Eisenia

Eisenia andrei
Eisenia fetida (= Eisenia foetida)
Eisenia fetida andrei
Eisenia veneta

1. Eisenia andrei

[ref. ID; 2170]

Test system

Accumulation and elimination of various chlorinated chemicals in earthworms in field-contaminated soil

Strains

Adult with a clitellum (weight 311+/-40 mg).

Toxicants

Field contaminated soil (approximately 8 kg topsoil was gathered at one site in the Volgermeerpolder, The Netherlands).
Chlorobenzenes (1,2,4-trichlorobenzene, 1,3,5-trichlorobenzene, 1,2,4,5-tetrachlorobenzene, 1,2,3,4-tetrachlorobenzene, pentachlorobenzene, hexachlorobenzene), Polychlorobiphenyl (PCB) (2,2',4,5,5'-pentachlorobiphenyl (PCB101), 2,3,3',4,4'-pentachlorobiphenyl (PCB105), 2,3',4,4',5-pentachlorobiphenyl (PCB118), 2,2',3,4,4',5'-hexachlorobiphenyl (PCB138), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153), 2,3,3',4,4',5'-hexachlorobiphenyl (PCB156), 2,3,3',4,4',5'-hexachlorobiphenyl (PCB157), 2,3',4,4',5,5'-hexachlorobiphenyl (PCB167), 2,2',3,4,4',5,5'-heptachlorobiphenyl (PCB180)).

Temperature

18 degrees C.

Measurements/observations

Mortality, body weight, accumulation.

Evaluations

Biota-to-soil accumulation factor (BSAF), elimination rate.

[ref. ID; 4479]

Test system

Availability

Samples

Sediments samples were taken on 45 contaminated sites in The Netherlands.

Measurements/observations

PAHs (Acenaphthalene, Anthracene, Chrysene, Fluoranthene, Fluorene, Naphthalene, Phenanthrene, Pyrene, Benzo[a]anthracene, Benzo[a]pyrene, Benzo[b]fluoranthene, Benzo[k]fluoranthene, Dibenzo[a,h]anthracene, Benzo[ghi]perylene) concentration in E. andrei tissue.

Evaluations

Biota-to-soil accumulation factor (BSAF), bioconcentration factors (BCF), elimination rate.

[ref. ID; 4572]

Test system

Acute and chronic toxicity

Strains

From Carolina Biological Supply (Burlington, NC, USA). Adult earthworms (wet wt, 300-600 mg) having a well-developed clitellum.

Toxicants

CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexazaisowurtzitane).

Test design

Soil 1: A sandy-type natural forest soil sample taken in the Montreal area.
Soil 2: The Sassafras sandy loam soil sample.
Soil 3: Artificial soil (70% (wt/wt) grade 4010 silica sand, 20% colloidal kaolinite clay, and 10% 2-mm sieved Canadian sphagnum peat according to the Organisation for Economic Cooperation and Development Guideline).

Acute toxicity (lethality test): 200 g of dry soil, 3 replications; using the U.S. Environmental Protection Agency Method (EPA/600/3-88/029); exposure period 14 days.
Chronic toxicity (reproduction test): 500 g of dry soil + food (2 g of dry cereal/weak), 4 replications; using The International Organization for Standardization Method 103390; exposure period 56 days.

Measurements

Acute toxicity: Number of surviving.
Chronic toxicity: Number of surviving, individual weight (wet biomass), number & weight of hatched and nonhatched cocoons, number & weight of juveniles.

Evaluations

Acute toxicity: NOEC & LOEC (using the ToxCalc program: Dunnett's multiple-comparison test).
Chronic toxicity: EC20 & EC50 (using the Linear interpolation with bootstrapping).

[ref. ID; 4958]

Test system

Reproduction test (28 days and 56 days) and NRRT assay

Strains

Adult E. andrei, were obtained from Carolina Biological Supply (Burlington, NC, USA), (ranging from 300 to 600 mg wet weight) having a well-developed clitellum were used.

Toxicants

HMX, RDX, TNT, Tetryl.

Test design

Test soil was contaminated with different PNOs such as HMX, RDX, TNT and its by-products. ISO (1998) method.

Measurements/observations

NRRT assay

28 days: Survival number and body weight of adult worms.
56 days: Number of cocoons, number of hatched cocoons, number of juveniles and biomass of juveniles.

[ref. ID; 5976]

Test system

Bioassays (28 day)

Strains

Mature (clitellate) worms.

Toxicants/concentrations

Pb(NO3)2, final soil Pb-spike concentration 2,000 mg/kg.

Temperature

20+/-degrees C, constant light.

Test design

Standard protocol: American Society for Testing and Materials (1997). Standard guide for conducting laboratory soil toxicity or bioaccumulation tests with Lumbricid earthworm Eisenia fetida. E 1676-97. In Annual Book of ASTM Standards, Vol 11.05. Philadelphia, PA, pp 1056-1074.
Soil: 21 soils with a wide range of soil properties (pH, OC, FEAL, and CEC).

Measurements/observations

Number of worms and cocoon, weight of worms, lead concentration in earthworm tissue.

Evaluation

IC (Internal concentration).

[ref. ID; 5977]

Test system

Chronic earthworm test (28-day)

Strains

The worms were taken from a breeding culture kept at ECT Oekotxikologie (Germany) since 1994. Only adult worms (with clitellum) with a fresh weight between 300 and 600 mg were used.

Toxicants/concentrations

Zinc nitrate-tetrahydrate (Zinc concentrations: 63, 125, 250, 500, and 1,000 mg/kg dry-weight soil).

Temperature/light conditions

18-22 degrees C, 400-800 lux (16:8-hr light:dark photoperiod)

Test design

International Organization for Standardization [ISO 11268-2].
10 worms in each of the four replicates were exposed to zinc nitrate in the test soil (OECD artificial soil and nine natural soils) for 28 days.

Measurements/observations

Numbers of juveniles.

Evaluations

NOEC, LOEC, EC10, EC50.

[ref. ID; 5980]

Test system

Acute and chronic test

Strains

Fully clitellated adults from in-house cultures.

