Aporrectodea

Experiment 1. Earthworms were collected nonpolluted forest soil in Lepelstraat + field soil Epen (control: metal spike 325 mg Cd/kg soil and 3,100 mg Zn/kg soil as metal acetate salts).
Experiments 2. Earthworms were collected nonpolluted sandy grassland soil in Boxtel.
Experiments 3. Two bioassays were conducted with A. caliginosa collected from two different sites: a soil from the Afferdensche en Deestsche Waarde, and a field soil Epen.

All experiments were conducted in glass jars (750 ml), with 400 g of moist soil and four earthworms in each jar. All soils were kept at moisture content of 80+/-4% of their maximum water holding capacity. The experiments were performed in a climate room at 80% relative humidity, 15+/-3 degrees C, and permanent illumination.

Measurements/observations

Subcellular distribution (granular fraction: tissue, cell membrane, and intact cell fraction: cytosolic fraction including proteins and microsomal fraction) of Cd, Pb, Ni, Ca, Cu and Zn in the earthworm.

[ref. ID; 5992]

Test system

Heavy metal concentrations in the tissues, ingesta and faeces

Collection site

Earthworms were collected by formalin extraction (20 L, 0.55%) from five separate 1 m2 sites situated on a longitudinal transect across the Cefn Parc Pb and Zn-mine, South Wales (O.S. grid ref. = ST 048822)

Test design

Earthworms were starved on moistened filter paper for 4 days by which time the alimentary canal was clear of ingested soil material. Animals were wet oxidized with concentrated (16N) "Analar" nitric acid, and analysed for Cd, Cu, Pb, and Zn by flame atomic absorption spectrophotometry.

Measurements/observations

Dry weight and tissue metal concentration of worms, metal concentrations of ingesta (crop contents) and egesta (faeces).

[ref. ID; 5994]

Test system

Effect of urbanization on earthworm community

Study sites

The study was carried out in Brussels, Belgium. A transect was demarcated along heavily trafficked streets running from the city centre to the suburbs, in which a decreasing gradient of urbanization was predicted. Within the transect, six public parks were chosen for the study.

Sampling method

Earthworms were extracted by an electrical octet method (Thielemann, 1986).

Measurements/observations

Average density and biomass of earthworms at individual sites, Pb, Cd, Cu, Zn, Ca, and Mg concentrations of worms.

[ref. ID; 5996]

Test system

Pb uptake by earthworms

Sample

Earthworms were collected from 2 sites.

Site 1. Near an old lead smelter in Tikkurila (The soil there is heavily contaminated with Pb from the smelter, which was in operation for nearly 60 years, until it was closed in the early 1980s and finally demolished in 1991).
Site 2. Pornainen (less Pb contaminated situated ‘control’ soil) about 25 km to the north-east of Tikkurila.

Sampling method

The worms were extracted by hand-sorting on a plastic sheet.

Test design

Experiments 1. Identification, counting and weighting of collecting earthworms
Experiments 2. Rearing experiments. Blocks of highly Pb contaminated soil were thawed and vertically cut into smaller units without destroying their structure, and the units were fitted into cylindrical plastic pots (diameter 8.5 cm, height 15 cm) to be used for rearing. The pots were fitted with caps of fine nylon net. During the rearing period the pots were kept inside a dark, ventilated chamber at temperature of +10 degrees C.

Measurements/observations

The number of species, the number of individuals and biomass, Pb concentration in earthworms and soil.

[ref. ID; 5998]

Test system

The effect of exogenous (soil) calcium on lead accumulation by earthworms

Collection site

Earthworms were collected from the shallow soil overlying the stony spoil heaps of 15 disused lead mines throughout Wales, Shropshire and Avon. Samples were also collected from an uncontaminated site (Dinas Powis) in South Wales.

Test design

Soil-feeding experiment: 3 soils were placed in a clean plastic box (23 cm x 23 cm x 12 cm) to a depth of 10 cm, respectively.

1. Cwmystwyth soil (a large volume of acidic, calcium-deficient and lead contaminated).
2. Calcium-supplemented (calcium concentration of approximately 6000 ug g-1 wet weight) Cwmystwyth soil.
3. Uncontaminated Dinas Powis soil.

15-20 worms were collected from Dinas Powis, were placed in the experimental boxes. The animals were maintained for 35 days in the dark at 4 degrees C, with soil moisture maintained at approximately 30%.

