Use of grasshoppers as test animals for the ecotoxicological evaluation of chemicals in the soil

Agriculture, Ecosystems and Environment, 16 (1986) 175--188 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

175

U S E O F G R A S S H O P P E R S AS T E S T A N I M A L S F O R T H E ECOTOXICOLOQICAL EVALUATION OF CHEMICALS I N ~rHE S O I L 1

GERHARD

H. S C H M I D T

Department of Zoology-Entomology, University of Hannover, Herrenhh'user Strasse 2, D-3000 Hannover 21 (F.R.G.) (Accepted for publication 20 February 1986) ABSTRACT Schmidt, G.H., 1986. Use of grasshoppers as test animals for the ecotoxicological evaluation of chemicals in the soil. Agric. Ecosystems Environ., 16: 175--188.

A method was worked out for an ecotoxicological evaluation of chemicals in the soil which makes it possible to compare laboratory and field results. Species of Acrididae were used as test animals, which deposit their eggs in the soil. The chemicals tested were HgCl~, urea, phenol, perylene, dichlorodiphenyl dichloro~thylen (DDD), methanol and ethyl acetate. The following results were obtained: insect numbers decreased in concentrations of more than 0.001% HgCl~ per dry sand in the laboratory tests and in more than 0.0001% under field conditions; mercury mainly affected the embryonic development taking place in the soil; urea >0.055 g N per kg dry sand decreased the number of deposited egg pods and the number of eggs laid; increasing amounts of urea also decreased the hatching of the larvae; all the other chemicals tested reduced the number of larvae developing into adults by 50% or more only in concentrations which could possibly be present in the environment.

INTRODUCTION M a n y o f t h e epigeic insect species d e p o s i t t h e i r eggs in t h e soil o f t h e i r h a b i t a t , a n d all t h e c h e m i c a l s p r e s e n t in t h e soil can i n f l u e n c e t h e d e v e l o p m e n t o f t h o s e eggs. I n t h e s u m m e r m o n t h s d u r i n g t h e s e c o n d b r o o d p e r i o d o f m a n y birds, acridids ( g r a s s h o p p e r s ) c a n r e p r e s e n t 2 0 - - 3 0 % o f t h e b i o m a s s o f a r t h r o p o d s in d i f f e r e n t e c o s y s t e m s o f E u r o p e w h i c h are n o t , o r o n l y m i n i m a l l y , s t r e s s e d b y c h e m i c a l s p r e s e n t as fertilizers, c o n t a m i n a n t s o r pesticides. A c r i d i d s are i m p o r t a n t p r e y f o r n u m e r o u s birds as well as f o r s o m e m a m m a l s , r e p t i l e s a n d a m p h i b i a b e c a u s e o f t h e i r r e l a t i v e l y high individual weight. T h u s , t h e y p l a y an i m p o r t a n t role in t h e f o o d c h a i n o f s t o r k s , f a l c o n s a n d b u z z a r d s ( J o h n s t o n , 1 9 5 6 ; S m a l l e y , 1 9 6 0 ; Bullen, 1 9 6 6 ; M o o r e a n d B e l l a m y , 1 9 7 3 ; Wiens, 1 9 7 3 ; B e d d i n g t o n et al., 1 9 7 6 ; S m i t h , 1 9 8 0 ) . I n highly stressed a g r i c u l t u r a l areas, t h e p e r c e n t a g e a n d a b s o l u t e n u m b e r s 1Dedicated to Prof. Dr. Dr. h.e.B. Rensch, Miinster i. Westf. who celebrated his 85th birthday on January 21, 1985. 0167-8809/86/$03.50

© 1986 Elsevier Science Publishers B.V.