Temperature/light condition

20+/-2 degrees C, photoperiod of 16 hr of fluorescent and incandescent illumination and 8 hr of darkness.

Food

Approximately 5 ml of cooked oatmeal per week.

Toxicants

Arsenic, Cadmium, Copper, Nickel, Lead, Zinc.

Test design

Environment Canada. 2004. Biological test method: Tests for toxicity of contaminated soil to earthworms (Eisenia andrei, Eisenia fetida, or Lumbricus terrestris). Report EPS 1/RM/43. First draft as of June 2004, Ottawa, ON.
Soil: Contaminated soil (Sudbury, Rouyn-Noranda), Control (Artificial soil and Clay loam reference soil).
Test units consisted of 500-ml wide-mouthed glass mason jars filled with a mass of hydrated test soil equivalent to three-quarters the volume of the jar. 5 earthworms were added per test unit for lethality tests and 2 for reproduction tests.

Measurements/observations

Survival number of adult, number of juveniles and cocoons produced, and weight.

Evaluations

IC50, IC20, NOAEC, LOAEC.

[ref. ID; 6066]

Test system

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

Strains

Laboratory bred 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; 6076]

Test system

24-hr acute toxicity

Toxicants

Carbaryl, Paraoxon.

Temperature

24-27 degrees C.

Test design

The standard OECD tests.

Measurements

Survival, Cholinesterase activity.

[ref. ID; 6115]

Test system

Reproduction test (28-days and 56-days)

Strains

E. andrei, also known as E. fetida andrei, obtained from Carolina Biologial Supply (Burlington, NC).

Toxicants

HMX and 2-Chloroacetamide.

Test design

ISO (1996) draft method.
Artificial soil consisted of 70% (w/w) grade 4010 silica sand, 20% (w/w) colloidal kaolinite clay and 10% 2-mm screened Canadian Sphagnum peat. Approximately 1% (w/w) calcium carbonate was used adjust the pH of the wetted substrate to 6.0+/-0.5. Water-holding capacity 75%. 1-L glass jars (500g of soil dry-based). 4 replicates. Food (2g of dry cereal) added to the surface once weekly.

Measurements/observations

Number of hatched cocoon and non-hatched cocoon, number and biomass of juveniles, number of biomass of adult.

Evaluations

LC50, EC10, EC25, EC50, NOEC, LOEC using ToxCalc program.

[ref. ID; 6131]

Test system

Reproduction bioassay (28-days and 56-days)

Strains

From a commercial source. Clitellate adults (300-600 mg).

Soil samples

Three sites, East Park Grass, East Park Wood and Peascroft Park Wood represent partially remediated (clay capped) coal slag areas. Two sites, Ladymoor Nature Reserve and Bilston Gas Works, were an unremediated metal refining and coal gasification sites, respectively. The sites would be contaminated with metals and polycyclic aromatic hydrocarbons (PAHs).

Test design

ISO (1998) procedure.

Measurements/observations

Numbers of hatched and unhatched cocoons, number of juveniles emerging per fertile cocoon.

[ref. ID; 6138]

Test system

OECD-style toxicity (28 days) test

Strains

From Ecology Earthworms, Hubbards Hall Farm, Bentley, Ipswich, Suffolk, UK. Mature adult (average weights 0.84+/-0.04 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), 20 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; 6710]

Test system

Cytotoxicity assay

Strains

E. andrei were from synchronized cultures bred in the ecotoxicology laboratories of the Department of Botany and Zoology at the University of Stellenbosch, South Africa.

Toxicants/concentrations

CdCl2 3.75-50 mg Cd kg-1.

Test design

Run 1: Eight specimens of pre clitellate E. andrei that originated from culture exposed to Cd for more than 10 generations were exposed OECD soil (OECD guidline 222) 400g to different concentrations of cadmium. Three replicates per exposure concentration, four replicates for controls. Exposures period 6 weeks. Temperature 20 degrees C, relative humidity 60%, darkness. The earthworms were fed every second week with 5 g of fresh, urine-free cattle manure.
Run 2: Ten specimens were exposed for 9 weeks in 500 g soil samples collected at different ultramafic sites in Barberton area, Mpumalanga Province, South Africa, containing high concentrations of metals such as chromium, manganese and nickel.

Measurements/observations

Bradford assay, MTT assay, NRR assay.

[ref. ID; 6722]

Test system

Influence of soil texture and organic matter on the avoidance test

Strains

Test design

Test soil: The artificial standard soil formulated by the Organization for Economic Co-operation and Development was modified by using different proportions of the same components to prepare test media belonging to different texture classes (coarse, median, and fine) and organic matter (2, 5, and 10% each soil texture class). pH 6+/-0.5. Moisture content 50% water-holding capacity.
Avoidance test: International Organization for Standardization (ISO) guideline 17512-1.2. A plastic box (length 20 cm; width 12 cm; height 5 cm) divided in two sections by a cardboad divider inserted transversally. Each section was filled with 250 g dry weight soil. 10 worms. 20+/-2 degrees C, and a 16:8 hr light:dark photoperiod for 48 hr.

Measurements/observations

Number.

Evaluations

Using the Fisher exact test.

[ref. ID; 6736]

Test system

Effect of chemical leaching process (METIX-AC)

Strains

From Carolina Biological Supply Company, Burlington, NC.

Toxicants

Sewage sludge (the Montreal Urban Community (non-digested sludge produced by physico-chemical wastewater treatment), the Becancour municipal wastewater treatment plants (aerobically digested sludge produced in sequential biological reactors)) + cadmium nitrate, copper chloride and zinc chloride.

Test design

Metals were added separately (+Cd, +Cu, or +Zn) or together (+HM) to obtained final concentrations of 100 mg Cd/kg dry wet, 3000 mg Cu/kg dry wet, and 5000 mg/kg dry wet to the sludges. Dehydrated and neutralized sludge samples were mixed with a topsoil supplied by the Montreal Botanical Garden to obtain an application rate of 45 g dry sludge/kg dry wet.
14-day lethality tests: The EPA standardized procedure (USEPA, 1989). Ten worms weighting between 0.3 and 0.6 g were exposed to 200 g of dry reference topsoil, or to the different mixtures of topsoil and municipal sludge. Water content of sludge-soil mixtures was adjusted to obtain 85% of water holding capacity. Potassium chloride was used as the reference toxicants. Triplicate at 20 degrees C.