[ref. ID; 6012]

Test system

The effect of type of LAS, soil type, and sewage sludge on toxicity test

Strains

Juvenile (14-21 days old).

Toxicants

Linear alkylbenzene sulfonates (LAS): distribution of the linear alkyl chains was: C10 14%, C11 34%, C12 31%, C13 21%.

Na-LAS: An aqueous solution (Isorchem 113/S-Na, active-matter concentration 16.1% (w/w), average molucular weight 342 g/mol)
Ca-LAS: Crystalline (purities >99%, mean molucular weight 674 g/mol).
Mg-LAS: Crystalline (purities >94%, mean molucular weight 658 g/mol).

Test soils

Three different agricultural soil types were collected at Lundgaard (a sandy soil), Askov (a sandy loam), and Flakkebjerg (a clayey soil), all belonging to the Danish Institute of Agricultural Sciences.

Measurements

Juvenile suvival number and weight.

Evaluation

LC10, EC10, LC50, EC50, NOEC, LOEc.

[ref. ID; 6066]

Test system

Acute toxicity test (14-d), prolonged toxicity test (6-8 weeks), and field test (4 yr)

Strains

Field captured adult worm.

Toxicants

E 605 forte (active ingredient: parathion), Unden flussing (active ingredient: propoxur)

Test design

Acute toxicity test: OECD-guideline No. 207, temperature 20 degrees C.
Prolonged toxicity test: Temperature 20 degrees C.
Run 1. [Soil surface contamination]: Test substances (Parathion: a normal application rate of 210 ml ha-1 and a 10-fold overdose, Propoxur: a normal applicaion rate of 900 ml ha-1) were homogeneously sprayed on the soil surface of test boxes containing 2 kg (dry wt) of artificial soil 20 individuals of E. fetida.
Run 2. [Soil total contamination]: The amount of test substance (20 times the normal rate used in agricultural applications) according to the surface of the test box was mixed into the soil homogeneously.
Field test: An orchard without pesticide treatments for the last 4 yr was for field studies. Size of test plots was 14x14 m with 2 m wide guard rows. Each treatment was replicated four times and compared to untreated control plots. (Parathion: normal rate of 210 ml ha-1, propoxur: normal rate of 900 ml ha-1).

Measurements/observations

Acute toxicity test: Mortility.
Prolonged toxicity test: Live weight and reproduction.
Field test: Mean earthworm abundance and biomass.

Evaluations

Acute toxicity test: LC50.
Prolonged toxicity test: Multiple t-test by Tukey.

[ref. ID; 6072]

Test system

Effect of repeated low doses of biocides

Toxicant

Azinphos-methyl, Captan, and Glyphosate.

Temperature

12 degrees C.

Test design

The surface area of the culture was 10 cm2 with a volume 10 cm3 + 1 immature worm (ca. 200 mg). The biocides were used alone, in all possible paired combinations and combination of all three biocides (22 treatments including control), and were applied at intervals of 14 days. 6 replicates. Test period 100 days.

Measurements

Number and weight of worms.

[ref. ID; 6102]

Test system

Seasonal changes in the tissue-metal (Cd, Zn and Pb) concentration in earthworm in heavy metal polluted soil

Sampling site

Mature (clitellate) worm were collected monthly from a 9-m2 sampling site at Cefn Parc mine, near Llantrisant, S. Wales (O.S. grid ref. ST 048822).

Toxicants

Cd, Pb, Zn.

Measurements

Dry weight and Cd, Pb, and Zn concentration in worm.

Evaluations

Monthly differences in tissue-metal concentrations were evaluated statistically by Duncan's multiple-range test. Differences in tissue-metal burdens between diapause and pre- or post-diapause animals were statistically evaluated by the Mann-Whitney U-test.

[ref. ID; 6107]

Test system

The tissue distribution of Cd, Cu, PB, Zn, and Ca in earthworm living in a non-polluted and a heavy metal polluted soil

Sampling site

Mature (clitellate) Aporrectodea caliginosa were collected from an uncontaminated site (Dinas Powis, O.S. grid reference ST 46723) and a disused, non-ferrous metalliferous site that is well-vegetated and with a fairly deep soil profile (Llantrisant, O.S. grid reference ST 048822).

Measurements

Dry weight, and Cd, Cu, Pb, Zn, and Ca concentration in the tissue of earthworm.

[ref. ID; 6138]

Test system

OECD-style toxicity (28 days) test

Strains

From Walker Organics, West Creiglee Farm, Roddymore, Crook, County Durham, UK. Mature adult (average weights 0.71+/-0.12 g), 280 individuals.

