 24 Notiophilus aquaticus - - 28 N.substriatus - 17 28 N.biguttatus - - 27 Loricera pilicornis 13 13 8 Asaphidion flavipes - 25 13 Bembidion lampros 2 2 1 B.femoratum - 25 am• B.andreae 19 20 28 B.quadrimaculatum 3 5 5 B.obtusum 19 Trechus quadristriatus 7 8 14 Badister bipustulatus 18 Harpalus aeneus 11 6 9 H.rubripes 19 - - H.tardus 17 20 24 Horufipes 10 4 2 Anisodactylus binotatus 19 - 28 Amara plebeja - 20 21 A.similata 25 12 A.ovata - - 28 A.aenea 19 25 IMO A.familiaris 16 12 14 A.anthobia - 25 19 Aotibialis - 25 28 A.bifrons 14 15 7 138.

Table 39 List of Carabidae in Church Field (cont.)

Species 1965 1966 1967 (15.v-27.ix) (6.v-13.ix) (9.v-12.ix)

A.praetermissa 25 A.apricaria - 25 A.fulva 19 - 19 A.consularis - 17 18 A.aulica - 20 28 Pterostichus caerulescens 20 OM. P.niger 14 P.vulgaris 8 7 6 P.madidus 4 3 4 Abax parallelepipedus 19 17 24 Calathus fuscipes 1 1 3 C.melanocephalus 9 10 17 C.piceus - 25 111•111, Synuchus nivalis - 25 MN/ Agonum malleri 19 - 21 A.dorsale 6 14 10 Metabletus 12 10 11 foveatus 139. ih 1966 to 5th in 1967. However, in spite of these increases, the small diurnal carabid Bembidion lampros wag still the most abundant species in Four Acre Field and was either most abundant or second in abundance to Calathus fuscipes in Church Field. Examination of Appendices 22 to 27 shows that of the species which occurred in one field but not the other the majority were "rare" in 1965 and their presence can be accounted for by chance immigration, as for example by flight, from neighbouring fields or from the surrounding woodlands. In 1966 a few species "common" in Four Acre Field, were notably absent in Church Field; these were Anisodactylus binotatus, Notiophilus biguttatus and Pteros- tichus niger. Similarly, Calathus melanocephalus which was "common" in Church Field was absent in Four Acre Field. In 1967, as in 1965, the majority of the different species were "rare" ones. The tables indicate that the "rare" species, although often forming over 50 per cent of the species composition in both habitats never contributed to more than 10 per cent of the total number of individuals trapped. These results suggest that the carabid fauna in Four Acre Field and Church Field were not as different in species composition as at first indicated by Mountford's index of similarity. 3. Seasonal abundance (Spring and Summer) The seasonal abundance of some of the common species 1400

100 Bembidion lampros

0 female

80 • male

c=callow

20 EK E

ER W Bembidion quadrimaculatum

P 20

Amara plebea

20 PER MBERS 0 NU Wilpalus rufipes

20[ c

Loricera pilicornis

10[ eLsrslaisilus tglgEa O

III tit,' I May I June July August 1987 Fig. 26 Relative abundance of "common"every common"Carabidae from untreated potato plots in Four Acre Field; I May, 1967 to II September, 1967. 1414,

60 Bembidion lampros

0 female

40 III male

c= callow

o -

20E Bembidion guadrimaculatum K O

j palus rufipes

WEE 20[

C PS PER O 8 TRA ior Pterostichus vulgaris

PER 0L- S

20E Pterostichus madidus NUMBER

Calathus fuscipes

May I June I July I August 1907 Fig. 27 Relative abundance of "common"every common"Carabidae from untreated potato plots in Church Field; 9 May, 1967 to 12 September, 1967. 142.

in Four Acre Field and Church Field are shown in Figs.26 and 27. These indicate that the smaller species such as Bembidion lampros and B.quadrimaculatum, although ubiquitous, were commonest early in the season when plant cover was minimal or absent while the larger species such as Harpalus rufipes, Pterostichus vulgaris, P.madidus and Calathus fuscipes occurred in peak numbers later in the season when plant growth was well established. There was therefore a marked succession of species which may have been due partly to inter species competition and partly to the effects of plant growth creating a more favourable habitat for some of the species, especially the larger ones. 4. Soil Fauna other than Carabidae A list of some of the surface active animals caught in the potato plots in Four Acre Field during the 1967 cropping season is given in Appendix 28. This list is typical of the fauna active on the soil surface at both the experimental sites during the three years of sampling but is not comprehensive, 5. Carabidae in Treated Potato Plots - Effect of Pesticide Treatments on Numbers Table 40 summarises the effects of the thionazin soil treatments on the surface active carabids in Four Acre Field and Church Field during 1965 to 1967. Where the variance ratio of treatment against error was significant (see Appen-. dices 29-34) the differences between means of individual 143.

Table 40 Summary of Effects of Pesticide Treatments on Surface Active Carabidae in Four Acre Field and Church Field, 1965-1967

TREATMENT PITFALL TRAP CATCHES (4 Traps per Treatment) Method FOUR ACRE FIELD CHURCH FIELD Pesticide of Dos. Applic. Rate 1965 1966 1967 1965 1966 1967 *** *** *** Thionazin B'cast High 79 114 172 •••• 248 I ti Low *** ** *** 128 250 338 =NI II•1•• 253 ** It In-row High 347 378 404 598 990 242 * t I Low 432 588 466 776 932 580x Control (No treatment) 328 542 812 593 938 308

TOTAL 1314 1872 2192 1967 2860 1631

* * * Significantly fewer carabids than in untreated plots, P =<0.001 * * Significantly fewer carabids than in untreated plots, P =<0.01 * Significantly fewer carabids than in untreated plots, P =

x Significantly more carabids than in untreated plots, P =<0.05 144. treatments and controls were teeted by the least critical ratio (L.C.R.) test, Differences that were significant by this test are marked by either an asterisk or a cross. The greatest effects were observed in the thionazin broadcast treatments in the very acid (pH 4.5-4.8) sandy loam soil of Four Acre Field where numbers of Carabidae were drastically reduced throughout the growing season these effects can be seen clearly in Figs.28 - 30 respectively. Figs.31-36also show the effects of the in-row treatments at the two sites and Fig.37 illustrates the effects of the broadcast treatments in Church Field during the 1967 cropping season. The results in 1967 were not comparable with those in the two previous years in either Four Acre Field or Church Field, for two main reasons. In both Four Acre Field and Church Field the amounts of in-row thionazin were approximately 2.5 times greater than in other years. This significantly decreased (P = <0.05 and <0.01) numbers at both the dosage rates in Four Acre Field. In Church Field removal of the weeds from the treated and the control plots resulted in a great decrease in the amount of plant cover in the control plots and owing to extremely poor growth of the potato crop compared with growth in treated plots considerably fewer carabids were trapped. Thus, although pitfall trap catches in all but the in-row "low" treated plot were less than those in the control, the differences NUMBERS OF CARABIDAE PER 4 TRAPS 1%) • CO • 0 • 0 0 0 0 o• f ?! 0

3 0. 5 xi• 0...... -n 3 ...... oc . '-o— ...... C ......

...... "*"." ......

.• ...... • ...... • • ...... a a = ••• ...... 3 ; `0

Ut •..... :b ...... s o

1.• ...... A

'b .0 NUMBERS OF CARABIDAE PER 4 TRAPS 3 3 3

... 4

...... CrN B •

...... • Øb VI .. •I

-.-.---c..)...... -- .....c1 14'7

120 1965 Fig. 34 110 .0control —•—•— thionacm In.-row low 100 --- thfonaten In row high

70I `F / 80 1.

30 •

Mtn August September

1966 FIG. 35

100 1967 Fig. 36

May 1 hone I July • 1° Aututt iSeptember Figs. 34-36 Relative numbers of Carabidoe from thionazIn treated I untreated potato plots in Church Field, 1965 -1967. In-row treatments. swawwan lscoppoJg .L961'P IMALI DA40 re. 0 Rb 0 C Cr 5 a. 0 0 A 3 0 7 — 0 re. re. 0. —06 0 0 0 0 in 0 Irl• trs "7, r 7 . .

oa *- g 4 3 c cr

...... •...... • 0- •-...... • ' • ••••• •••••••..-- • • ...... • sow 0 NUMBERS OFCARABIDAEPER4TRAPS

..•• ww•• • •• ...... -

• -•••.• ..••••• ...... O • •••• • ..

.. •

.... •.. pa 0

...... '9f1T ••• . •...... 0 .. :0 ......

... • . 0 . • •

149. were not statistically significant (at P=<0.05) at the end p of the season. A striking feature of these results was that in four out of six occasions pitfall trap catches in the in-row treated plots at the "low" rate were higher than those in the controls although the differences were not always statistically significant. Reasons for this are put forward in the discussion. 6. Effects of Pesticide Treatments on Individual Species The effects of the thionazin soil treatments on the adults and larvae of the commonly occurring carabid species in Four Acre Field and Church Field during the years 1965 and 1966 are shown in Figs.38 and 39. These results are shown in greater detail in Appendices 35-40 - for the years 1965-67 inclusive. The results from the in-row "high" and the broadcast "low" treatments in Four Acre Field were omitted from the histograms and the tables for the sake of clarity since they represented effects intermediate between those of the apparently sub-lethal dosages in the localized in-row "low" treatment and the highly lethal broadcast "high" treatment. The figures and tables indicate that the species affected most by the treatments were the small ones of average weight 1.5-5.0 mg such as Bembidion lampros, B.quadrimaculatum and Trechus quadristriatus whereas the larger species of mean weight 50 mg or more such as Pterostichus madidus, P.vulgaris and Calathus fuscipes appeared to be least affected. This must have been due partly to differences in their seasonal 150.

❑ control no treatment A 111 thionazin in row low thionazin broadcast high

a. Clam =1111L-

a. 250 B Cl) wCC CO E 200

ram__ Bembidion Bembidion Trechus Harpalus Harpalus Pterostichus TOTAL lampros quadri— quadri— aeneus rufipes vulgaris madidus LARVAE maculatum striatus Fig. 38 Relative abundance of carabid adults a larvae from experimental plots in Four Acre Field. A 19 May to 27 Sept.I965 B 6 May to 13 Sept.I966 151.

❑ control no treatment A thionazin in row low thionazin in row high

0 mail _OIL EMI

320 a. ce - 240

ce a 160 Cr) CC m 80

240 C

160

O CIL Ammo. i. r ▪I Ca Bembidion Bembidion Trechus Harpalus Pterostichus Calathus TOTAL lampros quadri.. quadri- rufipes vulgaris madidus fuscipes LARVAE maculatum striatus Fig. 39 Relative abundance of Carabid adults t larvae from experimental plots in Church Field. A 15 May to 27 Sept. 1965 B 6 May to 13 Sept. 1966 C I May to 12 Sept.1967 152. abundance (see Figs.26 & 27) and partly to differences in

weight or size (see bioassay experiments - Part.II). There were some exceptions to this rule and notably in Asaphidion flavipes which was more resistant - and Harpalus, aeneus which was more susceptible - to thionazin than other species of similar weight. Asaphidion flavipes which is diurnal in habit, is capable of good flight and has large well-developed compound eyes which must enable it to see and avoid the traps. Observations indicate that it occurs in arable fields in much larger numbers than the pitfall traps suggest and unlike many other species it rarely burrows or hides in cracks in the soil owing to its cryptic coloration. A characteristic feature of animals with this coloration is that for the coloration to be effective the animals must be able to remain stationary, whenever required, in order to escape detection. It is probably for this reason that the insect is more "tolerant" to the pesticide treatments than other species of similar weight for it probably comes into less contact with the pesticide. Harpalus aeneus on the other hand hides in the soil much more readily in order to escape detection but in addition it is capable of releasing a very powerful chemical - probably formic acid (see Eisner and Meinwald, 1966) - when disturbed or attacked by a would- be predator. It may be that this mechanism is triggered off by the effects of the pesticide and if the beetle is confined in a burrow at the time it might hasten death or actually cause its own self destruction. Similar effects could 1534 often be produced in the laboratory if the beetle was manhandled and then placed in a closed tube or if it was confined with another large carabid such as Pterostichus, vulgaris or P.madidus. 7. Effect of Thionazin Soil Treatments on Crop Growth Table 41 summarises the effects of the thionazin soil treatments on crop growth as made by eye and attempts to correlate this with some of the carabid species trapped in the various plots (the latter data being extracted from Appendix 40). The effects on carabids of the pesticide soil treatments and of the amount of plant cover no doubt interact and it was impossible to assess their individual effects on the activity of the carabids. It is suggested, however, that whereas the reduction in numbers of the smaller species (which are, as a rule, active in the earlier part of the season i.e. at the time of the pesticide applications) are those due to "direct toxicity" and resulted in their mortalities, the increase in numbers of the larger species (which are more common later in the season - see Figs.26 and 27 ) are partly the result of sub-lethal dosages and partly of increased plant cover which stimulated their activity and hence their numbers trapped. Thus, although the pesticide treatments had some phytotoxic effects in the early stages of crop growth, and especially at the "high" dosage rates there was much better plant growth later in. the season because the potato cyst nematode, Heterodera rostochiensis 154.

Table 41 Effect of crop and weed growth on the activity of Carabidae in thionazin-treated and untreated potato plots in Church Field as shown by pitfall trap catches, 8 August, 1967

Potato Weed Cover Total Carabidae Treatment Growth after 9.v.67-12.ix.67 Hoeing 8.viii.67 8.viii.67 Small Large Control Very Sparse 162 70 (No treatment) poor Thionazin Moderate Dense 332 168 In-row 'Low' Thionazin Fair Sparse 104 72 In-row !High' Thionazin Good Sparse 67 136 Broadcast /Low' Thionazin Good Sparse 154 Broadcast 'High' 34

Pitfall Trap Catches Mean weight (2x2 traps per treatment) Type/Species Thionazin Thionazin (mg.) In-row Blcast ci!‘ Con. Low High Low High Small Carabids B.lampros 2.2 2.1 128 266 92 56 31 B.4-maculatum 1.3 1.4 16 46 6 7 2 L.pilicornis 15.0 16.5 18 20 6 4 1 Large Carabids C.fuscipes 52.4 78.5 18 48 8 39 44 H.rufipes 98.1 119.5 36 54 32 55 65 P.vulgaris 149.0 195.2 26 10 10 14 P.madidus 121.0 144.5 16 40 22 32 31

POTATO GROWTH: Good - Plants about 18 in.high, uniform, meeting across row Moderate - Plants about 12-18 in. high. Fair - Some plants stunted, not meeting across row. Very - Many plants absent, some stunted poor and yellow. 1554

was controlled. There is thus a compensatory effect on carabid numbers although this does not occur simultaneously or immediately. 8. Effect of Thionazin Treatments on the Sex Ratio of Trapped Carabids Experiments in the laboratory (see Part.II) indicated that male carabids were consistently more susceptible to pesticides than the females, a result associated with the size of the individuals, males being on average smaller than females. An attempt was therefore made to correlate these effects with pitfall trap catches in the field. Table 42', summarizes the effects of the treatments on the total species and Tables 43 and 44) on the abundant species in Four Acre Field and Church Field during the 1967 cropping season. Generally more males were trapped than females (Table 42) irrespective of treatment. These results are analysed in greater detail in the Discussion. 9. Direct Effects of Thionazin Soil Treatments on the Mortality of Carabidae In 1965 Carabidae were enclosed on thionazin treated soil in Four Acre Field approximately 3 weeks after the application of the treatments. All adult Bembidion lampros were killed within two hours on fairly dry soil of the "high" thionazin broadcast treatment whereas none were dead in untreated plots even after 24 hours. Similar results were obtained with the larger carabid species Nebria brevicollis except that it took 9 hours for all to die on the thionazin 156.

Table 42 Effect of thionazin soil treatments on Sex ratio of total species of Carabidae trapped at two sites in 1967

Sex Ratio c31 : Control In-row In-row B'cast B'cast Low High Low High Four Acre Field 1.38: 1.20: 1.55: 1.39: 1.18: 1.00 1.00 1.00 1.00 1.00 Church Field 0.81: 1.33: 1.05: 1.43: 1.34: 1.00 1.00 ' 1.00 1.00 1.00

Table 43 Sex ratio of the common species in Church Field, 9.v.67 - 12.ix.67

Sex Ratio e : Control In-row In-row B'cast B'cast Low High Low High Bembidion 0.73: 1.25: 1.56: 1.15: 1.21: lampros 1.00 1.00 1.00 1.00 1.00 Harpalus 0.50: 0.42: 0.33: 0.72: 0.41: rufipes 1.00 1.00 1.00 1.00 1.00 Calathus 3.50: 5.00: 3.00: 1.44: 1.44: fuscipes 1.00 1.00 1.00 1.00 1.00 Pterostichus 1.67: 4.00: 1.20: 2.56: 9.33: madidus 1.00 1.00 1.00 1.00 1.00

Table 44 Sex ratio of the common spec es in Folly. Acre Field, 1.v.67 - 11.ix.67

Sex Ratio di • Control In-row In-row B'cast B'cast Low High Low High Bembidion 1.17: 1.13: 1.80: 0.83: 0.60: fampros 1.00 1.00 1.00 1.00 1.00 Harpalus 1.18: 1.90: 0.70: 1.87: 1.86: rufipes 1.00 1.00 1.00 1.00 1.00 Amara plebeja 1.29: 1.30: 1.50: 1.25: 0.50: 1.00 1.00 1.00 1.00 1.00 tAigoanZaus 2.33: 1.00: 0.40: 1.00: 0.60: ..inotatus 1.00 1.00 1.00 1.00 1.00 157.

treated soil. In warmers damper conditions one month after application all adult Bembidion tetracolum died within 20 minutes on the high thionazin broadcast treatment. Labor- atory bioassays later confirmed that the beetles died much more rapidly on wet than on dry soils. Harris (1964 al b; 1967) has shown dry soil tends to inactivate the insecticide because it is adsorbed to soil colloids. The mortalities among carabids left in the pitfall traps for up to one week indicated the duration of the toxic effects of the thionazin soil treatments (Table 45). Very few or no live carabids were trapped in either of the two broadcast treated plots for a period of 6-7 weeks after treatment. Mortalities in the in-row treated plots did not differ appreciably from those in the controls and may be negligible although mortalities may have occurred in localized spots especially of species that live in the soil or burrow. Table 45 Proportion of carabids found alive in thionazin treated plots in Four Acre Field Dates of treatment : In-row applications - 28.1v.67; broadcast applications - 1.v.67• Period of sampling weekly from 1.v. to 11.ix.67 inclusive.

Pitfall Trap Catches (2 traps per treatment) NNW Weeks THIONAZ. THIONAZ. THIONAZ. THIONAZ. CONTROL after ByCAST B'CAST IN-ROW IN-ROW Treatment 'HIGH' 'HIGH' 'LOW' 0-5 0/12 2/12 17/27 36/51 23/36 5-10 9/31 31/50 33/85 29/67 98/154 10-15 13/27 62/64 38/42 42/62 110/123 15-19 14/16 39/43 43/48 43/53 77/93 0-19 36/86 134/169 131/202 150/233 308/406

158.

10. Long Term Effects of Thionazin Soil Treatments (i) Seasonal abundance (autumn)

Tables 46 and 47 list the species of carabids which were active on the soil surface during the early autumn months in the two arable fields and Appendix 41 gives an incomplete list of the remaining surface active soil fauna. These tables indicate a reduction in the number of species active at this time of the year and in particular of the adult carabid species which no longer greatly out number those which occur as larvae. The results show that the small carabid species, Trechus quadristriatus, replaces Bembidion lampros as the most abundant adult species in the autumn but that the carabid larvae and especially the surface active Nebria brevicollis were most abundant.

Table 46 Pitfall trap records of Carabidae active in autumn in Four Acre Field, showing order of abundance of adult and larval species in 56 traps, (1 = most abundant; 2 = second most abundant, etc.) 1965 1966/67 Species 10 Nov-27 Dec 15 Nov.66-3 Jan.67 A. Adults Nebria brevicollis 4 2 Notiophilus substriatus 3 5 N.biguttatus 6 7 Asaphidion flavipes 7 Bembidion lampros 2 3 B.femoratum 6 B.tetracolum 8 Trechus quadristriatus 1 Bradycellus harpalinus 5 Anisodactylus binotatus 7 Pterostichus madidus 8 159. Table 46 cont.

1965 1966/67 Species 10 Nov-27 Dec 15 Nov.66-3 Jan.67 B. Larvae Nebria brevicollis 1 1 Leistus ferrugineus 6 Harpalus rufiyes 3 2 Amara aulica 6 A.bifrons 5 Amara spp. 5 Calathus fuscipes 2 3 Pterostichus vulgaris 4 4

Table 47, Pitfall trap records of Carabidae active in autumn in Church Field, showing order of abundance of adults and larvae in 48 traps

1965 1966/67 12 Nov-27 Dec. 15 Nov.66-3 Jan.67 A. Adults Nebria brevicollis 3 3 Notiophilus aquaticus 6 Nesubstriatus 2 2 N.biguttatus 6 5 Loricera pilicornis 6 Bembidion lampros 4 5 B.obtusum 5 Trechus quadristriatus 1 1 Bradycellus harpalinus 5 Amara aenea 6 Amara sp. 6 Pterostichus madidus 6 4 Calathus melanocephalus 5 B. Larvae Carabus violaceus 5 Nebria brevicollis 1 1 Harpalus rtifj.---;.22§ 3 2 Amara aulica 4 5 Amara sp. 4 Pterostichus vulgaris 4 Calathus fuscipes 2 3 160.

(ii) Residual effects of thionazin on total relative, numbers Pitfall traps were used to assess longer term effects as well as short term effects (Appendtces 42-45, Pigs.40-43). Table 48 summarizes the total number of carabids trapped in the thionazin treated plots for 8 weeks after harvesting the potato crop in 1965 and 1966. Trapping started at

about the same time and for the same length of time in both years but only 3 weeks after harvesting in 1965 and 6-7 weeks after harvesting in 1966. The evidence on persistence and leaching of thionazin residues in sandy loam soil (Part IV) suggests that the decreased numbers of carabids trapped in the thionazin treated plots in Four Acre Field in 1965 was caused by the pesticide and not the physical disturbance of the soil after harvesting i.e. thionazin which had been leached from the surface soil during the summer was again exposed by cultivation. These effects, however, must have been greatly lessened by the time pitfall trapping began in 1966 for although fewer were caught in treated plots than in the control the differences were not significant at P=<0.05. Table 48 RESIDUAL EFFECTS OF THIONAZIN SOIL TREATMENTS Summary of results in Four Acre Field and Church Field

Total Pitfall Trap Catches Time of Site from 4 traps per treatment Sampling Control In-row In-row Becast B'cast Low High Low High 10.xi- Four Acre * ** ** * * 27.xii.65 Field 40 15 9 12 5 15.xi.66- It 42 34 18 26 20 3.i.67 12.xi- Church 31 62x 33 27.xii.65 Field 15.xi.66- 40 44 28 IMP •••11. 3.i.67 161.

Table 48 cont. KeY * Significantly fewer carabids than in untreated plots (*at P =<0.05, ** at P =<0.01) x Significantly more carabids than in untreated plot at P =

Tables 49 and 50 list the species which were active in the treated and control plots during this period and it seems that the larvae were more vulnerable than the few adults which were trapped. The possible effects of sub-lethal dosages occurred in the in-row "low" treated plots in Church Field as indicated by significantly more (at P =<0.05) caught in this treatment than in the controls (Tables 48 and 50). 162.

14 O 0-- control thionazin broadcast low thionazin broadcast high Fig. 40

12 1965 • 10 •

8 .t5

LU 2 41k... N.N...... cc u.I o

(.0 10/XI 15/X1 22#1 gxo efCIl 131811 20/101 243185

cr 24 0 • control thionazin broadcast low I thionazin broadcast high Fig. 41

a 18 w CL 1966 Ls) cc 14 LU CO 2 z 12

0f 1111 taixit i0/x8 rpm 3/1/87 Figs.40 & 41 Relative numbers of adult a larva Carabidae from thionazin broadcast treated a untreated plots in Four Acre Field to show possible long term effects of treatments applied in the spring. 163.

