Advertisement

The bait-lamina earthworm test: a possible addition to the chronic earthworm toxicity test?

  • Stephan Jänsch
  • Adam Scheffczyk
  • Jörg RömbkeEmail author
Original Paper
  • 560 Downloads

Abstract

The bait-lamina method is a functional test method which is successfully used in field monitoring studies to assess the feeding activity of soil organisms. This endpoint addresses an important soil function and service: nutrient cycling. Therefore, the test has recently been standardized by the International Organization for Standardization. In the approach presented here, the bait strips were used in the laboratory as an additional endpoint in the standard earthworm reproduction test, regularly performed for the registration of chemicals as well as in soil assessment. The combined test was performed with the model chemicals zinc nitrate and tributyltin-oxide (TBT-O) in Organization for Economic Co-operation and Development artificial soil and eight natural soils. It was checked whether the results, available after 1 week, could be used to predict the effects of the chemicals on earthworm reproduction, available after 8 weeks. Out of 15 comparisons of EC50 values made in this study, in six of them there was no difference. In four cases the feeding rate was more sensitive and in five cases it was the other way around. The bait-lamina earthworm test (BLET) could be performed in the laboratory either as a screening tool for estimating the range of chronic ecotoxicity of chemicals or for testing the habitat quality of potentially contaminated soils (e.g. as part of a quick “on-site” analysis). Further research, mainly the preparation of a standard stand-alone BLET, its application on chemicals with different mode-of-actions, and the definition of a reference substance as a positive control, is needed.

Keywords

Ecotoxicology Eisenia andrei Feeding activity Functional test Chemicals Soil quality 

Introduction

The bait-lamina method was originally developed for measuring the biological activity of soils (Von Törne 1990). It is based on an optical evaluation of the feeding on small portions of a thin-layered bait substrate exposed to edaphic soil processes (Kratz 1998). This method has the advantage of being able to detect effects on the soil fauna in a short time and with little effort. In ecotoxicology, the bait-lamina method has mainly been used to monitor the effects of chemicals on the feeding activity of the soil biocoenosis in the field (Federschmidt and Römbke 1994; Van Gestel et al. 2001; Simpson et al. 2012). At least four groups of 16 bait-lamina are inserted into the upper soil layer and, depending on the soil, evaluated for the feeding activity (percentage of empty holes) after a period of 10–20 days. The method has also already been applied in the assessment of terrestrial model ecosystems (TME), where four strips per TME were inserted and evaluated after 2 weeks (Förster et al. 2004). Except for some preliminary work in the 1990s (Kampmann 1994), the method has not been used in ecotoxicological laboratory tests so far.

Earthworms are important in the soil due to their ability to change their habitat or even create new ones for other organisms through various activities: thus they are considered to be “ecosystem engineers” (Jones et al. 1994). They fulfill various ecologically important functions in soil, thus finally lead to an improved soil structure, increase of water infiltration (Edwards and Lofty 1977), often to the formation of a humic layer close to the soil surface and to an increased yield in orchards or grassland (Van Groenigen et al. 2014). Even the activity of the compost worms of the genus Eisenia results in the formation of water-stable aggregates (Schrader and Zhang 1997). The species E. andrei is recommended in various international standard guidelines (ISO 1998; OECD 1984, 2004), because it is easy to culture and breed. E. andrei is reproducing hermaphrodically, but usually two partners are necessary (Dominguez et al. 2003). For these reasons earthworms are very suitable ecotoxicological test organisms.

In contrast to the bait-lamina method, the earthworm reproduction test is a common laboratory requirement for assessing the effects of chemicals (pesticides, metals, pharmaceuticals, etc.) (OECD 2004). The overall approach of this work was to add bait-lamina strips to the earthworm reproduction test in the laboratory. This combination, called bait-lamina earthworm test (BLET), was used to test two model chemicals (a metal and a tin-organic chemical) in OECD (Organization for Economic Co-operation and Development) artificial soil (OECD 1984) and eight natural field soils (all of them originating from Germany). These two substances have been selected since they represent two very different chemical classes and thus, mode-of-actions. In addition, their effects on soil organisms (besides earthworms also springtails) and plants any were already studied in a wide range of soils, including those used in this study (e.g. Römbke et al. 2006a, 2007). Especially the latter is known as being toxic for annelids and, in particular, snails (Moore et al. 1991; Giusti et al. 2013).

In particular, two key questions were addressed when performing this combination of the two test methods:
  1. 1.

    Is it possible to use the results of the BLET, available after 1 week, to predict the range of effects of a test chemical on earthworm reproduction, available after 8 weeks? If so, the BLET test could be used as a range-finding test for the earthworm reproduction test.

     
  2. 2.

    Could the BLET be used for determining the effects of potentially contaminated field soils on the functional endpoint “feeding rate”? In this case, it could be used for the assessment of soil habitat quality.

     

Materials and methods

Description of the bait-lamina strips

The bait-lamina strips used are made of polyvinyl chloride (PVC) and have a length of 120 mm, width of 6 mm and thickness of 1 mm. They have a pointed tip at the lower end. In the lower part (85 mm) of each strip, 16 bi-conical apertures of 1.5 mm diameter are drilled that are 5 mm apart from each other (Fig. 1). The target soil depth of the first aperture is 0.5 cm and that of the 16th aperture 8.0 cm.
Fig. 1

Filling of bait-material into the bait-lamina strips

Preparation of bait-lamina strips

The bait substrate comprised 70% cellulose powder, 25% finely ground wheat bran and 5% activated carbon powder. The components were homogeneously mixed and stirred with deionized water to a viscid paste. The paste was filled into the holes of the bait-lamina from both sides in various cycles (Fig. 1). The strips were air-dried for at least 24 h prior to use.