176

are greatly reduced as in Franconia (Schmidt and Baumgarten, 1 9 7 4 / 7 7 ) and in the Apennines near Florence (unpublished). In grassland biotops o f Lower Saxony in 1981, acridids were only collected on fallow meadows close to a military area where t h e y made up just 3--7% o f the biomass o f arthropods. The diversity of acridids and other insect groups was strongly reduced by treatment with chemicals (Tischler, 1979; Odum, 1980). Heavy metals and persistent pesticides accumulate in the soil and along the f o o d chain can exterminate whole populations o f distinct species (Wieser, 1970; Schmidt, 1986). Our biozoenotic investigations demonstrated that acridids cannot tolerate typical pesticides used as insecticides and herbicides, and react very sensitively to heavy metals and fertilizers in the soft. This did n o t depend on the natural changes of the populations caused b y climatic factors. To determine the tolerance limits and toxicity of chemicals in the soil we have to compare investigations in the laboratory with observations in the field. It was our aim to develop a biotest for the laboratory which could be applied to explain field data. Test animals must breed continuously and have many of both sexes present all the time so that n o t all acridids are suitable. All species living in Central Europe are unsuitable because of their winter diapause of 4--5 months which can be broken only b y a long vernalisation period at 0°C or lower temperatures. Around the Mediterranean sea the species Acrotylus patruelis (HerrichS c h ~ f e r , 1838) and Aiolopus thalassinus (Fabricius, 1781) are w i t h o u t diapause and can be multiplied very quickly under laboratory conditions. A breeding stock can be easily collected from the sandy and moist regions around the shores of Italy and Greece. Populations of A. thalassinus occur near the lake of Neusiedl in Austria and in some moist areas of South Germany, b u t these populations have a facultative diapause and are therefore less suitable. The relevance o f the results using the two species w i t h o u t egg diapause to species with diapause was determined b y comparing A. patruelis and A. thalassinus with the Central European species Oedipoda caerulescens (Linnaeus, 1758), Glyptobothrus biguttulus (Linnaeus, 1758) and Glyptobothrus brunneus (Thunberg, 1815) in experiments with the same chemical t r e a t m e n t of the substrate in simulated long-time overwintering conditions. METHODS

Acrotylus patruelis and A. thalassinus from Sabaudia (Latina) and Cesenatico near the Adriatic sea were bred at r o o m temperatures of 22-27~C in cages 56 cm long X 26 cm wide X 63 cm high (Fig. 1) (Schmidt, 1981). To reduce the mortality of the larvae, not more than a b o u t 60 insects should be grown in one cage. The mortality is greatest in the first instar

177

8

.'." .-:..: .:::.:.:

. :: ":.: -.?

:'.:."

":'.:."':'(:".'.'.'.~

I l: : ::!~:i :

ii::!!: ~Jl!!d!i!5::i

"-'.':'>:'U~ "

'J

i

"

i iiii;,:;!~T" :.:.:::.

--;

....

i il,i

:-: :i:

ii

Fig. 1. Diagram of a terrarium for growing larvae and allowing egg laying by adults and which can be used for testing chemicals; (1) water flask with a communicating pipe; (2) hydroculture system; (3) feeding plate; (4) pot for egg deposition; (5) petri dish with wet sand; (6) nylon gauze for moulting; (7) radiator for lighting and heating; (8) glass door.

and on average, 5 0 - 6 0 % of the larvae became adults. The adults are removed immediately to different cages and used for experiments after 8 - 1 0 days (at the end of the pre~viposition time). F r o m each generation, 10 pairs were used to continue the breeding of the next generation. O. caerulescens and the Glyptobothrus species were n o t bred in captivity, 30 adults of each sex being caught in the field in August for each test. A b o u t 20--30 adult females 2--6 weeks old of the bred species were kept with the same number of males in a tray of plexiglass 60 X 30 X 30 cm high t o p p e d by a 50--60-cm high w o o d e n box with one or two sides of plexiglass, the other sides and the roof being nylon gauze for ventilation (Fig. 1). On the b o t t o m of the cage there was a hydroculture box with untreated seedlings of wheat or barley which could easily be changed. The other part of the b o t t o m was covered by a 1-cm layer of coarse-grained dry sand. The cages were heated and lit by an internal reflector bulb (Osram Concentra 60 or 75 Watt) under the roof of the upper box in one third of the area. It was situated so t h a t the seedlings were protected by a narrow angle o f radiation and the temperature reached 40--42 ° C on the sand underneath. The temperature gradient consistently decreased to 28°C in all directions during the light period (L/D 12 h). Such a temperature gradient is necessary for successful breeding experiments.