Toxicants/concentrations

Pb(NO3)2 1000, 3000, 4000, 5000, 7500 and 10 000 mg Pb kg-1.

Test design

OECD-style test: 1 L plastic containers (500g dry weight soil (Kettering loam, purchased from Barrycroft Stores Limited, Kettering, Cambridgeshire, UK), moisture content of the soil to 50% of the total WHC (water holding capacity), 15 degrees C, four replicates.

Measurements/observations

Mortality, weight of worms, total Pb in earthworm tissue.

Evaluations

LC50 using Probit analysis on SPSS statistical software version 10.01.
EC50 using USEPA (1984) Environmental Research Laboratory, Duluth MN 55804 USA, linear extrapolation method for sublethal toxicity.

[ref. ID; 6688]

Test system

Metal uptake

Strains

From unpolluted control soil.

Soil sample

Ultramafic soil (including Cr, Mn, Ni): In the Barberton area (Mpumalamga, South Africa), Kaapsehoop Chrysotile Mine (Kaapsehoop 1-3), Barberton Nature Reserve, Agnes Mine, Songimvelo Nature Reserve.
Unpolluted control soil: University of Stellenbosch, South Africa.

Test design

Plastic containers (15 cm x 10 cm x 5 cm) filled with 400 g dry soil, moistened to a moisture content 60-65%. 10 individuals per container. Worms did not receive food for the first 3 months. After that, worms were fed with 10 g of chick food (house brand, Agrimark Stellenbosch, South Africa) on a biweekly basis. Temperature 20 degrees C, darkness.

Measurements/observations

Metal (Al, As, Cd, Cr, Co, Cu, Fe, Mg, Ni, Pb, Zn) concentration in worm body.

[ref. ID; 6713]

Test system

Flooding response

Strains

From the 'Afferdensche en Deetsche Waarden'(ADW), a floodplain system along the river Rhine, in the central part of the Netherlands.

Toxicants

Zinc, cadmium, copper.
Flooded soil: Soil was collected from the top 0-10 cm horizon in an 'Afferdensche en Deetsche Waarden'(ADW).

Test design

Pot experiment: 44 plastic flowerpots (18 cm diameter, height 18.5 cm) 100 mg kg-1 zinc (Zn(NO3)/3H2O), 5 mg kg-1 cadmium (Cd(NO3)/4H2O), 20 mg kg-1 copper (Cu(NO3)2/3H2O). On top of the soil, grass (Lolium perenne) was sown. At 12 degrees C with continuous light, during 42 days. In the flooded treatments, the water level was raised to 5 cm water above the soil surface.

Run 1: Polluted combined with flooded soils + A. caliginosa 8 N and L. rubellus 3 N.
Run 2: Non-polluted combined with flooded soils + A. caliginosa 5 N, L. rubellus 3 N, and A. chlorotica 3 N.
Run 3: Polluted combined with non-flooded soils + A. caliginosa 8 N and L. rubellus 3 N.
Run 4: Non-polluted combined with non-flooded soils + A. caliginosa 5 N, L. rubellus 3 N, and A. chlorotica 3 N.

Moisture preference experiment: 8 glass aquaria (31 cm x 19 cm x 20 cm) were dividing five equal compartments with four Perspex partitions (height 13 cm). The aquaria were filled to a depth 11 cm with field soil at different moisture contents (35%, 45% (field capacity), 55%, 65% (saturated), 65%+ (saturated and an extra water layer) w/w). At 12 degrees C with continuous light, during 9 days.

Run 1: A. caliginosa 5 N (3 N adult + 2 N juveniles, FW 1.69 +/- 0.21 g per compertment).
Run 2: L. rubellus 5 N (3 N adult + 2 N juveniles, FW 1.28 +/- 0.12 g per compertment).
Run 3: A. chlorotica 3 N (2 N adult + 1 N juveniles, FW 0.51 +/- 0.08 g per compertment).

Health experiment: 40 small plastic buckets (diameter 12 cm, height 14 cm) were filled with 0.7 cm3 field soil. Two adult of each of the species A. caliginosa, L. rubellus and A. chlorotica added to each bucket (moisture content (35%, 45%, 55%, 65%, and 65%+). At 12 degrees C with 12 hr light, during 42 days.

Measurements/observations

Worm number and weight.