Measurements/observations

Bioaccumulation of Cd, Cu, Zn in earthworms.

[ref. ID; 6744]

Test system

Effect of time and mode of depuration on tissue copper concentration

Strains

From Ecology Earthworms, Hubbards Hall Farm, Bentley, Ipswich, UK. Individuals with fully clitellate (mean fresh weights: 410+/-2.56 mg).

Toxicants/concentrations

Cu(NO2)3/3H2O: 250 mgCu kg-1.

Test design

Culture period 28 days.

Soil: Kettering sandy loam soil is similar in its properties to the OECD standard soil. Moisture content 50%.
Worm: Eighty earthworms were added to 12 kg of the treated Kettering loam. 20+/-1 degrees C, constant darkness.
Depration method: 1) Depuration time (0, 24, 36, 48, and 72 hr). After depuration the filter papers (Whatmann no. 540) were dried at 40+/-1 degrees C for 24 hr and re-weighed. The earthworms were rinsed in ultrapure deionised water, dried on tissue paper and immersed for 20sec in boiling ultrapure water. 2) After depuration for 0, 24, 36, 48 or 72 hr, the earthworms was dissected following immersion in boiling water for 20sec. The earthworms were slit open to expose the alimentary canal, which was cut from above the crop to the bottom of the intestine. Each earthworm was pinned open onto a wax board and a small brush was used to carefully remove the soil particles present in the crop, gizzard and intestine.

Measurements/observations

The concentration of Cu and Ti in worm tissues.

[ref. ID; 6817]

Test system

Effect on life-cycle parameters

Strains

From Carolina Biological Supply (Burlington, NC). Adult E. andrei (a mean wet weight ranging from 490 to 580 mg) having a well-developed clitellum were used.

Toxicants

TNT, RDX, HMX.

Test design

A sandy-type clean forest soil was obtained from a local supplier and had the following physical characteristics: 82.8+/-1.1% sand (50-2,000 um), 9.2+/-1.8% silt (2-50 um), 8.0+/-2.0% clay (<2 um); 14.3% moisture, 3.8% organic matter content, and pH of 7.6+/-0.4.
The effects of explosives-spiked soils on growth and reproduction of E. andrei were assessed using the ISO (1998) method. Survival and growth of adult earthworms were determined after 28 days of exposure, whereas reproduction parameters, including cocoon production and hatching and juvenile survival and growth, were measured after 56 days.

Experimental design

Glass jars (including 500 g of soil dry weight (60% water holding capacity) and 10 worms) were closed using lids with 1.6-mm air holes. Food (2 g dry cereal) was added to the surface of the test matrix at the beginning of the experiment and then once weekly.

Measurements

Number and weight of adult, juvenile and cocoon.

[ref. ID; 6831]

Test system

Cocoon production toxicity tests

Strains

Adult earthworms with a well-developed clitellum. Worm ages ranged between 8.5 and 15.5 weeks, and individual weights were between 170 and 582 mg.

Toxicants/concentrations

Cadmium chloride (0, 10, 18, 32, 56, 100 mg Cd/kg), chromium(III) nitrate (0, 10, 32, 100, 320, 1000 mg Cr/kg), paraquat dichloride (0, 20, 45, 100, 200, 450, 1000 mg/kg), parathion-ethyl (0, 10, 18, 32, 56, 100, 180 mg/kg), fentin (triphenyltin chloride) (0, 0.32, 1, 3.2, 10, 32 mg/kg), benomyl (0, 0.1, 0.32, 1.0, 3.2 mg/kg), pentachlorophenol (PCP) (0, 5, 10, 20, 40 60 mg PCP/kg dry soil), carbendazium (o, 0.6, 1.92, 6.0 mg/kg), phenmedipham (0, 1.62, 5.18, 16.2, 51.8, 162 mg/kg).

Test design

Artificial soil (dry weight, 10% sphagnum peat, 20% kaolin clay, ca. 69% fine sand, pH 6.0+/-0.5).
Reproduction toxicity test were performed as described by Van Gestel et al. (1989). Each test was performed at five or six test concentrations and a control. The tests had four jars per concentration, each jar containing 10 earthworms. Jars were loosely covered with a glass petri dish to prevent moisture loss by evaporation and incubated at 20+/-5 degrees C in an illuminated climatic chamber (ca. 400 lx). Exposure period 3 weeks.

Measurements/observations

Adult, juvenile and cocoon number and weight, cocoon hatchability (for 5 weeks in untreated artificial soil as described by Van Gestel et al. (1988).

Evaluations

LC50 using the trimmed Spearman-Karber method (Hamilton et al., 1977/1978).
EC50 values for the effect of the chemicals on cocoon production were determined according to a logit model.
NOEC using the Williams test (Williams 1971, 1972) and Student's t test or by analysis of variance (ANOVa) using the software package Genstat 5.

[ref. ID; 6835]

Test system

Biomarker

Strains

From a vermiculturist (V. R. Pell, Worm-Hive Organics, Newark, UK).

Toxicants

CuCl2

Test design

Neutral-red retention assay. Eisenia andrei were exposed to an increasing range of soil copper concentrations (mgCu kg-1 soil dry wt: 4 (control), 20, 40, 80, 160, 320) for a period of 28 days in clear containers (15x8x6 cm) with ventilated lids. Each container was filled with 600 g of copper-loaded forest soil with a gravimetric water content of 15% (approximately 50% of its soil water-holding capacity). Five replicates of each test concentration. 10 mature worms (mean weight +/-SE, 520+/-6.7 mg) per container. 3 g of oven-dried (70 degrees C, 24 hr) horse manure was remoisted with 10 ml of glass-distilled water and added weekly to the surface of the soil in each container. The containers were maintained at 15 degrees C, 70% ambient humidity, and with a 12/12-hr 295/25 lux lighting regime.