Earthworms

The earthworms used in these tests, Eisenia andrei (Lumbricidae), came from a synchronized culture, were adult, had a fresh weight between 300 and 600 mg, and were at least 2 months but not more than 1 year old. The worms were fed with rolled oats at least every 2 weeks and occasionally with cow dung. Worms selected for the test were acclimatized in the respective soil under test conditions for at least 24 h before starting the test.

Soils

The OECD artificial soil (OECD 1984) and eight natural soils, being representative for Germany, were selected and characterized for these tests (Jessen-Hesse et al. 2005). The sampling locations of these soils are given Fig. 2. Individual properties of these soils are compiled in Table 1. Soil samples taken at the respective sites were air-dried, sieved (5 mm mesh size) and stored in 25 l plastic buckets at room temperature, for no longer than 3 months. In the tests, they were moistened to 40–60% of the maximum water-holding capacity (WHCmax). The amount of soil used in the tests per vessel was 500 g dry weight (DW).
Fig. 2

Locations in Germany where the test soils have been collected

Table 1

Physico-chemical characterization of the tested soils (eight natural field soils and OECD artificial soil)

Soila

WHCmax (ml/kg)

pH (CaCl2)

OC (%)

N (%)

C/N

CEC (cmol +/kg)

Sand (%)

Silt (%)

Clay (%)

OECD

631

6.0

4.7

0.07

67.1

8.9

75.4

16.6

8.04

BRG

601

4.9

2.34

0.29

8.1

14.1

13.6

56.7

29.7

BWZ

307

3.8

1.54

0.05

30.8

3.3

81.3

13.6

5.1

GGI

232

5.5

0.94

0.06

15.7

2.0

80.5

15.7

3.82

HAG

611

5.2

2.64

0.28

9.4

13.2

12.8

62.3

24.9

SBG

653

5.8

3.37

0.33

10.2

11.8

27.0

47.1

25.9

SHA

474

7.4

2.22

0.16

13.9

19.8

7.79

69.7

22.5

SOE

483

6.6

1.63

0.16

10.2

13.8

1.97

83.0

15.0

LUFA St. 2.2

500

6.1

2.7

0.19

14.2

7.9

76.9

16.3

6.84

WHCmax maximum water-holding capacity, OC organic carbon, CEC cation exchange capacity (Jessen-Hesse et al. 2005)

aAbbreviations and locations of soils: OECD artificial soil according to OECD (1984), BRG Breddewarden, Lower Saxony, BWZ Weitzgrund, Brandenburg, GGI Raddusch, Brandenburg, HAG Frankfurt-Harheim, Hesse, SBG Schmallenberg, North Rhine-Westphalia, SHA Schafstädt, Saxony-Anhalt, SOE Soest, North Rhine-Westphalia, LUFA St. 2.2 Landwirtschaftliche Untersuchungs- und Forschungsanstalt 2.2 standard soil, Speyer, Rhineland-Palatinate; all locations are in Germany

Test chemicals

Two model chemicals were selected to cover a broad range of chemicals properties: Zinc nitrate-tetrahydrate (Zn(NO3)2 × 4H2O) with a molar mass of 261.44 g/mol was obtained from Merck (Darmstadt, Germany; CAS–19154-63-3). In addition to the treatments with zinc nitrate, four vessels were spiked with 1900 mg nitrate/kg DW soil (as potassium nitrate). This concentration was equivalent to the nitrate concentration when testing the highest zinc nitrate concentration.

Bis(tri-n-butyltin)oxide (TBT-O; C24H54OSn2) with a molar mass of 596.07 g/mol was obtained from Crompton (Bergkamen, Germany; CAS-No. 56-35-9). Aqueous solutions of each test substance were mixed into the soils. Each treatment (=concentration) was set up separately.

Test performance

In the chronic earthworm tests (ISO 1998), ten worms in each of the four replicates were exposed to the spiked test soil for 28 days at 18–22 °C and 400–800 lx (light regime 16:8 h). Test vessels were polystyrene cups with a base area of 110 × 155 mm and a height of 60 mm (Bellaplast 590, Polarcup, Alf, Germany). Five concentrations of zinc nitrate were selected according to literature data (Römbke et al. 2006a): 181, 362, 725, 1449 and 2898 mg Zn(NO3)2/kg DW soil, corresponding to 63, 125, 250, 500 and 1000 mg zinc/kg DW soil. For TBT-O, five concentrations per soil were selected according to range-finding tests: for OECD artificial soil 3.2, 10.0, 31.6, 100 and 316 mg TBT-O/kg DW soil, for LUFA St. 2.2 soil 1.0, 3.2, 10.0, 31.6 and 100 mg TBT-O/kg DW soil, and for all other soils 0.32, 1.0, 3.2, 10 and 31.6 mg TBT-O/kg DW soil. The adult worms were fed with finely ground cow manure (free of growth promoters, nematicides or similar veterinary pharmaceuticals, provided by Jesus Bruderschaft e.V. Gnadenthal, Hünfelden, Germany). Before starting the experiment an amount of 5 g food per 500 g soil DW was mixed into the test soil. After the start of the experiment, food was first provided one day after application of the test item and introduction of the adult worms. Thereafter, the adult worms were fed weekly during the first 4 weeks of the test. An amount of 5 g of food moistened with 10 ml deionized water was spread on the soil surface of each test container. If the food remained uneaten the ration was reduced on demand. After removing of the adults on day 28, a further 5 g of food moistened with 10 ml deionized water was mixed into the soil of each test vessel. Mortality, biomass and morphological or behavioral changes of the adult worms were recorded after 28 days and the number of juvenile earthworms after 56 days by a heat-extraction method (water bath 60 °C).