14 .0.••••• control thionazin in-row low 0- thionazin in-row high Fig 42

12 \ II 1965 A I • / \ / \ / \ 10 \ i • / \ / N:... / I ... 4 \V ./ 8 i A ...R.., i :: ** I. 4 .•.• r .•-•.. La :1 2 oI 6 a. cn 0- a. • 12/m 15/X 22/x1 30/xi 13/x I i 20/x I 27/435 CC - 18 control thionazin in-row low thionazin in-row high Fig. 43 La a, 14 0. 1966 U) CC co 12 i z 10

k. 2

0_ 'M 1,-• -_• 141 2,/XI 28/21 (Om Ilixn ispot 27/xi i skew Figs.42 & 43 Relative numbers of adults larva Carabidae from thionazin in-row treated a untreated plots in Church Field to show possible long term effects of pesticide treatments applied in the spring. 164. Table 4.9 Residual effects of thionazin soil treatments on surface active Carabidae 7-8 months after the date of application in Four Acre Field: (1965, 4 traps per treatment; 1966, 2 traps x 2 per treatment

PITFALL TRAP CATCHES Species 10 Nov-27 Dec.1965 15 Nov.66-3 Jan.67 In-row B'cast In-row B'cast Con. L. H. L. H. Con. L. H. L. H. A.Adults Nebria brevi- coins 1 1 4 Notiophilus substriatus 3 2 1 4 N.biguttatus Asaphidion flavipes 1 Bembidion lampros 2 Trechus spp. 2 3 1 2 16 8 6 8 4 77747;Triatus &/or T.obtusus) Bradycellus harpalinus 2 Anisodactylus binotatus 1 B.Larvae Nebria brevi- coins 19 8 5 5 3 4 10 8 10 10 Leistus ferrugineus 2 Harpalus rufipes 2 1 1 12 8 4 2 Amara sp. 1 1 1 Calathus fuscipes 9 2 1 6 4 2 Pterostichus vulgaris 1 1 4 2 ** ** ** TOTAL 40 15 9 12 5 42 34 18 26 20 * * Significantly fewer carabids than in untreated plot at P =<0.01 Significantly fewer carabids than in untreated plot at P =<0.05 165.

Table 50 Residual offsets of tinionazin soil traatmants on surface active Carabidae 7-8 months after the date of application in Church Field (1965, 4 traps per treatment, 1966, 2.x 2 traps per treatment) x Significantly more carabids than in untreated plot at P =<0.05 PITFALL TRAP CATCHES

Species 12 Nov-27 Dec.65 15 Nov.66-3 Jan.67 In-row In-row Control Low High Control Low High

A. Adults Nebria brevi collis 1 2 Notiophilus substriatus 1 1 1 2 4 Loricera pilicornis 1 Bembidion lampros 1 Trechus spp. (quadristriatus) 5 8 5 10 8 4 Bradycellus. harpalinus 2 Amara sp. 1 Pterostichus maaidus 4- Calathus melano- cephaius B. Larvae Nebria brevicollis 19 33 20 14 20 14 Harpalus rufipes 1 5 4 6 4 Calathus fuscipes 5 12 5 4 6 2 Pterostichus madidus 2 Total 31 62x 33 40 44 28

166. 11. Effect of Fumigant Soil Treatments on Surface Active Carabids (a)Direct effects Figs. 44-47 and Appendix 46 summarize the effects of the three soil fumigants, ethylene dibromide chloropicrin and Dazomet on the carabids in Four Acre Field and Church Field respectively. Few species and individuals were caught so statistical analysis of the results was impracticable. However, the results at both sites were similar and were repeated in the second year of treatment so they were probably valid. All treatments decreased numbers of carabids trapped, probably from direct toxicity. Dazomet appeared to be the most and ethylene dibromide the least toxic. Effects of the fumigant treatments on individual species are recorded in Appendices 47-48, (b)Longer term effects The longer term effects of the fumigant treatments were assessed in the spring and summer of 1967 by pitfall trapping (Appendices 49 & 50 and summarized in Table 51).

Table 51 The residual affects of fumigant sail treatments applied 9-11 November, 1966, as shown by numbers caught per two traps

Site Pitfall Trap Catches (Period of (Derived from mean log(n+1)) Trapping) E.D.B. Chlorop. Dazomet CONT- Low High Low High Low High ROL Four Acre Field * *** ** 1 May-11 Sep.67 21.6 14.5 36.1 24.0 24.7 18.8 39.7 Church Field 9 May-12 Sep.67 23.6 16.6 18.1 23.8 23.9 15.1 23.4 3

t en tm control ea chloropicrin low 3 chloropicrin high tr er p s 2

2 trap 1 A I er p

s 0 ber

Num --o•••• control q - -a- - E DB low 3 - 1 —o—E DB high

2 -

......

29Ix 10/xi 16/xi 221x1 11xit °hit 13/xot 2c4xii 27ixii 1965 Fig, 44 Effect of soil fumigants on the relative abundance of carabid adults a larvae in Four Acre Field, 29 Oct. to 27 Dec.,1965 1 6 8 . 8 n —0.— control A —0-- Dazomet low —o-- Dazomet high

4 P.: \

11. .. . 2 ti.%,.. Ck \\ / ‘ %. '... % )3 \ : / •••.. / 0 — Ve;:14 0 173 0 4-I , a I I I I I I 4 C (9 E 4.., 0 Lt.- 6 - ....a•-•• control 4-' 4% --4,-- chloropicrin low

t_. % —a— chloropicrin high tla1 %. CL 4. V) 4 - :% ... Q. % '%.... 0 .A. a : • 4-1 \ : •i& 2 - --,.... (NI \ -. i i\ t- CL O - in L 1 (1/ Jra E z7 6 Ek ---0-- control ...\ --0-- E D B low \\ —0— E DB high %. \ 4

''.. 2

0 .* 13

a 9/xi 15/xi 22/xi 29/x1 6/xii 19/x n 27/au 1968 Fig. 45 Effect of soil fumigants on the relative abundance of carabid adults a larvae in Four Acre Field, 9 Nov. 1966 to 3 Jan., 1967

169.

0 ...0..- control —0-- Dazomet low —0-- Dazomet high ., r. 4 ..„ „ . b 0...... 0... : 2 .... . ▪ ...6 A ,Q,.. 0 ..., o- crw —.:1 o b f I I 1 I a

t 4 en tm

8 ....e.... control ...6- chloropicrin low trea —6-- chloropicrin high ..-'4. er

p 4 s

2 2 trap

p O - 1' a t I bers Num

8 —0.— control --0-- E D B low —0— E D B high •..09.. F3'.'. • 4 • .b

2/xi 12 15/xi 23/xi 3Cyx1 6/xii 13/xii 20/xli 27/xii 1985 Fig. 46 Effect of soil fumigants on the relative

abundance of carabid adults at larvae In Church Field, 2 Nov. to 27 Dec.,1965

170 .

9.5 Q. control --0--Dazomet low --0— Dazomet high

4:7\cv-0- - O ▪C

is? 9.5 ••••es•—• control L chloropicrin low 4-1 chloropicrin high **. C).1 CI V) Q. 4 •

2 L—

• o CV ▪E Z 8.5 Ck. —O.—control --0--E D B low \ —0-- E D B high

LI. ;I /

n : r / 11%

0 a a 10/XI 15 xi 224 xi 29/to 0/xii 13/xii 19/xii 271xii 311167 1966 Fig. 47 Effect of soil fumigants on the relative abundance of carabid adults & larvae in Church Field, 10 Nov.1966 to 3 Jan..1967 171.

Table 51 cont.

* * * Significantly fewer carabids than in untreated plots at P < 0.001 * * Significantly fewer carabids than in untreated plots at P < 0.01 * Significantly fewer carabids than in Untreated plots at P< 0.05

The 'high' rates of Dazomet and ethylene dibromide decreased numbers most. Chloropicrin at the 'high' and ethylene dibromide at the 'low' rate also decreased numbers, the latter result being contrary to the results obtained immediately following the fumigant application in the autumn. Differences between means of these treatments and the controls were significant (at P<0.05) in Four Acre Field and not Church Field but Fig. 48 indicates that but for the scarcity of carabids in the control plots (as a result of the complete absence of plant cover) the differences in Church Field would also have been statistically significant. The common species trapped in these plots during the growth of the potato crop were the same as those recorded in the thionazin treated plots (Tables 38 and 39). However, the following previously unrecorded species of Carabidae were trapped; all were "rare" or "occasional" species: Four Acre Field: Bembidion femoratum*, B.tetracolum? larva, Amara anthobia, A.lunicollis, A.praetermissa, A.aulica, Pterostichus cupreus, Abax parallelepipedus* and Agonum mullerio

Church Field: pembidion femoratum*, Amara aenea*, A.apricaria*, Pterostichus vernalis, Calathus piceus*, Synuchus nivalis*, Dromius linearis, Demetrias atricapillus (* species recorded in previous year)

172.

O0) 30—

ci)

43 20— E

E 10 0 .4- cn

from

e 30,- B bida a

Car 20 f o

a) 10 E

—

low bi h low hihLlow high . ethylene chloropicrin Dazomet con— dibromide trol

Fig. 48 Longer term effects of soil fumigants on the relative abundance of carabid adults & larvae: Summary of 18 to 19 weekly catches made I May to 12 Sept.,I967 from 2 traps per treatment. A. Four Acre Field B. Church Field 173. DISCUSSION

It is generally agreed that the more varied the habitat conditions the greater is the number of species inhabiting the habitat and the less varied the habitat the fewer the species of which some will tend to be relatively abundant. Arable fields are characterized by extreme simplicity, either bare soil or mono-culture. This simpli- fication of the environment is an essential aspect of crop cultivation and it is therefore inevitable that this will decrease the number of animal species present in the habitat. Of the species of Carabidae recorded in this study (see list, Part I ) and of those recorded by Greenslade (1961) for the whole of Silwood Park (about 100 species) only about a quarter (e.g. 25 species) would seem to be sufficiently common to be considered as potentially valuable predators. These include: Carabus violaceus, Nebria brevicollis, Notiophilus substriatus, N.biguttatus, Loricera pilicornis, Clivina collaris, Asaphidion flavipes, Bembidion lampros, B.tetracolum, B.andrea, B.quadrimaculatum, Trechus quadristriatus, Harpalus rufiLta, H.aeneus, Anisodactylus binotatus, Amara plebeja, A.similata, A.familiaris, A.bifrons, Pterostichus niger, P.madidus, P.vulgaris, Calathus fuscipes, C.melanocephalus, Agonum dorsale and Metabletus foveatus. These species are not listed in special order and, from what is known of their feeding habits, not all are regarded as beneficial. Information on the feeding habits of certain species 174. is contradictory. For instande, Amara species are said to be mainly or entirely phytophagous, feeding on plant (weed) seeds and pollen (Davies, 1953; Hossfeld, 1963; Kaczmarek, 1963) and yet A.familiaris is known to be a predator of cabbage root fly eggs and larvae (Coaker and Williams, 1963) and A.plebeja and A.tibialis, as predators of the wood cricket Nemobius sylvestris (Gabbutt, 1959). Similarly, Davies (1953, 1959), describes Calathus fuscipes as feeding on corn in the ear and on strawberries whereas Kaczmarek (1963) describes it as n euzoophagous" i.e. feeding exclusively or almost exclusively on animal food. Briggs (1957, 1965) has shown that Harpalus rufipes, H.aeneus and P.madidus can be important pests of strawberries and that larvae of Harpalus rufipes feed on the germinating seeds of several plants and especially on those of fat hen, Chenopodium album; yet Gersdorf (1937) found that H.rufipes and H.aeneus prefer-

red horse meat to lettuce seed in laboratory tests. However, numerous records exist of carabids as predators in arable fields, the following being especially noteworthy; Scherney (1955 and 1961), Wishart et al.(1956), Fox and McCellan (1956) Skuhravy (1959), Hughes (1959), Hughes and Mitchell (1960), Wright et al.(1960) and Dempster (1967). Dawson (1965) in a study of the ecology of eight species of Fenland Carabidae describes the feeding habits of all these species as being "extraordinarily catholic" and this may well apply to most carabids in general. Even such species as Notiophilus spp. 175.

which were thought to feed exclusively on Collembola (Schaller 1949 in Skuhravy, 1959; Davies, 1953) have been shown to have a wider diet (Gabbutt, 1959). The list of species of surface active organisms other than carabids trapped in Four Acre Field (Appendix 28) may therefore include many of the living prey upon which carabids feed. This was indicated by the fact that many weavils which fell into the traps such as Rhinonchus brucoides and R.castor were often found partially eaten. Also attacked and eaten were chrysomelid beetles such as Chaetocnema concinna and adult click beetles Agriotes obscurus. Worker honey bees which visited the potato plants during periods of flowering and which fell into the traps were invariably eaten. In the laboratory adult Harpalus rufipes and other large carabid species such as Pterostichus vulgaris and P.madidus were voracious predators of noctuid and other lepidopterous larvae, although in most cases the prey did not seem to be actually sought out for but were only attacked after contact. Smaller carabid species such as Bembidion lampros and Asaphidion flavipes were seen to feed on live (but perhaps injured?) epigaeic Collembola such as Tomocerus sp. Small earthworms or earthworms which had previously been attacked by larger carabid species were also eaten by Bembidion lampros. These few examples therefore indicate the importance of carabids as either predators or scavengers in arable fields 176. and suggests that their usefulness as predators - in spite of their general feeding habits - should not be under- estimated when assessing the use of pesticide treatments for the control of crop pests. That most of the commonly occurring species of carabids lived all the year round in arable fields was deduced by the capture of their larvae. Thus, Notiophilus substriatus, N.biguttatus, N.rufipes, Loricera pilicornis, H.rufipes, Anisodactylus binotatus, Amara bifrons, A.plebeja, A.familiaris (and probably other Amara species) and Agonum dorsale were captured as larvae during the summer months (June, July and August) while Carabus violaceus, Nebria brevicollis, Leistus ferrugineus, Harpalus rufipes, Amara aulica, Pterostichus madidus, P.vulgaris,and Calathus fuscipes larvae were captured during the early autumn and winter months. Although the proportion of larvae to adults trapped was not high in the summer months and did not exceed 9% of the total catch (Appen- dices 22-27) this is not surprising for Sharova (1957) has shown that the majority of these species live mainly in the natural interspaces of the soil. It is interesting to note, however, that after the addition of lime to the soil in 1967 (to correct the extremely acid soil pHs of 4.5-5.5) there was a considerable increase in the number of larvae caught relative to those in previous years. In Four Acre Field, for example, numbers trapped, increased from 1.2% and 2.6% in 1965 and 1966 respectively to 8.7% in 1967, while in Church 177. Plaid nuMbers increased from 1.2% and 0.2% in 1965 and 1966 respectively to 3.1% in 1967. These results suggest that some larvae are relatively unsuccessful in very acid soils. When assessing the effects of the pesticide treatments on the carabids, two factors have to be taken into consider-

ation. These are that the numbers of beetles trapped will

vary with breeding seasons and with the daily activity of the individuals (these effects are indicated in Figs.28-37). van der Drift (1959) and Vlijm et al. )1961) have shown that the major peaks of activity are correlated with breeding periods while Grdm (1959), Tipton (1960), Briggs (1961), Greenslade (1961) and Mitchell (1963a) have all shown that variation in -activity during the breeding seasons are correlated with temperature and perhaps with other climatic factors. Greenslade (1961, 1964a) found that ground vegetation can also impede movement as catch tends to vary inversely with the resistance to horizontal movement presented by the ground cover. Since a comparison of the numbers of carabids trapped in the treated and control plots was used as the criterion in assessing the toxicity of the pesticides then it was essential that all other factors in the plots should remain either constant or of equal intensity. These conditions were almost fully satisfied in the first two years of treatment but in the third year, owing to the poor growth of the potato crop in the untreated controls, removal of the weeds from the plots in Church Field caused an 178. appreciable difference in the amount of plant cover present in the treated and control plots. The result of thiswas that although the number of carabids trapped in the thionazin treated plots could be compared with those in the controls in the early stages i.e. before the potato plants became visible, a comparison in the later stages was not really valid. It is suggested therefore that it was for this reason that the relative numbers of carabids trapped in the broadcast treated plots in Church Field were not significantly less than those in the controls, as they clearly were in Four Acre Field (compare Figs.30 & 37 ). Removal of the weeds in Church Field must have reduced the incidence of such species as Amara spp. and Harpalus rufipes, which were abundant in Four Acre Field. That species distribution is associated with ground cover has also been noted by Rivard (1964, 1965) and Pearson and White (1964). A recurring feature of the thionazin treatments was that in the plots treated with the pesticide applied in the rows at the "low" rate of 2 lb.a.i./acre, more carabids were trapped than in the untreated controls. The differences were not always statistically significant at P =0.05 but it is suggested that these increases were due primarily to sub- lethal dosages. This phenomenon has been observed in other soil organisms such as Lumbricidae, Enchytraeidae, Nematoda, Collembola and Acari following the use of D.D.T. and B.H.C. (from Satchell„ 1955). Boudreaux (1963) cites cases where 179. not only organic pesticides but also inert residues such as dust, talc and fibres could cause plant mites to increase in numbers, all in the absence of predators. The following reasons may explain the increase in numbers of carabids: (1)A stimulation of nerve activity and a disturbance of physiological processes as a result of pick-up of sub-lethal dosages by contact action i.e. a "direct" effect of the pesticide on the activity of the beetle.

(2)A reduction in the amount of animal food causing an increase in searching activity for suitable food i.e. an "indirect" effect of the pesticide on food.

(3)An increase in plant growth resulting in a more favourable habitat as regards plant cover and micro-climate.

The thionazin treatments did not appear to alter the sex ratios of the carabid species trapped and in most cases males outnumbered females during the summer months in both the treated and the control plots (Tables 42-44). Rivard (1966) obtained similar results in untreated arable fields in Canada and considered that the males were either more numerous or more active than the females. Grum (1962 and 1967) postulates, however, that sex ratios give an indication of a populationT sliving space. For example, where the sex ratio is equal or contains more males to females this is about the centre of the living space; where the females predominate this is the periphery and in this region will also be found the centre of the living space of the larvae. 180. This interesting hypothesis might therefore be used to determine breeding sites of particular species and to map species distribution within given habitats if it is substantiated. The immediate or short term effects of the pesticide treatments were quite obvious and need no further elaboration but the long term effects were more difficult to assess. In spite of applications of high dosages of thionazin in three consecutive seasons, there was an upward trend in the total number of carabids trapped (Table 40). It is not known for example whether the thionazin treatments affected the reproduction rate or to what extent larval individuals were more susceptible to the pesticide than the adults. Bioassays in the laboratory (see Part II) suggested that although egg laying may have been stimulated, the eggs appeared to be sterile for no larvae ever emerged from them. Although organophosphorus pesticide residues are comparatively transient in the environment,their persistence will vary in different soils and in different climates. Thus, although the toxic effects of the thionazin treatments had practically disappeared in all the plots towards the end of the cropping season, cultivation of the soil after removal of the crop made available residues which had apparently leached into the deeper layers of the soil, with the result that there was renewed activity of the pesticide and a corresponding decrease in the numbers of carabids trapped. 181.

That long term effects could not be demonstrated for periods beyond 7-8 months is not surprising considering the comparatively small area of soil treated to the total area of the fields, plus a knowledge of the activity and foraging range of these beetles (see Part I). Since barriers did not exist between the plots, the beetles were able to migrate freely from plot to plot and any reductions which may have occurred as a result of the pesticide treatments could be amply compensated for by the influx of new individuals from untreated soils. Whether similar results would be obtained on a much larger scale - as in normal farming practices - is not certain but it appears probable that effects are likely to be more severe. The preliminary investigation on side-effects of the soil fumigant treatments revealed that all the pesticides were toxic to carabids and in particular Dazomet. Since the pesticides were applied in the autumn, the immediate effects on the carabid fauna were not so drastic because the colder soil and air temperatures reduced both carabid activity and diffusion of the fumigant in the soil. In these conditions, Dazomet proved to be the most toxic and ethylene dibromide the least toxic of the three fumigants. However, when the long term effects of the fumigant treatments were studied, ethylene dibromide proVed to be as toxic or even more toxic than Dazomet. This effect can be satisfactorily correlated with the molecular weights of the 182. fumigants. Dazomet, for example, had the lowest molecular weight (162) and could therefore diffuse more rapidly than either chloropicrin (1401.1ft.164) or ethylene dibromide (Mol.Wt.188). Diffusion is important in the penetration by a gas of a given substrate but is also an important factor in the loss of a fumigant from a treated space. Ethylene dibromide with the lowest molecular weight must have diffused less rapidly than either Dazomet or chloropicrin and although it at first appeared to be the least toxic,- it persisted longer and eventually proved to be as toxic as Dazomet. 183.

SUMMARY AND CONCLUSIONS

The following are the results of 3 years field work, 1965-67.

1. Totals of 42 and 45 different species of adult Carabidae were recorded from two arable fields respectively during three years of pitfall trapping in thionazin treated and untreated plots. The most species recorded during any one cropping season were 34 & 35 in Four Acre Field and Church Field respectively, but of these only about 12 species were "common" or "very common" and likely to be potentially valuable predators.

2. The species from the two arable fields appeared to differ appreciably when compared by an index of similarity but, since most of the species which were absent from one or the other of the two sites formed less than 10% of the total relative numbers, in spite of constituting over 50% of the total species, these differences became insignificant.

3. The most common species trapped in the two arable fields, were Bembidion lampros, B.quadrimaculatum, Trechus quadristriatus, Harpalus aeneus, H.rufipes, Loricera pilicornis, Amara bifrons, Pterostichus vulgaris, P.madidus, Calathus fuscipes, and C.melanocephalus.

4. There was a marked succession of species which may have been due partly to inter species competition and partly to the effects of plant growth. Bembidion lampros, 184. B.quadrimaculatum and Trechus quadristriatus were spring breeders, although larvae were never caught in pitfall traps (but numerous callow adults were trapped in the autumn). This resulted in two peaks of adult activity and abundance during the growth of the crop. Harpalus rufipes, Pterostichus vulgaris, P.madidus and Calathus fuscipes were all autumn breeders and larval overwinterers and generally there was one peak in adult abundance - during the autumn.

5. Data from pitfall traps give valid comparisons of relative abundance but are not of much use in determining absolute populations and hence were not used in calculating the number of insects per acre. This was especially true for animals other than Carabidae which fell into the traps. The available literature suggests that many of those ovganisms may be important prey for carabids.

6. The thionazin broadcast treatments at either 10 or 40 lb a.i. per six-inch deep apre considerably decreased the number of individuals and species trapped. It is concluded this is due to direct toxicity for there is no evidence that the beetles are repelled by the pesticide treatments. The pesticide remained toxic throughout the season but particularly in the first 8 weeks after treatment. The effects of the in-row treatments were not so obvious but it is concluded that small species such as Bembidion quadrimaculatum and Trechus quadristriatus are killed by the "high" dosage rate of 8 lb a.i./acre at the beginning of the season, 185. whereas the "law" rate of 2 lb a.i./acre causes numbers of individuals trapped to increase, particularly the smaller ones; this is attributed primarily to sub-lethal effects on activity.

7. Some exceptions to the above rule were noted particularly Asaphidion flavipes which is more resistant, and Harpalus aeneus which is more susceptible - to the thionazin treatments than other species of similar sizes. These differences are attributed to differences in behaviour. Thus, Asaphidion flavipes, with its cryptic coloration, rarely sought refuge in the soil and only moved in short bursts. This probably resulted in less uptake of pesticide than would occur in other species of similar size. Harpalus aeneus on the other hand actively hid in soil to escape detection - where it probably came into greater contact with the pesticide - but in addition it was capable of releasing a powerful repellent which could in some circumstances e.g. when confined in the soil - cause its own death.

8. The thionazin treatments caused phytotoxicity to potatoes at the "high" rates of application but because of better crop growth at the end of the season (control of eelworm), this increased the numbers trapped of the larger carabid species because they are attracted to vegetation.

9. Evidence suggested that thionazin may increase egg laying but the eggs are sterile or if initially viable are later killed by the pesticide. Larvae appeared to be 186.

particularly susceptible to the pesticide for although a few were trapped in the controls very few if any were caught in the plots treated with the broadcast applications.