Measurements

Food consumption, number of adult, cocoon and juvenile, weight, hatchability. Neutral red retention time. Cu concentration in worm, food, and soil.

[ref. ID; 6842]

Test system

Impact of soil characteristics on the environmental bioavailability of heavy metal

Strains

Toxicants

Dutch field soils.

Test design

Uptake of As, Cd, Cr, Cu, Ni, Pb and Zn in 20 Dutch field soils (moderately contaminated sites) and in OECD artificial soil (OECD 1984 protocol).
Plastic jars (350 ml) were filled with 200 g of homogenized fresh soil at pF 2 humidity, and then 4 adult animals (15 weeks old) were added. 20+/-1 degrees C under constant illumination. No food. Exposure period 0, 1, 2, 3, 4, 7, 14, 21, 28, 42, and 63 days.

Measurements/observations

Body weight, metal concentrations in the worms.

Evaluations

BSAF

[ref. ID; 6857]

Test system

Prediction of metal accumulation

Strains

Adult worm.

Toxicants

Twenty soils were collected at moderately contaminated sites in the Netherlands. Pore water was collected from field-wet soils, immediately after field sampling.

Test design

The soil samples were put in 1-L glass jars, closed with a glass petri dish. Temperature (20 degrees C) under constant illumination. No food. Exposure period 3 weeks.

Measurements/observations

Metal (As, Cd, Cr, Cu, Ni, Pb, Zn) concentrations in worms and body weight.

Evaluations

Bioconcentration factors (BCFs).

[ref. ID; 6865]

Test system

Acute toxicity

Strains

From Carolina Biological Supply Co. (Burlington, NC). Adult E. andrei (ranging from 300 to 600 mg wet weight) having a well-developed clitellum were used.

Toxicants

2,4,6-Trinitrotoluene (TNT).

Test design

Artificial OECD-Type soil consisted of 70% (w/w) grade 4010 silica sand (Indusmin), 20% (w/w) colloidal kaolinite clay (CAS: 1332-56-7), 10% (w/w) 2-mm screened Canadian sphagnum peat, according to the OECD method (1993). Total organic carbon content of the artificial soil was 1.30% (w/w; 2.24% organic matter content). pH 6.0+/-0.5. Sandy-type forest soil was sampled from a clean site and had the following pedological characteristics: 3% clay, 92% sand, 5% silt; 17.4 and 4.24%, moisture and organic matter contents, respectively; pH 5.88, CEC of 2.6 meq/100 g dry soil.

Filter Paper Contact Test: Series 1 TNT in acetonitrile (solvent vehicle), Series 2 TNT aqueous solution. 10 replicates and five different concentrations (plus negative control and solvent vehicle control). 48 and 72 hr exposure period.
Soil Toxicity Tests: Two types of soil (forest and OECD). Using the protocol of the US EPA (1989) based on the method proposed by Edwards (1984; Goats and Edwards, 1982) and the OECD guidelines (1993). Experimental period 7 and 14 days.

Measurements

Mortality and changes in biomass (body weight).

Evaluations

NOEC, LOEC, CV, LC10, LC25, LC50.

[ref. ID; 6873]

Test system

Development of an alternative artificial soil and chronic toxicity

Strains

Age-synchronized adult E. andrei (450-500 mg wet weight) obtained from a culture kept at the Department of Zoology, University of Ruhuna, Matara, Sri Lanka.

Toxicants

Carbendazim, Carbofuran, Chlorphyrifos.

Test design

Modified artifical soils (MAS) were prepared according to the standard guidelines of OECD (1984) and ISO (1998), but replacing the (10%) sphagnum peat with a similar amount of finely ground non-composted coco peat, composted coco peat, paddy husk or saw-dust. Temperature 20 and 26 degrees C.

Measurements/observations

Mortality and weight change.

Evaluations

LC50, EC10, EC50.

[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%. 10 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; 6927]

Test system

Bioaccumulation

Strains

From Carolina Biological Supply. Clitellated earthworms weighing from 425 to 690 mg.

Toxicants

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and [14C]RDX.

Test design

Nonlabeled RDX: ASTM standard guidelines for soil bioaccumulation studies with some modifications. Instead of plastic containers, glass containers (250-ml glass jar) were used to avoid adsorption of RDX to the container walls. A natural soil (fine loamy, siliceous, mesic semiactive, Typic Hapludult) collected from a grassland field on the property of the U.S. Army Aberdeen Proving Ground. Two grams of dry cereal were added to each test unit. Sampling time were 0.25, 1, 2, 4, 7 and 14 days.
[14C]RDX: Test units were placed into separate earthworm accumulation microcosms constructed from clear polycarbonate vacuum desicators (inner diameter, 23 cm).

Measurements/observations

Worm weight. Tissue RDX or [14C]RDX concentration in worm.

Evaluations

BAF.

[ref. ID; 6948]

Test system

The growth and sexual development of juvenile earthworms

Strains

Juvenile earthworm.

Toxicants/concentrations

Cadmium chloride (0, 10, 32, 100, and 320 mg/kg dry soil), copper chloride (0, 10, 32, 100, and 320 mg/kg dry soil), pentachlorophenol (0, 0.32, 1, 4, 8, 16, and 32 mg/kg dry soil).

Test design

All tests were carried out in an artificial soil, with a dry weight of 10% peat, 20% kaolin clay, ca. 69% sand, and 0.5-1% CaCO3. The chemicals were mixed homogeneously through the soil. Soil equivalent to 400 (for the pentachlorophenol test) or 500 g dry weight was placed in 1-litre glass jars. Finely ground (<0.5 mm) cow dung was used as a food source, and 10 juvenile worms were added to each jars. Four replicates per concentration.

For the Cd test, the artificial soil was made up with finely ground (<0.5 mm) peat and a moisture content of 35% (w:w). Soil pH 6.7-6.8. 23 degrees C in the dark.
For the test with Cu 1 mm sieved peat was used, and the soil moisture content was 55% (w:w). Soil pH 6.2. 20 +/- 1 degrees C under continuous illumination (200-400 lux).
For the test with pentachlorophenol 1 mm sieved peat was used, and the soil moisture content was 55% (w:w).