The bait-lamina were applied in parallel to the chronic earthworm tests. Each test vessel received two bait-lamina strips (i.e. 32 holes) at test start that were transversally inserted so that all holes were beneath the soil surface (Fig. 3). After 1 week, the strips were removed and the number of empty holes were optically assessed (Fig. 4).
Fig. 3

Use of bait-lamina strips in test vessels of the standard earthworm reproduction test

Fig. 4

Visual examination of the feeding activity on bait-lamina strips

Data assessment

The statistical calculations for all tests were performed with the program ToxRat, Version 2.09 (TOX-Rat 2003). For the calculation of the NOEC (no observed effect concentration) and LOEC (lowest observed effect concentration) values (reproduction, feeding activity on bait-lamina), data were checked for normality (R/S test, Kolmogorov–Smirnov test) and variance homogeneity (Cochran’s test). Depending on the results, either the Welch t test for inhomogeneous variances with Bonferroni adjustment, the Williams t test (monotonous dose–response) or Dunnett’s t test (non-monotonous dose–response) were used (EC 2007). In case the data were not normally distributed, the U test according to Mann–Whitney (significance level corrected for multiple comparisons) was applied. The significance level for the parametric tests (one-sided) was p ≤ 0.05. For the calculation of the LC50 (median lethal concentration), EC10 (10% effective concentration) and EC50 (median effective concentration) values (reproduction, feeding activity on bait-lamina), a linear regression was performed via probit analysis.

As an indicator of the power of the hypothesis tests the minimum detectable difference (MDD) in  % of the control value was calculated for the NOEC values (TOX-Rat 2003). For EC50-values the 95% confidence limits were determined, as far as possible. To investigate the precision and robustness of the different endpoints and compare the results of the two test systems used, the arithmetic means with standard deviation and the coefficient of variation (CV) in % were calculated. Variance of the results is also presented by the minimum and maximum values and the factor between the minimum and maximum value. With respect to the distribution of the single values in addition to the arithmetic mean, the geometric mean was calculated to compare the endpoints.

Results

Validity criteria

According to the ISO 11268-2 (1998) guideline, test results are valid in case the following criteria are fulfilled: adult mortality ≤10%, number of juveniles >30 per test vessel, and the coefficient of variation ≤30%. These validity criteria were fulfilled in the controls of all tests with both test substances (Römbke et al. 2006a, 2007). In the case of the bait-lamina field test ISO 18311 (ISO 2016) requires that at least 30% of the baits are pierced in the control vessels. This criterion was fulfilled in all tests.

Zinc-nitrate

The test results for the reproduction of earthworms in zinc nitrate spiked soils are summarized in Table 2 (for further details see Scheffczyk et al. 2014). Depending on the soil type the NOECs varied between 181 and 1449 mg Zn(NO3)2/kg DW soil, i.e. by a factor of eight. The EC10-values varied by a factor of 11, i.e. between 118 and 1289 mg Zn(NO3)2/kg DW soil. The NOEC- and EC10-values were similar (i.e. at most by a factor of two apart). The EC50-values differed between 422 and 1903 mg Zn(NO3)2/kg DW soil, i.e. there the factor between them is only 4.5. When comparing the NOEC-, EC10-, and EC50-values determined in the nine tests performed, only in two cases (BWZ, SHA) were both former values lower than the latter by a factor of two. The mean of the MDD was 29% and the CV was smallest for the EC50 (38%).
Table 2

Results of the earthworm reproduction test (Römbke et al. 2006a); all values in mg Zn(NO3)2/kg DW soil and comparison with OECD artificial soil (excluded from statistics)

Soil

EC10

EC50 (95% confidence limits)

NOEC

LOEC

MDD (%)

OECD

1110

1731 (ND)

725a

1449a

26a

BRG

1106

1361 (1346–1372)

725a

1449a

31a

BWZ

118

422 (194–791)

181a

362a

29a

GGI

527

864 (844–885)

725a

1449a

40a

HAG

1090

1439 (ND)

725a

1449a

20a

SBG

773

1903 (ND)

1449a

2898a

38a

SHA

427

1297 (1130–1496)

362b

725b

21b

SOE

1289

1804 (1788–1820)

1449a

2898a

24a

LUFA St. 2.2

478

1083 (690–1727)

725b

1449b

31b

Statistics

 Mean

726

1272

792

1585

29

 Min

118

422

181

362

20

 Max

1289

1903

1449

2898

40

 Factor

10.9

4.5

8.0

8.0

 

 SD

406

484

454

907

 

 CV (%)

56

38

57

57

 

 Geometric mean

593

1167

664

1329

 

