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Development and standardization of test methods for the prediction of sublethal effects of chemicals on earthworms
Soil Biol. Biochem. Vol. 29, No. 3/4. pp. 635-639.
1997 1997 Elsevier Scmce Ltd in Great Britain. All rights reserved 0038-07 17/97 %I 7.00 + 0.00 0
Pergamon
Printed
PII: S0038-0717(%)00030-2
DEVELOPMENT AND STANDARDIZATION OF TEST METHODS FOR THE PREDICTION OF SUBLETHAL EFFECTS OF CHEMICALS ON EARTHWORMS HARTMUT
KULA* and OTTO LARINK
Zoologisches Institut, TU Braunschweig, Pockelsstrasse IOa, D-38092 Braunschweig, Germany (Accepted 7 Febuary 1996)
Summary-The main purpose of the European cooperative research project SECOFASE (“Development, improvement and standardization of test systems for assessing sublethal effects of chemicals on fauna of the soil ecosystem”) is to develop test systems for the early detection and evaluation of sublethal effects of chemicals on the main soil living invertebrate groups. The effects of dimethoate (insecticide), copper (heavy metal) and linear alkylbenzene sulfonate (anionic detergent) as model chemicals are investigated in two standard test soils, the OECD artificial soil and the LUFA 2.2 soil. We present preliminary results of the sub-project “Lumbricidae”. Acute and sublethal effects of dimethoate and copper on the standard test organism Eisenia fetidu as well as Eisenia andrei were investigated in order to select suitable test conditions. Cocoon production and numbers of juveniles were the most sensitive sublethal factors whereas sensitivity of body weight development varied. Acute effects of dimethoate to Eiseniu fetida, Aporrectodeu caliginosa, A. rosea and Allolobophora chlorotica showed only small differences within one order of magnitude. In a characterization of dimethoate degradation by residue analysis in both soils a faster disappearance of dimethoate was observed in LUFA 2.2 soil. 0 1997 Elsevier Science Ltd
tide), copper (heavy metal) and linear alkylbenzene sulfonate (anionic detergent). Our paper presents preliminary results of the sub-project ‘Lumbricidae’. Acute and sublethal effects of dimethoate and copper were investigated in order to select suitable conditions for the development of the test system. Most of these experiments were conducted with Eiseniu fetidu, the standard organism for ecotoxicological testing with earthworms. The degradation behaviour of dimethoate in the two different soils was characterized by a residue analysis.
INTRODUCTION There is an urgent
need for the development
of test
procedures for assessing the ecological effects of chemicals in the terrestrial environment. The main purpose of the European cooperative research project SECOFASE (“Development, improvement and standardization of test systems for assessing sublethal effects of chemicals on fauna of the soil ecosystem”, 1993-1996) is to develop test systems for the early detection and evaluation of sublethal effects of chemicals on the main soil living invertebrate groups (Lokke and Van Gestel, 1993; Wiles et al., 1994). The following taxa are investigated in ten sub-projects: Lumbricidae, Enchytraeidae, Nematoda, Collembola, Oribatida, Gamasida, Isopoda, Diplopoda, Chilopoda and Staphylinidae. The test methods will be based on common principles for test conditions and endpoints for sublethal effects. As a result a test battery for soil fauna species representative of European soil ecosystems will be developed which can be used especially for regulatory purposes. OECD artificial soil (OECD, 1984) and LUFA Speyer 2.2 soil were chosen as standard test soils. Three model test chemicals representing different chemical classes are compared: Dimethoate (insecti-
MATERIALS AND METHODS Eisenia fetida, A. rosea
E. andrei,
Aporrectodea
caliginosa,
and Allolobophora chlorotica were used as test animals. Eiseniu spp. were laboratory bred, all other test animals were hand collected from an uncontaminated orchard. An important difference between the two test soils is the organic C content. The OECD artificial soil (OECD, 1984) consists of 70% quartz sand, 20% kaolin clay, 10% sphagnum peat and calcium carbonate to adjust the pH to 6.0 + 0.5. The organic C content is about 5.8%. The LUFA 2.2 soil is a commercially-available (LUFA Speyer, Germany) natural sandy soil with a pH of 5.8 and an organic C content of 2.3%.