Soil pH increased during the test from 5.8 to 6.3. 20+/-3 degrees C under continuous illumination (200-400 lux).

Measurements/observations

Worm weights and sexual development (tubercula pubertata (sub-adult) and clitella (adult)).

Evaluations

NOEC using an analysis of variance and the Williams test (Williams 1971, 1972).
EC50 using a logit model.
LC50 using the trimmed Spearmann-Karber method (Hamilton et al. 1977/1978).

[ref. ID; 6978]

Test system

2-wk LC50

Strains

Toxicants

3-chlorophenol (MCP), 3,4-dichlorophenol (DCP), 2,4,5-trichlorophenol (TCP), 2,3,4,5-tetrachlorophenol (TeCP), pentachlorophenol (PCP), 2,4-dichloroaniline (DCA), 1,2,3-trichlorobenzene (TCB).

Test design

Soil type: OECD artificial soil, two different sandy soil, and peaty soil.
Tests carried out at least in duplicate, with at least 5 concentrations and a control.

Evaluations

LC50 using trimmed Spearman-Karber method. Quantitative structure-activity relationships (QSARs).

[ref. ID; 7018]

Test system

Strains

Adult worms with a clitellum.

Toxicants

Hexachlorobenzene, Pentachlorobenzene, 1,2,3-Trichlorobenzene, 1,2,3,4-Tetrachlorobenzene.

Test design

Elimination kinetics: 50 worms were exposed to soil (OECD artificial soil, water content 35%) contaminated with 1,2,3-Trichlorobenzene (7.4 mg/kg) and 1,2,3,4-Tetrachlorobenzene (13.2 mg/kg), and another 50 worms were exposed to soil contaminated with Pentachlorobenzene (13 mg/kg) and Hexachlorobenzen (51 mg/kg). After 7 days, worms were transferred to uncontaminated soil. They were placed in groups of eight worms per jar. Temperature 17 degrees C.
Bioaccumulation: The accumulation of Hexachlorobenzene (10 mg/kg dry soil), Pentachlorobenzene (10 mg/kg dry soil), 1,2,3,4-Tetrachlorobenzene (10 mg/kg dry soil) in earthworms (weight 298+/-42 mg) kept in soil was determined by analyzing five earthworms at various exposure times (0, 2, 4, 7, 10, 13, 17, 24, 31, 40, and 49 days).

Measurements/observations

Chlorobenzenens concentration in worm.

Evaluations

Elimination kinetics: Half-lives for the elimination of chlorobenzenes.
Bioaccumulation: BSAFs, BAFs, and BCFs.

[ref. ID; 7030]

Test system

14-d LC50 and biotransformation

Strains

Mature adults with a clitellum and a wet mass between 30-600 mg.

Toxicants

2,4,6-Trinitrotoluene (TNT).

Test design

Temperature (20+/-2 degrees C) and light (cool white, 400 lux, photoperiod of 16 light hr:8 dark hr).

Filter paper experiment: Individually placing 20 worms into separate glass tubes lined with filter paper (60 cm2) and containing 1 ml of an aqueous solution of TNT 55 (mg/L). Exposure period 48 hr.
Soil exposure experiment: 1-L glass containers with perforated caps and chemically inert and porous geotextile. 10 worms per glass jar (200 g dry wt test soil, soil moisture content 20% v/W). TNT concentration 4-200 mg/kg. Triplicate. Exposure period 8 hr-14 day. Control soil is a field-collected, noncontaminated forest soil.

Measurements/observations

Number and weight of worm. 2,4,6-Trinitrotoluene and metabolites concentration in worm tissue.

Evaluations

LC50 with ToxCalc v.5.0.18 using the maximum likelihood probit analysis.

[ref. ID; 7082]

Test system

Intestinal uptake

Strains

Adult with a clitellum.

Toxicants

2,2',4,4',5,5'-Hexachlorobiphenyl (PCB 153), Hexachlorobenzene, Octachloronaphthalene, Pentachlorobenzene, and 1,2,3,4-Tetrachlorobenzene.

Test design

Uptake from food (cow manure). 1,000 ml jars contained 310 g artificial soil with a water content of 35%.

Single oral dose: At the start, 5 worms (total weight 1584+/-131 mg) were fed by adding 570 mg manure (on dry weight basis) that was contaminated with a nominal concentration of 105 ug/g 1,2,3,4-Tetrachlorobenzene, 108 ug/g Pentachlorobenzene, 103 ug/g Hexachlorobenzene, 100 ug/g PCB 153, 100 ug/g Octachloronaphthalene. Sampling times were 3, 5, 8, 12, and 16 hr and 1 and 2 days.
Multiple oral dose: The uptake of penta- and hexachlorobenzene from food by earthworms was studied by feeding the worms twice a week with contaminated manure. 5 worms (total weight 1820+/-4 mg) were fed 570 mg (on dry weight basis) on the first day of the week and 340 mg on the fourth day of the week. The manure was contaminated with 203 ug/g pentachlorobenzene and 198 ug/g hexachlorobenzene on dry weight basis. Exposure period 10 weeks.

Measurements/observations

Body burdens of test chemicals in worms.

Evaluations

Uptake efficiency, uptake rate constant, and biomagnification factors.

[ref. ID; 7088]

Test system

Bioaccumulation test

Strains

From in-house cultures at Stantec Consulting Ltd., Guelph, Ontario, Canada. Sexually mature adult worms, each with a clitellum and weighing 395.9+/-57.8 mg wet wt.

Toxicants/concentrations

Cadmium chloride hemipentahydrate: Cd 5 mg/kg soil dry. Zinc chloride: Zn 100 mg/kg soil dry.

Test design

Test method and procedure were standardized by following the draft OECD guidance (Egeler et al. 2009).
Soils were prepared by spiking an artificial soil with the metal salt and allowing the mixture to equilibrate under test condtions before adding earthworms to the soil. After addition of earthworms to the test soils for a period of time (i.e., the uptake phase), the earthworms were transferred to clean control soil for a period of time (i.e., the elimination phase).