EC10 10% effective concentration, EC50 median effective concentration, NOEC no observed effect concentration, LOEC lowest observed effect concentration, MDD minimum detectable difference, ND not determinable, SD standard deviation, CV coefficient of variation. See Table 1 for abbreviations and locations of soils

aDunnett’s t test for homogenous variances

bWilliam’s t test for homogenous variances

The test results for the feeding activity in soils spiked with zinc nitrate are summarized in Table 3. Due to technical reasons, no bait-lamina was inserted alongside the earthworm tests with zinc-nitrate in OECD artificial soil and LUFA St. 2.2. In the remaining seven tests performed, it was found that the NOECs covered a very broad range, between <181 and ≥2898 mg Zn(NO3)2/kg DW soil, i.e. differing at least by a factor of 16 apart. EC10 values varied between 43.9 and ≥2898 mg Zn(NO3)2/kg DW soil, with an even higher factor between them (66). The EC50 values differed less, just by a factor of 9.7, covering a range between 299 and >2898 mg Zn(NO3)2/kg DW soil. The NOEC- and EC10-values were similar (i.e. at most factor two apart) except for soil SOE where the EC10 was by a factor of seven lower than the NOEC. When comparing the NOEC-, EC10- and EC50-values only for the soils BWZ and SOE (EC10 only) the former two were clearly lower than the latter. In all other cases, they were less than a factor of two apart. For the GGI-soil the difference could not be determined because statistically significant effects were already observed at the lowest test concentration of 181 mg Zn(NO3)2/kg soil DW. In SHA-soil even the NOEC was higher than the highest test concentration of 2898 mg Zn(NO3)2/kg soil DW. The mean of the MDD was 33% and the CV was smallest for the LOEC (71%).
Table 3

Results of the bait lamina test; all values in mg Zn(NO3)2/kg DW soil and comparison with OECD artificial soil (excluded from statistics)

Soil

EC10

EC50 (95% confidence limits)

NOEC

LOEC

MDD (%)

OECD

NT

NT

NT

NT

NT

BRG

530

1113 (968–1281)

725a

1449a

38a

BWZ

184

396 (352–445)

181b

362b

23b

GGI

43.9

299 (ND)

<181a,c

181a

25a

HAG

817

1325 (1203–1460)

725a

1449a

32a

SBG

1412

2291 (2089–2511)

1449a

2898a

31a

SHA

>2898

>2898 (ND)

≥2898a

>2898a

40a

SOE

103

711 (506–1000)

725a

1449a

39a

LUFA St. 2.2

NT

NT

NT

NT

NT

Statistics

 Mean

855

1290

983

1527

33

 Min

43.9

299

<181

181

23

 Max

>2898

>2898

≥2898

>2898

40

 Factor

66

9.7

16

16

 

 SD

1022

978

946

1076

 

 CV (%)

120

76

96

71

 

 Geometric mean

394

973

656

1076

 

EC10 10% effective concentration, EC50 median effective concentration, NOEC no observed effect concentration, LOEC lowest observed effect concentration, MDD minimum detectable difference, ND not determinable, NT not tested, SD standard deviation, CV coefficient of variation. See Table 1 for abbreviations and locations of soils

aDunnett’s t test for homogenous variances

bWilliam’s t test for homogenous variances

cLowest concentration significantly different compared to the control; the lowest concentration was used for the calculation of the mean values

Based on the EC50 values (the most robust and thus suitable endpoint), the experiences gained in this contribution with zinc-nitrate are summarized as follows (Fig. 5). Results are classified as different as long as their confidence intervals do not overlap. In six tests for which toxicity values could be determined for both earthworm reproduction and feeding activity no difference in the sensitivity of both test endpoints was found in two soils (BWZ, HAG). In three soils the BLET was more sensitive (BRG, GGI, SOE), and in one soil (SBG) the earthworm reproduction test was more sensitive. However, in only two cases (GGI, SOE), did the EC50 values differ by more than a factor of two.
Fig. 5

Comparison of the EC50 (median effective concentration) values for the 8-week earthworm reproduction and the 1-week bait-lamina earthworm tests in soils spiked with zinc nitrate

TBT-O

Chronic endpoints for effects on the reproduction of earthworms in TBT-O spiked soils are summarized in Table 4 (for further details see Scheffczyk et al. 2014). Depending on the soil type the NOECs varied between <0.3 and 1.0 mg TBT-O/kg DW soil, i.e. by a factor of ≥3.3. The EC10-values varied by a factor of 83.3, i.e. between 0.03 and 2.5 mg TBT-O/kg DW soil. The EC50-values differed between 0.5 and 4.7 mg TBT-O/kg DW soil, i.e. the factor between them is only 9.4. When comparing the NOEC-, EC10- and EC50-values determined in the nine tests performed, in almost all cases (except HAG, SHA and SOE), both former values were by a factor of two lower than the latter. NOEC- and EC10-values were at most a factor of two apart in five soils (OECD, BWZ, GGI, SBG, LUFA St. 2.2). In four soils (BRG, HAG, SHA, SOE) the EC10 was more than a factor of two higher than the NOEC. In the test in GGI soil the NOEC was lower than the lowest concentration tested. The mean of the MDD was 19% and the CV was smallest for the EC50 (45%).
Table 4

Results of the earthworm reproduction test (Römbke et al. 2007); all values in mg TBT-O/kg DW soil and comparison with OECD artificial soil (excluded from statistics)

Soil

EC10

EC50 (95% confidence limits)

NOEC

LOEC

MDD (%)

OECD

3.9

13.4 (10.1–17.8)

3.2b

10.0b

20b

BRG

2.1

4.7 (3.0–6.5)