10. The long term effects of the thionazin treatments were relatively difficult to assess because of the small size of the plots and the continual migration of individuals from untreated areas. However, in spite of a noticeable decrease in the toxic effects after 8 weeks, there was evidence of renewed mortalities after the lifting of the potato crop. Pesticide residues must therefore have leached into the deeper layers of the soil but were once again made available by the turning over of the soil. These effects, however, did not seem to be as long lasting as the original treatments and it was unlikely that residues of any significance persisted to the following spring.

11. The toxic effects of the soil fumigants were correlated with their molecular weights. Dazomet with the lowest molecular weight proved to be the most toxic soon after application in the autumn. Ethylene dibromide had the largest molecular weight and although it was initially the least toxic, it persisted longer and later proved to be as toxic as Dazomet.

12. The results suggest that all the pesticides except thionazin applied in the row at 2 lb a.i./acre are deleterious to Carabidae but since their toxic effects were comparatively short lived they should not prove a serious hazard if 187. they are not used too intensively at too high rates. Whether these short-lived pesticides can have any serious longer term effects cannot be forecasted from the present experiments but only by doing large scale field experiments over a number of years may this question be resolved. 188. PART IV Persistence of Thionazin Residues in Soil, Water and Carabids CONTENTS page INTRODUCTION 189 MATERIALS AND METHODS 191 Apparatus and Reagents 191 1. Estimation of Thionazin Residues in Soil 192 (a) Treatment of soil samples for residue experiments 192 (b) Analysis of residues 195 (i)Determination of soil moisture 195 (ii)Determination of soil pH 195 (iii)Selection of extraction solvent 195 (iv)Extraction procedure 195 (v)Clean-up 196 (vi)Determination of thionazin by gas chromatography 197 (c) Results 198 2. Thionazin Residues in Water 213 (a) Method of leaching 213 (b) Analysis of residues 213 (i)Extraction 213 (ii)Determination of thionazin by gas chromatography 214 (c) Results 214 3. Thionazin Residues in Carabidae 217 (a) Treatment of beetles prior to extraction 217 (b) Analysis of residues 1 218 (i)Clean-up 219 (ii)Determination 220 (iii) Confirmation by thin layer chromatography . 221 (c) Results 222 DISCUSSION 224 SUMMARY AND CONCLUSIONS 233 189. INTRODUCTION

A study of the ecological aspects of soil applied pesticides on Carabidae would, not be complete without some measurements of the pesticide residues in the environment and in the beetles themselves. Experiments were therefore done to determine the persistence, rate of loss and distribution of thionazin residues in soil and the uptake by carabids with a view to correlating biological effects and residue concentrations. Since thionazin has a measured solubility in water of about 0.1% (w/v) at room temperature it seems likely that leaching of the soil by water will play an important role in the distribution of the pesticide. Thionazin released from the granules will be partly dissolved in the soil water in equilibrium with thionazin adsorbed on the soil particles. Downward percolation of water would be expected to transfer the pesticide slowly downwards to the deeper layers of the soil. Superimposed on this movement will be inevitable losses through evaporation at the soil surface, decomposition by bacteria and other micro-organisms or by simple chemical hydrolysis. All these processes will be affected by the soil temperature. Measurements have therefore been made of the levels of thionazin remaining at different depths inside treated soil samples stored under natural conditions and in the laboratory! 190. Leaching experiments were done in the laboratory with inter- mittent leaching to simulate a variable rainfall as might occur naturally. Concentrations of thionazin were measured in the leachates. Thionazin residues were also determined in carabid beetles in contact with treated soil for varying times. Thin layer and gas-liquid chromatography were used to analyse the residues. 191.

MATERIALS AND METHODS.

Apparatus : Gas chromatography The analytical instrument employed in these studies was a Varian Aerograph, Series 1200 with electron capture detector containing a 250 me tritium ionization source operated at 90 volts D.C. across the detector and a 1mV recorder. The column consisted of a 1/8 in. o.d. x 1 meter stainless steel tube packed with Chromosorb W (60/80 mesh) containing 5% silicone grease 30 and conditioned for several days at 210°C with a flow of nitrogen gas. The operating parameters were : column temperature, 160°C; injector temperature, 175°C; detector temperature, 170°C. The carrier gas consisted of nitrogen (oxygen and water-free), flow rate, 30-53 ml./min.

Reagents: The following "Analar" grade solvent were used; benzene, acetone and redistilled hexane; anhydrous sodium sulphate was used for drying the extracts. A pesticide standard was made by dissolving 10 mg. of pure thionazin in 100 ml. of redistilled hexane. This solution contained 100 micrograms (gg.) toxicant per millilitre (ml.). One ml. of this solution was diluted to 100 ml. to give a solution containing 1 gg. per ml. for use in subsequent gas chromatography. Solutions were stored at 0°C when not in use. 192,

1, Estimation of Thionazin Residues in Soil

) Treatment of soil samples for residue experiments Treated soils were prepared as previously described for laboratory bioassay tests in Part II and 100g. samples of air-dried soil were placed in the test containers. Distilled water was then added to the soil to give a moisture content of 15% by weight (which was equivalent to 76% of the field capacity as shown in Table 10) and the containers were placed in constant temperature rooms at 7°C + 0.5°C and 22°C + 0.5°C respectively. In order to evaluate the effects of aeration, half the containers were provided with air- tight lids, thus cutting down evaporation to a minimum, while the other half were left open. Forced air circulation was provided in each of the C.T. rooms and resulted in the complete evaporation of water from the soils in the open containers within a week at both the high and low temperatures This water loss was compensated for by the Addition of distilled water in fine droplet form from a spray gun without stirring the soil and thus avoiding losses of pesticide residue by physical disturbance of the soil. Two replicate ' containers per treatment were removed at weekly intervals for a total of 5 to 6 weeks, each container being sealed and stored in a deep freeze at —10°C until required for extraction of the thionazin residues.

Treated soils for leaching experiments were prepared as described previously and placed into containers consisting 193.

of modified plastic pill boxes with their "tops" and "bottoms" cut out arid replaced by fine mesh cotton/linen gauze (Field Experiment I) or nylon/terylene gauze (Field Experiment II and Laboratory Experiment ) which were then built up to form columns of 3i in. in depth and 2 in. in diameter. In this way layers of half or one inch of soil could be separated adequately while still forming an intact column. Treated soils, which were initially air-dried, were mixed with distilled water to give a moisture content of 15% by weight and were compacted into the containers giving 25g. in the half inch sections and 50g. in the one inch sections or a total of 175g. per column.

In Field Experiment I the sectioned columns were placed into trenches 3- in. deep, 4 in. wide and 48 in. long in a ploughed field in which large scale field experiments were simultaneously being done. The containers were surrounded by coarse, washed gravel to prevent untreated soil from splashing into the columns during periods of rain. In Field Experiment II the columns of treated soil were buried to the soil surface in a 6 ft. by 6 ft. area containing pure coarse sand to a depth of one foot. In both experiments four replicate columns were removed at 1, 2, 4, 8, 16, 32 and 64 days. Layers from appropriate depths were bulked, placed in polythene bags, labelled and stored in a deep freeze at —10°C until required for extraction. 194. During these experiments a continuous record was kept of soil temperatures at a depth of 1i-2 inches as measured by a mercury-in-steel thermograph. Rainfall data was obtained from the Field Station meteorological record- ings.

In the laboratory experiment, the set up was identical to that described in both the field experiments but the treated soils were kept in a constant temperature room at 15°C 0.5. OPIM 0 and were watered at 4-day intervals with 50 ml. aliquots of distilled water (equivalent to 23.5 mm. of rainfall). Soil leachates were initially filtered through glass wool, those from any four replicates at each time interval being bulked and stored in screw-cap glass bottles at 0°C.

Excess water had to be added to get sufficient leachate to pass through the soil columns. Thus, of the 50 ml. of distilled water added to each column, only 10-15 ml. were collected, the remainder being held by capillary forces, and surface tension within the soil pores and on the soil particles. Watering at 4-day intervals also allowed the soil to dry out during the intervening periods and prevented excess puddling of the soil which otherwise would have made leaching difficult and removal of the soils from the containers messy and inefficient. 195. (b) Analysis of residues (i)Determination of soil moisture In order to express results on the customary dry- weight basis the moisture contents of the treated soils were calculated prior to the extraction of residues by measuring the water loss of samples dried in an oven at 110-130°C for 24 hours.

(ii)Determinations of soil pH pH was measured by a glass electrode in soil suspensions (10 g. in 25 ml. water) using a Pye Model 60 pH meter.

(iii)Selection of extraction solvent Skrentny (private communication) has found a mixture of benzene/hexane/acetone (8/1/1) to be very effective for extraction of pesticides from soil. The acetone is added to facilitate mixing of the soil with the solvents. In practice, however, it was found that soil with water content greater than 20% tended to clump on mixing resulting in a less efficient extraction. The soils were therefore air- dried for a few hours if they contained excess water. It was realised, however, that such drying might intensify degradation of the pesticide and the procedure was only used when unavoidable. (iv)Extraction procedure Fifty grams of treated soil of known moisture content weals weighed in a 500 ml. screw-cap glass bottle of known 196. tare. 100 ml. of a benzehe/hexane/acetone mixture (8/1/1) were added and the boil-solvent mixture was allowed to stand for a few hours.' The mixture was then thoroughly homogen- ized for 15 minutes using a M.S.E. high speed homogenizer. After homogenization,the soil/solvent mixture was allowed to settle and any solvent which had evaporated was replaced (by addition to a previously recorded mark on the bottle) to restore the original soil/solvent ratio. The solvent was then decanted into a 100 ml. screwcap glass bottle containing anhydrous sodium sulphate to remove any traces of water. The bottle was sealed first with a strip of aluminium foil and then with the plastic screwcapt and was stored at 0°C until required for analysis.

This method of extraction gives almost theoretical recovery using pure thionazin (Skrentny, private communication) but 85-98% efficient extraction of thionazin when applied as a granular formulation. This wide range of recovery was later shown to be due to variability of the formulation. (v) Clean-up After extraction, the organic solvent containing the pesticide was coloured yellow by various pigments from the soil. These co-extractives, however, did not interfere with the G.L.C. analysis. Ashurst soil being light and sandy could therefore be analysed by G.L.C. without clean-up but other soils rich in organic matter would probably require clean-up especially if analysed by electron capture gas chromatography. 197. (vi) Determination of thionazin b gas chromato ra•h (G.L.C.) Aliquots of 1-5 µ1. of extract were injected into the gas chromatograph using a Hamilton micro-litre syringe. Smaller volumes were avoided as much as possible because of the increased errors of injection volume but 5µ1. gave reproducible results. As a rule, a minimum of two equal aliquots were injected from each extract providing differences in peak height were no greater than 5 per cent.

Reference chromatograms for any one set of soil extracts were prepared by injecting into the gas chromatograph 1-5 µ1. of a stock solution prepared from the pure pesticide. As thionazin gave a characteristic, sharp-pointed, narrow peak, measurement of peak height was preferred to that of peak area although the latter measurement may give more accurate results (Scott and Grant, 1964). Standardization was carried out several times during the course of a given set of extracts to check retention time and peak response. The retention time for thionazin under the stated conditions of column length, type of stationary phase, with a column oven temperature of 160°C and a carrier gas flow rate of 30 ml./min. was 1 min., 40 sec.

The concentrations of thionazin in the soil extracts were corrected to a dry weight basis using the appropriate factor calculated from the moisture content. If, for example, soil moisture content = 17% by weight, the conversion factor = 1- 10017 = 1- 0.17 = 0.83 and residues are divided 198. by this factor. The amount of thionazin in the soil r4r ; extract, as shown by the peak height, is obtained froth the standard curve. The concentration of thionazin in parts per million of soil extract is obtained by dividing the amount of thionazin found (in gg.) by the weight of oven dry soil injected (in g.).

The results from the experiment to determine the effects of temperature, soil moisture content and aeration on the persistence or loss of thionazin residues in Ashurst sandy loam soil are summarized in Table 52.

The 14.12 p.p.m. of thionaziri from the freshly treated Ashurst sandy loam soil represented an 86-90% recovery from soil treated with thionazin formulated as 5% active ingredient on Attapulgite granules at a dosage calculated to give 16 p.p.m. of thionazin. The actual thionazin content of the granules was found to vary considerably from the stated figure.

The results from this table are also represented graphically (Fig.49). It can be seen from this graph that thionazin residues fell rapidly during the first 3-4 weeks and thereafter more slowly. The persistence and metabolism of thionazin in soil have been studied by Getzin and Rosefield (1966) and by Getzin (1967) who obtained very similar results using radioactive labelled material. From the graph it is /99. Key to Table 52

* 1. CLOSED VESSELS - were vessels with air- tight lids to prevent excessive loss of water by evaporation. No addition of water was made after the initial preparation.

2. OPEN VESSELS - were vessels without lids to allow for aeration of soils. Loss of water by evaporation occurred freely and was compensated for by addition of distilled water.

3. CONC. - Concentration - mean of two replicates.

4. pH - Mean of two replicates.

5.S.M. - Soil moisture - mean of two replicates - % by weight. 200.

Table 52 Persistence of thionazin in Ashurst sandy loam at various temperatures, soil moisture contents and amounts of aeration

220C ± 0.5°C Time CLOSED VESSELS1 OPEN VESSPTs2 (weeks) Cone pa41- S.NI.5 Conc. pH S.M. ppm. ppm. 0 14.12 6.8 16.9 14.12 6.8 16.9 1 9.85 6.7 14.9 10.74 6.6 7.8 2 8.70 6.5 15.1 10.60 6.5 13.8 3 7.19 6.4 14.0 9.68 6.5 15.0 4 7.18 6.3 13.8 9.32 6.4 14.3 5 6.67 6.4 12.5 9.08 6.5 14.0 6 5.61 6.3 12.3 - -

7°c + 0.5°0 Time CLOSED VESSELS OPEN VESSELS (weeks) Conc. pH S.M. Conc. pH S.M PPm. PPm. 0 14.12 6.8 16.9 14.12 6.8 16.9 1 12.95 6.9 16.1 13.57 6.5 13.3 2 12.86 6.7 16.3 12.46 6.5 14.3 3 11.56 6.8 15.7 12.68 6.6 15.5 4 11.89 6.6 15.1 12.50 6.6 15.5 5 10.88 6.5 15.4 12.09 6.6 16.0 6 10.29 6.5 16.4 201.

N C 0

O C 4O ea

0 A 7°C open vessels ♦ 7°C closed vessels O 22°C open vessels • 22°C closed vessels

1 2 3 4 5 6 Time (weeks)

Fig. 49 Effect of temperature on the persistence of thionazin in sandy loam soil in open & closed vessels 202. pbssible to obtain a comparative estimate of the loss of thionazin residues in various environmental conditions (Table 53). The slowest rate of loss occurred at the low temperature (7°C .4- 0.3°C) and continuous ventilation and was 12.5% after 28 days. The rate of loss after 28 days was 2.1 times faster, at the same temperature in closed vessels; 7.3 times faster at 22°C + 0.5°C in open vessels and 13.3 times faster at 22°C 4- 0.5°C in closed vessels. These differences increased somewhat with time but remained in the same order (Table 53).

Similar results were obtained in the two field and one laboratory experiments to determine the persistence of thionazin at different depths inAshurst sandy loam soil (Tables 54, 55 & 56;Figs.50(1-3)51(1-5) & 52). In these experiments thionazin disappeared most rapidly from the top half inch layer and the main cause appeared to be leaching rather than chemical or biological degradation. Sorption of pesticides to the particles of a sandy loam soil is not as powerful as in silt or clay loams (Getzin & Chapman, 1959; Lichtenstein & Schulz, 1959; Roberts, 1963; Getzin & Rosefield 1966) and there is a relationship between soil type, porosity and pesticide movement (Swanson et al., 1934). These factors satisfactorily explain the downward movement of thionazin by the leaching action of water. In the field experiments, loss of the pesticide was greatest in the top half inch and in the lower 2i-34 inch 203. Table 53 The rate of loss of thionazin residues in Ashurst sandy loam soil as affected by temperature, soil moisture and aeration

Time (clays) after beginning of experiment Per Cent 22°C + 0.5°C 7°C + 0.5°C Loss Moist soils Moist soils Moist soils Moist soils of in closed in open in closed 'in open Thionazin vessels vessels vessels vessels

12.5 2.10 3.85 13.65 28.0 (13.3x) (7.3x) (2.1x) (1x)

25.0 6.00 12.25 38.85 (6.5x) (3.2x) (1x)

37.5 12.25 (40.251x)5 (3.3x)

50.0 25.2 (1x) 204.

Key to Tables 54-56

S.M.%= Soil moisture content (% by weight)

Conc.= Concentration of thionazin as determined by G.L.C. with electron capture detector. N.B. Cones. At 1-64 Days = Uncorrected for per cent rocovetias

Table 54

N.B. Conc. At 0 Days = 95-98% recovery of applied dosage. pH = almost constant over range 6.3-6.5

Table 55 N.B. Conc. At 0 Days = 84-86% recovery of applied dosage. pH = almost constant over range 6.0-6.3

Table 56 N.B. Conc. At 0 Days = 84-86% recovery of applied dosage. pH = almost constant over range 6.0-6.3 205. Table 54 FIELD EXPERIMENT I Persistence of thionazin residues at different depths in sandy loam soil (12.v.67 - 15.vii.67)

Time in Days Depth 0 1 2 4 (inches) S.M Conc. S.M. Conc. S.M. Conc. S.M. Conc. PPm. PPm. PPm. PPm. 16.5 10.78 4.5 10.25 11.0 10.65 22.0 8.18

3-13 16.5 10.78 14.3 11.24 13.9 10.65 23.4 12.99

14-24 16.5 10.78 14.2 10.72 13.6 10.83 26.6 14.17

23-31 16.5 10.78 14.2 11.24 13.8 11.79 25.6 13.18

Mean conc. in 0-33 10.78 10.86 10.98 12.13 inches Time in Days 58 16 32 64 Depth (inches) S.W. Conc. S.M. Conc. S.M. Conc. S.M. Conc. % PPm. % PPm. % PPm. % PPm. 0-3 18.7 6.95 22.0 2.48 2.0 1.00 4.0 0.57

1 el l 7- 1 7 23.7 10.35 28.6 6.89 7.8 2.65 16.8 0.83

13-24- 24.2 10.89 29.2 7.59 20.0 2.60 18.2 0.82

23-33 22.4 8.15 27.3 5.53 15.6 2.63 15.0 0.73 Mean conc. in 0-33 9.09 5.62 2.22 0.74 inches 206. Table 55 FIELD EXPERIMENT II Persistence of thionazin residues at different depths in Ashurst sandy loam soil (14.vi.67 - 17.viii.67)

Time in Days Depth 0 1 2 4 (inches) S.M. Conc. S.M. Conc. S.M. Conc. S.M. Conc. PP111. % PPm. Mom. PPm. 0-4 16.5 9.50` 2.0 6.12 2.0 7.41 1.5 5.06'

16.5 9.5q 13.3 8.30 8.4- 8.67 3.0 7.78 14-24 16.5 9.50 15.2 8.73 15.2 8.80 15.4 7.97 24-34 16.5 9.50 14.7 9.12 15.8 9.32 16.3 8.05 Mean conc. 8.07 8.55 7.22 _g4 16.5 9.50 Inches

Time in Days Depth 6 16 32 64. (inches) S.M. Conc. S.M. Conc. S.M. Conc. S.M. Conc. % ppm. % PPm. % 0-4 1.8 5.14 3.3 3.39 1.5 1.35 11.5 0.92 4-14 2.5 7.07 21.9 7.17 6.6 4.61 22.8 1.73 14-24 14.4 6.18 25.2 8.47 26.6 4.49 26.6 2.32

24- -34- 16.8 7.29 27.4 6.47 26.4 3.88 24.1 1.75 Mean conc. 0-3i 6.42 6.38 3.58 1.68 inches 207. Table 56 LABORATORY EXPERIMENT Persistence of thionazin residues at different depths in Ashurst sandy loam at 15°C + 0.5°C

Time in Days Depth 0 1 2 4- (inches) S.M. Conc. S.M. Conc. S.M. Conc. S.M. Conc. PPm. PPm. PPm. PPm. 04 16.5 9.50 8.1 9.89 5.7 10.38 4.0 9.23 2-1 16.5 9.50 14.6 8.06 13.6 10.07 13.2 8.53 14-2i 16.5 9.50 13.4 8.65 14.1 9.42 13.5 9.07 2-1-33- 16.5 9.50 13.7 10.26 9.5 8.92 7.8 8.88 Mean conc. 0-3i 9.50 9.2 9.70 8.93 inches

Time in Days Depth 8 16 32 64 (inches) S.M. Conc. S.M. Conc. S.M. Conc. S.M. Conc. % PPm. % PPm. 04 16.5 9.82 6.3 5.97 9.3 2.44 3..1 1.05 1-1f 17.6 9.11 21.9 7.03 25.3 5.09 19.5 2.61 1i-2i 16.2 8.22 21.1 7.22 23.3 6.12 18.8 3.64- 24.-34- 11.1 8.97 19.3 8.22 24.1 7.50 12.3 4.56 Mean conc. 0-3i 9.03 7.11 5.29 2.9? inches 208. 15 .0.4epth 0 -0-5 in. 14 j -6-depth 05-t-5 in. -0-depth 1.5-65 in. -9-depth 5-5-3-5 in. E 0. a. C 11

C o 9

O 7

C a

16 22 64

30 -0-- maximum •—•-• mean 111+. minimum

0 0 * 4 6 16 as 64

o- r"---1 I 0 16 *4 II* 40 46 66 64 Time (days) Fig. 50.1-3 Persistence of thlonazin at different depths In sandy loam soil In Four Acre Field. Field experiment I. 12 May to 15 July,1967. 50.1 Breakdown curves 50.2 Soil temperature ('C) at depth of 2 in. 50.3 Rainfall data 209. lor -0-depth 0 -OAS en. -6-depth 05-14 en. -0-depth 1-5-2.5 en. -4-depth 2.5-1.5 en. 0.. 0. C N aC O 8 .0

C O 4 a C ▪(I P C O 2

18 32 84

.-0— maximum — mean 60 minimum

0 0 2 4 8 IS 92 84

50e-

‘"a E so

0 so 0 CC

0 rt 18 114 32 40 45 56 64 Time (days)

Fig. 51.1-3 Persistence of thionazin at different depths in sandy loam soil in Four Acre Field. Field experiment 11.5 June to 17 Aug.,1967. SI. I Breakdown curves SI.2 Soil temperature (°C) at depth of 2 in. 51.3 Rainfall data Fig. 52Persistence ofthionazin atdifferent depthsinsandy loam soil

Concentration of thionazin (ppm) 8 2 6 0 4 in laboratory conditions at15°C. 0

Time (days) 32

- - -b.-depth -1 0 0-depth 7- - depth0-0-5in. depth 0.5-1-5 2.5.-3-5 1.5-2.5 in. in. in. 64 211. layer whereas in the laboratory experiment the loss was

greatest in the top half inch but least in the lowest

layer (Table 57).

Table 57 The "half life" of thionazin residues (in days)

in Ashurst sandy loam soil at various depths.

Calculated Half Life (Days)

Treatment Date . Depth (inches) (1967) 1-i4 11-24 21-32

Field Experiment I 12.V-15.VII 11 22 23 16 Field Experiment II 14.VI-17.VIII 10 31 31 27 Laboratory Experiment 22 37 49 62

This result can be explained by the higher overall temperatures experienced in the soils exposed to natural conditions and by a possible enhancement of leaching effects in the field experiments owing to contact of the soil columns with other soil beneath.

The other main difference between the field and laboratory experiments was in the rate of loss of the pestivs, cide. Thus, in spite of the considerable leaching which occurred in the laboratory experiment, the mean loss of the 12 —0— field experiment 12.v.67-15.vii.67 E O. —0— laboratory experiment at 15°C a- 10

I I 0 O 4 8 18 32 64 Time (days)

Fig. 53 Relative persistence of thionazin in sandy loam soil in field a laboratory conditions. 213. pesticide in the 31 inch column of soil was less than half that in Field Experiment I, the "half life" of thionazIn residues in the laboratory soil being 39 days as compared to 17 days in the soil in the field (from Fig.53).

2. Thionazin Residues in Water (a)Method of leaching Leaching was done as described previously using 50 ml. aliquots of distilled water per 32 in. column of soil at regular periods of 4 or 8 days. The soil leachates as collected were yellow but appeared to be free from solid soil particles.