Uptake phase (21 days): 50 g of each test soil and the control soil were allocated to 125-ml glass test vessels (soil depth ~5cm). One worm per vessel. Test vessel incubated under room temperature (22.5+/-1.5 degrees C), a light regime of 16:8-hr light:dark, the ligth intensity ~150 lux, maximum holding capacity 60%.
Elimination phase (21 days): Worms pre-exposed during the uptake phase were transferred to clean soil.

Measurements/observations

Metal concentration in worm tissues and soil.

Evaluations

BAFs.

[ref. ID; 7089]

Test system

Bioaccumulation test

Strains

Earthworm acclimation: Six kilograms of Webster soil (2.4% organic carbon, 35.6% clay, pH 5.5) was spiked with Cd(NO3)2 solution to achieve a soil Cd concentration of 20 mg/kg (dry wt) and a moisture content of 50% of the Webster water-holding capacity (48%), and left overnight for equilibration at 20+/-2 degrees C. E. andrei were exposed to the acclimation substrate for 28 days.

Toxicants

Cd(NO3)2

Test design

224-d bioaccumulation test was conducted in test soils with both acclimated and unacclimated worms according to procedures for bioaccumulation tests provided by Environment Canada (2004).

Test soil: Webster soil were spiked with Cd(NO3)2 to achieve total soil Cd concentrations of 20 and 100 mg/kg (dry wt). Control was unamended Webster soil.
Bioassays: Ten worms were placed on the surface of the soil (200 g dry wt) in each test chamber (glass mason jars; 500 ml; Ball), and test chambers were sealed with a perforated metal lid (one hole, ~2.0 mm, to allow gas exchange) and screw collar. The test chambers were maintained under continuous fluorescent lighting at 20+/-2 degrees C. Worms were fed with 25 g of separated dairy solids (Ohio Agricultural Research and Development Center) once a week.

Measurements/observations

Cd concentration in worm (whole and subcellular fractions).

[ref. ID; 7158]

Test system

Influence of soil organic matter content on elimination rate

Strains

Adult with a clitellum.

Toxicants/concentrations

Hexachlorobenzene (10.7+/-1.3 mg/kg), Pentachlorobenzene (10.9+/-0.9 mg/kg).

Test design

Three types of OECD artificial soil were used, differing in 2mm-sieved peat (OM) content: 3%, 10%, and 20% OM. Because peat is the major water holding component of the soil, the absolute water content differs with varying organic matter content.
Prior to the elimination, three groups of 40 worms each were exposed to OECD artificial soil (10% OM, 1000 mL jars containing 300 g soil (dry weight basis)) contaminated with pentachlorobenzene and hexachlorobenzene for 7 days.
After 7 days, worms were transferred to three uncontaminated OECD artificial soils differing in their OM content. The worms were placed in groups of five per jar containing 200 g soil (or 150 g soil for the 20% OM) and sampled at several times until 34 days of elimination. Every 10 days the remaining worms were transferred to new uncontaminated OECD soil. All tests were carried out at temperature 18 degrees C in complete darkness.

Measurements/observations

Hexachlorobenzene and Pentachlorobenzene concentrations in worm.

2. Eisenia fetida (= Eisenia foetida)

The two acute toxicity tests using Eisenia fetida recommended by the OECD (1984) and EEC (1985) have become routinely used in the risk assessment and regulation of new and existing chemicals. (ref. ID; 1915)
Eisenia fetida is an ultra epigeic species (living almost entirely in organic matter) currently used as the standard earthworm in terrestrial ecotoxicology test in the European Union. The current Organisation of Economic and Cooperative Development acute earthworm toxicity test (OECD, 1984) also uses the earthworms E. fetida/andrei Bouche as biological monitors for testing effects of contaminants on soil biota. These species are readily available through suppliers and are easy to culture in laboratories. The two species are very similar, both in their physiology and their mode of life. However, many European species of earthworm behave differently to E. fetida/andrei. Differences in the mode of life of earthworm species may affect the exposure of earthworms to contaminants, and consequently the accurate risk assessment of contaminated sites. (ref. ID; 6138)
E. fetida is a litter dwelling species of epigeic earthworm feeding mainly on organic particles. (ref. ID; 6703)
E. fetida was selected as the reference earthworm in the international toxicity tests (International Standard Organization 1993, 1998; OECD 2004) because it is robust and can easily be cultured in large quantities in the laboratory; it matures in 8 weeks; exhibits a higer reproductive rate and a shorter generation time than other species and is responsive to a wide range of toxicants. This reasoning holds equally well for accumulation studies and in addition E. fetida is now readily available from commercial suppliers (though it should never be assumed that the identity of the species correct or that only one species is present in the supply), researchers have experience using the earthworm and a large quantity of data exists on its response to metals. However, the choice of E. fetida in toxicity and accumulaion studies has been a source of criticism, principally because it is not a natural soil species. It inhabitats organic rich habitats such as compost and manure heaps (Bouche 1972). Additionally some authors have found that E. fetida is less sensitive to contaminants than other species (Langdon et al. 2005; Spurgeon & Weeks 1998) and that accumulation is species dependant (Langdon et al. 2005; Spurgeon & Hopkin 1996). (ref. ID; 6739)

[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

Lethality test (14d-LC50): Ten worms were placed in glass containers with 500 g dry mass of a soil-like substrate composed of a mixture of sphagnum peat, kaolinite clay, industrial quartz sand in a dry-weight ratio of 1:2:7. The pH (1N KCl) was adjusted to 6.0+/-0.5 with calcium carbonate. The water content was kept at 55% on a dry mass basis. Temperature 15 degrees C. Forty worms were tested at each test concentration.
Reproduction test (14d-EC50): Similar above experimental conditions, however, some modification following; a) the replacement of the artificial soil with a natural sandy soil (Kooyenburg) containing 3.7% organic matter, 1.4% clay, and pH (1N KCl) 4.8, and b) the supply of coarsely groud air-dried leaves of alder, Alnus glutinosa, for food.