[ref. ID; 6745]

Test system

Metal bioaccumulation

Floodplain site

Three sites along the rivers Nieuwe Merwede and Waal. The sites are located on gradually sloping riverbands, and are subjected to periodic inundation.

Collection organisms

Collection was random within the 10x10 m plot. At least 5 individuals of each pedo-ecological group were taken. After taking to the laboratory, the earthworms were allowed to defaecate on wet filter paper for 48 hr. Wet weights were determined, followed by storage in freezer at -18 degrees C until analyses.

Measurements/observations

As, Cd, Cr, Cu, Pb, Zn, Mn, Fe, Ca concentration in earthworms.

[ref. ID; 6786]

Test system

The applicability of CBRs as a practical tool in soil quality assessment of contaminated sites

Strains

From unpolluted grassland sites in the region of Wageningen. Clitellated adult stage.

Toxicants

CuSO4/5H2O

Test design

Batches of five adults worms in 600 ml volume of soil (an arable sandy soil from Kooyenburg, and a silty clay loam from Oostelijk Flevopolder) were incubated in a climate chamber with a constant temperature of 15 degrees C and continuous light. Crushed leaves of alder (Alnus glutinosa) were added on top of the soil surface as a suitable food source. Experiment period was 4 weeks.

Measurements/observations

Survival and cocoon number.

Evaluations

Treatment effects and dose-response data were analyzed using the Genstat 5, release 4.2, statistical package.

[ref. ID; 6858]

Test system

Life-cycle and biomarker responses to zinc in four earthworm species (Eisenia fetida, Lumbricus rubellus, and Lumbricus terrestris)

Strains

From an unpolluted pasture on the University of Reading campus, Reading, United Kingdom, by digging and hand sorting. Adult worm with mean wet weight 724 mg.

Toxicants/concentrations

Zn[NO3]2/6H2O aqueous solutions: 0, 190, 350, 620, 1200, 2000, and 3600 ug/g.

Test design

Zinc exposures were conducted in a natural soil-based test system. The soil used was a mixture of a commercially available sandy loam soil (Rockalls, Wokingham, Berkshire, UK) and commercially available Sphagnum peat (Bullrush Ltd, County Tyrone, UK). One kilogram of the soil mix was added to each experimental a container (plastic boxes 220x160x80 mm), with four replicate containers used for each test concentration. Water-holding capacity 60%. 6 worms per container. During exposure period, the test containers were covered to limit water loss and kept in constant light at 15 degrees C for 42 days. Finely ground fresh horse manure (dried and rewetted to 75% water content) was added as source of food (4 g dry weight per weekly to each container) in all tests.

Measurements/obsevations

Life-cycle parameter: Survival, weight change, and cocoon production rate.
Biomaker: Neutral-red retention by coelomocytes lysosomes.
Zinc conconcentration in worm tissues.

Evaluations

Significant differences in parameters were calculated using analysis of variance (ANOVA). When, differences were found, Tukey's multiple comparison test was used to determine differences between specific treatments.

LC50 by the log-probit method using the MicroProbit 3.0 statistical software package.
EC50 using the linear interpolation technique within the ICp 2.0 software system.
Sublethal sensitivity index (SSI) were determined from calculated effect concentration (EC10) values. SSI = LC50/EC10.

[ref. ID; 6891]

Test system

Standarization of test methods for acute and sublethal effects of chemicals

Strains

From an uncontaminated orchard.

Toxicants

Dimethoate, copper, and linear alkylbenzene sulfonate.

Test design

An important difference between the two test soils is the organic C content. The OECD artifical 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%.

Acute toxicity test: According to OECD guideline no. 207 (OECD, 1984).
Reproduction toxicity test: The test substance was mixed homogeneously with 500 g (dry wt) of soil. The soil was put into 1 L plastic boxes which were closed with a transparent plastic lid with small holes for ventilation. Water holding capacity 50%. 4 worms per jar. Finely ground cattle manure was spread on the soil surface as food source. Food was renewed weekly. Experimental period 28 days. Dimethoate was not mixed into the soil but applied superficially in order to investigate the effect of different type of exposure.

Measurements/observations

Acute toxicity test: Body weight, occurrence of external injuries, general behaviour (ex. burrowing).
Reproduction toxicity test: Mortality, body weight development, cocoon production and numbers of hatched juveniles.

Evaluations

Acute toxicity test: LC50 using the trimmed Spearman-Karber method (Hamilton et al., 1977).
Reproduction toxicity test: Analysis of variance (ANOVA) was applied results. Significant differences (P < /_ 0.05) between control and treatment were determined by Tukey's multiple t-test.