(b)Analysis of residues (i) Extraction Soil leachates were removed from cold storage at 0°C and allowed to thaw out overnight at room temperature. Some sedimentation occurred and care was taken not to disturb this sediment when removing aliquots for pesticide determination. The pH was determined on 25 ml. of each leachate using a glass electrode. Thionazin residues were then extracted from a further 25 ml. of soil leachate as described by Lichtenstein et al (1966) but using "Analar" grade benzene instead of the recommended solvents. Leachates were extracted twice in a 500 ml. separating funnel, the benzene extracts being transferred to a screw-capped glass bottle and treated with a little anhydrous sodium sulphate. Emulsions were rarely troublesome but could be broken down 214.

by thq addition of a "pinch" of salt. The bottles were then sealed And stored at 0°C until required.

(ii) Determination of thionazin by gas chromatography The benzene extracts from the soil leachates did not require clean-up or concentration prior to G.L.C. analysis. Injections of 3 gl. of extract gave deflections ranging from 39% to 15% of full scale. After the tenth consecutive soil leaching, however, when residue concentrations became low? 5-7 gl. had to be injected to obtain sufficient sensitivity.

(c) Results The method of extraction described above gave a 92-108% recovery from thionazin formulated as granules on Attapulgite clay. The results from the soil leachates are summarized in Table 58 and in Fig.54.

From' data in Table 58 it was calculated that the total amount of thionazin leached by 550 ml. of water (equivalent to 259 mm. rainfall) was equal to 154.1-231.5 gg. or 7.9-11.8% of the applied dosage. The amount of thionazin remaining in the soil after the 64th day was 31.3% of the applied dosage (from Table 56) and hence 56.9-60.8% of thionazin could not be accounted for and were presumably lost by causes such as bacterial and chemical degradation as well as by evaporation from the soil surface. The amount lost by leaching is not the maximum estimate because ro Soil Time of Number of Soil- Concentration Amount of Ageing Leachings Water of Thionazin Thionazin in CO (days) per Column pH in Leachate 10-15 ml. of (p.p.m.) Leachate (11;.) so o c+ 0" 0 H. Treated* 4 1 6.9 2.13 21.3-32.0 0o 1-... 0 8 0 It 8 2 7.1 5 25.7-38.6 P • 0 O w N ti 12 3 7.1 2.42 24.2-36.3 & 0'1 1-1. 4 it 16 4 6.7 2.07 20.7-31.1 ro ro E ro ti 20 5 6.6 1.46 14.6-21.9 M • 4 11 24 6 6.7 1.29 12.9-19.4 P 0 0 0 0. %.11 tt 28 7 7.2 0.99 9.9-14.9 1Po ro o B 14. il 8 7.2 0.95 9.5-14.3 p 0 32 o H. 0 Ft -- 0 It 40 9 8.1 0.80 8.0-12.0 ri-o 0 1-0 ro H. It 10 0.47 4.7-7.1 48 7.5 o 0 1-a• 2.6-3.9 0 0 il 56 11 8.6 0.26 ro 1+ c+ Control 0 1 7.0 0.00 0 0 ln & ro ri P ci- * 1.96 mg. thionazin in 175 g. soil ro CD Fig. 54Amountof thionazinleachedfroma3.5in. column ofsoilby50ml. 1 0 . U r7 0 re c 1.5 C C

in leac hate 2 3-0 2-0 00 0.5 1.0 0 aliquots of distilledwater. 1

• 2

••1 Number a 3

• • 11 4

O—O thionazin leachings (50m1)with time ( %vow 5

4,— 8 / —0 pH / /

/ 9

4 days)

• • N NI 10

/ /

/ 11 9.0 8 0 7.0 8.0 217. leaching was terminated after 56 days. The leaching did not simulate natural rainfall during typical summer months at Ashurst Lodge as can be seen from Figs.50.3 and 51.3 although the total amount applied was only 1.5 times as great as the total rainfall recorded for May and June of 1967 which amounted to 175 mm.

It would appear from a comparison of Field Exper- iments I and II that rain had its greatest effect on leaching of the pesticide in the first weeks after application of treatment. Also as leaching progressed beyond the 32nd day the alkalinity of the water gradually increased (Fig.54)for reasons not altogether clear which would favour chemical degradation of the pesticide. In these circumstances it does not appear that the loss of thionazin in solution in water would greatly exceed the observed maximum figure of 11.8%.

3. Thionazin Residues in Carabidae (a) Treatment of beetles prior to extraction Beetles of known sex and species were put on Ashurst sandy loam soil containing 16 p.p.m. of thionazin applied as granules. Conditions were identical to those described for bioassay tests in Part II but the• insects were exposed for a maximum period of 24 hours at 15°C + 0.5°C. During this period most, if not all, of the treated insects were killed whereas no mortalities occurred in the untreated controls. The beetles were then removed, placed into well 218. labelled glass tubes and kept in a deep freeze at -10°C until required for extraction and determination of thionazin residues.

(b) Analysis of residues Methods for detecting pesticide residues in dead bees have been investigated for more than 10 years at Rothamsted Experimental Station (Needham et al., 1966; Needham - private communication, 1968) but in no case have residues of organophosphate pesticides been extracted although much evidence of organophosphorus poisoning has been obtained by non-

specific methods such as esterase-inhibition tests. Davis and Harrison (1966) briefly described a method for the extraction of chlorinated hydrocarbon pesticide residues from soil invertebrates (including Carabidae) and the method described below is one which incorporated some of these ideas and also included other general procedures recommended by de Faubert Maunder et al. (1964) and Egan et al.(1964) for gas chromatography determinations and Abbott and Thompson (1966) and Lichtenstein et al. (1966) for thin layer chromatography determinations. The beetles were first washed thoroughly in a stream of distilled water to remove adhering soil particles and were then dried on blotting paper. Samples of any one species and sex were bulked and weighed on an electric, air-

damped, aperiodic balance to the nearest milligram (mg.). The beetles were then placed in a 25 ml. extraction bottle 219. and 20 ml. of solvent (benzene/hexane/acetone mixture in the ratio of 8/1/i) were added. The sample was macerated for 15 minutes (although 5 minutes may have been sufficient) in an electric M.S.E. homogenizer after which any solvent which had evaporated (as indicated by comparison of the levels with a previously recorded mark) was compensated for by addition of analytical grade benzene. The mixture was allowed to settle and the solvent decanted into a 25 ml. screwtop glass bottle and allowed to dry over anhydrous sodium sulphate. (i) Clean-up The extracts appeared to be clear and colourless but in fact contained too many impurities or co-extractives for satisfactory analysis of thionazin residues by gas chromatography without prior clean-up. The extracts were therefore concentrated to 1 ml. by evaporation of the solvent under a current of air and were then cleaned by the sweep co-distillation method of Storherr and Watt (1965). This rapid clean-up procedure was originally devised for organophosphate pesticide residues in crude crop extracts prior to estimation by G.L.C. and proved satisfactory for the beetle extracts. Briefly, the procedure involved injecting concentrated beetle extracts into a heated glass tube packed with glass wool at 175°C. The vaporized extract was carried through the column in a flow of nitrogen gasp the pesticide passing through freely while other extraneous 220. Materials were deposited on the glass wool. The pesticide on emerging from the glass tube was condensed in a Teflon tube coiled in an ice bath and was collected in 1-2 ml. of solvent (i.e. benzene) in a 10 ml. graduated test tube. The extract was concentrated or diluted as necessary and chromatographed. This method was reputed to give 89-101% recoveries of pesticide. Unfortunately, with beetle extracts, interfering compounds still persisted which tended to mask the thionazin peak, making identification of the pesticide somewhat doubtful. Several additional methods of clean-up were also attempted such as chromatography through columns of partially de-activated alumina (de Faubert Maunder et al. 1964) or partially de-activated "Nuchar" or mixtures of "Nuchar" and Fuller's Earth (Call - private communication) but none was completely satisfactory for adequate clean-up. (ii) Determination By fortification of the control extracts and by comparison of the chromatograms from the fortified and treated beetle extracts it was possible to show presence of thionazin in extracts although such data alone was not sufficient to prove the presence of thionazin. The following satisfactory clean-up procedure was then developed which both confirmed the presence of thionazin and enabled quantitative estimates of residues to be made. This method involved the use of

* A Wilkens Instrument & Research Inc. product, Walnut Creek, California. 221. thin layer chromatography (T.L.C.) in conjunction with G.L.C. for quantitative evaluation of the separated pesticide residues.

(iii) Confirmation by thin layer chromatography

Apparatus: This was as described by Abbott and Thomson (1966).

Reagents: The stationary phase on the chromatoplate consisted of silica gel G (Camag) with calcium sulphate binder. The mobile solvent was a mixture of hexane, chloroform and methanol (7:2:1).

Visualization reagents: Plates were sprayed successively with a palladium chloride solution (0.5g. palladium chloride and 2 ml. conc, HC1 in 98 ml. of acetone),and 5N sodium hydroxide.

Method: Extracts were concentrated to about one drop under a stream of air. Using freshly made pipettes, extracts were spotted 2.5 cm. away from the lower edge of a chromatoplate. The plate was developed at room temperature until the solvent front reached about 2.5 cm. from the upper edge (time approximately 30 minutes). After removal of most of the mobile solvent by evaporation, the plate was sprayed successively with the palladium chloride and the 5N sodium hydroxide solutions. 222. Residues from the carabid extracts were, however, so small that they were below the lower limits of detectab- ility by this method (which is in the region of 0.1-1.0pg as compared to 0.1-1.0 nanograms*by G.L.C.). As insufficient pesticide was present in the carabid extracts to give a visible spot with the visualization reagents used, the area of silica gel suspected of containing the pesticide was not treated with these reagents but was scraped out into a test tube and the pesticide eluted out for further examination by G.L.C. Redistilled hexane proved inadequate for elution and was replaced by acetone followed by partitioning into redistilled hexane. This yielded an extract of sufficient purity for quantitative estimates by G.L.C.

(e) Results Under the stated conditions, thionazin had an Rf value of 0.57. Spotting of standard solutions of pure thionazin on to the chromatoplates as described above gave recoveries of 91% but in practice estimated recoveries of between 57-62% were obtained from the beetle extracts. This may have resulted from an underestimate of the suspected layer of the silica gel containing the pesticide but an overestimate of this layer was purposefully avoided in order to pick up as few interfering compounds as possible. If more time and more beetles had been available it is possible that more accurate results could have been attained. The results of these analyses are summarized in Table 59. * 1 ng. = 1 x 10-9g.

223. Table 59 Thionazin extracts from Carabidae kept on treated soil

Speciesl Sex Mean Treatment2 Thionazin3 Conc. fresh Residues of residues weight recovered per beetle (mg.) (p.p.m.) (pg.)

A.dorsale Ae 9.5 Residual 3.11 0.03 contact G.fuscipes c?? 40.0 0..46 0.02 -H.rufipes ?& 83.0 IT 0.04 C.fuscipes 58.0 0.02 V°t 0.34 H.rufipes 8810 0.03 P.madidus el? 105.0 re 0.19 0.02

1. Adult beetles of undetermined age. 2. Exposed for 24 'hours to 100g. of Ashurst sandy loam soil treated at 16 p.p.m.(=1.6mg) with 5% granular thionazin (active ingredient). Soil moisture content was 15% by weight; temperature 15°C 0.5°C; relative humidity 96%1 overhead strip lighting with a 16/24 hour time period. 3. Determined by G.L.C. with electron capture detector and uncorrected for per cent recovery. 224. DISCUSSION

There are three major ways in which pesticides may disappear from the soil. These are (1) by physical removal from the area of interest as, for example, by leaching or volatilisation; (2) by destruction such as by chemical or biological decomposition and (3) by uptake by plants and other living Organisms. From the known physico- chemical properties of this pesticide it might be expected that one of the pathways of its degradation would be by hydrolysis to produce the sodium salt of 2-pyrazinol. That this occurs has been shown by Kiigemaji and Terriere (1963) but there is little evidence to show that this compound persists long in the soil or that it has any significant biological activity (Getzins 1967). It would appear from field and laboratory experiments on the persistence of thionazin at different depths in Ashurst sandy loam soil that one of the causes of its disappearance from the soils irrespective of degradation processes, is downward leaching in solution in water. Thus, although little appreciable loss of the pesticide was observed in the first 8-16 days, there was a distinct change in its concentration at the different depths examined, concentrations in the second, third and fourth layers being at or above those of the mean and that in the topmost layer being distinctly below that of the mean (Figs.50.11 51.1 & 52)0 This suggests that loss of pesticide was due more to leaching 225. than to volatilization which does occur at 22°C and 75-100% R.H. as shown by bioassay tests (see Part II). The loss of thionazin from soil showed a curvilinear relationship with time typical of insecticide breakdown in soil. This procesS is influenced by (1) the nature of the pesticide and its physical and chemical properties (2) the formulation of the pesticide, the nature of the carrier and the binding agent being of importance (Barthel et al., 1960; Mulla and Axelrod, 1960) (3) the soil type, especially organic content and (4) climatic factors, especially temper ature and rainfall, The pesticide is probably released more readily from the larger pores of the granules at first and more slowly from the finer pores and this could account for the greater availability and breakdown initially. Loss of the pesticide after desorption from the granules can, however, also be modified by biological and chemical degradation. In this respect the gradual decrease in soil pH from 6.8 to 6.3 observed in the treated soils in closed vessels is perhaps significant (Table 52). This was perhaps because the compound was being hydrolysed to phosphoric acid as one of its degradation products but it may also have been due partly to an increase in CO2 - as a result of increased microbial activity. Fey (1966) has shown that when malathion and sumithion (01 0-dimethy1-0.-(3-methyl-4-nitrophenyl) phosphorothioate) are incorporated into a variety of soils, stimulation of respiration occurs which is associated with 226. an Inca-ease in microtlora population. This, however, is most likely an amendment effect i.e. the killing of certain species permitting others to increase. Getzin (1967), on the other hand, has shown that one of the products of the degradation of thionazin in soil is CO2, as much as 60% of 14 the C from the parent labelled compound being recovered as C140 2 after 20 weeks, Although no experimental work was carried out on the metabolic products of thionazin in this study, the above data on soil pH may be regarded as corroborative evidence that acidic compounds are produced which are probably the result of either chemical or biological degradation processes. The effects of temperature and aeration on moist treated soils were some of the more important environmental factors studied, both of which affect persistence in the soil. Thus, the combination of high temperature and adequate moisture had the greatest effect on the disappearance of thionazin from the fairly acid Ashurst sandy loam soil (Fig.49) whereas low temperatures and dry conditions had the opposite effect. Aeration in combination with high soil moisture contents probably assists in the disappearance of the pesticide from the soil as a result of increased volatilization but in the absence of additional water it causes drying out of the soil with a consequent decrease in disappearance presumably because the pesticide is absorbed more strongly to the dry soil particles and also because chemical decomposition is decreased. 227• /A addition to these physical and cheWical effects, microbial activity is also stimulated by the presence of water (Lichtenstein and Schulz, 1960) which further assists in the degradation of the pesticide in the soil. Movement of the pesticide in soil is governed by a number of factors, the most important of which are (1) the chemical nature of the pesticide and its physical properties such as solubility in water and volatilization in air which bears a relationship to its vapour pressure; (2) soil type and topography; (3) uptake or transport in living organisms such as burrowing animals and in the roots of plants and (4) by the action of man through his cultivations. Consideration of the chemical nature of the pesticide affords an explanation as to why some exhibit more movement in soil than others. It is well known, for example, that chlorinated hydrocarbon pesticides move very little in soils owing to their low solubilities in water and to their relative involatility. Hence their prolonged persistence in the environment. Organophosphorus pesticides, are much more soluble in water and will therefore be leached more readily than will the chlorinated hydrocarbons. As an example, Lichtenstein (1958) showed that parathion leached through soil more readily than did lindane which is less water soluble. Some organophosphorus pesticides, however, move more readily through soil in the gaseous phase than in solution on account of their greater volatility (Burt et al., 1965) which explains 228. why, fyr examples phorate and disulfoton aro more effective is dry soils than monazon and dimethoate which move mainly in solution in water. Formulations might also be expected to affect movement and if persistence and movement are closely inter-related, which they appear to be, then pesticides from granular formulations should move less than, for example, from emulsions which in turn will move less than fine dusts or wettable powders. Soil type encompasses a number of factors which all may affect movement. These include particle size of soil, the order being coarse sand > fine sand > silt > clay. Sfirface area is inversely related to particle size. The surface area influences the amount of pesticide which can be adsorbed and is modified by organic matter and soil moisture, the former tending to increase and the latter to decrease adsorption. Harris (1964) has shown that in moist soils, inactivation of the pesticide was proportional to the organic content of the soil while in dry soils, inactivation was related to the adsorptive capacity of the mineral fraction. Particle size also affects the pore space which is regulated by the compactness of the soil. The pore spaces, like the surface area, are modified by organic matter and soil moisture, the greater the organic matter the greater the pore spaces while the greater the soil moisture, the less are the pore spaces. Pore spaces affect movement of the 229. pesticide in the vapour phase although this factor is probably not very important for thionazin (Call, private communication) but Pore size determines water permeability, hence leaching. Thus, the more compact the soil, the less the movement and vice versai This is one reason why cultivations assist in the removal of pesticide residues from the soil (see Lichtenstein & Schulz, 1961). Topography is of importance for less soluble pesticides (i.e. the chlorinated hydrocarbons) because it assists in the movement of these pesticides by surface run off during periods of rainfall. Little work seems to have been done on this aspect of the movement of pesticides in soil but Lichtenstein (1958) has demonstrated the downward leaching of lindane on sloping plots, 1.3 to 2.2 times more pesticide being recovered in the lower half as compared with the upper half of the plot (slope 5° to 15°). Edwards (1964) obtained similar results when drenches of DDT were applied to steeply sloping plots but differences in the upper and lower halves of the plots were not significant although there was insecticide in untreated soil lower down the slope. The movement of pesticide residues from soil into crops has been extensively studied by a number of workers including Getzin and Chapman (1959), Lichtenstein (1959, 1960), Lichtenstein et al. (1962, 1964, 196511 19652, 19653) and Wheatley (1965) to name but a few. Briefly, the results from these investigations show that movement of pesticides 2500 from soils into plat iS greatest from sandy soils which are relatively free of organic matter and that relatively small amounts are absorbed from agricultural soils. Formulation also affects uptake, more pesticide being taken up from soil treated with emulsions than from that treated with granules. In general uptake by plants is only a small fraction of the amount applied to the soil. It might therefore be expected that a compound like thionazin with its demonstrated solubility in water will move rapidly through soil and be taken up by the roots of plants. Thionazin, has known systemic properties but there is little evidence as yet to show that it is actively taken up into plants in sufficient amounts to cause significant losses from soil, or that it persists long in the plant tissues. Very little work seems to have been done on the movement of pesticides in or from soils on animals. Minute amounts will of course be sorbed on to soil invertebrates and in particular on earthworms which actively ingest soil in their burrowings but no estimate has been made of how much loss of pesticide this could cause. The fourth major way in which pesticides can move through soil is by mechanical action such as by cultivations. There are several ways in which this movement can be studied including the use of iron filings (Morrison and Crowell, 1952), fluorescent tracers (Staniland, 1961; 1964a; 1964b), radioactive compounds (Lichtenstein, 1958) and more recently 231. by gas chromatography. Depending on the type of implement used pesticides can be moved in all directions in the soil. Edwards (1964) found it extremely difficult to mix insecticides deeply into field soils and in all cases it would appear that no matter how thorough the mechanical mixing, pesticides remain mostly in the top 3 to 6 inches of soil.

The factors affecting movement of pesticides in soils have been briefly described for comparison with results obtained from the leaching experiments. It might therefore be expected that movement of thionazin in soil (and in soil water) would be least in heavy clay soils with much organic matter and greatest in light sandy foams with little organic matter. The fact that thionazin is soluble in water to the extent of 0.1% will affect its distribution in soil quite considerably so that not only will it be readily available for uptake by the roots of plants but it will spread by leaching and diffusion into all the soil pores in its vicinity. Any organisms living in these pore spaces, including carabids, will therefore be affected not only by direct contact with the pesticide particles but also by contact with the pesticide in solution in water as well as by fumigation from the pesticide in the vapour phase, although these effects might be expected to be very slight. This explains why carabid species which burrowed into moist soil were much more susceptible to the pesticide than those which only crawled on the surface. 232. The beetle extracts indicated that the rate of pick up of thionazin was approximately the same for all the species tested, irrespective of size. As a result, a smaller species would pick up proportionately more pesticide per unit weight than a larger species - which could contribute to their greater susceptibility. The amounts found in the animal were extremely small in relation to the amount applied to the soil and would therefore not be expected to cause any appreciable loss from the soil. However, they may be of significance if the beetles were to be eaten by scavengers or larger predators for it has been shown that residues of chlorinated hydrocarbon pesticides can and do accumulate in the fat bodies of invertebrates or the organs of birds (Davis, 1966; Walker et al., 1967). These residues, although perhaps not lethal on their own, may affect the metabolism or reproduction of these animals and may eventually have serious consequences. 233. SUMMARY & CONCLUSIONS

1. Experiments were done to determine the persistence and recovery of thionazin (0,0-diethyl 0-2-pyrazinyl phosphorothioate from a treated sandy loam soil; from the soil water and from adult carabids kept on the treated soil. Soils treated at 16 po p.m. (32 lb A.I./6-in.acre) were aged in controlled environmental conditions in the laboratory as well as in variable conditions in the field. Residues were determined by gas chromatography with electron capture detector.

2. Thionazin disappeared most rapidly from hots moist soil (22°C and 76% of field capacity) and least rapidly from cold, dry soil (7°C and variable moisture content). The loss of thionazin from soil showed a curvilinear relationship with time, being relatively more rapid in the earlier stages. This was partly attributed to processes of adsorption and desorption which could be modified by a number of factors such as (1) formulation (2) soil type and (3) climatic factors as well as by microbial and chemical degradation. It appeared that adsorption was reversible at first but as the pesticide was released from the granules and became dispersed in the deeper layers of soil the forces of adsorption increased and those of desorption decreased. 3. Disappearance of residues occurred most rapidly in the top half inch of soil and was attributed mainly to leaching. It was estimated that as much as 11.8% of the applied dosage 234. could be lost in soluticin in rain water after 64 days with rainfall totalling 259 mill. Leaching accounted for approximately 17% of the total ambunt of thionazin lost. 4. The "half life" Of thionazin residues in soil was 17 days in the field experiment as Compared to 39 days in a laboratory experiment at a constant temperature of 15°C 0.5°C and with periodic leachings at 4 to 8 day intervals. It is concluded that loss of pesticide residues in soil cannot be explained on a limited number of simple factors alone but are the result of a number of factors which are complex and interlinked and which at times are complementary and at others in opposition to one another. 5. Small amounts of thionazin were recovered from beetles killed by contact with treated soil. The rate of pick up appeared to be approximately the same for all the species tested, irrespective of size and could help to explain the greater susceptibility of the smaller species. 6. The significance of thionazin residues in the environment is discussed and it is concluded that the downward movement of the pesticide in moist, light sandy loam soils would be considerable and could account for the greater susceptibility of those species of carabids which burrowed than of those which rarely burrowed. 235.