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; 1915]

Test system

14-days acute toxicity and 21-days population growth rate

Strains

Commercial supplier; adult with fully clitellate, mean weight 260 (190-480 mg).

Toxicants/concentrations

ZnNO3/6H2O (0, 190, 350, 620, 1200, and 2000 ug Zn/g dry weight soil).

Test design/concentrations

Artificial soil medium (70% sand, 20% kaolin clay and 10% organic matter (as Sphagnum peat), pH 6.0+/-0.5 by the addition of powdered calcium carbonate.
Influence of temperature: 15, 20, 25 degrees C, 4 replicates.

Measurements/observations

Mortality, cocoon production rates.

Evaluations

LC50, EC50.

[ref. ID; 3310]

Test system

56-days sublethal toxicity

Strains

This strain were obtained from a stock maintained in this laboratory over several years, originating from garden compost heaps. Freshly hatched earthworm of the same age (+/- 4 days, kept without feeding, mean weight 3.5+/-0.6 mg) were used.

Toxicants/concentrations

Copper oxychloride (= Virikop). Cu concentrations 0, 3.3, 10, 33, 100, and 330 mg Cu/kg-substrate.

Temperature

25 degrees C.

Test design

The substrate used consisted urine-free cattle manure that had been previously dried, ground, and sieved (particle size 500 < /_ 100 um). The gravimetric water content of substrate was adjusted to 78%. 4 replicates.

Measurements/observations

Cocoon production, earthworm growth, maturation time, survival rate, reproduction success, total number of hatchlings produced.

Evaluations

Difference between the control and the contaminated variants were checked for significance by means of analysis of variance (ANOVA).

[ref. ID; 3313]

Test system

Acute and sublethal toxicity

Toxicants

Mancozeb (Dithane M-45 wettable powder).

Test design/concentrations

Acute toxicity: 14-days

Strains; Adult with an average biomass of 200 to 600 mg.
Method; OECD Guideline 207 (OECD, 1984): Artificial soil consisted of 70% silica sand, 20% kaolin clay and 10% peat moss thoroughly mixed and pH 6.0+/-0.5 with the addition of calcium carbonate.
Concentration; 0, 400, 800, 1200, 1600, and 2000 mg/kg.
Measurements/observation; Mortality.
Evaluation; LC50 by probit analysis.

Sublethal toxicity: 10 weeks

Strains; 25 days old with an average biomass of 100 to 200 mg.
Experimental conditions; The substrate used was urine-free cattle manure, which was sundried and ground to give a particle size between 0.5 and 1.0 mm, moisture content 75% and pH 7.5-8.0. 5 replicates.
Concentration; 0, 8, and 44 mg/kg.
Measurements/observations; Weight and production of cocoons and juveniles.
Evaluations; Any significant differences for growth and reproduction calculate by ANOVA.

[ref. ID; 4447]

Test system

Acute & chronic toxicity assays

Strains

From a commercial earthworm breeding farm.

Toxicants

Arsenate.

Test design/concentrations

Artificial soil (OECD Guideline 207, 1984) + 2% (dry weight) finely ground cow drug + Na2HAsO4 (0, 10, 18, 32, 56, and 100 mg As/kg dry wt) x 4 replications, constant illumination (400-800 lux). Temperature 20+/-1 degrees C.

Measurements/observations

Mortality, number of cocoons.

Evaluations

EC50 by the probit method, LC50 by the moving average method, NOECs by Kruskal-Wallis ANOVA followed by post-hoc multiple comparisons.

[ref. ID; 4582]

Test system

Acute toxicity

Toxicants/concentrations

Ortho Groundclear Total Vegetation Killer (5% glyphosate by mass (as isopropylamine salt): herbicide), toxicant concentration: nominal concentration recommended for application (Groundclear:water=1:4), 1/10, 1/100, 1/1,000, 1/10,000, water x 5 replications (N=30 worms per treatment).

Test design

Run 1: 21 plastic boxes (commercial potting soil 2 L + 20 worms). 82 ml aged tap water (control), 82 ml nominal solution of Groundclear, 82 ml of 1/10th the nominal solution of Groundclear; final soil moisture of approximately 40% by mass at a temperature 16 degrees C. 24 hr. The numbers of dead worms counted.
Run 2: Plastic boxes separated into two halves by a plastic divider. One side was placed 1 L of potting soil moistened with aged water, and on the other was placed 1 L of soil moistened with a nominal solution of Groundclear. After the plastic divider was removed, twenty worms were placed at the mid-point where the two soil types were adjacent. 24 hr (N=7 replications). The numbers of worms present in each soil type were counted.
Run 3: Worms were placed in potting soil moistened either with aged tap water (control) or a nominal solution of Groundclear. After 10 hr of exposure, worms were removed. The worms placed at one end of a plastic box 30 cm in length and floored with a paper towel that had been moistened with aged tap water. A 40 watt white then was positioned at the end of the box in which the worms had been placed, and we counted the number moving >20 cm away from the light/heat source within 30 min.

Evaluations

Run 1: 48 hr mortality (two-tailed Kruskall-Wallis one-way ANOVA).
Run 2: 24 hr locomotive activity (two-tailed Kruskall-Wallis one-way ANOVA & two-tailed Wilcoxon signed-ranks test).
Run 3: Apparent stimulation of avoidance response (two-tailed, Mann-Whitney U test).

[ref. ID; 4631]

Test system

Comet (single cell gel electrophoresis) Assay

Strains

Eisenia fetida Savigny 1826, Synchronized adult.

Toxicants

NiCl2

Test design/concentration

In Vitro: Exposure concentration (0, 2, 6, 12 ug/ml), 10 replications, 1 hr in the dark at 4 degrees C.
In Artificial Soil Water: Using the protocol provided by Kiewiet & Ma (1991). Exposure concentration (0.0, 0.0049, 0.0087, 0.0175, 0.025 mg/l), 48 hr in the dark at 20 degrees C.
In Substrate: Exposure concentration (0, 60, 240, 480 mg/kg), 2 replicates. 200 g of washed, dried cattle manure in a 180x110x85-mm plastic container to attain a moisture 75%. The prepared substrates were left for 48 hr in a climate-controlled room at 20 degrees C and 70% RH to stabilize.