[ref. ID; 6963]

Test system

The effect of Ca2+, Mg2+, Na+, H+, EDTA and DOC on copper toxicity

Strains

The worms were sampled in a sandy, uncontaminated soil. Average wet weight 381+/-7 mg.

Toxicants

Cu2+.

Test design

Temperature 15+/-2 degrees C under continous illumination (1500 Lux). No food.

Liquid medium: Worms were exposed in 1 L of an aqueous Steiner nutrient solution to a range of Cu2+ concentrations. pH 4-8. Exposure period 1 week.
Solid medium: Three hundred grams inert quartz sand was used and the flow rate of the modified nutrient solution was 1 L per day. Exposure period 7 days.
Contaminated field soils: About 300 g of soil. Exposure period 28 days.

Measurements/observations

Mortality.

Evaluations

LC50 using the trimmed Spearman-Karber method. Development of biotic ligand model.

[ref. ID; 7048]

Test system

The impact of bioturbation on the distribution and availability of zinc in a soil profile

Strains

A. caliginosa was collected from an natural grassland soil in a floodplain near Druten, Gelderland, the Netherlands.

Toxicants

Non-polluted soil and 10-year aged zinc spiked soil (approximately 500 mg/kg dry soil) were collected from an earlier semi-field experiment, carried out by Smit et al. (1997).

Test design

Soil columns (Perspex columns (n=24, diameter 10 cm, length 40 cm) with some small holes on top and bottom to allow air and water flow). 24 perspex columns were filled with non-polluted soil to a depth 23 cm, followed by a polluted layer of 5 cm and finally a 2 cm layer of non-pollted soil on top. In 12 columns, five eathworms with an average weight 220 mg (SD +/-40 mg; n=60) were introduced, the other 12 columns did not recieved. The columns were placed in a dark climate room at a temperature of approximately 12 degrees C. Sampling time 80, and 175 days.

Measurements/observations

Number of individuals in 5 cm thick depth layers. Total zinc concentration of casts.

2. Aporrectodea caliginosa tuberculata

[ref. ID; 6065]

Test system

Bioaccumulation and biotransformation

Sampling site

The field samples were collected from the area of the sawmill "Sikoniemi" (near the town of Kuopio in Central Finland). The sawmill was abandoned 28 yr ago, and at present there is a dense birch forest in the area.

Toxicants

Chlorophenol.

Test design

Laboratory experiments: Soil was taken from a hayfield which had not been ploughed for several years (organic matter content 47% (pH 5.0) and 4.7% (pH 5.8)) + Chlorophenol + food (clean grass) + 10 worms, 16+/-1 degrees C.

Measurements

2,3,4,6-Tetrachlorophenol, Pentachlorophenol and their metabolites in worms.

3. Aporrectodea icterica

[ref. ID; 5994]

Test system

Effect of urbanization on earthworm community

Study sites

Sampling method

Earthworms were extracted by an electrical octet method (Thielemann, 1986).

Measurements/observations

Average density and biomass of earthworms at individual sites, Pb, Cd, Cu, Zn, Ca, and Mg concentrations of worms.

4. Aporrectodea longa

Aporrectodea longa (Ude) is abundant and widely distributed in fertile soils. It is a comparatively large, slow-growing species that consumes large amounts of soil, much of which is deposited as surface casts, in creating an extensive burrow network. It also consumes dead organic matter taken from the soil surface. As a deep-burrowing surface feeder it is classified as an anecic species. (ref. ID; 6137)

[ref. ID; 505]

Test system

Acute lethality tests (OECD 1984/EEC 1985)

Strains

Sexually mature.

Toxicants and Reference standard chemical

Chlorpyrifos and chloracetamide (ClCH2CONH2).

Test design

Measurements/observations

Body weight and number.

Evaluations

14d-LC50 according to the trimmed Spearman-Karber method (Hamilton et al. 1977). 14d-EC50 using the statistical software package GENSTAT 5. NOEC applying analysis of variance.

[ref. ID; 5992]

Test system

Heavy metal concentrations in the tissues, ingesta and faeces

Collection site

Test design

Measurements/observations

Dry weight and tissue metal concentration of worms, metal concentrations of ingesta (crop contents) and egesta (faeces).

[ref. ID; 6066]

Test system

Acute toxicity test (14-d), prolonged toxicity test (6-8 weeks), and field test (4 yr)

Strains

Field captured adult worm.