*Author for correspondence. 635
636
Hartmut Kula and Otto Larink
Acute toxicity test The acute toxicity was investigated in order to determine appropriate concentrations for the testing of sublethal effects. The acute toxicity tests were conducted according to OECD guideline no. 207 (OECD, 1984). In addition, body weight development, occurrence of external injuries and general behaviour, such as burrowing, were recorded. In tests with the indigenous species, a reduced number of test animals (l-4) were used per replicate. LC,,, was calculated with the trimmed SpearmanKarber method (Hamilton et al., 1977). Reproduction toxicit? test
)
Sublethal effects were investigated in reproduction toxicity tests (C. A. M. Van Gestel, unpubl. Ph.D. thesis, University of Utrecht, 1991; H. Kula, unpubl. Ph.D. thesis, University of Braunschweig, 1994). Each concentration had 4 replicates. The test substance was mixed homogeneously with 500 g (dry wt) of soil ( = total soil contamination). Soil moisture was adjusted to 50% of the water holding capacity. The soil was put into 1 1 plastic boxes which were closed with a transparent plastic lid with small holes for ventilation. Ten (E. fetidu) or 4 (A. caliginosa) test animals were introduced into the soil substrate. Finely ground cattle manure was spread on the soil surface as food source. Each week the feeding activity was checked visually by a semiquantitative estimation of the food residues (feeding activity 0: none, 1: low, 2: medium, 3: high). Food was renewed weekly. After 28 days exposure mortality, body weight development, cocoon production and numbers of hatched juveniles were determined. The cocoons were transfered to Petridishes containing moist filter paper. The total number of hatching juveniles as well as the number of infertile cocoons were evalutated during the following weeks. In a modified test design (BBA, 1994) dimethoate was not mixed into the soil but applied superficially (= soil surface contamination) in order to investigate the effect of different type of exposure. Numbers of cocoons were not estimated with this test design. Only the number of surviving juveniles after 8 weeks of exposure was determined. A reproduction toxicity test with E. jktidu and dimethoate was combined with a residue analysis (in cooperation with U. Heimbach, K. Metge and J. Siebers, Biologische Bundesanstalt fiir Land- und Forstwirtschaft, Braunschweig). The initial concentration of dimethoate was 26.9 mg a.i. kg-’ dry wt of soil. Test animals were introduced 1, 4 and 8 days after incorporation of the chemical into the soil substrate. Samples for residue analysis were taken from test boxes where test animals had been introduced 1 day after contamination. Waterextractable and water and acetone-extractable residues were determined using standard gas chromatography-techniques (DFG, 1991).
Table I. Acute toxicity of dimethoate (LC,) Artificial soil (mg a.i. kg E.
LUFA 2.2 soil
’dry wt test substrate)
fertda
adult Juvenile
207 142
98 90
179 IO”’
47 32”
A. caliginosa
adult - juvenile A. chlorottca - adult
191
316
89
13
A. rOSea - adult
* Preliminary results from range finding test.
Analysis of variance (ANOVA) was applied to the results. Significant differences (P I 0.05) between control and treatments were determined by Tukey’s multiple t-test.
RESULTS
Acute toxicity test Results of the acute toxicity tests are shown in Table 1. In general it was difficult to estimate the number of dead animals in tests with dimethoate as this chemical paralysed the test animals even at very low concentrations (10-32 mg kg- ‘). The animals responded very slowly to a mechanical stimulus. The body appeared contracted and rigid. According to the guideline these test animals have to be registered as alive. Test animals showing these symptoms when left in the soil substrate often died within 1-2 weeks after the end of the test. Reproduction toxicity test Differences in the amount of dimethoate residues between the two soils were observed in the reproduction test combined with the residue analysis. After 22 days residues in LUFA 2.2 soil were about half those in the in artificial soil (Fig. 1). In total, 31-39% of the initial concentration were found in
Fig. 1. Dimethoate residue analysis in artificial soil and LUFA 2.2 soil Initial concentration at day 0: 26.9 mg a.i. kg-’ dry wt soil substrate, all replicates with IO adult E. fetida, 4 replicates per treatment, WAE = water/acetone extraction, WE = water extraction.
637
The prediction of sublethal effects of chemicals on earthworms Table 2. Acute and sublethal effects of dimethoate on Mortality WI Art. soil Control
Body weight development
I%1
Eisenia
fefida
in two soils with different concentrations
Feeding rate
Number of cocoons 51.4 * 6.6
0
120.7 f 7.0
2.8
Di (day I)$ Di (day 4) Di (day 8) LUFA 2.2 Control
4 0 3
105.0 * 14.9 113.5 f 1.3 99.2 + 16.4
1.6 2.0 2.4
2
124.1 _+8.6
2.9
Di (day 1) Di (day 4) Di (day 8)
14 3 13
93.1 + 16.3’ 116.8 + 12.4 95.0 f 13.7*
1.9 2.3 2.2
I .2 + 2.7* 6.8 + 4.9’ 7.3 + 4.3* 23.4 _+7.2 0’ 2.0 + 2.4’ 3.0 k 0.8*
Infertile cocoons
I”hl
Number of juveniles
2
187.8 + 24.7
50 7 3
2.4 f 5.4* 19.0 + 19.8* 25.0 f 15.3’
27
54.0 _+24.6
0 50 8
0* 4.3 & 8.5: 6.3 f 1.3’
:Di = treated with 26.9 mg a.i. dimethoate kg-’ at day 0 (test animals introduced at days I, 4 and 8); ‘Feeding rate: semiquantitative rating 0,1,2,3. Mean of 4 replicates; * P < 0.05, multiple f-test by Tukey.