1.0b

3.2b

21b

BWZ

0.3

2.0 (1.3–3.0)

0.3b

1.0b

26b

GGI

0.03

0.5 (0.01–1.3)

<0.3a,c

0.3a

34a

HAG

2.4

4.1 (ND)

1.0a

3.2a

16a

SBG

0.6

2.5 (1.5–4.0)

0.3b

1.0b

8b

SHA

2.4

3.9 (ND)

1.0b

3.2b

20b

SOE

2.5

4.1 (ND)

1.0b

3.2b

17b

LUFA St. 2.2

1.0

3.1 (2.9–3.1)

1.0b

3.2b

11b

Statistics

 Mean

1.4

3.1

0.74

2.3

19

 Min

0.03

0.5

<0.3

0.3

8

 Max

2.5

4.7

1.0

3.2

34

 Factor

83.3

9.4

3.3

10.7

 

 SD

1.04

1.4

0.36

1.3

 

 CV (%)

73

45

49

57

 

 Geometric mean

0.8

2.6

0.64

1.8

 

EC10 10% effective concentration, EC50 median effective concentration, NOEC no observed effect concentration, LOEC lowest observed effect concentration, MDD minimum detectable difference, ND not determinable, SD standard deviation, CV coefficient of variation. See Table 1 for abbreviations and locations of soils

aDunnett’s t test for homogenous variances

bWilliam’s t test for homogenous variances

cLowest concentration significantly different compared to the control; the lowest concentration was used for the calculation of the mean values

The test results for the feeding activity in soils spiked with TBT-O are summarized in Table 5. In the nine tests performed, it was found that the NOECs covered a range between 0.3 and 3.2 mg TBT-O/kg DW soil, i.e. differing by a factor of 10. EC10 values varied only slightly more, i.e. 0.3 and 5.4, with a factor of 18 between them. The EC50 values differed less, just by a factor of 6.1, covering a range between 1.4 and 8.5 mg TBT-O/kg DW soil. In the nine tests performed, the NOEC- and EC10-values were similar (i.e. at most by a factor of two apart). Comparing the NOEC-, EC10- and EC50-values in only three cases (OECD, GGI, SHA) the former two were clearly lower than the latter. In all other soils (BRG, BWZ, HAG, SBG, SOE, LUFA St. 2.2) the EC50 was less by a factor of two higher than the corresponding EC10 and/or NOEC. In the soil HAG, the EC50 was even slightly below the corresponding NOEC due to the high MDD of 43%. For the SHA-soil the difference could not be determined because statistically significant effects were already observed at the lowest test concentration of 0.3 mg TBT-O/kg soil DW. The mean of the MDD was 34% and the CV (%) was similar for all endpoints (47–54%).
Table 5

Results of the bait lamina test; all values in mg TBT-O/kg DW soil and comparison with OECD artificial soil (excluded from statistics)

Soil

EC10

EC50 (95% confidence limits)

NOEC

LOEC

MDD (%)

OECD

5.3

16.0 (13.4–19.2)

3.2a

10.0a

13a

BRG

5.4

6.9 (ND)

3.2b

10.0b

33b

BWZ

4.5

8.5 (ND)

3.2b

10.0b

28b

GGI

3.0

7.9 (5.2–12.0)

3.2b

10.0b

35b

HAG

2.1

3.1 (2.5–3.9)

3.2b

10.0b

43b

SBG

2.7

3.7 (2.5–5.4)

3.2b

10.0b

45b

SHA

0.3

1.4 (1.0–1.9)

<0.3b,c

0.3b

22b

SOE

2.0

3.4 (3.0–3.9)

1.0b

3.2b

29b

LUFA St. 2.2

4.1

7.4 (5.9–9.2)

3.2b

10.0b

36b

Statistics

 Mean

3.0

5.3

2.6

7.9

34

 Min

0.3

1.4

<0.3

0.3

22

 Max

5.4

8.5

3.2

10.0

45

 Factor

18

6.1

10

33

 

 SD

1.6

2.7

1.2

3.9

 

 CV (%)

54

51

47

49

 

 Geometric mean

2.4

4.6

2.1

5.6

 

EC10 10% effective concentration, EC50 median effective concentration, NOEC no observed effect concentration, LOEC lowest observed effect concentration, MDD minimum detectable difference, ND not determinable, SD standard deviation, CV coefficient of variation. See Table 1 for abbreviations and locations of soils

aWilliam’s t test for homogenous variances

bDunnett’s t test for homogenous variances

cLowest concentration significantly different compared to the control; the lowest concentration was used for the calculation of the mean values

Again, the EC50 values are used as basis for the comparison between the results of the earthworm reproduction tests and in the BLET with TBT-O (Fig. 6). Results are classified as different as long as their confidence intervals do not overlap. Of the nine comparable tests, no difference in the sensitivity of both test endpoints was found in four soils (OECD, HAG, SBG, SOE). In one soil the BLET was more sensitive (SHA), and in four soils (BRG, BWZ, GGI, LUFA St. 2.2) the earthworm reproduction test was more sensitive. In four cases (BWZ, GGI, SHA, LUFA St. 2.2), the EC50 values differed by more than a factor of two.
Fig. 6

Comparison of the EC50 (median effective concentration) values for the 8-week earthworm reproduction and 1-week bait-lamina earthworm tests in soils spiked with tributyltin-oxide

Discussion

Test performance and validity criteria

No technical difficulties were observed when performing the tests (e.g. earthworms were not stressed by the inclusion of the strips). Methodologically, the test design chosen here was determined by the requirements of the earthworm reproduction test according to the ISO guideline 11268-2 (ISO 1998). A corresponding guideline was since also published as OECD guideline 222 (OECD 2004). Regarding validity, all criteria as defined in these guidelines have been fulfilled. The same is true for the BLET as originally defined for field investigations according to ISO guideline 18131 (ISO 2016).