178 The humidity in the cage was increased b y a shallow bowl filled with water and a cotton pad. As further food, the hoppers received lettuce, fruits, wheat bran, carrots, protein-rich dog f o o d and a variety o f wild plants. The number of eggs deposited, number of eggs per pod, percentage of larvae hatching from the eggs and the percentages of larvae becoming adults were recorded. For egg deposition, a series of 9 plastic boxes, each 7.5 X 7.5 X 5 cm in size, were placed near the hydroculture b o x directly under the lamp. All boxes were filled up with washed sand of 0 . 3 4 - 0 . 4 3 mm grain size and 12% moisture. One of the boxes was used for the control w i t h o u t any treatment, the others being treated with different concentrations (Fig. 2) of chemicals dissolved in the added water or mixed with dry sand before adding water if insufficiently water soluble. When it was necessary to know the limiting toxicity more closely, the concentrations were graduated over a smaller effective range. Each experimental series lasted 14 days. During this period, the boxes used for egg deposition stood in the terrarium daffy from 09.00 to 12.00 h and from 14.00 to 17.00 h under L/D o f 12 h because o f the concentrated egg laying during these periods. On t h e other hand, a strong desiccation of the substrate was avoided b y these short periods. The water content of the substrate was controlled daily, any loss being compensated by adding pure water. After each interruption o f egg deposition (twice daily), the position of the boxes was changed in a planned sequence. Thus, no b o x was favoured b y a higher temperature and mass laying.

C

0.1

0,5

I~

5

10

50

100

500

Fig. 2. Arrangement of the boxes for egg laying in the laboratory tests. The numbers indicate the concentration steps (rag kg -~) mainly used for the test chemicals to ascertain the toxic concentration. Each box was filled with 300 g dried sand plus 40 ml water = 340 g wet test substrate; grain size was 0.34--0.43 ram; C = control box without treatment.

After a 2-week period, the deposited eggs of A. patruelis and A. thalassinus were incubated in situ in the boxes at 30°C. The hatching of the larvae was checked daily for 5 weeks and recorded. Each b o x was kept in a plastic bag so that the substrate did not dry out, with cellucotton in each bag to

179

reduce the condensation of water on the walls of the plastic bag. This is necessary because the young larvae stick to the water very easily and die. After an incubation time of 5 weeks, the egg pods and remaining eggs from which no larvae hatched were counted under water. Between 4 and 8 replicates for each test chemical were tested in parallel or in series. The data were evaluated b y the t-Test. In some cases only 2 or 3 replicates were used and hence only mean values are reported as preliminary values. After 4 - 6 weeks oviposition, the eggs of O. caerulescens and Glyptobothrus spp. were stored in the sand boxes in a refrigerator at 4--5°C until the following March. This vernalisation was necessary to break the diapause and to increase the hatching to a b o u t 70%. The larvae were hatched, as above, for 2 weeks at 30°C. They developed into adults in 6 weeks. All the larvae which hatched at 30°C and survived at least 24 h were reared in cages as in Fig. 1, except that the b o t t o m s were covered b y a 2-cm layer of moist sand. A separate breeding cage was used for each chemical concentration and the control. The mortality was relatively high during the first instar, which is normal. The population density should n o t be more than 60 larvae per cage to ensure a high number of adults and to be sure that only test chemicals influence this survival. The whole larval development phase lasts 30--.35 days on average in the t w o species w i t h o u t diapause (Schmidt, 1980). RESULTS

pH: species A. patruelis As a result of the possible changing of pH values b y the different chemicals, it was necessary to show if pH influenced egg deposition and development. The pH values of the soil were prepared b y different phosphate buffer solutions, b u t the sandy substrate used had a high buffer capacity and had to be treated with 10% phosphoric acid solution and washed to get a neutral reaction, before buffering to a constant pH value. TABLE I The influence of pH on t h e f o r m a t i o n of egg pods in A. patruelis (H.-S.) pH of t h e sand 3