artificial soil, compared to 15-24% in LUFA 2.2 soil. The difference between water-extractable and water and acetone-extractable residues was constant in artificial soil with lO-15%. For LUFA 2.2 soil similar results were obtained only for the last three sampling dates. The residue analysis was conducted with a rather high dimethoate concentration of 26.9 mg a.i. kg- ’to ensure the occurrence of effects. This also explains the mortality observed in some treatments (Table 2). In both soils cocoon production was reduced almost completely when test animals were introduced 1 day after incorporation of the chemical. In test animals introduced on days 4 and 8, reduction still was about 90%. Cocoon production of control animals in LUFA 2.2 soil was only 50% that of artificial soil. In addition, the percentage of infertile cocoons was very high in LUFA soil 2.2. Significant effects on body weight development were observed only in LUFA 2.2 soil. A reduction of the feeding rate at the beginning of the test was observed in all treatments in both soils. During the last 10 days of the test treated animals restarted feeding. The effects of a different exposure of dimethoate was investigated in artificial soil at concentrations corresponding to the NOEC. E. fetidu was compared to E. undrei. Regarding numbers of juveniles total
soil contamination and soil surface contamination showed similar results for E. fetidu (Table 3). Significant effects on body weight development were observed with total soil contamination only. E. andrei was slightly more sensitive to total soil contamination than E. fetidu (Table 4). Significant effects on body weight development were again observed only with total soil contamination. Sensitivity differences between both species were also registered in total soil contamination tests with copper. With E. fetidu similar results were obtained in both soils (Table 5) whereas E. undrei was very susceptible in LUFA 2.2 soil (Table 6). In artificial soil E. undrei showed a slight, but not significant increase in numbers of juveniles at 3.2, 10 and 32 mg kg- ‘. Significant reductions occurred only at 320 mg kg ‘. In contrast to this, numbers of juveniles were significantly reduced at 3.2 mg kg- ’in LUFA 2.2 soil (Table 6). Body weight development was not influenced in both species even at the highest concentration tested. Results of most of the experiments with LUFA 2.2 soil indicated suboptimal conditions for the test animals. Cocoon production and hatching success generally were lower than in the artificial soil. Therefore the effect of soil moisture on body weight development and reproduction of E. fetidu and A.
Table 3. Sublethal effects of dimethoate on Eisenia firida in artificial soil. No test animal died Dimethoate (mg kg - ‘) Control Total contamination 2.5 5.0 1.5 Surface contamination 2.5 5.0 7.5 Mean of 4 replicates;
??
Body weight development WI
Number of cocoons
Number of juveniles
99.5 f 10.5
44.0 + 8.3
189.0 + 36.7
94.5 * 7.4 96.3 f 1.9 82.5 f 2.4*
49.5 * 7.3 32.5 * 3.1 18.3 k 4.7’
177.0 f 47.0 124.3 + 21.2 67.3 f 28.7’
99.3 * 10.0 101.3 * 5.7 97.5 f 4.9
ND ND ND
147.5 f 47.9 121.5 f 35.9 95.0 k 12.9’
P 5 0.05, multiple r-test by Tukey. ND = not determined
638
Hartmut Table 4. Sublethal Dimethoate
(mg kg
and Otto
Body weight development [“hl
‘)
Number
soil. No test animal
of cocoons
Number
died of juveniles
133.8 _+ 2.9
16.5 + 3.0
39.8 * 8.0
134.0 + 7.3 114.5 f 4.2’ 83.3 f 9.3*
16.3 k 5.1 3.5 + 3.0’ 0.3 + 0.5’
38.3 + 15.1 7.8 + 8.4* 0*
135.8 f 12.8 127.5 ? 3.8 114.8 * 10.2
ND ND ND
58.8 + 14.7 29.3 ? 16.0 8.5 + 11.1’
* P 2 0.05. multiple
Table 5. Sublethal
Larink
on Eisenia andrei m artificial
effects of dimethoate
Control Total contamination 2.5 5.0 7.5 Surface contamination 2.5 5.0 7.5 Mean of 4 replicates;
Kula
I-test by Tukey. ND = not determined
effects of copper on Eisenia /&da.
No test animal
died
Body weight development
Ku1 (mg kg ‘)
Number
[%I
Artificial soil Control 3.2 IO 32 IO0 320 LUFA 2.2 soil Control 3.2 IO 32 100 320 Mean of 4 replicates;
* P
of cocoons
Number
of juveniles
141.2 137.4 146.0 147.1 144.7 148.2
+ k i f f f
4.4 8.0 5.9 5.0 8.0 7.4
34.5 29.5 32.8 25.0 26.3 3.8
+ i + k f f
4. I 4.2 4.8 4.2* I.O* I .3*
97.3 75.8 97.0 80.0 49.5 7.5
+ f + f + +
145.9 145.3 136.8 144.5 148.1 135.1
_t f i f + :
4.6 I.7 4.3 4.8 8.8 4.2
10.3 6.0 8.8 4.8 4.3 0.5
_+ 2.4 + 0.8 f 3.4 _+ 2.l* + 3.0* z 1.0”
5.3 3.5 2.8 I.8 0.3
f f i f +
< 0.05, multiple
Table 6. Sublethal
I I.5 17.7 16.2 12.9 18.1* I.31
0.9 2.6 1.7 2.9 0.5* I .ort 2.0*
r-test by Tukey.
effects of comxr
on Eisenio andrei. No test animal
died
Body weight development Number
[%I Artificial soil Co”ttol 3.2 IO 32 100 320 LUFA 2.2 soil Control 3.2 IO 32 100 320 Mean of 4 replicates;
I.6 3.8 8.9 I .7
3.9 1.3 4.8 4.0 6.0 6.7*
120.0 144.0 142.8 140.0 120.5 46.5
f f f f f f
+ 6.3 _+ 7.3 + 7.4 rt 9.7 * 5.9 f 7.7
49.5 41 .o 34.3 37.0 17.8 2.8
f + + + + i
7. I 5.9 7.3’ 4.6’ 3.6* 2.2*
155.3 114.3 105.8 107.0 49.8 5.5
+ 9.0 + 24.3* + 26.2’ _+ 13.9’ + 8.8* f 4.5’
* P < 0.05, multiple
Table 7. Effects of soil moisture
52 57 61 66
i * f +
12.7 19.2 24.3 28.5 15.6 17.9’
I-test by Tukey.