Comparison of test sensitivity

In the context of the application of the BLET as a screening tool, its sensitivity compared to the more complex and longer lasting earthworm reproduction test needs to be discussed. There is no information in the scientific literature since the BLET has not been used in laboratory ecotoxicological tests so far. Here, the two EC50 values were considered to be different in case the respective 95% confidence limits did not overlap.

So far, no distinct difference regarding sensitivity of one of the two endpoints (reproduction and feeding activity) has been identified. Out of 15 comparisons made in this study, in six cases there was no difference. In four cases the feeding rate was more sensitive and in five cases it was earthworm reproduction. In fact, the two values did only once differ by a factor of more than five; in almost all comparisons they differed by a factor of less than three. Therefore, the use of the BLET as a screening tool and/or range-finding test can be recommended since comparable effects can already be detected after 1 week (chronic earthworm test: 8 weeks). However, since the results may depend on the respective mode-of-action of the test chemicals, a more profound assessment of the BLET is needed on the basis of further testing experience with more substances belonging to different classes.

Influence of soil properties

Soil properties clearly influence the outcome of ecotoxicological tests with soil organisms. This is either because they have a direct effect on the animals and their activities (e.g. Ma and Bonten 2011), or (probably even more) due to their impact on the bioavailability of soil contaminants. In the case of zinc, this relationship has been substantiated: For the earthworm Eisenia fetida, pH and OM content are most important when tested in a range of artificial soils with different pH values and OM contents (Spurgeon and Hopkin 1996). In the earthworm tests presented here, the correlation between zinc toxicity and soil factors like pH, OC content and CEC was not significant, which might be caused by the fact that the tested soils had very different properties, while in the work cited above only artificial soils with different adjusted pH values and OM contents were tested (Römbke et al. 2006a). Therefore, it is not clear whether the availability of zinc indirectly played a role in the effects of zinc on the feeding activity.

Compared to heavy metals, few studies assessing the influence of soil properties on the toxicity of organic chemicals to soil invertebrates have been performed (e.g. Van Gestel 1992). As the pore water concentration of non-ionizable substances depends on the organic matter content of the soil, effects on organisms should decrease with increasing organic matter content of the soil (Lock et al. 2002). While there are examples that this relationship is not always true (e.g. for Lindane (log Kow = 3.85; Lock et al. 2002), in the case of TBT-O (log Kow = 3.74) a clear and significant correlation between EC50 values (i.e. a chronic endpoint) and organic matter content could be shown (Römbke et al. 2007) for earthworms. When discussing the modeling of the uptake of polycyclic aromated hydrocarbons (PAH) in soil, Jager (2003) also mentions that sorption of “difficult” substances like (organo-)metals in soils cannot be predicted on the basis of organic matter content alone, meaning that more measurements with a broad range of soils and, probably, more species are necessary.

Test improvement

In case the BLET is to be used as a stand-alone laboratory trial, several parameters of the test design may be adjusted, e.g. in order to reduce variability or increase sensitivity and statistical power. In particular, these parameters are
  • Test duration: what is the minimum test duration to achieve a sufficient control feeding activity and what test duration is too long so that differences in feeding activity are no longer detected or the advantage of a short-term screening method gets lost? In the field, it is recommended to run the test until at least 30% but preferably not all bait slits are pierced. For example, at tropical sites with an average air temperature of about 26 °C a test duration of 1 week was chosen (Römbke et al. 2006b). This test duration was also sufficient in our laboratory tests which were performed at a temperature of 18–22 °C.

  • Addition of food: before starting the test, it was discussed whether—in contrast to the standard earthworm reproduction test—no additional food besides the bait-lamina substrate should be incorporated into the soil to avoid distracting the worms from feeding on them. However, the results gained here show that despite the availability of food the earthworms fed on the bait-lamina. This is true in tests in which the relationship between the endpoint feeding rate and earthworm reproduction is studied, but in short-term tests feeding is not recommended to get clearer results.

  • Number of animals and bait strips per test vessel/size of test vessels: it may be useful to expose the earthworms individually or in smaller groups than 10 and use smaller test vessels with less soil and possibly one only bait strips. This would further reduce testing demands and might even reduce variability and increase statistical power since the number of replicates may in turn be increased.

Regular test use

When using the BLET for the identification of appropriate concentrations in tests with chemicals, a problem could occur in case the respective chemical does affect the reproduction and the feeding activity of the earthworms at very different concentrations. However, this possible discrepancy is mitigated by the fact that the 1-week BLET would be designed as a range-finding test. When referring to standard guidelines, such tests are usually performed at concentrations with a spacing factor of ten (e.g. OECD 2004). The effect values (i.e. the EC50 values) found in this study for the two different endpoints were with one exception (far) less than a factor of 10 apart, meaning that the effects observed in the BLET would mostly still result in a suitable range of concentrations for the earthworm reproduction test. However, in order to assure that the range-finding results of the BLET are robust, further studies with chemicals with different mode-of-actions have to be performed.