4

5

6

7

8

9

10

11

N u m b e r of deposited egg p o d s Experiment 1 Experiment 2

4 6

9 4

2 5

3 3

4 3

2 6

12 6

6 8

4 2

Mean n u m b e r s

5

6.5

3.5

3

3.5

4

9

7

3

180

The experiments were carried out between pH 3 and pH 11 and Table I shows the results. In all cases, 80--100% of the embryos hatched and pH had no influence. Experiments could, therefore, be carried o u t b e t w e e n pH 3 and 11 in sandy soil with comparable results, assuming that the other species react similarly.

Mercuric(II)chloride (Merck) (a) A. patruelis The percentage of larvae hatching was significantly (P < 0.05) reduced if the dose exceeded 1 mg Hg per kg dry soil. The variation of hatching increased with higher concentrations until no hatching occurred (Fig. 3). The egg laying females were evidently unable to distinguish b e t w e e n the different HgC12 concentrations because t h e y laid egg pods and eggs equally in all concentrations. The proportion o f larvae developing to adults did not differ from that of the control until concentrations of > 5 mg Hg per kg, when it became a little lower. % 100

A. patruelis 1"--7 A. thalassinus

80' >

60'

~" t,O, u L-

20

C

0.121

0.605

1.21

6.05

12,1

60.5

121

605

HgCI 2 mg per kg dry s0it Fig. 3. I n f l u e n c e o f d i f f e r e n t HgCl~ c o n c e n t r a t i o n s o n t h e larval h a t c h i n g r a t e (in %) in c o m p a r i s o n w i t h t h e c o n t r o l (C, w i t h o u t t r e a t m e n t ) ; *P < 0 . 0 5 ; m e a n values o f 5 replicates in b o t h cases.

(b) A. thalassinus Predominantly mercuric chloride affected the percentage of larvae hatched from the eggs (Fig. 3), so that their number was reduced in 1 mg Hg per kg dry soil and significantly so (P < 0.05) at 5 mg per kg and above. The females of this species were also unable to distinguish among the

181

different HgCI2 concentrations. A slight increase in the number o f egg pods was observed with increasing concentration of the test chemicals in the soil, but the number o f eggs per pod remained constant ( 8 - 1 1 ) . Larval mortality was increased at 0.605 mg HgCI~ per kg dry soil (cf. Schmidt, 1984).

(c ) O. caerulescens In species with a winter diapause of the eggs, it was possible to get only one test replicate per year. The results of the experiments from 1 9 8 0 to 1982 were similar to those o f the t w o other species: The females deposited numerous egg pods in all HgC12 concentrations and did not distinguish between them. Hatching of n

egg pods

I roll

20,

B eggs/pod

18, 16,

14.

12,

10'

6

4 **

C

0.0055

Q0275

0.055

0.23-0.275 Q46-057 2.75

.4

5.5

gN per kg d r y soil Fig. 4. Influence o f urea on the deposition o f egg pods and eggs per pod in A. patruelis, related to nitrogen content per kg dry sand; * * P < 0.01, significant; * * * P < 0.001,

highly significant from control (C); mean values o f 8 replicates.

182 larvae was decreased to less than 50% in 6.05 mg HgC12 per kg dry sand and in 10 times higher concentrations, no larvae hatched. The percentage of larvae developing into adults was decreased to less than 50% in concentrations of more than 0.12 mg HgC12 per kg.