numbers of juveniles were highest with the highest soil moisture content. Test animals of A. cafiginosa (Table 8) increased slightly in body weight in the wetter soil, but differences were not significant. Due to the inactivity of the test animals nearly no cocoons were produced.
on body weight development
Body weight development [“/I 73.0 74.5 78.4 75.8
of juveniles
f * * f k f
I.8
caliginosa was investigated in LUFA 2.2 soil. Water content was adjusted between 23% and 29% of dry wt of soil, corresponding to a water holding capacity between 52% and 66%. In E. fetida (Table 7) no differences were found in body weight development. Cocoon production and
Soil moisture [%I of WHC
Number
40.3 41.3 43.8 40.5 38.8 21.3
113.5 + lll.o*6.6 108.3 f 108.4 i 112.2 + 114.6 + 107.0 105.8 108.3 106.2 116.8 119.9
of cocoons
Number
I.4 6.6 0.4 2.3
Mean of 4 replicates; * P 5 0.05, multiple r-test by Tukey ‘Feeding rate: semiquantitative rating 0.1.2.3.
and reproduction
of cocoons
44.oio 56.0 f 2.8* 45.5 i 0.7 65.5 f 0.7*
of E./&da
Number 187.5 251.0 208.0 289.5
of juveniles + + + t
0.7 7.1’ 2.8’ 2. I *
in LUFA
2.2 soil Feeding I 2 3 3
rate’
The prediction of sublethal effects of chemicals on earthworms Table 8. Effects of soil moisture on body weight development and reproduction of A. Soil moisture [%I of WHC 52 51 61 66
Body weight development [%I 91.3 f 98.3 f 100.5 + 109.3 -+
coli&xa
639 in
Number of cocoons
Animals in resting phase [%I
0.5 * 0.7 1.5 &-0.7 0.5 * 0.7 0
30 50 30 0
4.3 5.8 2.8 16.1
LUFA 2.2 soil Feeding rate 0.5 0.5
I I
Mean of 4 replicates; * P < 0.05, multiple l-test by Tukey. ‘Feeding rate: semiquantitative rating 0.1.2.3.
DISCUSSION
Sensitivity differences between E. fetida and the endogeic mineral soil species A. caliginosa, A. rosea and A. chlorotica were small and within one order of magnitude. This is in accordance with Heimbach (1985) and Van Gestel (lot. cit.). In most cases E. fetida is reported to be the least susceptible species. The LC, in LUFA 2.2 soil usually was lower than in artificial soil. This could be explained by a higher bioavailability of the pesticide due to the lower organic C content of LUFA 2.2 soil. The faster disappearance of dimethoate from LUFA 2.2 soil could be explained by a higher microbial activity in contrast to the artificial soil. The differences in microbial activity will be investigated in further experiments. Concerning sublethal effects cocoon production and numbers of juveniles were the most sensitive biological response. Body weight development was a rather insensitive response, especially when testing copper. With dimethoate, however, sensitivity was equal to cocoon production and numbers of juveniles. In order to recognize possible sublethal effects which might have an influence on the ecological fitness of the test species more than one biological response should therefore be evaluated. Dimethoate and copper are suitable model test chemicals. Dimethoate shows a considerable acute toxicity especially to the indigenous earthworm species while sublethal effects occur at low concentrations. Copper in contrast has a moderate acute toxicity, but sublethal effects can also be observed at low concentrations. Acute and sublethal effects of the third test chemical (LAS) still have to be investigated. A data base on species specific sensitivity of adult and juvenile life stages of earthworm species belonging to different ecological categories will be elaborated.
Optimum conditions for the use of LUFA 2.2 soil will be investigated in order to ensure the quality criteria set up in the SECOFASE work manual (Lokke and Van Gestel, 1993).
Acknowledgements-The
funding for this work
provided by the EU Programme Environment (contract no. ERB-EVSV-CT92-0218).
was 199&1994
REFERENCES
BBA (1994) Richtlinien Bir die Zulassung von Pflanzenschutzmitteln im Zulassungsverfahren; Teil VI, 2-2; Auswirkungen von Pganzenschutzmitteln auf die Reproduktion und das Wachstum von Eisenia fetida/Eisenia andrei. Saphir-Verlag, Ribbesbiittel. DFG (1991) Methodensammhmg zur Rtickstandsanalytik von PBanzenschutzmitteln der Deutschen Forschungsgemeischaft: Bestimmung von Omethoat und Dimethoat 236-(42)-l. VCH, Stuttgart. Hamilton M. A. , Russo R. C. and Thurston R. V. (1977) Trimmed Spearman-Karber method for estimating median lethal concentrations in toxicity bioassays. Environmental Science and Technology 11, 714-719. Heimbach F. (1985) Comparison of laboratory methods using Eisenia foetida and Lumbricus terrestris, for the assessment of the hazard of chemicals to earthworms. Zeitschrift fdr Pflanzenkrankheiten 186193.
und Pflanzenschutz
92,
Lokke H. and Van Gestel C. A. M. (1993) Manual of SECOFASE. Development, improvement and standardization of test systems for assessing sublethal effects of chemicals on fauna of the soil ecosystem. National Environmental Research Institute, Denmark. OECD (1984) Guideline for testing of chemicals no. 207. Earthworm, acute toxicity tests. Adopted 4 April 1984. Wiles J. A., Kammenga J. E. and Lokke H. (1994) Progress Report 1993 of SECOFASE, Second Technical Report. Development, improvement and standardization of test systems for assessing sublethal effects of chemicals on fauna of the soil ecosystem. National Environmental Research Institute, Denmark.