GENERAL DISCUSSION

An ever increasing world population requires that more food should be produced to feed more people before the population outstrips subsistence and Malthus's predictions made in 1798 come true. This can be achieved in two major ways (1) by increasing the area of arable or cultivated land (or by exploitation of other possible food sources from the environment e.g. the sea) or (2) by increasing food production from existing areas of cultivated land. In the latter category comes the use of pesticides to control pests attacking crops and considerable increases in yield have been achieved this way. To. quote a few examples, the control of cocoa capsids in Ghana by lindane spraying was shown to increase the yield of cocoa by about 600% in the third year of spraying and control of grasshoppers in Saskatchewan in 1949 and 1950 is estimated to have produced a saving of 90 million dollars for an expenditure of 2.14 million dollars (Boon, 1966). In general, the benefits obtained from pesticides cannot therefore be doubted but these haventt been achieved without some detrimental and perhaps serious consequences. Thus, the development of resistance by pest organisms is becoming a serious problem and evidence of the contamination of the environment as well as of pesticidal effects on non target organisms (especially predators and parasites) is also accumulating at a rapid rate (see Newsom, 1967). Some of 236. these problems can be overcome by the use of more specific control agents such as the use of chemo-sterilants in conjunction with suitable baits or attractants, or of insect pathogens, anti feeding compounds, etc. From a practical point of view, however, until these alternative methods have been shown to work, pesticides must remain the most economic and rapid method of controlling pests of agricultural importance and are likely to be used in still greater quantities in the future. Very few pesticides yet produced have no side-effects on organisms other than those against which they were applied. Carabids, being omnivorous in habit and widely distributed are among the organisms which are likely to be most affected by pesticide treatments. Pitfall trapping in this study have shown that many species of Carabidae may be active in arable fields much of the year although of these, only about 15-25 are present in sufficient numbers to be considered as potentially valuable predators. However, in spite of their rapid locomotion and of their morphological characteristics (especially their heavy chitinization) which makes them suitable as predators, they were killed by dosages needed to kill pests(see toxicity tests, Part II). Laboratory results have been confirmed by results in the field and-vice versa. Considering the mobility of the insects and the relatively small size of the field plots it is surprising that results have been so clear cut in demonstrating the toxicity and 237. persistence of the pesticides to the beetles. The results suggest therefore that effects would be much more striking in large areas of treated crops. Although the thionazin broadcast treatments of 10 and 40 lb/acre were higher than normal dosages for the control of insect pests,they indicate the dangers of using higher dosage rates than necessary. This situation may arise if, for instance, a pest species develops some resistance to an otherwise potent pesticide. The present thesis leaves several questions still unanswered. These include the following:- What effects, if any, would result if Carabidae were eliminated from arable fields? (This problem has been partially answered by Wright et al. (1960) who demonstrated that cabbage root fly attack increased in aldrin treated plots and in plots where carabid movement was restricted by mechanical barriers, and by Dempster (1967) who has shown the importance of adult Harpalus rufipes as a predator of Pieris rapae larvae in Brussels sprouts). How important are Carabidae in affecting populations of other organisms in the crop habitat? What immediate or long term effects do pesticides have on eggs, larvae and pupae of Carabidae? What effects, if any, would carabid adults add larvae suffer from eating food contaminated with pesticide residues? These questions could therefore provide an ecological basis for further work on the possible side effects of pesticides in the environment. 238.

ACKNOWLEDGEMENTS

I wish to thank Professor O.W. Richards, F.R.S. and Professor T.R.E. Southwood for facilities in the Entomology Department at Imperial College Field Station, Ascot. I am indebted to Mr. M.J. Way for supervising this work, for his helpful advice and encouragement and for his criticisms of the manuSCript. Thanks are due to Dr. F. Call and to Mr. R.F. Skrentny for much helpful advice with gas chromatography and to Dr. G. Murdie and Mr. J. Dunwoody for statistical advice and help with computer analyses. Aokknowledge•- ment is made to Messrs G.E.J. Nixon and J.W. Coles of the British Museum (Natural History) for identification of hymenopterous and nematode parasites respectively, and to Dr. M.L. Luff and Mr. O.W. Steele for help in the identification of Carabidae and Staphylinidae respectively. Mrs. van Emden was of much assistance in translating numerous German scientific papers. Thanks are also due to Mr. H. Devitt for photographing all the figures and to Miss M. Wendon for typing the thesis. I am grateful to the Ministry of Overseas Development for a two-year grant and to my parents for their constant moral and financial support.

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WALKER, C.H., HAMILTON, G.A. & HARRISON, R.B. (1967) Organochlorine insecticide residues in wild birds in Britain. J.Sci.Fd_Agric.la : 123-129 WALKER, J.O. (1963) A glasshouse trial to compare the biological efficiency of disulfoton when impreg- nated onto various inert carrier granules. Haywood Chemicals Ltd., Tech.Rep.56 WAY, M.J. & SCOPES, N.E.A. (1965) Side-effects of some soil-applied systemic insecticides. Ann.appl. Bio1.55 : 340-541 WEBER, G. (1953) The macrofauna of light and heavy arable soils and the effect on them of plant-protection substances. Z.Pfl.Ernaher.D4n.61 : 107-118 WELLS, A.L. & GUYER, G. (1967) New soil insecticides for the control of potato-infesting wireworms. J.econ.Ent.62 : 441-444 WHEATLEY, G.A. (1965) The assessment and persistence of residues of organochlorine insecticides in soils and their uptake by crops. Ann.appl.Biol.55 : 325 WILLIAMS, G. (1958) Mechanical time-sorting of pitfall captures. J.Anim.Ecol.27 : 27-35 WILLIAMS, G. (1959) Seasonal and diurnal activity of Carabidae with particular reference to Nebria, Notiophilus and Feronia. : 309- 330 WISHART, G., DOANE, J.F. & MAYBEE, G.E. (1956) Notes on beetles as predators of eggs of Hylemyia brassicae (Bouche). Can.Ent.88 : 634-639 WOODVILLE, H.C. (1967) Control of narcissus bulb fly, Merodon equestris F. Control of narcissus bulb fly damage in the south-west. Pl.Path.Supp1.1.16.(1) : 38-39 WRIGHT, D.W. (1956) Entomology report, Rep.Nat.Veg.Res.sta• Wellesbourne (1955) p.47 WRIGHT, D.W. (1962) Entomology report. Rep.Nat.Veg.Res.sta. Wellesbourne (1961) p.9 WRIGHT, D.W. (1965) Alternatives to organochlorine insecticides for the control of the carrot fly and the cabbage root fly. Ann.appl.Bio1.15. : 337-340 256

WRIGHT, D.W., HUGHES, R.D.1 & WORRALL, J. (1960) The effect of certain predators on the numbers of cabbage root fly (Erioischia brassicae (Bouche)) and on the subsequent damage caused by the pest. Ann.appl.Bio1.48 t 756-763

ADDENDUM

FLEMING, W.E. & BAKER, F.E. (1935) The use of carbon disulfide against the Japanese beetle. Tech.Bull.U.S.Dep.Agric.no.478

257.

APPENDIX

The follbwing symbols have been used in the analysis of variance tables and represent the following quantities. df = degrees of freedom SS = Sums of squares MS = Mean square F = Variance Ratio = Significant difference at P<0.05 or 5% ** = Significant difference at P<0.01 or 1% *** = Significant difference at P<0.001 or 0.1%

The following legend has been used to represent pesticide treatments:

A1H = Thionazin, broadcast at high dosage rate A1L . it ft If low tt It It ti II tt A2H .--- in-row high A2L . tt ft ti low ft tt AO = ft control (No treatment)

BH . Ethylene dibromide, high dosage rate

BL = tr It low

BO tl If control (No treatment)

CH = Chioropicrin, high dosage rate

tt it CL = low rt

CO = ft' control (No treatment)

DH = Dazomet, high dosage rate

DL IT low tt IT DO = control (No treatment) 258.

APITNDIX 1 Relative abundance of Carabidae from bare soil in Hill Bottom and Four Acre Field 1 March, 1966 to 31 May, 11966

NUMBER PER TRAP Species Hill Bottom Four Acre Field Adults Nebria brevicollis 0.4 2.7 Notiophilus substriatus 1.4 1.6 N.rufipes 0.1 N.biguttatus 0.4 2.3 Loricera pilicornis 1.3 1.9 Clivina collaris 0.4 Asaphidion flavipes 2.6 2.9 Bembidion lampros 92.5 72.8 B.bruxellense 0.1 B.tetracolum 2.0 5.9 B.femoratum 1.6 4.8 B.andreae 8.4 12.1 B.quadrimaculatum 17.8 1.9 B.obtusum 0.1 B.lunulatum 0.1 Trechus quadristriatus 0.4 1.1 Harpalus aeneus 1.3 1.9 H.tardus 0.1 H.rufipes 0.1 0.1 Acupalpus meridianus 0.1 0.1 Bradycellus harpalinus 0.3 Anisodactylus binotatus 0.2 0.2 Amara plebeja 0.1 A.similata 0.1 A.aenea 0.2 A.familiaris 0.1 A.anthobia 0.1 Pterostichus caerulescens 0.2 0.1 P.vernalis 0.1 259.

APPENDIX 1 cont.

NUMBER PER TRAP Species Hill Bottom Four Acre Field

P.nigrita 0.1 P.strenuus 0.1 P.madidus 0.2 Calathus melanocephalus 0.1 Agonum mulleri 0.2 0.1 A.dorsale 1.1 1.3 Metabletus foveatus 0.3 0.5 Larvae: Carabus violaceus 0.1 Nebria brevicollis 4.7 2.2 Notiophilus sp. 0.2 Harpalus rufipes 0.1 Calathus fuscipes 0.3 0.1

TOTAL 138.8 117.9

Mortalities due to Phaenoserphus viator and P.pallipes in third instar Nebria › brevicollis larvae from Four Acre Field and Hill Bottom ro

SITE DATE OF NUMBER CAUSE OF DEATHS NUMBERS H CAPTURE EXAMINED P.viator P.pallipes Other PUPATING factors FOUR 27.1,66 67 24 35 8 ACRE 1.11.66 112 33 4 37 38 FIELD 8.11.66 26 7 6 13 15.11.66 16 2 2 7 5 i.iii.66 19 1 1 9 8 8.111.66 21 14 7 15.iii.66 9 8 1 22.111.66 5 1 3 1 N 29.111.66 5 4 1 oN 5.1v.66 7 3 4 . 12.1v.66 5 5 19.1v.66 1 1 3.v.66 1 1 Total 294 68 7 127 92 HILL 28.11.66 26 7 10 9 BOTTOM 7.111.66 21 12 9 14.111.66 15 10 5 21.111.66 19 15 28.i4? i.66 18 12 6 4.1.3r66 23 10 13 11.iv.66 26 14 12 18.1v.66 34 11 23 Total 182 7 94 81

APPENDIX 3 Toxicity of thionazin in sandy loam soil to Bembidion tetracolum (4) at 15°C + 0.5°C. Pilot experiment. Estimation of the minimal effective or "critical" dose

CONC. SEX PROBIT REGRESSION LINE p.p.m. x Slope (b) L.T.50 (log hours) CHISQ. D.F. 32 a 0 5.02 0.06 14.95 + 2.09 0.05 + 0.009 4.59 6 16 1. 4.93 0.59 5.77 7 1.04 0.60 + 0.024 0.38 5 8 a 4.84 0.77 5.13 7 1.17 0.80 -7 0.035 1.35 2 5.30 1.40 4.08 + 0.68 1.32+ 0.039 2.59 4 2 a 7 4.99 1.67 3.72 + 0.73 1.67 + 0.036 3.68 1 g 4.98 2.34 4.82 7 0.68 2.34 -7 0.030 1.27 5 0.5 g 0 4.57 2.74 1.80 7 0.34 2.98 7 0.069 7.56 10

APPENDIX 4 Toxicity of thionazin in sandy loam soil to Bembidion tetracolum (oo 00) at 15°C ± 0.5°C. Estimation of the minimal effective dose

CONC. SEX PROBIT REGRESSION LINE Slope (b) L.T.50 (log hours) CHISQ. D.F. 2.0 5.07 1.18 9.55 + 1.91 1.17 + 0.013 1.00 6 24.0 4.84 1.22 8.19 -7 1.20 1.24 + 0.015 1.17 8 4.98 1.69 10.63 7 1.76 1.69 0.017 0.77 4 1.0 5.28 2.06 6.17 + 0.97 2.01 0.021 3.28 8 0.5 Dr7s. 0.5 N.S.

N.S. Not significant from control mortality APPENDIX 5A Toxicity of thionazin in sandy loam soil to Bembidion lampros (&'& ??) at 15°C 0.5°C. Estimation of the minimal effective or "critical" dose

.•=01•011.m.1.1•110•16 Conc. Sex TINE (HOURS) FOR PER CENT KILL 13.11/4MAk 7 13 20 27 33 40 47 53 60 67 73 80 87 93 100 16 ed% 1.0 1.2 1.3 1.5 1.7 1.9 2.0 2.2 2.3 16 1.2 1.3 1.5 1.7 1.9 2.0 2.2 2.3 2.5 8 tM 2.2 2.5 2.8 3.0 3.3 3.5 3.8 4.5 5.0 8.0 8 2.8 3.0 3.3 3.5 3.8 4.0 4.5 5.5 6.0 7.0 14 4 4:3V1 9.0 10 11 12 13 14 15 24 28 4 9.0 10 11 12 14 15 24 30 w 2 30 24 36 48 72 96 120 144 (3-% 2 24 36 48 72 96 120 4,o 3d' 120 168 240 336 504 672 1 120 240 336 504 672 0 ac? 336 672 0

263.

APPENDIX 5B Probit regression lines for time/mortality data]m Appepdix 5A

Conc. Sex PROBIT REGRESSION LINE p.p.m. SLOPE (b) L.T.50 y x (log hours) CHISQ D.F.

16 crc 5.22 0.20 9.16+1.33 0.17+0.02 0.95 7 1.17 7 16 3? 5.27 0.24 9.31+1.41 0.21+0.02 8 ge?' 5.13 0.53 6.91+0.85 0.51+0.02 9.15 13 8 7? 5.40 0.67 4.06+0.52 0.58+0.05 12.17 16 4 giP 5.09 1.11 6.03+1000 1.09+0.02 3.16 8 2.05 9 4 V? 5.09 1.14 5.54+0.81 1.12+0.02 2 8? 5.10 1.75 4.11+0.74 1.72+0.04 0.32. 4 1.04 4 2 ?q 5.19 1.69 4.85+0.85 1.65+0.04 1 ac? 4.66 2.50 2.27+0.59 2.65+0.07 1.13 4

1 n 4.93 2.47 3.02+0.62 2.50+0.05 1.90 4 APPENDIX 64 Toxicity of thionazin in sandy loam soil to Pterostichus vulgaris (aN8c ?y) at 15°C + 0.5°C. Estimation of the minimal effective dose

Conc. Sex TIME (HOURS) FOR PER CENT MORTALITY p.p.m. 7 13 20 27 33 40 47 53 60 67 73 80 87 93 100 8 3`.3% 22 24 26 28 3o 32 48 60 72 84 8 48 6o 72 96 120 4 c* 48 6o 72 84 96 120 144 168 4 120 144 192 240 456 844 o n 120 360 844 o 77 844

APPENDIX 6B Probit regression lines for time/mortality data in Appendix 4A

Conc. Sex PROBIT REGRESSION LINE p.p.m. SLOPE (b) L.T.50 (log hours) CHISQ

8 cid+ 4.96 1.53 5.14 + 0.75 1.54 + 0.02 4.05 9 8 n 5.26 i.86 6.3o 4.: 1.34 1.82 4. 0.03 1.76 4 4 8T 5.11 1.92 6.58 + 1.03 1.91 + 0.02 0.88 6 4 77 4.53 2.44 1.79 + 0.47 2,70 + 0.09 3.22 7 APPENDIX 7A Toxicity of thionazin in sandy loam soil to Bembidion tetracolum as affected by soil moisture (concentration of a.i. = 16 p.p.m., temperature 15'C ± 0.5°C S.M% = Soil Moisture (% of sticky point); oc = S.M. Control S.M. SEX TIME (HOURS) FOR PER CENT MORTALITY 6 13 20 27 33 40 47 53 60 67 73 80 87 93 100 0 did' 3.7 4.0 4.3 4.7 5.0 5.7 6.3 6.7 7.0 7.5 9.0 0 9? 3.7 4.3 5.0 5.7 6.o 6.3 6.7 7.0 7.5 9.0 5 d'd 4.0 6.5 7.5 8.o 8.5 9.0 10 11 12 13 24 5 99 9.0 10 11 13 14 24 10 45151 3.3 3.5 4.0 4.3 4.5 4.8 5.5 7.0 10 19 3.5 4.5 5.5 6.0 6.5 7.0 9.0 10 15 Ar 2.0 . _2.5 2.8 3.0 3.3 3.5 3.8 4.0 4.3 15 9 3.3 3.5 4.0 4.3 4.5 4.8 5.0 5.5 6.0 8.0 20 de' 2.0 2.5 2.7 2.9 3.0 c'N 20 99 3.3 3.5 4.0 4.5 4.8 5.0 .5.3 v.) 25 thr 2.5 2.8 3.0 3.3 3.5. 4.0 25 99 3.0 3.3 3.5 3.8 4.0 4.3 4.8 5.0 6.0 6.5 30 dU 1.2 1.5 1.7 2.0 2.2 2.3 2.5 207 2.9 30 q9 2.0 2.5 2.7 2.9 2.4 3.5 4.0 4.5 4.8 40 dtr 1.2 1.5 1.6 1.7 202 2.7 40 99 2.7 2.9 3.0 3.3 3.5 4.0 4.3 4.8 50 d&' 1.7 1.8 1.9 2.0 2.1 2.2 2.5 2.5 50 99 2.1 2.2 2.3 2.5 2.7 2.9 3.0 3.2 3.3 3.7 4.3 60 d& 1.6 1.7 1.8 1.9 1.9 2.0 2.2 2.3 2.5 2.7 60 99 2.0 2.1 2.2 2.3 2.5 2.7 3.0 3.2 8o db.' 1.9 1.9 2.1 2.2 2.3 2.5 3.0 80 99 1.9 2.1 2.2 2.3 2.5 2.7 2.9 3.2 3.3 95 dtt4 2.0 2.3 2.3 2.8 3.0 3.3 95 99 2.0 2.3 2.5 3.0 3.3 3.5 3.8 4.0 100 crcr 1.7 1.9 2.0 2.1 2.3 2.7 2.9 3.2 4.3 100 22 2.5 2.7 2.9 3.2 3.3 4.o 4.3 oc croft 3.0 5.0 6.0 7.0 oc 9? 4.0 5.0 6.0 7.0 8.0 9.0 12 13

APPENDIX 7B Probit regression lines for time/mortality data in Appendix 7A Soil Sex Moisture PROBIT REGRESSION LINE (% Sticky _ Slope L.T.50 CHISQ. Point) x (b) (log hours) ... D.F. 5 d' d' 4.8o 0.91 6.55 + 0.685 0.94 0.015 10.18 17 5 2 9 4.59 1.09 8.05 7 1.665 1.14 0.021 1.76 5 10 see 5.19 0.63 11.52 + 1.586 0.010 7.78 10 lo 0.61 %/ 9 4.69 0.77 7.23-7 0.882 0.82 0.016 5.56 12 15 0 dr 5.23 0.50 9.69 + 1.557 0.47 0.014 4.96 7 15 2 7 5.05 0.67 9.87 + 1.159 0.67 0.011 4.43 12 20 ear' 5408 0.41 14.21 + 2.280 0.41 0.009 2.99 6 20 q 5.49 0.62 8.52 + 1.503 0.56 0.017 2.09 9 25 ew e 5.47 0.48 13.17 + 2.607 0.44 0.013 1.31 5 25 5.25 0.61 9.17-7 1.308 0.59 0.013 3.13 10 IN 30 ,,T 907 a. %) ‘) 4.97 0.28 9.27 + 1.159 0.29 0.012 4.72 11 cr% 30 q. 5.03 0.49 9.89-7 1.136 0.49 0.011 3.93 12 • 40 a d' 5.24 0.24 9.35-7 1.306 0.21 0.012 4.55 to 40 5+18 0.54 11.97 7 1.746 0.52 0.010 2.43 8 50 cede 4.97 0.30 24.08 7 3.576 0.30 0.006 3.6o 7 50q q 5.22 0.44 10.28 + 1.440 0.42 0.011 2.61 10 60 ce d' 5.12 0.29 15.77-7 2.247 0.28 0.008 3.77 9 6o 5.05 0.39 15.40 + 2.182 0.39 0.008 2.36 80 4:$ 31 5.21 0.34 14.88-7 2.371 0.32 0.009 2.87 8 8o g ?. 5.17 0.38 11.24 + 1.598 0.37 0.010 5.63 9 95 ear 5.63 0.38 9.76 7 2.417 0.32 0.023 0.88 . 4 95 2 2 5.19 0.46 9.95 + 1.513 0.44 0.013 3.51 7 10o ce a?' 5.04 0.37 10.07 + 1.109 0.36 0.010 5.47 14 100 5.18 0.49 13.44 7 2.239 0.47 d 0.010 4.78 7 APPENDIX 8 Toxicity of thionazin in sandy loam soil to Bembidion tetracolum (n) as affected by soil compaction. Concentration of a.i. 16 p.p.m., temperature 15°C + 0.5°C, soil moisture 76% of field capacity.

Compaction PROBIT REGRESSION LINE of soil SLOPE L.T.50 CHISQ DP 7 x (b) (log hours) Uncompacted 5.19 0.40 11.74+1.135 0.38+0.007 1.93 19 Compacted 5.03 0.46 18.48+2.075 0.46+0.005 2.80 14

APPENDIX 9A Toxicity of thionazin in sandy loam soil to Nebria brevicollis as affected by light and temperature (Concentration of a.i. 16 p.p.m., soil moisture 76% of field capacity) T = Treated; C = Control; * Callow adults Temp• Hours Sex TINE (HOURS) FOR PER CENT MORTALITY (°C) light per day 7 13 20 27 33 .4o 47 53 6o 67 73 8o 87 93 100

7.T 24/24 48 72 84 96 108 120 132 168 7.T 24/24 6o 84 96 132 144 168 192 216 240 288 336 7.T 0/24 48 54 60 72 ' 84 108 100T 12/24 24 26 30 38 48 54 6o 72 10.T 12/24 24 26 28 30 34 48 54 60 64 72 15.T 16/24 8 9 10 11 12 13 14 15 15.T 16/24 12 13 14 15 16 19 22.T 24/24 5 6 7 9 10 11 12 22.T 24/24 7 8 9 10 11 12 13 15 16 17 APPENDIX 9A cont.