Urea (H2N-CO-NH2) (Merck): A. patruelis Urea significantly (P < 0.05) reduced the deposition rate of oothecae (egg pods) and eggs in concentrations greater than 0.0275 g N per kg dry soil until finally only some e m p t y egg pods were produced (Fig. 4). Egg deposition was affected more than the production o f pods. The reduction o f the number of eggs and pods with increasing concentrations of urea was affected probably by ammonia which was liberated from the urea b y the enzyme urease in the soil. Ammonia may be perceptible by the females (Schmidt, 1983). The number of larvae hatched from the deposited eggs was less affected (Fig. 5) because of the fast decomposition of urea in the soil. Only treatment with high doses of urea (more than 0.2 g N per kg dry soil) prevented the development of the embryos. Besides the damage b y liberated ammonia, the egg shell may have been penetrated b y urea and the development stopped as occurred in grillid eggs (Hogan, 1961, 1962).

100

80 6O

2O

C

0.0055 0.0275 0.055 023-0275 0/,6-057 g N perkg dry soil

Fig. 5. Influence of urea on the hatching rate of larvae (in %) of A. patruelis in comparison to the control (C, w i t h o u t treatment); **P < 0.01; m e a n values o f 7 replicates.

Phenol: A. patruelis Phenol (99.5%, Riedel de Haen) was tested in 3 replicate experiments in different concentrations. The mean values are given in Table II. During a period of 14 days, there was no difference in egg deposition or in hatching of the embryos as larvae in treated and untreated soil. The

183

proportion of larvae developing into adults was reduced to about 50% with more than 6.05 mg phenol per kg dry sand. A further increase (more than 1.2 g kg -1) of the phenol concentration was not possible because of the toxicity of phenol vapour to the depositing females. Measurements of the UV absorption at 275 nm of the water extract o f the purified sand showed a high phenol content. -Under these experimental conditions, the present and added phenol was rapidly decomposed and after 14 days was not detectable even in the sand with the highest concentration added. The lack of effect of phenol on the embryos may be due to very fast decomposition in the soft. T A B L E II Average e f f e c t o f phenol on the deposition of egg pods and eggs in A. patruelb~; 3 series of experiments C o n c e n t r a t i o n per kg dry sand Control N u m b e r of egg pods Eggs p e r p o d

Percentage of h a t c h e d larvae N u m b e r of larvae used for experiments

(C)

0.121 mg

0.605 mg

1.210 mg

6.050 mg

12.10 mg

60.50 mg

0.121

0.605

1.210

g

g

g

4.3 14

3.5 10,5

2 26.5

5 11.5

3.5 17

4 14.5

6.6 19.5

6.3 16

3 12

6 17

78

61

77

66.5

75

79

93

79

86

98

61

38

49

33

39

48

108

106

31

97

Atrazin (2-Chloro-4-ethylamino-6-isopropylamino.l,3,5-triazin): A. patruelis Experiments (two replicates only) used different concentrations of atrazin in aqueous solutions although the solubility of atrazin is low. Thus, it was not possible to try percentages higher than about 2.5 mg per kg dry soft. Table III gives the results. T A B L E III Mean effect of atrazin on the egg deposition and embryonic development a f t e r l i q u i d a p p l i c a t i o n in 2 s e r i e s o f e x p e r i m e n t s Concentration Control

per kg dry sand (mg)

0.061

0.12

0.60

1.21

2.42

20 12

15 12

21 12

12 14

19 11

28 10

98

98

98

99

99

89

223

149

157

186

211

235

(c) Number of egg pods Eggs per pod Percentage of hatched larvae Number of larvae used for experiments

in A . p a t r u e l i s

184 TABLE IV Effect of different concentrations of atrazin on the deposition of egg pods, eggs and the embryonic development of A. patruelis after dry application I Concentration in dry sand mg kg -1 Control Experiment O