1997 1997 Elsevier Scmce Ltd in Great Britain. All rights reserved 0038-07 17/97 %I 7.00 + 0.00 0
Pergamon
Printed
PII: S0038-0717(%)00030-2
DEVELOPMENT AND STANDARDIZATION OF TEST METHODS FOR THE PREDICTION OF SUBLETHAL EFFECTS OF CHEMICALS ON EARTHWORMS HARTMUT
KULA* and OTTO LARINK
Zoologisches Institut, TU Braunschweig, Pockelsstrasse IOa, D-38092 Braunschweig, Germany (Accepted 7 Febuary 1996)
Summary-The main purpose of the European cooperative research project SECOFASE (“Development, improvement and standardization of test systems for assessing sublethal effects of chemicals on fauna of the soil ecosystem”) is to develop test systems for the early detection and evaluation of sublethal effects of chemicals on the main soil living invertebrate groups. The effects of dimethoate (insecticide), copper (heavy metal) and linear alkylbenzene sulfonate (anionic detergent) as model chemicals are investigated in two standard test soils, the OECD artificial soil and the LUFA 2.2 soil. We present preliminary results of the sub-project “Lumbricidae”. Acute and sublethal effects of dimethoate and copper on the standard test organism Eisenia fetidu as well as Eisenia andrei were investigated in order to select suitable test conditions. Cocoon production and numbers of juveniles were the most sensitive sublethal factors whereas sensitivity of body weight development varied. Acute effects of dimethoate to Eiseniu fetida, Aporrectodeu caliginosa, A. rosea and Allolobophora chlorotica showed only small differences within one order of magnitude. In a characterization of dimethoate degradation by residue analysis in both soils a faster disappearance of dimethoate was observed in LUFA 2.2 soil. 0 1997 Elsevier Science Ltd
tide), copper (heavy metal) and linear alkylbenzene sulfonate (anionic detergent). Our paper presents preliminary results of the sub-project ‘Lumbricidae’. Acute and sublethal effects of dimethoate and copper were investigated in order to select suitable conditions for the development of the test system. Most of these experiments were conducted with Eiseniu fetidu, the standard organism for ecotoxicological testing with earthworms. The degradation behaviour of dimethoate in the two different soils was characterized by a residue analysis.
INTRODUCTION There is an urgent
need for the development
of test
procedures for assessing the ecological effects of chemicals in the terrestrial environment. The main purpose of the European cooperative research project SECOFASE (“Development, improvement and standardization of test systems for assessing sublethal effects of chemicals on fauna of the soil ecosystem”, 1993-1996) is to develop test systems for the early detection and evaluation of sublethal effects of chemicals on the main soil living invertebrate groups (Lokke and Van Gestel, 1993; Wiles et al., 1994). The following taxa are investigated in ten sub-projects: Lumbricidae, Enchytraeidae, Nematoda, Collembola, Oribatida, Gamasida, Isopoda, Diplopoda, Chilopoda and Staphylinidae. The test methods will be based on common principles for test conditions and endpoints for sublethal effects. As a result a test battery for soil fauna species representative of European soil ecosystems will be developed which can be used especially for regulatory purposes. OECD artificial soil (OECD, 1984) and LUFA Speyer 2.2 soil were chosen as standard test soils. Three model test chemicals representing different chemical classes are compared: Dimethoate (insecti-
MATERIALS AND METHODS Eisenia fetida, A. rosea
E. andrei,
Aporrectodea
caliginosa,
and Allolobophora chlorotica were used as test animals. Eiseniu spp. were laboratory bred, all other test animals were hand collected from an uncontaminated orchard. An important difference between the two test soils is the organic C content. The OECD artificial soil (OECD, 1984) consists of 70% quartz sand, 20% kaolin clay, 10% sphagnum peat and calcium carbonate to adjust the pH to 6.0 + 0.5. The organic C content is about 5.8%. The LUFA 2.2 soil is a commercially-available (LUFA Speyer, Germany) natural sandy soil with a pH of 5.8 and an organic C content of 2.3%.
*Author for correspondence. 635
636
Hartmut Kula and Otto Larink
Acute toxicity test The acute toxicity was investigated in order to determine appropriate concentrations for the testing of sublethal effects. The acute toxicity tests were conducted according to OECD guideline no. 207 (OECD, 1984). In addition, body weight development, occurrence of external injuries and general behaviour, such as burrowing, were recorded. In tests with the indigenous species, a reduced number of test animals (l-4) were used per replicate. LC,,, was calculated with the trimmed SpearmanKarber method (Hamilton et al., 1977). Reproduction toxicit? test
)
Sublethal effects were investigated in reproduction toxicity tests (C. A. M. Van Gestel, unpubl. Ph.D. thesis, University of Utrecht, 1991; H. Kula, unpubl. Ph.D. thesis, University of Braunschweig, 1994). Each concentration had 4 replicates. The test substance was mixed homogeneously with 500 g (dry wt) of soil ( = total soil contamination). Soil moisture was adjusted to 50% of the water holding capacity. The soil was put into 1 1 plastic boxes which were closed with a transparent plastic lid with small holes for ventilation. Ten (E. fetidu) or 4 (A. caliginosa) test animals were introduced into the soil substrate. Finely ground cattle manure was spread on the soil surface as food source. Each week the feeding activity was checked visually by a semiquantitative estimation of the food residues (feeding activity 0: none, 1: low, 2: medium, 3: high). Food was renewed weekly. After 28 days exposure mortality, body weight development, cocoon production and numbers of hatched juveniles were determined. The cocoons were transfered to Petridishes containing moist filter paper. The total number of hatching juveniles as well as the number of infertile cocoons were evalutated during the following weeks. In a modified test design (BBA, 1994) dimethoate was not mixed into the soil but applied superficially (= soil surface contamination) in order to investigate the effect of different type of exposure. Numbers of cocoons were not estimated with this test design. Only the number of surviving juveniles after 8 weeks of exposure was determined. A reproduction toxicity test with E. jktidu and dimethoate was combined with a residue analysis (in cooperation with U. Heimbach, K. Metge and J. Siebers, Biologische Bundesanstalt fiir Land- und Forstwirtschaft, Braunschweig). The initial concentration of dimethoate was 26.9 mg a.i. kg-’ dry wt of soil. Test animals were introduced 1, 4 and 8 days after incorporation of the chemical into the soil substrate. Samples for residue analysis were taken from test boxes where test animals had been introduced 1 day after contamination. Waterextractable and water and acetone-extractable residues were determined using standard gas chromatography-techniques (DFG, 1991).