Temp. Hours Sex TIME (HOURS) FOR PER CENT MORTALITY (°C) light per day 7 13 20 27 33 40 47 53 60 67 73 80 87 93 100

7.0 24/24 de 7.0 24/24 99 84 7.0 0/24 de io.c 12/24 *Elie 10.0 12/24 *99 15.0 16/24 cre 15.0 16/24 g9 22.0 24/24 cror 15 22.0 24/24 Q9

T = Treated C = Control * Callow adults

APPENDIX 9B Toxicity of thionazin in sandy loam soil to Nebria brevicollis as affected by light and temperature (concentration of a.i. 16 p.p.m., soil moisture 76% of field capacity)

Temp. Hours Light Sex PROBIT REGRESSION LIVE (°C) per 24 hrs. y x Slope (b) L.T.50(log his Chisq. DF

7 24/24 e 5.20 1.98 7.74 1.064 1.95 + 0.017 2.95 8

7 It 4.77 2.20 4.41 0.561 2.25 + 0.024 3.92 12

7 0/24 d d' 5.38 1.80 7.92 1.540 1.75 0.021 0.88 5

10 12/24 ed* 4.95 1.59 7.68 0.921 1.60 + 0.016 2.57 9 10 0 4.98 1.60 5.86 0.773 1.60 + 0.019 2.22 9 9 * 15 16/24 071 437 5.13 1.05 12.50 1.900 1.04 + 0.011 2.38 6

15 It 5.38 1.16 15.64 2.663 1.13 + 0.010 2.24 6 o 7 22 24/24 cad' 5.20 0.90 8.23 1.304 0.88 + 0.017 1.58 6 22 o 5.15 1.06 7.84 1.077 1.04 + 0.015 2.62 9

* Callow adults. APPENDIX 10A Toxicity of thionazin in sandy loam soil to PtersosichUs vulgaris as affected by soil pH (Concentration of a.i. = 16 p.p.m.r temperature 15°C + 0.5°C)

CaCO3 pH Time Sex TIME (HOURS) FOR PER CENT MORTALITY g/Kg. (weeks) soil 7 13 20 27 33 40 47 53 60 67 73 8o 87 93 100

0.T 4.8 0 6.5 7.0 7.5 8.5 9 r5 11 0.T 4.8 O 99 6.5 8.0 8.5 9.0 10 10.5 11 11.5 5.7 0 df 6.5 8.0 8.5 9.0 10.0 12.0 1.T 5.7 0 29 6.5 7.0 7.5 8.0 9.0 9.5 10.5 11.5 12.0 5.T 6.6 0 as 6.5 7.5 8.0 10.0 10.5 5.T 6.6 O 29 6.5 7.0 8.5 10.5 11.0 11.5 12.0 50.T 6.9 0 6.5 7.5 8.5 9.0 50.T 6,9 0 99 6.5 7.5 8.0 9.0 9.5 10 10.5 11 O.0 4.8 0 ect" O.0 4.8 0 22 50.0 6.6 0 01/1 50.0 6.6 0 29 0.T 4.9 1 99 5 11 12 13 14 15 16 1.T 6.0 1 22 10 11 12 13 14 15 16 5.T 7.6 1 99 8 10 11 12 13 14 15 16 50.T 7.6 1 99 5 6 7 8 9 10 11 15 0.0 4.9 92 50.0 7.6 1 0.T 4.9 .2 (AP 3 4 5 7 8 9 1.T 6.o a dtp 5 6 7 8 9 10 11 12 13 15 5:T 7,6 2 dd 5 6 7 8 9 10 11 12 15 50.T 7.6 2 ea 4 5 6 7 8 10 11 14 0.0 4.9 2 dir 3 50.0 7.6 2 are T = Treated C = Control

APPENDIX 10B. Probit regression lines for time/mortality data in Appendix 10B

pH Time Sex CaCO3 PROBIT REGRESSION LINE g/Kg. (means) (weeks) L.T.50 soil y SLOPE (b) log hrs. CHISQ DF o 4.8 0 du, 5.69 0.92 6,48+1.69 0.81+0.03 1.91 8 o 4,8 o 99 5.19 0.94 7.4671.41 0.92+0.02 5.72 .9 1 5.7 0 de 5.62 0.94 5.49+1.31 0.8370.03 1.58 10 10 1 5.7 0 79 5.23 0.95 7.77+1.30 0.92+0.01 4.29 5 6.6 o 0.11, 5.94 0.90 6.30+2,09 0.7570.05 2.05 7 . 1.01+0.02 3.61 10. 5 6.6 o T9 4.75 0.97 6.5671.3o 5o 6.9 0 dIP 5.90 0.88 7.0073.39 0.7570.07 2.26 4 50 6.9 0 99 5,11 0.93 10.42+1.71 0.92+0.01 4.42 8 o 4.9 1 99 4.63 1.04 4.17+0.78 1.12+0.03 11.80 10 1 6.0 1 5.08 1.08 11.6171.68 1.07+0.01 3.10 7 99 6 5 7.6 1 99 5.08 1.09 10.7571.79 1.0870.01 1.48 5o 7.6 1 Oo 5.55 0.92 5.76+0.91 0.8270.03 2.73 9 o 4.9 2 de 5.24 0.75 8.74+1.4o 0.72+0.02 3.08 5 1 6.0 2 &a% 5.21 0.97 7.27+0.97 0.94+0.02 1.71 9 5 7.6 2 dVi 5.27 0.96 7.11+0.93 0.92+0.02 2.19 9 5o 7.6 2 de 5.33 0.89 6.1370.83 0.8/i70.02 1.72 9

APPENDIX itA Comparative toxicity of thionazin in sandy loam soil to various carabid species Concentration of a.i. 16 p.p.m. temperature 15°C 4. 0.5°C, soil moisture. 15% by weight of air dry soil

SPECIES SEX TIME (HOURS) FOR PER CENT MORTALITY 7 13 20 27 33 40 47 53 60 67 73 80 87 93 100 T.quadristriatus Tdd 1.0 1.1 1.2 1.3 1.4 1.5 2.0 2.3 2.5 3.0 II IT Tp9 10 1.1 1.2 1.3 1.40 2.0 2.3 2.5 3.0 4.0 it II cse it It B.lamprosl TZC 1.0 1,2 1.3 1.5 1.7 1.9 2.0 2.2 2.3 tt Tp9 1.2 1.3 1.5 1.7 1.9 2.0 2.2 2.3 2.5 0 Ced‘ 336 672 It C29 A.flavipes Tee 9.0 10 11 12 13 14 15 20 24 n T99 11 13 14 15 20 24 26 28 48 TT Cd'cr tt C99 B.andreae TaV 1.0 1.3 1.5 1.7 1.9 2.0 2.2 tt T?2 1.3 1.5 1.7 1.85 2.0 2.2 2.3 2.5 tt Cad` It C99 B.tetracolum2 Tad` 1.7 1.8 1.9 2.00 2.1 2.2 2.3 2.3 tt T99, 2.1 2.2 2.3 2.50 2.7 2.9 3.0 3.2 3.3 3.7 4.3 II C&3 TT C9 A.dorsale Tded` 1.5 1.8 2.0 2.3 2.3 3.0 It T9,9 2.0 2.3 2.50 2.8 . 5 If Ced4 tt Cp9 L•pilicornis TS& 2.0 2.1 2.2 2.75 2.3 2.3 2.4 2.5 2.8 O 99 2.3 2.4 2.5 2.6 2.7 2.9 2.9 3.0 3.3 3.3 3.6 II cde tt C?9 APPENDIX 11A coati. T = Treated C = Control

SPECIES SEX TIME (HOURS) FOR PER CENT MORTALITY 7 13 20 27 33 40 47 53 60 67 73 80 87 93 100 Heaeneus3 Tde .1.8 2.0 2,3 2.5 2.8 3.0 3.3 It T?? 1.8 2.0 2.3 2.8 3.0 3.3 3.5 4.0 ft calf ft ? N.brevicollis4 Toll) 8 9 10 11 12 13 14 15 ft TT44 12 13 14 15 16 19 tt It C C.fuscipes Tdd 4.5 5.0 5.5 6.0 7,o 8.0 9.0 10 It T9? 4.0 5.5 6.o 8.0 12 13 IT 7.0 9.0 It Cd):11 c99 ••••7n H.rufipes Tcfcr 4.5 5.0 5.5 6.0 7.o \.ei It T12 4.5 5.o 6.o 7.o 8.0 9.o It C& It cu.. P.madidus T?e. 9 10 11 12 13 15 16 24 It T29 11 13 14 15 16 24 It cee It CR? P.vulgaris TM 7 8 9 10 11 13 14 15 22 It T9? 13 15 22 24 28 32 It Cde It C29 A.parallele- VrolP 7 7.5 8 9 to 11 12 15 18 pipedus T2a 7.5 9 11 12 15 18 24 It CoSir IT C

1. Appendix 4 2. Appendix 7 3. Appendix 13 4. Appendix 9 APPENDIX 11B Probit regression lines, for time/mortality data in Appendix /1,6,

SPECIES SEX PROBIT REGRESSION LINE X SLOPE (b) L.T.50 CHISQ D.P. .(log hours) Trechus quadristriatus ddt 5.26 0.23 6.68+0.94 0.19+0.02 3.82 9 It 92 5.18 0.27 5.917o.76 of 2470.02 1.77 11 Bembidion lamprosi crd' 5.22 0.20 9.1671.33 0.17+0.02 0.95 7 11 tt 99 5.27 0.24 9.3171.41 0.21+0.02 1.17 7 Asaphidion flavipes de` 4.90 1.11 8453+1.58 1.12+0.02 0.96 7 ft Ir 9? 4 6 93 1.26 6.070.86 1.2770.02 2.34 9 Bembidion andreae ecr 5.00 0.22 10.25+1.64 0.2270.01 7.46 6 It II 99.5.15 0.27 12.50+1.92 0.2670.01 1.50 6 Bembidion tetracolum2 d&' 4.97 0.30 24.08+3.58 0.30+0.01 3.60 7 It II 9q 5.22 0.44 10.28+1.44 0.42+0.01 2.61 10 Agonum dorsale (Ye 5.18 0.32 13.4172.15 0.3070.01 1.66 5 II It 9 5.22 0.41 15.25+_2.49 0.3970.01 0.67 3 Loricera pilicornis d? 5.25 0.36 23.86+3.38 0.35+0.01 3.39 8 It It '97 5.25 0.46 18.83+2.14 0.44+0.01 2.25 14 Harpalus aeneus3 d'' 5.20 0.38 10.2771.78 0.3670.01 3.47 5 II It -9R 5.10 0.43 8.1171.28 0.42+0.02 3.14 7 Nebria brevicollis4 db't 5.13 1.05 12.5671.90 1.04+0.01 6 il It 2.38 97 5.38 1.16 15.6472.66 1.1370.01 2.24 6 Calathus fuscipes cee, 5.28 0.79 7*49710,34 0.75+0.02 1.96 6 11 It i>9 5.03 0.87 7,070,87 0.87+0.02 3.34 10

Harpalus rufipes db.) 5.09 0.71 12.6772.37 0.70+0.01 6.24 It It V9 5.14 0.73 10.07+1.64 0.72+0.01 4.77 6 Pterostichus madidus (Air 5.21 1.09 7.13+1042 1.06+0.02 8.04 7 II It 92 4.74 1.14 8.6971.86 1.1770.02 1.45 5 Pterostichus vulgaris o'cr 4.93 1.05 .6.74+107 1.06+0.02 3.40 8 11 .99 4.92 1.28 7,9471.09 1.2970.02 2.50 6 Abax parallelepipedus ddr 5.04 0.99 6.52+1.12 0.9670.02 1.29 7 It II ?9.4.69 1.08 6.7971.00 1.13+0.02 1.98 7

1. Appendix 4 2. Appendix 7 3. Appendix 13 4. Appendix 9

APPENDIX 1,2 Toxicity of thionazin in sandy loam soil as affected by age of the insect. Test insect: Harpalus aeneus, Concentration a.i. = 16 p.p.m., temperature 15°C+0.5°C

AGE (Approx.) Sex PROBIT REGRESSION LINE y SLOPE (b) L.T.50 CHISQ D.F. log hours Callow newly emerged ddt 5.41 0.30 15.91+4.01 0.27+0.01 0.49 2 5.20 0.38 10.27+1.78 0.3670.01 3.47 Mature, second year dd 5.27 0,41 6.6471.19 0.37+0.02 1.89 7 tt fi 5.10 0.45 8.1171.28 0.4270.02 3.14 7

APPENDIX 13 Effect of starvation on the mortality of Bembidion tetracolum on thionazin treated soil. Concentration of a.i. = 2 p.p.m; temperature 15°C + 0.5°C

DAYS STARVATION Sex PROBIT REGRESSION LINE

(before test) y x SLOPE (b) L.T.50 CHISQ D.F. log.hours 4 ore 5.39 1.33 10.21+1.11 1.29+0.01 11.88 14 db" 5.47 1.38 11.94+1.43 1.3470.01 6.99 12 0 ?2 5.32 1.38 11.10+1.26 1.3570.01 2.03 12 0 7? 5.19 1.42 9.2571.39 1.4070.01 2.48 11

APPENDIX 14 Effect of starvation on the mortality of Agonum dorsale on thionazin treated soil. Concentration of a.i. = 16 p.p.m., temperature 15°C 0.5°C

DAYS STARVATION Sex _ PROBIT REGRESSION LINE (before test) y SLOPE (b) L.T.50 CHISQ D.P. log hours

4 DoT 5.18 0.33 10.45+1.72 0.31+0.01 4.42 5 0 as 5.20 0.27 13.91+2.73 0.2670.01 2.32 3 4 5.19 0.53 17.63+3.39 0.31+0.01 0.74 3 0 5,24 0.55 11.95+2.22 0.537b.01 0.55 4 APPENDIX 15A Fumigant toxicity of thionazin in sandy loam soil to Nebria brevicollis as influenced by temperature. Concentration of a.i. = 16 p.p.m; Soil moisture: 15% by weight; hours light per 24 hours: 0 T = Treated C = Control TEMP. TYPE OF SEX TIME (HOURS) FOR PER CENT MORTALITY (°C) VESSEL 7 13 20 27 33 40 47 53 6o 67 73 80 87 93 100 7 Closed T cid' 144 168 192 216 240 264 312 360 504 624 7 It c dl? 144 22 tt T (3'd" 9 10 11 12 13 15 17 22 it T in 13 14 16 18 24 26 28 48 22 ft C crcr 22 tt C oo 24 22 Closed T ckf 10 12 13 14 15 16 22 Open T dEr 24 26 28 32 72 96 22 Closed C dos 22 Open C cre 36 48 72 96

APPENDIX 15B Probit regression lines for time/mortality data in Appendix 15A

TEMP. TYPE OF Sex PROBIT REGRESSION LINE (°C) VESSEL y ; SLOPE L.T.50 CHISQ D.F. log hours 7 Closed dcr 4.94 2.38 6.57+0.99 2.39+0.02 6.28 8 22 " (1) 8'cr 5.11 1.11 10.9171.64 1.10+0.01 4.55 7 22 tt io? 5.19 1.29 5.2870.78 1.25+0.02 7.34 10 22 Closed(2) die 5.16 1.10 12.89+2.28 1.09+0.01 2.90 5 22 Open ckr 5.17 1.60 1.23+0.59 1.45+0.12 8.20 6 APPENDIX 16A Persistence of thionazin in sandy loam soil as determined' by bioassay tests with Bembidion tetracolum at 15°C+0.5°C I. Determination of mortalities on soils of known concentrations

Conc. Sex TIME ( HOURS) FOR PER CENT MORTALITY p.p.m. 7 13 20 27 33 40 47 53 60 67 73 8o 87 93 loo 7 4.5 5.0 5.5 6.o 6.5 7.o 8.o 7 6.0 6.5 7.5 8.5 9.5 10 11 12 6 ?),E„ 5.5 7.5 8.0 8.5 9.5 10.0 11 11.0 12 14 15 23 5 6.0 6.5 7.5 8.0 8.5 9.0 9.5 10 5 9? 9.0 10 11 12 12 13 14 16 23 4 00 9.5 12 12 15 16 23 3 076" 13 14 16 23 3 99 23 24 25 28 30 33.0 36 0 oat?' 0 99 II. Determination of mortalities on soils treated at 8.0 p.p.m. and aged at 15°C 4. 0.5°C Time Type of TIME (HOURS) FOR PER CENT MORTALITY (Weeka) container Sex 7 13 20 27 33 40 47 53 60 67 73 80 87 93 100 O Closed T dre 3.0 3.2 3.3 3.5 3.9 4.7 0 Open T cit? 3.o 3.2 3.3 3.5 3.9 4.0 O Closed T 4.o 4.3 4.7 5.0 6.0 6.5 7.0 7,5 8.0 O Open T 4? 4.7 5.0 5.5 6.o 6.5 7.0 O Closed C db' O Open C 92 1 Closed T db4 6.5 7.0 7.3 8.5 9.o 9.5 10 11 12 1 Open T drop 3.5 4.5 6.5 7.o 7.3 8.0 8.5 9.0 9.5 10 I Closed T 99 7.3 8.5 9.5 12 13 15 16 18 24 1 Open T 92 7.5 8.5 9.5 11 12 13 14 16 17 18 24 i Closed C oil 1 Open c tf2 APPENDIX 16A cont. T = Treated C = Control

Time Type of Sex PER CENT KILL (weeks) container 7 13 20 27 33 40 47 53 60 67 73 80 87 93 100 2 Closed T de 9.0 12 14 15 16 17 19 20 2 Open T edr 9. o 10 11 12 13 15 16 17 18 19 2 Closed T 13 16 18 19 21 22 24 33 34 2 Open T 419 i8 19 20 21 22 23 24 33 2 Closed 2 Open Cd 3 Closed Td" 21 22 23 25 26 27 3 Open T ecr 21 22 23 24 25 26 3 Closed T 26 27 28 30 32 34 36 38 48 3 Open T 25 26 27 28 29 30 32 34 36 38 3 Closed 3 Open C d8 r. Time Type of Sex PER CENT KILL (weeks) container 10 20 30 40 50 60 70 80 90 100 4 Closed I" &I 35 37 39 42 48 4 Open T dkfi - 24 30 33 35 37 39 4 Closed 48 54 6o 4 Open T 743 48 54 60 Closed C dt2 4 Open C

APPENDIX 16B Persistence of thlonazin in sandy loam soil as determined by bioassay tests with Bembidion tetracolum at 15°C + 0.5°C I. Determinations of L.T.501 s on soils of known concentration

Conc. Sex PROBIT REGRESSION LINE *me p.p.m. y x SLOPE (b) L.T.50 CHISQ D.P. log hours 7 cre 5.29 0.77 11.48+1.91 0.75+0.01 1.24 6 7 VC?5.12 0.93 10.89+1.41 0.92+0.01 6.71 10 6 qq 4.90 1.01 6.07+0.73 1.03+0.01 2.82 16 5 dtil 5.13 0.89 12.36+1.94 0.8870.01 5.39 7 5 71 4.84 1.08 9.41+1.40 1.09+0.01 4.10 10 4 4.42 1.13 8.0044.28 1.2070.02 5.55 9 3 a 4.31 1.19 15.3873.33 1.24+0.02 2.65 3 3 5.50 1.42 12.44+2.59 1.38+0.01 1.16 8

II. Determination of L.T.504 s at weekly intervals on soils treated at 8 p.p.m. and aged in open and closed containers at 15°C+0.5°C

Time Type of Sex WM, PROBIT REGRESSION LINE (weeks) container y x SLOPE (b) L.T.50 CHISQ D.F. log hours O Closed did' 5.54 0.54 15.61+3.09 0.50+0.01 5.22 7 O Open dre 5.30 0.52 25.72+4.76 0.51+0.01 3.11 5 O Closed 4i 5.03 0.76 9.79+1.38 0.75+0.01 5.46 8 O Open In 4.98 0.76 i5.41+2.82 0.76+0.01 3.52 1 Closed dice' 4.91 0.93 11.0971.60 0.94+0.01 4.07 9 1 Open dad" 5.05 0.84 8.0470.98 0.84+0.01 14.75 13 1 Closed trit 4.72 1.10 6.0770.73 1.1470.02 3.54 14 1 Open 73 4.89 1.09 7.62+0.87 1.10+0.01 1.88 13

APPENDIX 16B cont.

Time Type of Sex PROBIT REGRESSION LINE (weeks) container x SLOPE (b) L.T.50 CHISQ D.F. log hour6 2 Closed d'd 4.97 1.17 9.90+1.31 1.18+0.01 8.41 10 2 Open ea 4.98 1.15 8.9471.28 1.1570.01 7.28 9 2 Closed 7q 4.69 1.31 8.9371.16 1.3470.01 4.68 12 2 Open (9. 4.85 1.32 17.0473.47 1.33+0.01 0.95 6 3 Closed die 5.41 1.37 16.3873.80 1.35+0.01 2.69 5 3 Open AP 5.31 1.36 26.9875.46 1.35+0.01 1.22 4 3 Closed 4.91 1,50 13.09.72.09 1.50+0.01 2.17 8 3 Open LI 4.86 1.48 16'.1672.30 1.49+0.01 2.17 8 4 Closed 4"0 4.97 1,58 32.96.18.94 1.58+0.01 0.14 3 4 Open db" 4.79 1.48 11.03t2.12 1.5070.01 5.53 10 4 Closed 49 5.17 1.72 21.79t7.20 1.71'0.01 0.31 1 4 Open (;)? 5.34 1.70 31.60+12.24 1.69+0.01 0.02 1 APPENDIX 17 Persistence of thionazin in sandy loam soil treated at 8 p.p.m. and aged at 1.5°C + 0.5°C in semi-open plastic containers as determined by bioassay with Bembidion tetracolum

Sex PROBIT REGRESSION LINES • Time (weeks) y x SLOPE (b) L.T.50 CHISQ D.P. log hours 4 d's. 5.19 1.46 8.22+1.42 1.44+0.02 1.10 4 13 or? 5.14 1.74 9.7771.40 1.73+0.01 12.90 10 21 el? 5.32 2.53 3.02+0.28 2.43+0.02 20.55 19 APPENDIX 18A Toxicity of phorate (Thimet) in sandy loam soil at 15°C 0.5°C to various species of carabids

Species Sex Conc. TIME (HOURS) FOR PER CENT MORTALITY (adults) p.p.m. 6 li 17 22 28 33 39 44 50 56 61 67 72 78 83 89 94 100 T.4..atrisitue 4 32 1.o 1.1 1.3 1.4 n t i6 1.0 1.1 1.3 1.4 H 49 4 2.3 2.8 2.9 3.0 3.1 n d9 1 3.8 4.3 4.5 4.8 5.o 5.3 5.8 6.o 6.8 u 8? o

Species Sex Conc. TIME (HOURS) FOR PER CENT MORTALITY (adults) p.p.m. 8 17 25 33 42 5o 58 67 75 83 92 100 N.brevicollis de 32 2.3 2.5 2.6 2.7 2.8 3.0 3.3 ii die 16 2.8 3.1 3.3 3.3 3.5 3.8 4.3 n et0 4 5.3 5.5 6.o 6.3 6.8 7.3 8.o n de 1 17 18 19 21 n d'e o n 9.9 32 2.8 3.o 3.1 3:3 3.3 5.o n 9T 16 3.3 3.8 40 4.8 5.3 n n 4 5.8 $.8 8.o 9.o 18 if V 1 18 20 23 25 26 27 n 99 0 APPENDIX 18A cont.

Species Sex Conc. TIME (HOURS) FOR PER CENT MORTALITY (adults) p.p.m. 7 13 20 27 33 4o 47 53 60 67 73 80 87 93 100 dc` 32 1.2 1.3 1.3 1.4 1.5 1.6 1.7 1.9 cilds 16 1.6 1.7 1.8 1.9 2.0 2.2 2.5 4.6 11 dd 4 4.0 4.5 6.o 6.5 7.o 8.o 15 11 d'd' 1 15 21 24 28 30 39 48 72o It crci/ 0 11 w 32 1,6 1.7 1.8 1.9 2.0 2.2 2.3 2.5 w It 77 16 1.8 1.9 2.2 2.3 2.5 3.0 4.o 4.5 ‘.)4oD 11 4 6.o 6.5 8.o 15 16 21 . 11 74 1 28 30 39 72 120 240 720 11 77 0

APPENDIX 18B Probit regression lines for time/mortality data in Appendix 16A

Species Sex Conc. PROBIT REGRESSION LINE p.p.m. y SLOPE (b) L.T.50 CHISQ D.F. . log hours Trechus quadristriatus ay. 32 5.36 0.02 21.46+6.41 0.01+0.01 3.43 2 16 5.12 0.05 17.6673.64 0.0470.01 1.22 2 It tt 4 5.47 0.45 16.9573.41 0.4270.01 5.47 3 tt tt 1 5.22 0.70 16.51+2.11 0.69+0.01 3.63 9 Nebria brevicollis 32 5.48 0.43 16.90+3.24 0.4070.01 1.39 7 It It 16 4.93 0.52 24.78+4.30 0.5270.01 5.02 7 It It 4 5.25 0.79 15.8273.31 0.7770.01 0.56 5 it tt 1 5.40 1.27 23.4678.45 1.25+0.01 1.66 2 rt tt 32 5.10 0.51 14.2273.60 0.50+0.01 2.97 5 11 it 16 5.19 0.59 13.41+3.23 0.5670.02 1.72 3 tt It 4 4.92 0.87 12.49+3.27 0.8670.02 0.12 3 11 it 1 5.33 1.37 18.76+3.72 1.3570.01 2."8 4 Pterostichus vulgaris 32 5.18 0.17 18.09+2.62 0.16+0.01 1.22 7 6 16 5.06 0.28 16.0672.56 0.2770.01 1.35 tt tt 4 5.19 0.30 8.5271.52 0.2870.01 31.86* 9 VI 1 4.8o 0.41 8.8071.24 0.437001 2.34 9 tt tt 6 32 4.91 0.77 6.2571.24 0.7970.02 1.23 tt 11 1.55 6 16 4.95 1.00 6.5570.84 1.01+0.02 tt tt 11 4 5.04 1.43 3.2370.52 1.4270.03 90.08* It 11 1 4.88 1.83 1.92+0.35 1.90+0.07 1.45 6

* Denotes heterogeneity

APPENDIX 1.9A Persistence of phorate (Thimet) in sandy loam soil at 15°C as determined by bioassay with Pterostichus vulgaris

Conc. Time Sex TIME (HOURS) FOR PER CENT MORTALITY p.p.m. (weeks) 7 13 20 27 33 40 47 53 6o 67 73 8o 87 93 too 32 4 ecr 1.5 2.0 2.5 3.o 4.0 5.0 32 2.5 3,o 3.5 4.0 5.0 16 tt (cs $i 2.5 3.0 4.0 5.0 6.0 8.o 16 ?7 3.0 3.5 5.0 6.o 8.0 19 do" 21 24 30 48 72 120 336 480 4 1(9. 8 19 30 48 72 120 336 48o 576 672 do" 72 120 480 576 672 tt Fx 120 480 576 336 480 576 672 0 tt 48 336 480 672 ‘.n 32 22.4 d& I 7 8 9 10 11 13 21 32 10 11 12 13 21 24 36 48 16 It 13 21 24 36 72 96 120 16 It 96 120 144 168 192 240 288 0 ft & 120 0 It 9? 286.