When using BLET for the quick determination of the effects of potentially contaminated field soils it is clear that the endpoint feeding rate could be influenced by the respective soil properties. Although so far only soils from Germany have been used, this influence has probably been well covered for Central Europe considering the quite large differences in soil properties such as pH, organic carbon or clay content. However, this might not be true for other regions with very different soil properties—a difference which has been highlighted in various ecotoxicological investigations, e.g. in the Mediterranean regions (Chelinho et al. 2011; Madani et al. 2015).

Regarding a standardization of this method the current lack of several validity criteria is problematic. A positive control (i.e. reference substance) needs to be identified that causes a defined reduction of the feeding activity under standardized test conditions. For the time being, the minimum control feeding activity of 30% as defined for the field bait-lamina test could be used. Additionally, a maximum permissible coefficient of variation for feeding activity in controls may be a useful validity criterion, but before this could be defined a meta-analysis of bait-lamina results is needed.

Conclusions

The results presented here can be summarized as follows:
  • It is possible to use the results of a BLET to predict the range of effects of a test chemical on earthworm reproduction, i.e. to use the BLET as a range-finding test. Further tests (running with both endpoints for one and 8 weeks, respectively) with more chemicals and different mode-of-actions are needed to confirm the relationship between both endpoints.

  • The BLET, running for just a week, can be used for the quick determination of the effects of potentially contaminated field soils on an important functional endpoint.

In case this test is going to be used in other regions than Central Europe, further testing with regional soils is needed to provide a robust basis for the evaluation and applicability of the results. The BLET is sensitive, delivers fast results and requires little resources and training. Although some effort has to be put in getting more experience with the BLET, in the future it might be used as an additional method in soil ecotoxicology.

In any case, the BLET that has a very good ratio between practical efforts and meaningful results would be a good addition for “on-site” test batteries (i.e. short-lasting and relatively simple test methods). In addition, the BLET would allow assessing the effects of a chemical on a soil function related to nutrient cycling, i.e. an extremely important function provided and/or in the field regulated partly by earthworms (e.g. Brussaard 2012; Keith and Robinson 2012).

Notes

Acknowledgements

This research was part of the “ERNTE”-project funded by the German Federal Ministry for Education and Research (R&D No. 0330300).