Table I. Acute toxicity of dimethoate (LC,) Artificial soil (mg a.i. kg E.
LUFA 2.2 soil
’dry wt test substrate)
fertda
adult Juvenile
207 142
98 90
179 IO”’
47 32”
A. caliginosa
adult - juvenile A. chlorottca - adult
191
316
89
13
A. rOSea - adult
* Preliminary results from range finding test.
Analysis of variance (ANOVA) was applied to the results. Significant differences (P I 0.05) between control and treatments were determined by Tukey’s multiple t-test.
RESULTS
Acute toxicity test Results of the acute toxicity tests are shown in Table 1. In general it was difficult to estimate the number of dead animals in tests with dimethoate as this chemical paralysed the test animals even at very low concentrations (10-32 mg kg- ‘). The animals responded very slowly to a mechanical stimulus. The body appeared contracted and rigid. According to the guideline these test animals have to be registered as alive. Test animals showing these symptoms when left in the soil substrate often died within 1-2 weeks after the end of the test. Reproduction toxicity test Differences in the amount of dimethoate residues between the two soils were observed in the reproduction test combined with the residue analysis. After 22 days residues in LUFA 2.2 soil were about half those in the in artificial soil (Fig. 1). In total, 31-39% of the initial concentration were found in
Fig. 1. Dimethoate residue analysis in artificial soil and LUFA 2.2 soil Initial concentration at day 0: 26.9 mg a.i. kg-’ dry wt soil substrate, all replicates with IO adult E. fetida, 4 replicates per treatment, WAE = water/acetone extraction, WE = water extraction.
637
The prediction of sublethal effects of chemicals on earthworms Table 2. Acute and sublethal effects of dimethoate on Mortality WI Art. soil Control
Body weight development
I%1
Eisenia
fefida
in two soils with different concentrations
Feeding rate
Number of cocoons 51.4 * 6.6
0
120.7 f 7.0
2.8
Di (day I)$ Di (day 4) Di (day 8) LUFA 2.2 Control
4 0 3
105.0 * 14.9 113.5 f 1.3 99.2 + 16.4
1.6 2.0 2.4
2
124.1 _+8.6
2.9
Di (day 1) Di (day 4) Di (day 8)
14 3 13
93.1 + 16.3’ 116.8 + 12.4 95.0 f 13.7*
1.9 2.3 2.2
I .2 + 2.7* 6.8 + 4.9’ 7.3 + 4.3* 23.4 _+7.2 0’ 2.0 + 2.4’ 3.0 k 0.8*
Infertile cocoons
I”hl
Number of juveniles
2
187.8 + 24.7
50 7 3
2.4 f 5.4* 19.0 + 19.8* 25.0 f 15.3’
27
54.0 _+24.6
0 50 8
0* 4.3 & 8.5: 6.3 f 1.3’
:Di = treated with 26.9 mg a.i. dimethoate kg-’ at day 0 (test animals introduced at days I, 4 and 8); ‘Feeding rate: semiquantitative rating 0,1,2,3. Mean of 4 replicates; * P < 0.05, multiple f-test by Tukey.
artificial soil, compared to 15-24% in LUFA 2.2 soil. The difference between water-extractable and water and acetone-extractable residues was constant in artificial soil with lO-15%. For LUFA 2.2 soil similar results were obtained only for the last three sampling dates. The residue analysis was conducted with a rather high dimethoate concentration of 26.9 mg a.i. kg- ’to ensure the occurrence of effects. This also explains the mortality observed in some treatments (Table 2). In both soils cocoon production was reduced almost completely when test animals were introduced 1 day after incorporation of the chemical. In test animals introduced on days 4 and 8, reduction still was about 90%. Cocoon production of control animals in LUFA 2.2 soil was only 50% that of artificial soil. In addition, the percentage of infertile cocoons was very high in LUFA soil 2.2. Significant effects on body weight development were observed only in LUFA 2.2 soil. A reduction of the feeding rate at the beginning of the test was observed in all treatments in both soils. During the last 10 days of the test treated animals restarted feeding. The effects of a different exposure of dimethoate was investigated in artificial soil at concentrations corresponding to the NOEC. E. fetidu was compared to E. undrei. Regarding numbers of juveniles total
soil contamination and soil surface contamination showed similar results for E. fetidu (Table 3). Significant effects on body weight development were observed with total soil contamination only. E. andrei was slightly more sensitive to total soil contamination than E. fetidu (Table 4). Significant effects on body weight development were again observed only with total soil contamination. Sensitivity differences between both species were also registered in total soil contamination tests with copper. With E. fetidu similar results were obtained in both soils (Table 5) whereas E. undrei was very susceptible in LUFA 2.2 soil (Table 6). In artificial soil E. undrei showed a slight, but not significant increase in numbers of juveniles at 3.2, 10 and 32 mg kg- ‘. Significant reductions occurred only at 320 mg kg ‘. In contrast to this, numbers of juveniles were significantly reduced at 3.2 mg kg- ’in LUFA 2.2 soil (Table 6). Body weight development was not influenced in both species even at the highest concentration tested. Results of most of the experiments with LUFA 2.2 soil indicated suboptimal conditions for the test animals. Cocoon production and hatching success generally were lower than in the artificial soil. Therefore the effect of soil moisture on body weight development and reproduction of E. fetidu and A.