Toxicants

E 605 forte (active ingredient: parathion), Unden flussing (active ingredient: propoxur)

Test design

Acute toxicity test: OECD-guideline No. 207, temperature 20 degrees C.
Prolonged toxicity test: Temperature 20 degrees C.
Run 1. [Soil surface contamination]: Test substances (Parathion: a normal application rate of 210 ml ha-1 and a 10-fold overdose, Propoxur: a normal application rate of 900 ml ha-1) were homogeneously sprayed on the soil surface of test boxes containing 2 kg (dry wt) of artificial soil 20 individuals of E. fetida.
Run 2. [Soil total contamination]: The amount of test substance (20 times the normal rate used in agricultural applications) according to the surface of the test box was mixed into the soil homogeneously.
Field test: An orchard without pesticide treatments for the last 4 yr was for field studies. Size of test plots was 14x14 m with 2 m wide guard rows. Each treatment was replicated four times and compared to untreated control plots. (Parathion: normal rate of 210 ml ha-1, propoxur: normal rate of 900 ml ha-1).

Measurements/observations

Acute toxicity test: Mortility.
Prolonged toxicity test: Live weight and reproduction.
Field test: Mean earthworm abundance and biomass.

Evaluations

Acute toxicity test: LC50.
Prolonged toxicity test: Multiple t-test by Tukey.

[ref. ID; 6137]

Test system

Biomaker model (comet assay and postlabelling analysis) of sublethal genotoxicity

Strains

Specimens were obtained commercially (Ecology Earthworms, Ipswich) or collected by excavation from neutral grassland on the Lancaster University campus.

Toxicants/concentrations

Benzo[a]pyrene (0.1, 1.0, 10.0 ppm) and/or lindane (10.0 ppm).

Test design

Soil was collected from the top 5-20 cm of a pasture field in Lancashire (UK). AN earthworm was added to 50 g of soil and maintained at 15 degrees C for 24 hr or 7 days. The feasibility of determining DNA damage induction (in the form of DNA SSBs (single-strand breaks) and carcinogen-DNA adducts) in the intestinal tissues (crop/gizzard and intestine) of worm.

Measurements/observations

The alkaline single cell-gel electrophoresis (comet) assay: DNA SSBs, comet tail lengths (CTLs) (um) measurement.
[32]P-Postlabelling analysis: DNA adduct spots.

[ref. ID; 6902]

Test system

Strains

From bait shops and acclimated in the laboratory for four months.

Toxicants

Parathion (Formulation E 605 forte Bayer, 50% a.i.) and Amitrole-Diuron (Ustinex).

Test design

OECD-Guideline 207 (OECD, 1984). Experimental period 28 days.
Soil substrate (artificial soil or loamy natural soil (5.6% sand, 75% silt, 19% clay; 2.1% organic substance; pH 6.0; air-dried and sieved)), temperature (20 or 10 degrees C), soil moisture (82% or 51% Max. water capacity), way of application (mixing pesticide into the soil or spraying onto the soil surface) 1000g (dry wt) soil and 5 worms per test container. Each container was supplied weekly with 2.5 g (dry wt) of a mixture of two-thirds air-dried, finely ground cow manure and one-third dried leaves of stinging nettle (Urtica dioica L.).

Measurements/observations

Mortality, weight, cocoon production, and behavioural changes.

[ref. ID; 7032]

Test system

Field assessment on reproduction (effect of soil temperature)

Toxicants

Benomyl.

Test design

The study area was a field, grown with grass since 1995, owend by the Danish Institute of Agricultural Sciences, Research Centre Foulum, Jutland, Denmark. The experiment was set up as a randomized block design with four blocks each consisting of three 15-m x 15-m plots. Plots were situated 15 m apart, and the four blocks were situated 30 m apart. Benomyl was applied by a tractor-mounted boom spray unit at 203 kPa and a tractor speed of 3.8 km/hr. Three treatments were applied, i.e., 0 (control), 0.5, and 1.0 (+400 L water) kg active ingredient (AI)/ha. During the following 2 days, the plots were irrigated with approximately 40 mm of precipitation in order to maximize incorporation of the fungicide into the soil. Soil temperature was measured at 5- and 15-cm depths. Experimental period 32 days.

Measurements/observations

Earthworm density in the soil.

Evaluations

Reproductive rate.