APPENDIX 22 A. Carabid species caught in untreated potato _plots in Four Acre Field - 19.v.65 27.ix.65

% of TOTAL CATCH (2 traps per plot) VERY COMMON COMMON RARE (>10%) (1-10%) (<1%) Bembidion lampros Harpalus rufives(6.7) Acupallaus (45.4) Trechus quadristriatus meridianus(0.6) (4.0) Amara spp.(0.6) Pterostichus Bembidion andreae (3.9 =era_pill- vulgaris (22.9) Calathus fuscipes (3.4 cornis (0.3) Aerostichus madidus Asaphidion (2.4) flavipes(0.3) Nebria brevicollis(1.8) Harpalus Harpalus aeneus (1.5) tardus (0.3) liembidion guadrimacul- AMETErallel- atum (1.5) epipedus(0.3) Anisodactylus binotatus Agonum dorsale (1.2) (O,3) Amara bifrons (1.2) Metabletus larvae (unidentified) foveatus (0.3) (1.2)

B. Carabid species caught in thionazin treated but not in untreated potato plots in Four Acre Field - 19.v.65 - 27.ix.65

SPECIES % of GRAND TOTAL CATCH(20 traps) Notiophilus biguttatus 0.2 Badister bipustulatus 0.1 Amara fulva 0.2 Pterostichus niger 0.1 Dromius melanocephalus 0.1 287. APPENDIX 23 A. Carabid species caught in untreated potato plots in Four Acre Field - 6.v.66 - 13,ix.66 L = Larvae % of TOTAL CATCH (2 traps per plot)

VERY COMMON COMMON RARE (>10%) (1-10%) (<1%) Bembidion lampros Harpalus rufipes Notiophilus sub- (48.3) (9.6) striatus(0.7) Pterostichus ICEIFEFEatus (0.7) Bembidion quadri- Amara similata vulgaris (11.8) maculatum (7.0) T0.7) Trechus quadristr- Loricera pili- iatus (5.2) cornis (L)(0.7) Nebria brevicol- Harpalus aeneus lis (0.4) (3.7) Clivina collaris Loricera pilicornis (0.4) (1.5) Bembidion femoratum (0.-4) Amara bifrons Harpalus tardus (1.5) (0.4) Harpalus rufipes (L) Acupalpus merid- (1.5) ianus (0.4) Amaraplebeja Anisodactylus bino- (0.4) tatus (1.1) Pterostichus Calathus fuscipes caerulescens (1.1) (0.4) P.ni er (0.4) .ma idus (0.4) Metabletus foveatus (0.4) Amara sp. (L) (0.4)

B. Carabid species caught in thionazin treated but not in untreatedeated potato plots in Four Acre Field, 6.v.66 - /untr

SPECIES % of GRAND TOTAL CATCH(10 traps) Carabus violaceus (0.1 Asaphidion flavipes 0.1 $embidion andreae 0.4 Amara fulva 0.2 Talathus piceus (0.2 Agonum dorsale (0.2 otiophilus sp. (L) (0.2 Agonum dorsale (L) (0.1

APPENDIX I213 Probit regression lines for time/mortality data in Appendix 19A

Conc. Time Sex PROBIT REGRESSION LINE p.p.m. (weeks) y x SLOPE (b) L.T.50 CHISQ D.F. log hours 32 01 de 5.18 0.17 18.09+2.62 0.16+0.01 1.22 7 32 99 5.06 0.28 16.0672.56 0.27+0.01 1.35 6 16 0 dfcP 3.19 0.30 8.5271.52 0.28+0.01 31.86 9 16 O _99 4.80 0.41 8.8071.24 0.43+0.01 2.34 9 4 0 4.91 0.77 6.25+1.24 0.79+0.02 1.23 6 4 9q 4.95 1.00 6.5570.84 1.01+0.02 1.55 6 1 0 d'cP 5.04 1.43 3.2370.52 1.42+0.03 90.08 11 1 0 99 4.88 1.83 1.92+0.35 1.90+0.07 1.43 6 32 4 &el 3.25 0.42 7.09+1.18 0.39+0.02 5.71 5 32 4 22 5.12 0.52 11.33+2.34 0.51+0.02 1.28 3 16 4 de 4.96 0.60 7.19+1.20 0.61+0.02 1.89 5 16 4 99 4.89 0.67 5.6371.11 0.6970.03 1.19 5 4 4 db" 4.97 1.69 2.4370.42 1.70+0.06 5.88 6 4 4 9q 4.86 1.93 1.52+0.19 2.02+0.07 4.50 10 1 4 de 1 4 7-9

6 32 22.4 dbl. 5.20 0.98 10.67+1.83 0.96+0.01 1.41 32 22.4 Ti 4.88 1.16 4.6670.71 1.1970.03 1.22 7 16 22.4 61,171 5.11 1.57 5.4570.55 1.5370.04 1.55 6 16. 22.4 94) 4.85 2.25 2.75I0.58 2.3070.04 1.00 8 1 (from Appendix 18) N.S. Not significant from control mortality

APPENDIX 20A Toxicity of disulfoton in sandy loam soil at 15°C 0.5°C to various species of carabids. Concentration of a.i. = 32 p.p.m.

Species Sex TIME (HOURS) FOR PER CENT MORTALITY (adults) 7 13 20 27 33 40 47 53 6o 67 73 80 87 93 100 Pterostichus vulgaris Td'd' 9 18 24 36 48 96 768 It T?? 36 48 72 96 768 It Code 240 768 Co.? 768 Agonum dorsale ToMP 6 7 8 8.5 9 T.?? 6 8 8.5 9 12 17 11 core 11 C?? TIME (HOURS) FOR PER CENT MORTALITY Species Sex 7 13 17 27 30 37 43 63 67 77 83 87 93 97 100 Bembidion lampros Tcfo." 18 24 30 36 48 72 96 120 T.?T 18 24 30 36 48 72 120 132 144 768 11 cedi cv7. 768

T = Treated C = Control APPENDIX 20B Probit regression lines for time/mortality data in Appendix 20A

Species Sex Conc. PROBIT REGRESSION LINE p.p.m. X' SLOPE (b) L.T.50 CHISQ D.P. log hours

P.vulgaris cre 32 5.34 1.61 1.77+0.31 1.42+0.08 8.34 8 ft 77 32 4.23 2.14 0.85+0.32 3.04+0.38 2.27 5 A.dorsale d&7 32 5.32 0.88 19.99+3.77 0087+0.01 1.25 3 It 32 8.71+1.67 0.954-0.02 2.30 5 ?? 4,81 0.93 B.lampros die 32 5.02 1.61 4.02+0,44 1.61+0.02 3.04 6 tt 99 32 5.18 1.67 2.85+0.25 1.60+0.03 4.33 11 APPENDIX 21A Toxicity of menazon in sandy loam soil at 15°C + 0.5°C to various species of carabids

Species Sex Conc. TIME (HOURS) FOR PER CENT MORTALITY p•p•m• 7 13 20 27 33 40 47 53 60 67 73 80 87 93 100

H.rufipes d'd' 512 12 48 168 336 432 504 576 672 tt 512 14x 18x 24x 35x 43x

11 d&' 256 14x 18x 24x 35x 41x 43x Sox 11 77 256 14x I8x 24x 35x 41x 43x 7ox 87x 11 AP 0 43x 63x 70x 84x 87x 133x 140x tt 0 7x 28x 87x 98x 133x 147x P.vulgaris d'd' 10, 000 4 5 7 8 9 10 12 14 22 tt vq. 10,000 5 7 12 14 22 24 25 it d'?' 0 79 0

x = x24 hours APPENDIX 2113 Probit regression line for time/mortality data in Appendix 21A

Species Sex Conc. PROBIT REGRESSION LINE 7c. SLOPE. (b) L.T.50 CHISQ D.F. log hours

H.rufipes WI 512 5.23 2.37 1.84+0.28 2.25+0.07 9.65 7 II 2.76 6.61+1.06 2.72+0.02 2.02 6 Y7 512 5.22 It (AP 256 5.19 2.81 4.40+0.75 2.774-o.o3 3.81 7 II 2.96+0.03 7.60 11 77 256 5.03 2.97 4.03+0.50 It dW' 10,000 5.14 0.89 5.46+o.85 0.86+0.02 1.578 P.vulgaris 10,000 4.75 1.09 4.64+0.60 1.15+0.03 6.63 9

293. APPENDIX 24 A. Carabid species caught in untreated potato lots in Four Acre Field - 1.v.67 - 11.ix.67 L = Larvae % of TOTAL CATCH (2 traps per plot) VERY COMMON COMMON RARE (>10%) (1-10%) (<1%) Bembidion Amara plebeja(9.6) Harpalus aeneus(0.7) n71177(717(41.1) Loricera pilicor- Notiophilus biguttatus nis (5.4) (L) (0.5) Harpalus Anisodactylus Trechus 4-striatus latiEta(11.8) otatus (4.9) (0.5) Amara bifrons(4.9) Anisodactylus bino- Bembidion quadri- tatus (0.5) maculatum(4.7) Pterostichus madidus Agonum dorsals (L) (0.5) (3.2) Calathus fuscipes(0.5) Pterostichus Notiophilus substriatus saris (2.5) (L) (0.3) Loricera pilicor Clivina collaris(0.3) nis (L) (1.7) Bembidion tetracolum Amara plebeja (L) (0.3) (1.7) B.andreae (0.3) Assimilata (1.2) Acupalpus meridianus Haryalus rufipes (0.3) (0 (1.0) Bradycellus verbasci (0.3) Amara aenea (0.3) A.familiaris (0.3) A.tibialis (0.3) A.bifrons (L) (0.3) Calathus melanocephalus (0.3) Metabletus foveatus (0.3)

B. Carabid species caught in thionazin treated but not in untreated potato plots in Four Acre Field - 1.v.67 11.ix.67 SPECIES % of GRAND TOTAL CATCH (10 traps) Carabus violaceus (0.3) Asaphidion flavipes (2.3) Harpalus tardus (0.2) Amara fulva (0.2) Pterostichus caerulescens (0.2) P.niger (0.6) P.vulgaris (L) (0.2) P.madidus (L) (0.2)

294. APPENDIX 25 A. Carabid species caught in untreated kotato plots in Church Field - 15.v.65 27.ix.65

% of TOTAL CATCH (2 traps per plot) VERY COMMON COMMON RARE (>10%) (1-10%) (<1%) Calathus Bembidion quadri.. ' Pterostichus vulgaris fuscipes (39.5) maculatum (9.3) (0.8) Bembidion Agonum dorsale(3.7) Loricera pilicornis Trechus 4-striatus (0.7) lamptvaa (27.5) (3.4) Carabus violaceus(0.3) Pterostichus madid- Harpalus tardus (0.3) us (2.7) Amara familiaris (0,3) Nebria brevicollis Badister bipustulatus (2.4) (0.2) Harpalus rufipes Amara bifrons (0.2) (2.2) Amara sp. (0.2) Metabaetus foveatus Abax parallelepipedus (1.9) (0.2) Harpalus aeneus(1.7) Calathus melanocephalus (1.5) Larvae (unidentified) (1.2)

B. Carabid species caught in thionazin treated but not in untreated potato plots in Church Field - 15.v.65 - 27six.65

SPECIES % of GRAND TOTAL CATCH (12 traps) Bembidion andreae (0.1) B.obtusum (0.1) Harpalus rubripes (0.1) Anisodactylus binotatus ( 0 . ) Amara aenea (0.1) A.fulva (0.1) Asaamsallat (0.1)

295.

APPENDIX 26 A. Carabid species caught in untreated _potato plots in Church Field - 6.v.66 - 13.ix.66

% of TOTAL CATCH (2 traps per plot)

VERY COMMON COMMON RARE (010%) (1-10%) (<1%) Calathus fuscipes Bembidion quadrimac- Loricera pilieornil (33.7 ulatum (8.1) (0.9) Bembidio n lampros Harpalus aeneus(2.8) Nebria brevicollis (17.1) Carabus violaceus (0.6) Pterosti chu s (1.9) Amara familiaris madidus (15.8) Trechus quadris- (0.6) Harpalus rufipes triatus (1.9) Metabletus foveatus (11.1 Calathus melanoeeph- (0.6) alus (1.9) Notiophilus sub- Pterostichus yule. striatus (0.4) Faris (1.1) Amara aulica (0.4) Asaphidion flavipes (0.2) Harpalus tardus (0.2 Amara bifrons (0.2) A.consularis (0.2) Harpalus rufipes (L) (0.2)

B. Carabid species caught in thionazin treated but not in untreated potato plots in Church Field - 6.v.66 13.ix.66

SPECIES of GRAND TOTAL CATCH (6 traps) Bembidion femoratum (0.1) B.andreae (0.2) Amara plebeja (0.6) A.similata (0.1) A.aenea (0.1) A.anthobia (0.1) A.tibialis (0.1) A.praetermissa (0.1) A.apricariek (0.1) Pterostichus caerulescens (0.2) Abas parallelepipedus (0.2) Calathus piceus (0.1) Synuchus nivalis (0.1) Agonum dorsale (0.6) Notiophilus sp. (L) (0.1)

296. APPENDIX 27 A. Carabid species caught in untreated potato plots in Church Field - 9.v.67 -12.ix.67

% of TOTAL CATCH (2 traps per plot) VERY COMMON COMMON RARE (>10%) (1-10%) (<1%) Bembidion Loricera pilocornis Nebria brevicollis (L) lampros (41*6) (5.8) (0.7) Harpalus Calathus fuscipes Notiophilus biguttatus rufipes (11.7) (5.8) (L) (0.7) Bembidion 4-macul- Bembidion andreae atum (5.2) (0.7) Pterostichus madidus Trechus 4-striatus (5.2) (0.7) Harpalus aeneus Harpalus tardus(0,7) (4.5) Amara plebeja (0.7) Metabletus foveatus A.ovata (0.7) (4.5) A.familiaris (0.7) Amara similata A.bifrons (0.7) (2.6f A.consularis (0.7) Agonum dorsale(2.0) Calathus melanceeP110 Asaphidion flavipes alus (0.7) (1.3) Agonum mulleri (0.7) Pterostichus vulgaris (L) (1.3)

B. Carabid species caught in thionazin treated but not in untreated potato plots in Church Field 9.v.67 - 12.ix.67

SPECIES % of GRAND TOTAL CATCH (14 traps) Notiophilus aquaticus, (0.1) N.substriatus (0.0 Anisodactylus binotatus (0.1) Amara tibialis (0.1) A.fulva. (0,5) A.aulica (0.1) Pterostichus niger (1.1) Abax parallelepipedus (0.3) Loricera pilicornis (0.1) Amara familiaris (L) (0.1) A.bifrons (L) (0.1) Pterostichus madidus (L) (0.1) 297. APPENDIX 28 List (incomrilete) of species collected in pitfall traps from 1 May to 11 Sept.1967 injootatoplots in Four_Acre Field. Number of traps = 28 R = Rare (1 or 2); 0 = Occasional (2-10); C = Common (10-50 V.C. = Very Common (>50)

COLEOPTERA: Staphylinidaet Stenus clavicornis (0); S.nanus (R); Aleochara bipustulata (0); Atheta sp. (TX.); Mycetoporus brunneus (0); Tachyporus hypnorum (V.C.); T.obtusus (R); Tachinus rufipes (C); T.laticollis (R); Megarthrus denticall776); Quedius cinctus (R); Q.fuliginosus (R); Philonthus lamin- atus (V.C.); Philonthus spp. (C); Xantholinus sp. (0); Bledius sp. (R); Oxytelus rugosus (C) Curculionidae: Barypithes pellucidus (R); Phytobius quadrituber culatus (0); Rhinonchus brucoides (C); R.castor (0); Ceuthorrhynchus spp. (C); C.erysimi (0); Gronops lunatus(R); Phytonomus arator (0); Scarabaeidae: Aphodius granarius (R); Geotrupes stercorarius(R); Helophoridae: Helophorus sp. (R) Sphaeridiidae: Megasternum boletophagum (C) Chrysomelidae: Chaetocnema concinna (V.C.) Cantharidae$ Cantharis pallida (R) Elateridae: Agriotes obscurus (0); A.pallidulus (0) Silphidae: Silpha tristis (R) Histeridae: Peranus (=Hister) bimaculatus (R) Byrrhidae: Cytilus sericeus (adults and larvae)(C) Coccinellidae: Coccinella 7-punctata (adults and larvae) (C) Mycetophagidae: Atomaria sp. (C) HYMENOPTERA: Formicoidea (ants) (0);Peoototrupoidea (e.g.Phaenoserphus sp.) (R); Apoidea (Worker Honey Bees) (0) 298. HEMIPTERAt Saldidid (digtSaldula sp.) (V.C.); Tingidae (R); Nabidae (0); Aphidad (0); Anthocoridae (e.g.Anthocoris nemorum) (R); LEPIDOPTERA LARVAE: Noctuidae (cutworms) (C)

DIPTERA LARVAE: Stratiomyidae (0); Tipulidae (R); Syrphidae (0) DIPTERA ADULTS: undetermined species (0)

N.B. Although quantitative records were kept of the animals in each of the plots these are not thought to be representative of the actual numbers present in the field and hence are categorized as either "rare", "occasional", "common", or "very common". The list is incomplete for no records were kept of Myriapoda (Chilopoda and Diplopoda), Annelida(earthworms), Arachaida (spiders, mites or harvestmen), Mullusca (slugs and snails) or various small or delicate, flying or jumping insects such as adult flies or Collembola. 299. APPENDIX 29 Pitfall trap e,atches of carabids from thionazin treated_plots iat weekly intervals in Four Aare Field 1965

Sample Date Pitfall Trap Catches Number (1965) (Total of 4 traps) A1H Al L A2H A2L AO

1 19-22.v 3 4 16 15 16 2 22-31.v 6 7 43 35 36 3 31.v-5.vi 8 1 81 93 74 4 5-14.vi 5 2 22 62 25 5 14-20.vi 3 5 23 19 6 20-28.vi 4 3 34 1302 12 7 28.vi-4.vii 0 2 15 13 12 8 4-12.vii 1 9 12 15 12 9 12-18.vii 4 2 6 10 6 10 18-26.vii 2 8 15 7 4 11 26.vii-1.viii 2 2 6 6 11 12 1-9.viii 8 22 6 23 16 13 9-16.viii 7 8 23 20 13 14 16-23.viii 2 17 11 34 20 15 23-31.viii 14 16 15 32 10 16 31.viii-6.ix 2 7 6 5 7 17 6-13.ix 2 8 3 9 15 18 13-20.ix 2 2 8 5 11 19 20-27.ix 4 3 2 6 9 TOTAL 19.v -27.ix 79 128 347 432 328 Analysis of Variance Source of Variation df ss MS F Dates 18 . 4,01 0.23 3.29 ** Treatments 4 5.37 1.34 19.14 * * * Error 72 4.74 0.07 TOTAL 94 14.12 Least Critical Ratio (L.C.R.) Pitfall Pitfall Trap Catches(Total of 4 traps) Catches A 11 A 11 A H A L AO L.C.R. 1 1 2 2 Mean actual *** *** *** values 3.33 5.07 12.82 16.13 13.88 1.99

300. APPENDIX 30 Pitfall trap ca.bches of carabids from thionazin treated plots in Four Acre Field, 1966

Sample Date Pitfall Trap Catches (Total of 4 traps) Number (1966) Al H AIL A2H A2L AO 1 6-13.v 4 4 2 4 8 2 13-20.v 10 12 92 88 64 3 20-27.v 2 4 0 12 4 4 27.v-3.vi 16 18 56 66 82 5 3-10.vi 2 8 32 44 68 6 10-17.vi 6 0 0 88 22 7 17-24.vi 0 16 12 10 12 8 24.vi-1.vii 2 6 22 26 64 9 1-8.vii 0 0 16 20 14 10 8-15.vii 4 8 52 24 20 11 15-22.vii 0 10 4 6 4 12 22-29.vii 4 6 16 16 18 13 29.vii-5.viii 4 38 6 8 4 14 5-15.viii 20 42 10 28 40 15 15-22.viii 22 32 32 58 48 16 22-31.viii 2 14 10 18 32 17 31.viii-6.ix 8 26 8 30 12 18 6-13.ix 8 6 8 42 26 TOTAL 6.v-13.ix 114 250 378 588 542

Analysis of Variance: Source of Variation df SS MS Dates 17 8.66 0.51 4.25 *** Treatments 4 5.79 1.45 12.08 *** Error 68 8.20 0.12 TOTAL 89 22.65

Least Critical Ratio (L.C.R.) Pitfall Trap Catches (Total of 4 traps)

H A L AO L.C.R. Al A1L A2H 2 Mean *** ** *** NumbersX 3.89 8.82 10.99 23.58 20.70 2.48 x Mean of 18 samples 301. APPENDIX 31 Pitfall trap catches of carabids from thionazin treated plots in Four Acre Field, 1967

Sample Date Pitfall Trap Catches Number (1967) (Total of 4(2x2) traps) Al H Al L A2H A2L AO 1 1-8.v 0 6 2 2 2 2 8-15.v 4 4 22 48 34 3 15-22.v 4 0 4 8 2 4 22-30.v 0 2 0 0 0 5 30.v-5.vi 16 12 26 44 34 6 5-12.vi 16 18 40 48 92 7 12-19.vi 6 10 20 26 50 8 19.26.vi 8 8 10 6 8 9 26.vi-3.vii 20 34 74 20 100 10 3-10.vii 12 30 26 34 58 11 10-17.vii 16 50 12 44 54 12 17-24.vii 6 4 6 10 32 13 24.31.vii 8 24 16 30 52 14 31.vii-7.viii 10 30 26 24 64 15 7-14.viii 14 20 24 16 44 16 14-21.viii 1,4 22 16 22 46 17 21-28.viii 10 36 28 20 50 18 28.viii-4.ix 6 4 22 32 40 19 4-11.ix 2 24 30 32 50 TOTAL 1 .v-14. ix 172 338 404 466 812

Analysis of Variance: Source of Variation df SS MS Dates 18 14.85 0.83 16.60 *** Treatments 4 3.29 0.82 16.40 *** Error 72 3.87 0.05 TOTAL 94 22.01

Differences between means of treatments:Least Critical Ratio (L.C.R.) Pitfall Trap Catches (Total of 4 traps) A H A L A H A2L AO L.C.R. 1 1 2 Mean *** *** ** * (at 0.10 Pitfall 6.73 11.88 14.89 17.66 27.38 1.79 Catches 302.

APPENDIX 32 Pitfall trap catches of carabids from thionazin treated plots in Church Field, 1965

Sample Date Pitfall Trap Catches 4 traps) Number (1965) (Total of A L AO A2H 2 1 15-22.v 39 40 38 2 22-31.v 34 38 30 3 31.v-8.vi 73 107 69 4 8-14.vi 55 49 45 5 14-20.vi 32 34 33 6 20-28.vi 6 19 18 7 28.vi-4.vii 8 20 17 8 4-12.vii 4 6 10 9 12-18.vii 9 9 11 10 18-26.vii 7 7 1 11 26.vii-1.viii 4 16 12 12 1-7.viii 9 12 4 13 7-16.viii 16 3.+ 10 14 16-23.viii 20 20 38 15 23.31.viii 49 97 52 16 31.viii-6.ix 62 119 91 17 6-13.ix 85 51 45 18 13-20.ix 46 54 48 19 20-27.ix 40 44 21 TOTAL 15.v-27.ix 598 776 593

Analysis of Variance: Source of Variation: df SS MS * * * Dates 18 7.69 0.43 14.33 Treatments 2 0.22 0.11 3.67 * Error 36 0.94 0.03

TOTAL 56 8.85 Least Critical Ratio (L.C.R.) Pitfall Trap Catches (Total of 4 traps) t, AO L.C.R. A2H Al Mean Pitfall Jatch 21.40 29.64 22.02 1.30 303.