Table 3. Sublethal effects of dimethoate on Eisenia firida in artificial soil. No test animal died Dimethoate (mg kg - ‘) Control Total contamination 2.5 5.0 1.5 Surface contamination 2.5 5.0 7.5 Mean of 4 replicates;
??
Body weight development WI
Number of cocoons
Number of juveniles
99.5 f 10.5
44.0 + 8.3
189.0 + 36.7
94.5 * 7.4 96.3 f 1.9 82.5 f 2.4*
49.5 * 7.3 32.5 * 3.1 18.3 k 4.7’
177.0 f 47.0 124.3 + 21.2 67.3 f 28.7’
99.3 * 10.0 101.3 * 5.7 97.5 f 4.9
ND ND ND
147.5 f 47.9 121.5 f 35.9 95.0 k 12.9’
P 5 0.05, multiple r-test by Tukey. ND = not determined
638
Hartmut Table 4. Sublethal Dimethoate
(mg kg
and Otto
Body weight development [“hl
‘)
Number
soil. No test animal
of cocoons
Number
died of juveniles
133.8 _+ 2.9
16.5 + 3.0
39.8 * 8.0
134.0 + 7.3 114.5 f 4.2’ 83.3 f 9.3*
16.3 k 5.1 3.5 + 3.0’ 0.3 + 0.5’
38.3 + 15.1 7.8 + 8.4* 0*
135.8 f 12.8 127.5 ? 3.8 114.8 * 10.2
ND ND ND
58.8 + 14.7 29.3 ? 16.0 8.5 + 11.1’
* P 2 0.05. multiple
Table 5. Sublethal
Larink
on Eisenia andrei m artificial
effects of dimethoate
Control Total contamination 2.5 5.0 7.5 Surface contamination 2.5 5.0 7.5 Mean of 4 replicates;
Kula
I-test by Tukey. ND = not determined
effects of copper on Eisenia /&da.
No test animal
died
Body weight development
Ku1 (mg kg ‘)
Number
[%I
Artificial soil Control 3.2 IO 32 IO0 320 LUFA 2.2 soil Control 3.2 IO 32 100 320 Mean of 4 replicates;
* P
of cocoons
Number
of juveniles
141.2 137.4 146.0 147.1 144.7 148.2
+ k i f f f
4.4 8.0 5.9 5.0 8.0 7.4
34.5 29.5 32.8 25.0 26.3 3.8
+ i + k f f
4. I 4.2 4.8 4.2* I.O* I .3*
97.3 75.8 97.0 80.0 49.5 7.5
+ f + f + +
145.9 145.3 136.8 144.5 148.1 135.1
_t f i f + :
4.6 I.7 4.3 4.8 8.8 4.2
10.3 6.0 8.8 4.8 4.3 0.5
_+ 2.4 + 0.8 f 3.4 _+ 2.l* + 3.0* z 1.0”
5.3 3.5 2.8 I.8 0.3
f f i f +
< 0.05, multiple
Table 6. Sublethal
I I.5 17.7 16.2 12.9 18.1* I.31
0.9 2.6 1.7 2.9 0.5* I .ort 2.0*
r-test by Tukey.
effects of comxr
on Eisenio andrei. No test animal
died
Body weight development Number
[%I Artificial soil Co”ttol 3.2 IO 32 100 320 LUFA 2.2 soil Control 3.2 IO 32 100 320 Mean of 4 replicates;
I.6 3.8 8.9 I .7
3.9 1.3 4.8 4.0 6.0 6.7*
120.0 144.0 142.8 140.0 120.5 46.5
f f f f f f
+ 6.3 _+ 7.3 + 7.4 rt 9.7 * 5.9 f 7.7
49.5 41 .o 34.3 37.0 17.8 2.8
f + + + + i
7. I 5.9 7.3’ 4.6’ 3.6* 2.2*
155.3 114.3 105.8 107.0 49.8 5.5
+ 9.0 + 24.3* + 26.2’ _+ 13.9’ + 8.8* f 4.5’
* P < 0.05, multiple
Table 7. Effects of soil moisture
52 57 61 66
i * f +
12.7 19.2 24.3 28.5 15.6 17.9’
I-test by Tukey.
numbers of juveniles were highest with the highest soil moisture content. Test animals of A. cafiginosa (Table 8) increased slightly in body weight in the wetter soil, but differences were not significant. Due to the inactivity of the test animals nearly no cocoons were produced.