5. Aporrectodea rosea

[ref. ID; 5994]

Test system

Effect of urbanization on earthworm community

Study sites

Sampling method

Earthworms were extracted by an electrical octet method (Thielemann, 1986)

Measurements/observations

Average density and biomass of earthworms at individual sites, Pb, Cd, Cu, Zn, Ca, and Mg concentrations of worms.

[ref. ID; 5996]

Test system

Pb uptake by earthworms

Samples

Earthworms were collected from 2 sites.

Site 1. Near an old lead smelter in Tikkurila (The soil there is heavily contaminated with Pb from the smelter, which was in operation for nearly 60 years, until it was closed in the early 1980s and finally demolished in 1991).
Site 2. Pornainen (less Pb contaminated situated ‘control’ soil) about 25 km to the north-east of Tikkurila

Sampling method

The worms were extracted by hand-sorting on a plastic sheet.

Test design

Experiment 1: Identification, counting and weighting of collecting earthworms.
Experiment 2: Rearing experiments. Blocks of highly Pb contaminated soil were thawed and vertically cut into smaller units without destroying their structure, and the units were fitted into cylindrical plastic pots (diameter 8.5 cm, height 15 cm) to be used for rearing. The pots were fitted with caps of fine nylon net. During the rearing period the pots were kept inside a dark, ventilated chamber at temperature of +10 degrees C.

Measurements/observations

The number of species, the number of individuals and biomass, Pb concentration in earthworms and soil.

[ref. ID; 6891]

Test system

Standarization of test methods for acute and sublethal effects of chemicals

Strains

From an uncontaminated orchard.

Toxicants/concentrations

Dimethoate, copper, and linear alkylbenzene sulfonate.

Test design

Acute toxicity test: According to OECD guideline no.207 (OECD, 1984).
Reproduction toxicity test: The test substance was mixed homogeneously with 500 g (dry wt) of soil. The soil was put into 1 L plastic boxes which were closed with a transparent plastic lid with small holes for ventilation. Water holding capacity 50%. 4 worms per jar. Finely ground cattle manure was spread on the soil surface as food source. Food was renewed weekly. Experimental period 28 days. Dimethoate was not mixed into the soil but applied superficially in order to investigate the effect of different type of exposure.

Measurements/observations

Acute toxicity test: Body weight, occurrence of external injuries, general behaviour (ex. burrowing).
Reproduction toxicity test: Mortality, body weight development, cocoon production and numbers of hatched juveniles.

Evaluations

Acute toxicity test: LC50 using the trimmed Spearman-Karber method (Hamilton et al., 1977).
Reproduction toxicity test: Analysis of variance (ANOVA) was applied results. Significant differences (P < /- 0.05) between control and treatment were determined by Tukey's multiple t-test.

[ref. ID; 6911]

Test system

Lead tolerance

Strains

A. rosea were collected, by hand digging an area of 10 m x 10 m, from a heavily shot area and an un-shot area.

Toxicants

Contaminated soil were collected a clay pigeon shooting site in Norfolk, UK. The site had been in operation since the 1960s.

Test design

The prepared soils (250 g wet weight) were placed in plastic beakers and a single earthworm added to each beaker (n=3). The beakers were then coverd with perforated laboratory film and kept in a cool dark cupboard. Moisture content 50% WHC. Experimental period 28 days.

Measurements/observations

Condition index, body weight and tissue Pb concentrations in worm.

[ref. ID; 7032]

Test system

Field assessment on reproduction (effect of soil temperature)

Toxicants

Benomyl.

Test design

The study area was a field, grown with grass since 1995, owend by the Danish Institute of Agricultural Sciences, Research Centre Foulum, Jutland, Denmark. The experiment was set up as a randomized block design with four blocks each consisting of three 15-m x 15-m plots. Plots were situated 15 m apart, and the four blocks were situated 30 m apart. Benomyl was applied by a tractor-mounted boom spray unit at 203 kPa and a tractor speed of 3.8 km/hr. Three treatments were applied, i.e., 0 (control), 0.5, and 1.0 (+400 L water) kg active ingredient (AI)/ha. During the following 2 d, the plots were irrigated with approximately 40 mm of precipitation in order to maximize incorporation of the fungicide into the soil. Soil temperature was measured at 5- and 15-cm depths. Experimental period 32 days.

Measurements/observations

Earthworm density in the soil.

Evaluations

Reproductive rate.