APPENDIX 33 Pitfall trap catches of carabids from thionazin treated plots in Church Field, 1966

Sample Date Pitfall Trap Catches Number (1966) (Total of 4(2x2) traps) A2H A2L AO 1 6-13.v 0 6 0 2 13-20.v 92 114 100 3 20-27.v 32 28 38 4 27.v-3.vi 80 56 54 5 3-10.v 72 62 54 6 10-17.v 82 64 38 7 17.24.v 10 12 4 8 24.vi-1.vii 42 28 24 9 1-8.vii 20 52 24 10 8-15.vii 28 34 28 11 15-22.vii 22 38 20 12 22-29.vii 34 54 30 13 29.vii-5.viii 92 32 18 14 5-15.viii 84 92 172 15 15-22.viii 44 72 60 16 22-31.viii 112 106 56 17 31.viii-6.ix 114 34 102 18 6-13.ix 30 48 116 TOTAL 6.v-13.ix 990 932 938

Analysis of Variance: Source of Variation: df SS MS Dates 17 8.86 0.52 13.00 *** Treatments 2 0.10 0.05 1.25 N.S. Error 34 1.48 0.04 TOTAL 53 10.44 301i.

APPENDIX 34 Pitfall trap catches of carabids from thionazin treated plots in Church Field, 1967

Sample Date Pitfall Trap Catches Number (1967) (Total of 4 traps) Al H A1Z A2H A2L AO 1 9-16.v 3 2 14 20 8 2 16-23.v 3 2 4 4 4 3 23-30.v 2 0 0 0 0 4 30.v-6.vi 11 20 46 26 42 5 6-13.vi 3 5 40 94 40 6 13-20.vi 5 6 12 28 22 7 20-27.vi 2 7 12 18 10 8 27.vi-4.vii 8 20 18 34 32 9 4-11.vii 11 9 8 62 16 10 11-18.vii 7 9 4 30 14 11 18-25.vii 11 10 2 4 4 12 25.vii-1.viii 17 12 6 30 16 13 1-8.viii 12 26 14 32 8 14 8-15.viii 29 17 20 40 14 15 15-22.viii 32 26 0 50 6 16 22-29.viii 26 31 16 18 40 17 29.viii-5.ix 26 37 12 48 26 18 5-12.ix 40 14 14 42 6 TOTAL 9.v-12.ix 248 253 242 580 308

Analysis of Variance: Source of Variation df SS MS F Dates 17 9.72 0.57 6.33 *** Treatments 4 1.72 0.43 4.78 ** Error 68 5.98 0.09 TOTAL 89 17.42 Least Critical Ratio (L.C.R.) Pitfall Trap Catches (Total of 4 traps)

AlH A L A2H A2L AO L.C.R. Mean Pitfall 5% Catch 9.62 9.89 8.57 22.39 11.94 1.59 305. APPENDIX 35 Relative numbers and species of Carabidae from thionazin treated and control plots in Four Acre Field, 19.v.65 - 27.ix.65

Total catch from 4 traps per treatment SPECIES Control In-row Broadcast "Low" "High"

Nebria brevicollis 6 2 1 Notiophilus biguttatus 2 Loricera pilicornis 1 2 Asaphidion flavipes 1 Bembidion lampros 149 224 10 B.andreae 12 2 Bouadrimaculatum 5 4 Treahus quadristriatus 13 9 1 Badister bipustulatus 1 Harpalus aeneus 5 12 1 H.tardus 1 H.rufipes 22 69 18 Acupalpus meridianus 2 1 Anisodactylus binotatus 4 7 Amara bifrons 4 2 A.fulva 3 Amara spp. 2 2 Pterostichus niger I P.vulgaris 75 63 34 P.madidus 8 10 3 Abax parallelepipedus 1 Calathus fuscipes 11 8 6 Agonum dorsale 1 1 1 Dromius melanocephalus 1 1 Metabletus foveatus 1 2 Larvae (undetermined) 4 7 Total numbers 328 432 79 Total species 19+ 20 12+ 306.

APPENDIX 36 Relative numbers and species of Carabidae from thionazin treated and control plots in Four Acre Field, 6.v.66 - 13.ix.66

Total catch from 2 x 2 traps per treatment Species Control In-row Broadcast "Low" "High" Carabus violaceus - 2 - Nebria brevicollis 2 2 - Notiophilus substriatus 4 2 - N.biguttatus 4 8 2 Eoricera pilicornis 8 8 2 Clivina collaris 2 - - Asaphidion flavipes - - 2 Bembidion lampros 262 270 26 B.femoratum 2 2 B.andreae - 6 B.ouadrimaculatum 38 62 - Trechus quadristriatus 28 48 - Harpalus aeneus 20 12 6 H.tardus 2 2 - Zrufipes 52 40 14 Acupalpus meridianus 2 2 - Anisodactylus binotatus 6 4 2 Amara plebeja 2 - - A.similata 4 2 2 A.bifrons 8 - - A.fulva - 2 2 Pterostichus caerulescens 2 2 - P.niger 2 - 2 P.vulpris 64 72 44 P.madidus 2 22 - Calathus fuscipes 6 8 6 C.piceus - - 2 Agonum dorsale - 2 - Metabletus foveatus 2 4 2 Harvalua rufipes (larvae) 8 - - Loricera pilicornis 4 - - Amara sp. 2 - - Notiophilus sp. - 4 - Agonum dorsale - 2 -

Total numbers 542 588 57 Total species 23 22 14 307. APPENDIX 37 Relative numbers and species of Carabidae from thionazin treated and control plots in Four Acre Field, 1.v.67 - 4.ix.67

Total catch from 4(2x2) traps per treatment Species Control In-row "Low" Broad- cast "High" A. L. A. L. A. L. Carabus violaceus 4 Notiophilus biguttatus 4+2* 2 Loricera pilicornis 44 14 12 10 8 4 Clivina collaris 2 4 2 Asaphidion flavipes 2 14 nembidion lampros 334 204 32 B.tetracolum 2 B.andreae 2 2 n.quadrimaculatum 38 14 4 Trechus quadristriatus 4 2 H.aeneus 6 18 8 H.tardus 2 2 H.rufipes 96 8 58 2 40 Acupalpus meridianus 2 2 Bradycellus verbasci 2 Anisodactylus binotatus 40 4 12 16 ara plebeja 78 14 46 10+2+ 6 A.similata 10 2 8 A.aenea 2 A.familiaris 2 6 A.tibialis 2 A.bifrons 40 2 12 A.fulva 2 Eterostichus caerulescens 2 ).niger 6 4 I!.vuipris 20 24 12 2 P.madidus 4 4 2 Calathus fuscipes 4 2 C.melanocephalus 2 Agonum dorsale 26 2 4 Metabletus foveatus 2

Total numbers 738 74 440 26 166 6 Total species 23 8 24 4+ 15 2

* N.substriatus + Species undetermined

A = Adults L = Larvae 3o8.

PPENDD; 38 Relative numbers and species of Carabidae from thionazin treated control plots in Church Field, 15.v.65 -.ix.6

Total from 4 traps per treatment Species Control In-row In-row "Low" "High" Carabus violaceus 2 6 5 Nebria brevicollis 14 26 21 Loricera_pilicornis 4 5 7 Bembidion lampros 163 212 178 B.andreae 1 T3.quadrimaculatum 55 61 25 B.obtusum - 1 Trechus quadristriatus 20 21 16 Badister bipustulatus 1 1 - Harpalus aeneus 10 8 9 H.rubripes - 1 H.tardus 2 1 1 H.rufipes 13 8 16 Anisoda*y.lus binotatus - . 1 Amara aenae - - 1 A.familiaris 2 4 2 A,bifrons 1 12 1 A.fulva - 1 - Amara spp. 1 2 7 1706stichus vulgaris 5 27 12 1%madidus 16 23 23 Abax parallelepipedus 1 Calathus fuscipes 234 297 /22; C.melanocephalus 9 21 12 Agonum mulleri 1 - A.dorsale 22 23 14 Metabletus foveatus 11 5 9 larvae (undetermined) 7 10 11 Total numbers 593 776 597 Total species 19+ 21+ 21+ 309

APPENDIX 39 Relative numbers and species of Carabidae from thipnazin treated and control plots in Chur2h, Field- 6.1%06.43.41x0.64

Total from 4(2x2) traps per treatment Species In-row In-row Control "Low" "High" Carabus violaceus 18 14 20 Nebria brevicollis 6 2 2 Notiophilus substriatus 4 2 toricera pilicornis 8 6 8 Asaphidion flavipes 2 - tembidion lampros 160 226 276 B. emoratum - 2 - B.andreae - 4 - B.T.iadrimaculatum 76 52 44 Trechus quadristriatus 18 34 10 Harpalus aeneus 26 26 32 H.tardus 2 2 H.rufipes 104 138 118 Amara piabeja - 2 2 A.similata - 2 A.aenea - - 2 A.familiaris 6 10 24 A.anthobia 2 - A.tibialis - - 2 A.bifrons 2 6 4 A,praetermissa - 2 - A.apricaria - 2 - A.consularis 2 2 2 A.aulica 4 - - Pterostichus caerulescens - 4 - l'.vulpris 10 30 30 P.madidus 148 134 160 Abax parallelepipedus - 2 4 Calathus fuscipes 316 188 198 C.melanocephalus 18 12 16 C.piceus - - 2 Synuchus nivalis - 2 - onum dorsale. - 6 10 Me abletus foveatus 6 22 18 Notiophilus sp. larva - 2 Harpalus rufipes larva 2 - -

Total numbers 938 932 990 Total species 20 27 24 310. APPENDIX. 40 Relative numbers and species of Carabidae from thionazin treated and control plots in Church Field, 9.v.67 12.ix.67

Total from 4 traps per treatment In-row In-row B'cast B'cast Species Control "Low" "High" "Low" "High" A L A L A L A L A L Carabus violaceus 4 2 1 Nebria —171767icollis. 2' 2 2 1 Notiophilus aquaticus 1 N.substriatus 2 N.biguttatus 2 2 1 Loricera pilicornis 18 20 2 6 4 1 Asaphidion flaviVes 4 2 4 2 6 Bembidion lampros 128 266 92 56 31 Beandreae 2 13.quadrimac- Ulatum 16 46 6 7 2 Trechus iTaTFITtriatus 2 4 2 5 2 Earpalus aeneus 14 6 12 6 3 H.tardus 2 4 H.rufipes 36 54 32 55 65 Anisodactylus binotatus 2 Amara plebeja 2 2 2 A.similata 8 2 4 3 3 A.ovata 2 A.familiaris 2 8 6 1 2 2 A.anthobia 2 2 1 2 A.tibialis 2 ilbifrons 2 26 6 9 1 10 A.fulva 5 A.consularis 2 2 4 A.aulica 1 Pterostichus PirUK 2 2 5 4 P.vus 4 26 10 10 14 Pymadidus 16 40 22 32 2 31 Aloax parallelepipedus 2 1 1 Calathus fuscipes 18 48 8 39 44 311. AP14MAX 40 cont.

Calathus melanoceppialus 2 4 2 4 Ationum mulieri 2 2 1 1 A.dorsale 6 4 4 2 6 7 Metabletus 'oveatus 14 4 8 1 1

Total numbers 300 8 574 6 242 - 248 5 248 Total species 22 3 23 2 24 - 22 3 26

A = Adults L = Larvae B'cast = Broadcast 312.

APPENDIX 41

List (incomplete) of species other than carabids collected in pitfall traps from November to December in Four Acre and Church Fields

R = Rare (1-2); 0 = Occasional (3-10); C=Common (10-50) V.C. = Very Common (>50) A.Four Acre Field (1965 and 1966) COLEOPTERA: Staphylinidae : adults (C), larvae (0) Curculionidae Phytonomus arator (R) Byrrhidae : Simplocaria semistriata (0) Cholevidae : Catops sp. (0) LEPIDOPTERA : larvae (undetermined species) (0) HYMENOPTERA : Formicoidea (ants) (R) DIPTERA : adults (R), larvae (0) DIPTERA : Syrphidae (0)

B.Church Field (1965 and 1966) COLEOPTERA Staphylinidae : adults (C), larvae (0-C) Curculionidae : larvae (R) Cantharidae : larvae (0) Elateridae : larvae (R) Byrrhidae : Simplocaria semistriata (0) Cholevidae : Catops sp. (R) HEMIPTERA Aphidae : adults (R) LEPIDOPTERA : larvae (0) DIPTERA : larvae (0) 313. APPENDIX 42 Pitfall trap catches of carabids from thionazin treated plots in Four Acre Field 7-8 months after treatment of soil A. After first year of treatment

Pitfall Trap Catches Sample Date (Total of 4 traps) Number (1965) A1H A1L A2H A2L AO 1 10-15.xi 1 2 0 5 14 2 15-22.xi 0 0 2 1 3 3 22.xi-1oxii 0 1 1 1 5 4 1-6.xii 1 2 0 2 .2 5 6-13.xii 0 2 0 3 3 6 13-20.xii 1 4 2 0 8 7 20-27.xii 2 1 4 3 5 Total 5 12 9 15 40

Analysis of Variance:- Source of Variation df SS MS Dates 6 0.416 0.069 1.255 N.S. Treatments 4 1.335 0.334 6.073 ** Error 24 1.327 0.055 Total 34 3.078

Least Critical Ratio (L.C.R.)

Pitfall Trap Catches (Total of 4 traps) AiL A2H A2L AO L.C.R.

Mean * * * * * * * * * Pitfall Catches 0.57 1.46 0.90 1.74 4.84 2.26

314.

APPENDIX 43 Pitfall trap catches of carabids from thionazin treated plots in Four Acre Field 7-8 months after treatment of soil

B. Second year of treatment

Pitfall Trap Catches Sample Date (Total of 4(2x2) traps) Number (1966/67) A1H Al I, A2H A2L AO 1 15-22.xi 6 14 6 18 24 2 22-29.xi 4 4 4 6 10 3 29.xi-6,.xii 0 2 4 2 0 4 6-13.xii 2 0 0 0 2 5 13-19.xii 4 4 2 2 2 6 19-27.xii 4 2 2 6 4 7 27.xii.66- 3.i.67 0 0 0 0 0 Total 15.x1.66- 20 26 18 34 42 3.1.67

Analysis of Variance Source of Variation df SS MS Dates 6 4.270 0.712 15.478 *** Treatments 4 0.078 0.020 0.435 N.S. Errors 24 1.100 0.046 Total 34 5.448 315.

APPENDIX 44 Pitfall trap catches of carabids from thionazin treated plots in Church Field 7-8 months after treatment of soil

A. First year of treatment

Pitfall Trap Catches Sample Date (Total of 4 traps) Number (1965) A2H A2L AO 1 12-15.xi 0 1 2 2 15-22.xi 3 13 8 3 22-30.xi 6 9 4 4 30.xi-6.xii 5 8 3 5 6-13.xii 10 12 4 6 13-20.xii 4 8 7 7 20-27.xii 5 11 3 Total 12.xi-27.xii 33 62 31

Analysis of Variance Source of Variation df SS MS Dates 6 0.978 0.163 5.62 * * Treatments 2 0.280 0.140 4.83 * Error 12 0.347 0.029 Total 20 1.605

Least Critical Ratio (L.C.R.) Pitfall Trap Catches (Total of 4 traps) L.C.R.(5%) A2HA2I" AO Mean Pitfall Catches 3.76 7.62 4.07 1.580 316. APPENDIX 45 Pitfall trap catches of carabids from thionazin treated plots in Church Fields 7-8 months after treatment of soil B. Second year of treatment

Pitfall Trap Catches Sample Date (Total of 4(2x2) traps) Number (1966/67) A2H A2 AO 1 15-22.xi 2 8 14 2 22-29.xi 6 16 6 3 29.xi-6.xii 4 6 4 4 6-13.xii 0 0 0 5 13-19.xii 10 14 12 6 19-27.xii 6 0 4 7 27.xii.66 0 0 0 3.1.67 Total 15.xi.66 28 44 40 3.1.67

Analysis of Variance Source of Variation df SS MS Dates 6 3.664 0.611 9.70 ** Treatments 2 0.028 0.014 0.22 N.S. Error 12 0.758 0.063 Total 20 4.450 APPENDIX 46 Summary of fumigant soil treatments on Carabidae in Four Acre Field and Church Field : 1965 and 1966

PESTICIDE & DOSAGE LEGEND DATE OF PERIOD OF TOTAL CATCH OF CARABIDAE RATE TREATMENT SAMPLING 1965 1966 1965 & 1966 1966 1965 GRAND 1965 1966 No. Spp No. Spp TOTAL A. FOUR ACRE FIELD Ethylene dibromide BH 29.x 11.xi 10.xi 15.xi 3 1 4 3 7 "High" to to ti tt BL " tt 27.xii 3.i.67 3 2 11 4 14 "Low" Chloropicrin *High" CH it 9.xi If ft 2 2 3 2 5 It "Low" CL It If ft It 3 3 6 3 9 Dazomet "High" DH tt n It tt 4 3 1 1 5 tt it II It n "Low" 'DI: -* - 5 3 5 Control (mean of 4 (A000, It it it " 11.5 8 16.75 8 28.25 plots) CO,DO) B. CHURCH FIELD Ethylene dibromide BH 2-3.xl 11.xi 12.xi 15.xi 4 4 .9 2 13 "High" to to it it 131, II II 27.xii 3.i.67 5 3 17 4 22 "Low" it ti Chloropicrin "High" CH IT 10.xi " it 4 3 6 2 10 ti "Low" CL tt tt " it 2 2 9 3 11 Dazomet *High" DH tt tt it it 3 2 4 2 7 If "Low" 'DI: II II II It 2 2 5 3 7 Control (mean of 4 (A0001 ft It It II 20 4 25 11 45 plots) CO,DO) 318.

APPENDIX 47 Pitfall trap catches in pesticide treated plots following application of soil fumigants. Four Acre Field; two traps per treatment

Pitfall Trap Catches Carabid Species E.D.B. CH. Dazomet Control (Mean of H L H L H L 4 plots) A.Adults(10 Nov-27 Dec '65) Nebria brevicollis 0.25 Notiophilus substriatus 0.50 N.biguttatus Bembidion lampros 1 1.00 Trechus sp. 1 1 0.75 Bradycellus harpalinus 1 B,Larvae (10 Nov-27 Dec'65) Nebria brevicollis 3 1 2 6.00 liarpalus rufipes 1 1.00 Amara sp. 0.25 Pterostichus vulgaris 1 1 0.50 Calathus fuscipes 1 1 1.25 Total Numbers 3 3 2 3 4 0 11.50 A.Adults(15 Nov' 66-3 Jan' 67) Nebria brevicollis 0.25 Notiophilus substriatus Bembidion lampros 1 0.25 Sotetracolum 0.25 TreChus sp. 1 4 1 1 1 3.25 Bradycellus harpalinus 1 0.50 B.Larvae(15 Nov'66-3 Jan'67) Nebria brevicollis 2 5 2 4 2 5.25 Harpalus rufipes 1 1 3.00 Amara aulica 1 Amara sp. 2 Pterostichus vulgaris 1.75 6alathus fuscipes 2.25

Total Numbers 4 11 3 6 1 5 16.75 Grand Total 7 14 5 9 5 5 28.25

CH. = Chloropicrin

H = High; L = Low 319. APPENDIX 48 Pitfall trap catches in pesticide treated plots following application of soil fumigants. Church Field; two traps per treatment

Pitfall Trap Catches C arabid E.D.B. CH. Daz. Control Species (Mean of H L H L H L 4 plots) A.Adults(12 Nov-27 Dec'65) Nebria brevicollis 1 Notiuhilus substriatus 1 0.75 Bembidion lampros 1 Trechus sp. 1 1 2 1 1 1.75 Amara sp. B.Larvae (12 Nov-27 Dec'65) Nebria brevicollis 2 2 1 13.25 Harpalus rufipes Amara aulica 1 Calathus fuscipes 2 1 1 1 4.25 Total numbers (adults & larvae) 4 5 4 2 3 2 20.00 A.Adults(1 Nov'66-3 Jan'67) Nebria brevicollis 2 1 1 1.50 Notiophilus substriatus 0.25 Bembidion lampros 1 1 B.obtusum 0.50 Trechus sp. 3 2 7 1 2 5.50 Bradycellus har alinus 0.25 B.Larvae(15 Nov'66-3 Jan'67) Carabus violaceus 0.25 Nebria brevicollis 6 8 3 3 1 9.50 Harpalus rufipes 4 2 4.25 Amara sp. 0.25 Eterostichus madidus 1.00 MIgUEETT7771777 1 1 1.75 Total numbers (adults & larvae) 9 17 6 9 4 5 25.00 Grand Total 13 22 10 11 7 7 45.00

CH. = Chloropierin L = Low

H = High Daz. = Dazomet 320. APPENDIX 49 Pitfall trap catches of carabids from fumigant treated plots in Four Acre Field, 1 May - 11 September, 1967

Pitfall Trap Catches Samp. Date (6(2x3) traps per treatment No. (1967) BL BH CL CH DL DH B0+C0+D0 1 1-8.v 6 0 0 6 3 12 23 2 8-15.v 30 33 18 93 90 30 58 3 15-22.v 3 0 3 0 0 3 13 4 22-30.v 6 0 3 0 0 0 3 5 30.v-5.vi 48 12 54 6 27 36 111 6 5-12.vi 111 93 204 126 99 84 90 7 12-19.vi 66 42 84 72 57 45 48 8 19-26.vi 9 0 27 3 0 6 9 9 26.vi-3.vii 177 102 81 201 105 90 61 10 3-10.vii 93 75 75 84 30 24 66 11 10-17.vii 39 33 75 27 57 27 44 12 17-24. vii 9 9 72 21 54 0 10 13 24-31.vii 27 57 39 66 72 39 67 14 31.vii-7.viii 27 81 30 39 48 18 64 15 7-14.viii 3 9 51 30 36 45 52 16 14-21.viii 33 3 51 42 63 9 43 17 21-28.viii 21 39 78 81 45 42 67 18 28.viii-4.ix 15 30 69 12 48 33 69 19 4-11.ix 3 9 75 24 24 30 58 Total 1.v-11.ix 726 627 1089 933 858 573 956

Analysis of Variance Source of Variation df SS MS Dates 18 32.91 1.83 16.64 *** Treatments 6 2.67 0.45 4.09 *** Error 108 11.90 0.11 Total 132 47.48

Least Critical Ratio Pitfall Trap Catches(6(2x3)traps per treatment L.C.R. BL BH. CL CE DL DH B0+C0 (P=. +DO 0.05) Mean actual * *** * ** values 20.58 13.49 35.14 23.04 23.72 17.84 38.72 1.63 conv. from log (n+1) 321.

APPENDIX 50 Pitfall trap catches of carabids from fumigant treatedplots in Church Field 9 May - 12 September, 1'67

Pitfall Trap Catches Sample Date (6(2x3) traps per treatment No. (1967) BL BH CL CH DL DH B0+00+ DO 1 9-16.v 36 39 18 39 15 9 27 2 16-23.v 12 9 3 3 3 0 3 3 23.,30.v 0 0 0 0 3 0 2 4 30.v-6.vi 135 90 138 96 108 54 59 5 6-13.vi 72 51 105 66 69 27 31 6 13-20.vi 57 30 15 72 60 42 12 7 20-27.vi 15 24 15 6 6 6 7 8 27.vi-4.vii 33 51 15 27 24 15 55 9 4-11.vii 18 30 30 36 18 9 19 10 11-18.vii 24 18 30 18 75 36 11 11 18.-25.vii 3 3 0 3 24 30 7 12 25.vii-1.viii 27 18 24 30 30 6 40 13 1-8.viii 27 18 18 69 15 33 36 14 8-15.viii 51 9 24 36 18 24 37 15 15-22.viii 12 0 18 45 30 21 34 16 22-29.viii 27 45 60 48 57 48 57 17 29.viii-5.ix 93 30 24 24 24 1.8 99 18 5-12.ix 12 9 27 30 21 15 53 Total 9.v-12.ix 654 474 564 648 600 393 589

Analysis of Variance Source of Variation df SS MS Dates 17 21.64 1.27 9.07 *** Treatment 6 0.81 0.14 1.75 N.S. Error 102 8.32 0.08 Total 125 30.77

Pitfall Trap Catches (9 May to 12 Sep., 1967) BL BH CL CH DL DH BO+CO+ DO Mean act. values converted 22.60 15.60 17.07 22.77 22.88 14.07 23.44 from log (n + 1)