0.01 E/O %

O

0.05--0.061

0.1--0.12

E/O %

O

O

E/O %

9 12 9 6

11 13 17 25

9

17

E/O %

1

12

10

100

--

11

9

100

2

8

12

98

--

4

14

96

3 4

10 14

16 18

99 80

96 87

15 16 14 26

94 75

14

94.2

91.5

11 16

91.3

Mean

8.5

8 13 10 21 9

17

100 96

91 80 91.8

1The 4 experiments demonstrate the variation of the values; O: number of egg pods; E]O: eggs per pod; %: percentage of hatched larvae. I n w a t e r soluble c o n c e n t r a t i o n s , a t r a z i n did n o t i n f l u e n c e t h e d e p o s i t i o n o f eggs a n d o o t h e c a e o r e m b r y o n i c d e v e l o p m e n t , b u t t h e d e v e l o p m e n t o f larvae i n t o adults w a s g r e a t l y d e c r e a s e d ; 13% o f t h e larvae d e v e l o p e d in t r e a t e d sand i n d e p e n d e n t o f t h e c o n c e n t r a t i o n a n d 60% in t h e c o n t r o l . F o r t e s t i n g higher c o n c e n t r a t i o n s o f a t r a z i n , d r y c h e m i c a l was m i x e d w i t h d r y s a n d a n d m o i s t e n e d . This a l l o w e d c o n c e n t r a t i o n s u p t o 10 m g p e r k g d r y s a n d t o b e t e s t e d ( T a b l e I V ) . T h e r e was still n o i n f l u e n c e o n egg d e p o s i t i o n a n d e m b r y o n i c d e v e l o p m e n t , b u t larval d e v e l o p m e n t w a s dec r e a s e d ( 1 6 % in t r e a t e d sand, 62% in t h e c o n t r o l ) .

Perylene (C2oH12, cyclic hydrocarbon): A. p a t r u e l i s T w o r e p l i c a t e e x p e r i m e n t s w e r e carried o u t w i t h d i f f e r e n t w a t e r s o l u b l e c o n c e n t r a t i o n s . T a b l e V gives t h e results. T h e s o l u b i l i t y o f p e r y l e n e in w a t e r is v e r y low. T h e c o n c e n t r a t i o n s t e s t e d did n o t i n f l u e n c e t h e r a t e o f r e p r o d u c t i o n . O n l y t h e larval d e v e l o p m e n t w a s r e d u c e d t o a b o u t 50%.

DDD (Dichlorodiphenyl dichloro ethane): A. p a t r u e l i s T w o r e p l i c a t e e x p e r i m e n t s d e m o n s t r a t e d n o e f f e c t o f D D D in w a t e r soluble c o n c e n t r a t i o n s o n t h e r a t e o f r e p r o d u c t i o n ( T a b l e VI), b u t o n l y 2 0 % o f t h e larvae f r o m D D D - t r e a t e d sand d e v e l o p e d t o a d u l t s c o m p a r e d t o 45% f r o m u n t r e a t e d sand.

185

0.5--0.6

1.0--1.21

2.0--2.42

5

O

E/O

%

O

E/O

%

O

E/O

%

4 8 10 8

9 19 16 16

100 99 93 93

9 10 8 14

9 13 16 21

100 99 100 90

12 16 3 9

12 8 14 17

88 89 85 80

8

15

10

15

10

13

85.5

94.5

97.3

10

O

E/O

%

15 19

9 14

--93 69

17

12

91.5

O

E/O

%

16 10

10 15

--97 80

13

13

93.4

TABLE V Effect of perylene dissolved in water on the egg deposition and embryonic development in A. patruelis (mean of 2 replicates)

Concentration per kg dry sand (mg) Control

Number of egg pods Eggs per pod Percentage of hatched larvae Number o f larvae used for experiments

0.012

0.33

0.061

13 16

18 14

13 15

12 13

93

92

96

94

175

224

187

143

TABLE VI Average effect of DDD dissolved in water on egg deposition and embryonic development in A. patruelis (2 replicates) Concentration per kg dry sand (mg) Control Number of egg pods Eggs per pod Percentage of hatched larvae Number o f larvae used for experiments

0.012

0.03

0.061

15 11

20 12

12 13

16 15

98

98

98

98

159

232

144

234

Methanol and ethyl acetate: A . p a t r u e l i s B o t h c h e m i c a l s are v e r y s o l u b l e in w a t e r . T w o r e p l i c a t e e x p e r i m e n t s w e r e c a r r i e d o u t w i t h c o n c e n t r a t i o n s o f 0 . 0 0 0 1 - - 0 . 6 m l p e r k g so i l . N o n e