6. Aporrectodea trapezoids

Endogenic earthworm. (ref. ID; 6763)

[ref. ID; 6763]

Test system

Strains

Adult earthworms were provided by Edwin Berry, National Soil Tilth Laboratory, USDA Agricultural Research Service, Ames, Iowa. The earthworms species used were Lumbricus rubellus (weighing 0.80-1.21 g), Aporrectodea trapezoids (weighing 2.15-2.57 g), Lumbricus terrestris (weighing 5.08-6.23 g).

Toxicants

Plasmid: pJP4, Donor: Alcaligenes eutrophus JMP222N, Recipient: Pseudomonas fluorescens C5t

Test design

Soil: A Hubbard loamy sand (Udorthenic Haploboroll).
Microcosm: The microcosm consisted of a polyvinyl chloride tube (50 by 10 cm diameter) which was cut into eight 5-cm segments. The remaining 10-cm segment served as the headspace.

Experimental design

Soil microcosms containing spatially segregated, coinoculated donor and recipient strains, inoculated control (donor and recipient strain only) soil microcosms, and uninoculated control soil microcosms. Triplicate microcosms for each of the four treatment (i.e., no worms, L. rubellus, A. trapezoides, and L. terrestris). 3 adult earthworms added to surface of the microcosm. (The final earthworms density was equivalent to approximately 100 earthworm per m2 of soil. 20 degrees C. Experimental period 2 weeks.

Measurements/observations

Vertical distribution of earthworms number. Donor, recipient, transconjugant bacteria number of casts and cocoon.

7. Aporrectodea tuberculata (Eisen)

Endogenic earthworm. (ref. ID; 6724)

[ref. ID; 6129]

Test system

Biomarker (Metallothioneins, Cytochrome P4501A monooxygenase, glutathione-S-transferase) to copper and zinc exposure

Strains & soils

Adults with a well developed clitellum (0.5-1 g in fresh weight).

Population samples 1 (polluted soil): Worms and soil were collected by hand at three distances (1, 2, and 4 km) from the Imatra Streel Oy Ab steel smelter (Imatra, Finland, 61 degrees 12'N, 28 degrees 48'E). The smelter was founded in 1937 in the present location. Till 1944 there was also a copper factory next to the smelter. Currently the smelter produces special steel by smelting recycled metal.
Population samples 2 (test soil): Worms and soil were collected for laboratory experiment without earlier metal exposure in Jyvaskyla, Finland (62 degrees 14'N, 25 degrees 44'E).
Population samples 3 (reference soil): Worms were living in deciduous forests.

Toxicants

CuCl2/2H2O, ZnCl2.

Laboratory experiments

The experiment was established in 192 pots, each 8 cm in length and 6 cm in inner diameter, which were kept in a climate chamber (15 degrees C, darkness). Each pot recieved 100 g of test soil (fresh weight) and one worm from either of the two A. tuberculata populations, with or without earlier metal exposure. For each population, pots of a control soil and soils with three combined copper/zinc concentration series (100/175, 200/350, 400/700 mg kg-1 in dry weight of soil) were established.

Measurements/observations

Metal concentration in earthworms (field samples: 48 individuals, laboratory experiment 192 individuals), Metallothioneins (MT) concentrations, Cytochrome P4501A(CYPIA) and glutathione-S-transferase (GST) activities.

[ref. ID; 6724]

Test system

The transfer of Cesium-137 in a Soil-Earthworm-Plant-Snail

Strains

Adult earthworms produced for fishing came from Mondial Peche (Besancon, France-Comte, France).

Toxicants

[137]Cs activity concentration was 130+/-4 Bq/kg dry mass.

Experimental design

Temperature was 24 degrees C during the day and 16 degrees C during the night.

Run 1: CS (container + soil + snail), CSL (container + soil + snail + litter), CSE (container + soil + snail + earthworms), and CSLE (container + soil + snail + litter + earthworms). Each container with earthworms was filled with 440 g (dry mass) of soil, and three earthworms weighing 5.23+/-1.59 g (fresh mass) were introduced. Containers without earthworms contained 140 g of soil. In CSL and CSLE treatments, 5 g (dry mass) of the litter was homogeneously deposited on the soil surface. 4 replicates. Containers is transparent polystyrene of 3,200 cm3.
Run 2: MS (microcosm + soil + snail), MSP (microcosm + soil + snail + plant), MSPE (microcosm + soil + snail + earthworm). In MSPE microcosms, eight adult earthworms weighing 4.50+/-1.16 g (fresh mass) were introduced. 4-7 replicates.

Measurements/observations

Number, weight, Cesium-137 activity concentration of worm.