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

References

  1. Brussaard L (2012) Ecosystem services provided by the soil biota. In: Wall DH et al (eds) Soil Ecology and Ecosystem Services, 1st edn. Oxford University Press, Oxford, pp 45–58CrossRefGoogle Scholar
  2. Chelinho S, Domene X, Campana P, Natal-da-Luz T, Scheffczyk A, Römbke J, Andrés P, Sousa JP (2011) Improving ecological risk assessment in the mediterranean area: selection of reference soils and evaluating the influence of soil properties on avoidance and reproduction of two oligochaete species. Environ Toxicol Chem 30:1050–1058CrossRefGoogle Scholar
  3. Dominguez J, Velando A, Aira M, Monroy F (2003) Uniparental reproduction of Eisenia fetida and E. andrei (Oligochaeta: Lumbricidae): evidence of self-insemination. Pedobiol 47:530–534Google Scholar
  4. EC (Environment Canada) (2007) Guidance document on statistical methods for environmental toxicity tests. Report EPS 1/RM/46Google Scholar
  5. Edwards CA, Lofty JR (1977) Biology of earthworms, 2nd edn. Chapman and Hall, LondonCrossRefGoogle Scholar
  6. Federschmidt A, Römbke J (1994) Erfahrungen mit dem Köderstreifen-Test auf zwei fungizidbelasteten Standorten. Braunschw Naturkdl Schr 4:675–680Google Scholar
  7. Förster B, Van Gestel CAM, Koolhaas JE, Nentwig G, Rodrigues JML, Sousa JP, Jones SE, Knacker T (2004) Ring-testing and field-validation of a terrestrial model ecosystem (TME)— an instrument for testing potentially harmful substances: effects of carbendazim on organic matter breakdown and soil fauna feeding activity. Ecotoxicology 13:129–141CrossRefGoogle Scholar
  8. Giusti A, Barsi A, Dugué M, Collinet M, Thomé JP, Joaquim-Justo C, Roig B, Lagadic L, Ducrot V (2013) Reproductive impacts of tributyltin (TBT) and triphenyltin (TPT) in the hermaphroditic freshwater gastropod Lymnaea stagnalis. Environ Toxicol Chem. 32:1552–1560CrossRefGoogle Scholar
  9. ISO (International Organization for Standardization) (1998) Soil quality—effects of pollutants on earthworms (Eisenia fetida). Part 2: determination of effects on reproduction. ISO 11268-2. GenevaGoogle Scholar
  10. ISO (International Organization for Standardization) (2016) Soil quality—method for testing effects of soil contaminants on the feeding activity of soil dwelling organisms—bait-lamina test. ISO 18311. GenevaGoogle Scholar
  11. Jager T (2003) Worming your way into bioavailability. Modelling the uptake of organic chemicals in earthworms. Dissertation, University of UtrechtGoogle Scholar
  12. Jessen-Hesse V, Römbke J, Jänsch S, Hund-Rinke K, Terytze K (2005) Auswahl und Charakterisierung von Böden zur Optimierung der Anwendung ökotoxikologischer Testmethoden bei der Beurteilung potentiell belasteter Standorte. Bodenschutz 4(05):110–116Google Scholar
  13. Jones CG, Lawton JH, Shachak M (1994) Organisms as ecosystem engineers. Oikos 69:373–386CrossRefGoogle Scholar
  14. Kampmann T (1994) Entwicklung eines standardisierten Labortests mit Köderstreifen für ökotoxikologische Prüfungen: erste Vorversuche. Braunschw Naturkdl Schr 4:681–686Google Scholar
  15. Keith AM, Robinson DA (2012) Earthworms as natural capital: ecosystem service providers in agricultural soils. Economology J II(Year II):91–99Google Scholar
  16. Kratz W (1998) The bait-lamina test—general aspects, applications and perspectives. Environ Sci Pollut Res 5:94–96CrossRefGoogle Scholar
  17. Lock K, De Schamphelaere KC, Janssen CR (2002) The effect of lindane on terrestrial invertebrates. Arch Environ Contam Toxicol 42:217–221CrossRefGoogle Scholar
  18. Ma W-C, Bonten LTC (2011) Bioavailability pathways underlying zinc-induced avoidance behavior and reproduction toxicity in Lumbricus rubellus earthworms. Ecotoxicol Environ Saf 74:1721–1726CrossRefGoogle Scholar
  19. Madani S, Coors A, Haddioui A, Ksibi M, Pereira R, Sousa JP, Römbke J (2015) Effects of contaminated soils from a former iron mine (Ait Amar, Morocco) on enchytraeids and predatory mites in standard laboratory tests. Ecotoxicol Environ Saf 119:190–197CrossRefGoogle Scholar
  20. Moore DW, Dillon TM, Suedel BC (1991) Chronic toxicity of tributyltin to the marine polychaete worm, Neanthes arenaceodentata. Aquat Toxicol 21:181–198CrossRefGoogle Scholar
  21. OECD (Organization for Economic Co-operation and Development) (1984) OECD guideline for testing of chemicals No. 207. Earthworm acute toxicity test. ParisGoogle Scholar
  22. OECD (Organization for Economic Co-operation and Development) (2004) Guidelines for the testing of chemicals No. 222. Earthworm reproduction test (Eisenia fetida/Eisenia andrei). ParisGoogle Scholar
  23. Römbke J, Jänsch S, Junker T, Pohl B, Scheffczyk A, Schallnaß H-J (2006a) Improvement of the applicability of ecotoxicological tests with earthworms, springtails and plants for the assessment of metals in natural soils. Environ Toxicol Chem 25:776–787CrossRefGoogle Scholar
  24. Römbke J, Höfer H, Garcia MVB, Martius C (2006b) Feeding activities of soil organisms at four different forest sites in Amazonia using the bait-lamina method. J Trop Ecol 22:313–320CrossRefGoogle Scholar
  25. Römbke J, Jänsch S, Junker T, Pohl B, Scheffczyk A, Schallnaß H-J (2007) The effect of tributyltin-oxide on earthworms, collembolans and plants in artificial and natural soils. Arch Environ Contam Toxicol 52:525–534CrossRefGoogle Scholar
  26. Scheffczyk A, Frankenbach S, Jänsch S, Römbke J (2014) Comparison of the effects of zinc nitrate and tributyltin-oxide on the reproduction and avoidance behavior of the earthworm Eisenia andrei in laboratory tests using ten soils. Appl Soil Ecol 83:253–257CrossRefGoogle Scholar
  27. Schrader S, Zhang H (1997) Earthworm casting: stabilisation or destabilisation of soil structure. Soil Biol Biochem 29:469–475CrossRefGoogle Scholar
  28. Simpson JE, Slade E, Riutta T, Taylor ME (2012) Factors affecting soil fauna feeding activity in a fragmented lowland temperate deciduous woodland. PLoS One 7(1):e29616. doi: 10.1371/journal.pone.0029616 CrossRefGoogle Scholar
  29. Spurgeon DJ, Hopkin SP (1996) Effects of variations of the organic matter content and pH of soils on the availability and toxicity of zinc to the earthworm Eisenia fetida. Pedobiologia 40:80–96Google Scholar
  30. ToxRat® (2003) Software for the statistical analysis of biotests. Copyright: ToxRat Solutions GmbH, AlsdorfGoogle Scholar
  31. Van Gestel CAM (1992) Toxicity of chemicals for earthworms: a review. In: Greig-Smith P, Becker H, Edwards PJ, Heimbach F (eds) Ecotoxicology of earthworms. Intercept, Andover, pp 44–54Google Scholar
  32. Van Gestel CAM, Van der Warde JJ, Derksen A, Van der Hoek EE, Veul MFXW, Bouwens S, Rusch B, Kronenburg R, Stokamn GNM (2001) The use of acute and chronic bioassays to determine the ecological risk and bioremediation efficiency of oil-polluted soils. Environ Toxicol Chem 20:1438–1449CrossRefGoogle Scholar
  33. Van Groenigen JW, Lubbers IM, Vos HMJ, Brown GG, De Deyn GB, Van Groenigen KJ (2014) Earthworms increase plant production: a meta-analysis. Nat Sci Rep 4:6365CrossRefGoogle Scholar
  34. Von Törne E (1990) Assessing feeding activities of soil-living animals. I. Bait-lamina tests. Pedobiologia 34:89–101Google Scholar

Copyright information

© Springer International Publishing Switzerland 2017

Authors and Affiliations

  1. 1.ECT Oekotoxikologie GmbHFlörsheimGermany

Personalised recommendations