186

o f the concentrations of chemicals affected embryogenesis or oviposition. Adult development was reduced by methanol to 47% and b y ethyl acetate to 33% in comparison with control values o f 54 and 66%, respectively. DISCUSSION AND E V A L U A T I O N OF THE BIOTEST

The results demonstrated the possibility of grasshoppers for evaluating chemicals in the soil in laboratory and simulated field conditions. A. patruelis can be used for the biotest as well as A. thalassinus; b o t h are similarly sensitive to mercury and can be bred throughout the whole year. A. thalassinus is a little bigger and more robust than A. patruelis and will oviposit in a wider range of soil types. Thus, different types of soil could be tested. The only problem is that the normal rate for larvae to develop into adults varied from 45 to 65% depending on the season, spring giving the highest percentage and autumn the lowest. Therefore, it is necessary to carry o u t testing with the same generation of the test insect. In the laboratory tests, the treatment b y chemicals lasted 3--4 weeks b y using continuously breeding species, whereas in overwintering experiments (with species distributed in Central Europe) the egg pods remained in contact with the chemicals 4--5 months at temperatures near to freezing point. Therefore, a direct comparison of the species with and w i t h o u t egg diapause is more difficult, b u t mercury chloride evidently had the same effect on the embryogenesis of diapausing and non~liapausing species. The mortality of the larvae was significantly greater in diapausing species and it seems that mercury influences the postembryonic development over several months. Similar results were obtained in the other diapausing species G. brunneus. For mercuric chloride, the limiting concentration is a b o u t 1.0 mg per kg d r y sand for species that breed continuously; for species with egg winter diapause the limiting concentration is 0.1 mg per kg. Kloke (1977) reported that there is often 0.1--1 mg mercury per kg of air dry soil and recommended 5 mg mercury per kg as a tolerable limit. The present investigations demonstrate that such high concentrations of mercury are highly toxic to acridids and it is n o t surprising that acridids have almost died o u t in areas where mercury is used agriculturally. The toxic effect of urea and other sources of ammonia used as fertilizers can be partly avoided b y the insects themselves. They can protect their offspring against injury if t h e y can select untreated places in the fields. Experiments carried o u t with the diapausing species O. caerulescens and G. biguttulus with different urea concentrations led to similar results. The liberated ammonia damaged the acridid e m b r y o in almost all these experiments. The fast decomposition of urea and ammonia carbonate in the experimental sand explains the lack of differences in tolerance to urea in species with and w i t h o u t egg diapause. This presupposes that the chemical is applied only once during the life cycle of the species, b u t in practice, in areas with

187

high application rates, eggs and e m b r y o s of insects are poisoned b y urea as well as ammonia salts used as fertilizers. When fertilizers are mainly used in springtime, practically all species of grasshoppers are affected in Central Europe because of the hatching of the larvae in May. In meadows and pasture-land, which are fertilized every year, almost no acridid can be observed. All the other chemicals tested had a latent effect in water soluble concentrations, so that t h e y affected the p o s t e m b r y o n i c stages. Normally a b o u t 5 0 - ~ 0 % of the larvae developed into adults, b u t with treatment, the percentage was reduced to as much as a quarter of that, although this d e p e n d e d on the persistence of the chemicals. F o r further evaluation of the toxic effects, investigations are necessary on a wider basis. It is well k n o w n that prolonged treatment leads to an accumulation of heavy metals and highly stable chemicals in plants and insects (Smith, 1980). Thus, if crop plants take up heavy metals and accumulate them at rates similar to those of the insects here, then the biotest which has been described will prove useful in the measurement of potentially toxic chemicals in the soil. ACKNOWLEDGEMENT

Supported b y grants of the Ministry o f Research and Technology of the Federal Republic of Germany, No. 037214.

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