on body weight development
Body weight development [“/I 73.0 74.5 78.4 75.8
of juveniles
f * * f k f
I.8
caliginosa was investigated in LUFA 2.2 soil. Water content was adjusted between 23% and 29% of dry wt of soil, corresponding to a water holding capacity between 52% and 66%. In E. fetida (Table 7) no differences were found in body weight development. Cocoon production and
Soil moisture [%I of WHC
Number
40.3 41.3 43.8 40.5 38.8 21.3
113.5 + lll.o*6.6 108.3 f 108.4 i 112.2 + 114.6 + 107.0 105.8 108.3 106.2 116.8 119.9
of cocoons
Number
I.4 6.6 0.4 2.3
Mean of 4 replicates; * P 5 0.05, multiple r-test by Tukey ‘Feeding rate: semiquantitative rating 0.1.2.3.
and reproduction
of cocoons
44.oio 56.0 f 2.8* 45.5 i 0.7 65.5 f 0.7*
of E./&da
Number 187.5 251.0 208.0 289.5
of juveniles + + + t
0.7 7.1’ 2.8’ 2. I *
in LUFA
2.2 soil Feeding I 2 3 3
rate’
The prediction of sublethal effects of chemicals on earthworms Table 8. Effects of soil moisture on body weight development and reproduction of A. Soil moisture [%I of WHC 52 51 61 66
Body weight development [%I 91.3 f 98.3 f 100.5 + 109.3 -+
coli&xa
639 in
Number of cocoons
Animals in resting phase [%I
0.5 * 0.7 1.5 &-0.7 0.5 * 0.7 0
30 50 30 0
4.3 5.8 2.8 16.1
LUFA 2.2 soil Feeding rate 0.5 0.5
I I
Mean of 4 replicates; * P < 0.05, multiple l-test by Tukey. ‘Feeding rate: semiquantitative rating 0.1.2.3.
DISCUSSION
Sensitivity differences between E. fetida and the endogeic mineral soil species A. caliginosa, A. rosea and A. chlorotica were small and within one order of magnitude. This is in accordance with Heimbach (1985) and Van Gestel (lot. cit.). In most cases E. fetida is reported to be the least susceptible species. The LC, in LUFA 2.2 soil usually was lower than in artificial soil. This could be explained by a higher bioavailability of the pesticide due to the lower organic C content of LUFA 2.2 soil. The faster disappearance of dimethoate from LUFA 2.2 soil could be explained by a higher microbial activity in contrast to the artificial soil. The differences in microbial activity will be investigated in further experiments. Concerning sublethal effects cocoon production and numbers of juveniles were the most sensitive biological response. Body weight development was a rather insensitive response, especially when testing copper. With dimethoate, however, sensitivity was equal to cocoon production and numbers of juveniles. In order to recognize possible sublethal effects which might have an influence on the ecological fitness of the test species more than one biological response should therefore be evaluated. Dimethoate and copper are suitable model test chemicals. Dimethoate shows a considerable acute toxicity especially to the indigenous earthworm species while sublethal effects occur at low concentrations. Copper in contrast has a moderate acute toxicity, but sublethal effects can also be observed at low concentrations. Acute and sublethal effects of the third test chemical (LAS) still have to be investigated. A data base on species specific sensitivity of adult and juvenile life stages of earthworm species belonging to different ecological categories will be elaborated.
Optimum conditions for the use of LUFA 2.2 soil will be investigated in order to ensure the quality criteria set up in the SECOFASE work manual (Lokke and Van Gestel, 1993).
Acknowledgements-The
funding for this work
provided by the EU Programme Environment (contract no. ERB-EVSV-CT92-0218).
was 199&1994
REFERENCES
BBA (1994) Richtlinien Bir die Zulassung von Pflanzenschutzmitteln im Zulassungsverfahren; Teil VI, 2-2; Auswirkungen von Pganzenschutzmitteln auf die Reproduktion und das Wachstum von Eisenia fetida/Eisenia andrei. Saphir-Verlag, Ribbesbiittel. DFG (1991) Methodensammhmg zur Rtickstandsanalytik von PBanzenschutzmitteln der Deutschen Forschungsgemeischaft: Bestimmung von Omethoat und Dimethoat 236-(42)-l. VCH, Stuttgart. Hamilton M. A. , Russo R. C. and Thurston R. V. (1977) Trimmed Spearman-Karber method for estimating median lethal concentrations in toxicity bioassays. Environmental Science and Technology 11, 714-719. Heimbach F. (1985) Comparison of laboratory methods using Eisenia foetida and Lumbricus terrestris, for the assessment of the hazard of chemicals to earthworms. Zeitschrift fdr Pflanzenkrankheiten 186193.
und Pflanzenschutz
92,
Lokke H. and Van Gestel C. A. M. (1993) Manual of SECOFASE. Development, improvement and standardization of test systems for assessing sublethal effects of chemicals on fauna of the soil ecosystem. National Environmental Research Institute, Denmark. OECD (1984) Guideline for testing of chemicals no. 207. Earthworm, acute toxicity tests. Adopted 4 April 1984. Wiles J. A., Kammenga J. E. and Lokke H. (1994) Progress Report 1993 of SECOFASE, Second Technical Report. Development, improvement and standardization of test systems for assessing sublethal effects of chemicals on fauna of the soil ecosystem. National Environmental Research Institute, Denmark.