Measurements

Coelomocytes.

[ref. ID; 4953]

Test system

Single- and multiple-occupancy test

Strains

Eisenia fetida (Savigny, 1826) obtained from Ecology Earthworms (Hubbards Hall Farm, Bentley, Ipswich, Suffolk, UK). Earthworms (fresh mass range 0.22-0.52 g, mean+/-SD=0.34+/-0.06 g, n=2) used in the test.

Toxicants/concentrations

Pb(NO3)2: Pb concentration: 0 (= control), 1000, 1800, 3000, 5000, 7500, and 10000 mg Pb/kg soil.

Temperature

20 degrees C.

Test design

The soil used in the tests was Kettering loam, obtained from Turf Management Systems (Iver Heath, Buckinghamshire, UK). This soil is almost identical to the OECD standard soil. The soil is texturally a silty loam and contains 2.8+/-0.7% (n=24) organic matter; the mineral fraction of the soil is 79+/-2% (n=3) quartz and 14+/-2% (n=3) clay (predominantly kaolinite).
The toxicity tests were based on the OECD earthworm acute toxicity test. The air-dried and sieved soils were weighed into plastic bags -500 g for the multiple-occupancy tests (four replicates) and 50 g for the single-occupancy tests (six replicates). Single earthworms were added to the 50 g soil masses and 10 earthworms to the 500 g soil masses.

Measurement/observations

Mortality, weight change, and metal uptake.

Evaluations

EC50, LC50, Median lethal concentrations (probit analysis with SPSS Version 10.1 (Chicago, IL, USA)).

[ref. ID; 4954]

Test system

14-d acute toxicity test

Strains

Eisenia fetida obtained from a commercial supplier (Vermitechnology Unlimited, Orange lake, FL, USA). Worms used in all tests were adults (>60 day old) with a clitellum.

Toxicants

Polycyclic aromatic hydrocarbons (PAHs): Naphthalene, Acenaphthylene, Acenaphthene, Fluorene, Phenanthrene, Anthracene, Fluoranthene, Pyrene, Benzo[a]anthracene, Chrysene, Benzo[b]fluoranthene, Benzo[k]fluoranthene, Benzo[a]pyrene, Indeno[1,2,3-cd]pyrene, Dibenz[a,h]anthracene, Benzo[ghi]perylene.

Temperature

19-25 degrees C.

Test design/concentrations

The soils used in this work included four PAH-contaminated soils (gasoline- and diesel-range organics) and seven lamp-black-containing PAH-contaminated soils obtained from manufactured-gas plant sites. Appropriate amounts of contaminated test soils were mixed with control soil to yield concentrations of 0, 6.25, 12.5, 25, 50, and 100% of each soil (200 g).
Methods used for 14-d toxicity testing with E. fetida were those outlined in ASTM method E 1676-97. 3 replicates. Continuously fluorescent light.

Measurement/observations

Number of surviving worms, PAHs concentrations of worm tissue.

Evaluation

LC50.

[ref. ID; 5974]

Test system

The 21-d earthworm reproduction test

Toxicants

Sb2(SO4)3, Sb2(C4H4O6)3/6H2O, BaO, Ba(NO3)2, Ba(C2H3O2)2, BaSO4, BeSO4/7H2O

Tests design

International Standard Organization [ISO 11268-2].

Measurements/observations

Adult survival and cocoon production.

Evaluations

NOEC, LOEC, EC20, EC50. [ref. ID; 5975]

Test system

Acute (14-d) & chronic (28-d, 56-d) toxicity tests

Strains

Originally were obtained from Carolina (Burlington, NC, USA), adult clitellate worms (0.3-0.6 g).

Toxicants

Sodium tungstate Na2WO4/2H2O, Lead nitrate Pb(NO3)2

Temperature & light condition

21+/-1 degrees C, continuous lighting.

Test design/concentrations

Soil: Artificial soil (OECD protocols: Guideline 207) & Field soil (silty loam soil of the Grenada-Loring soil series

Acute toxicity: In amber-glass, 60 ml, wide-mouth jars containing 40 g of either field soil or artificial soil. Each of the 10 concentration and the control had 10 individuals jars with one worm per jar.
Chronic toxicity; In clear-glass pint jars containing 250 g of field soil. Each of the 10 concentration and the control had five replicate containers with five worm per jar. Dried fermented alfalfa (2 g/container) was provided for food.

Measurements/observations

Number of adults, cocoon and juveniles.

Evaluations

LC50.

[ref. ID; 6001]

Test system

Intracellular Cr(IV) reduction

Strains

From a horse compost heap.

Toxicants

Potassium dichromate

Test design

In vitro experiments

Microsomal fraction: Microsomal preparations were incubated for 1 hr at 20 degrees C in the presence of potassium dichromate (20 uM Cr). Incubation mixture: microsomes 1 mg protein/ml, glucose 6-phosphate 10 mM, glucose-6-phosphate dehydrogenase 7 units/ml, MgCl2 10 mM, Mops (3-N-morpholinepropanesulfonic acid) 90 mM, NADP 0.3 mM (pH 7.4).
Mitochondrial fraction: Mitochondrial suspensions (0.5 mg protein/ml) were incubated in the presence of potassium dichromate (20 uM Cr) at 20 degrees C for 15 min.
Cytoplasmic fraction: Cytoplasmic fraction were incubated in the presence of potassium dichromate (20 uM Cr) at 20 degrees C for 20 min. Incubation mixture: 1) for DT-diaphorase assay, 2) for aldehyde oxidase assay.

In vivo experiments A Cr(VI)-enriched quartz sand was used as an artificial soil. This sand consisted of large-size granules (3-4 mm in diameter), which could not be ingested by earthworms. This procedure is conceptually similar to that used in the "contact filter paper test", but it has the advantage of recreating a more natural environment for earthworm. Artificial soil 400 g + dichromate solution 350 ug Cr/60 ml distilled water + earthworm 40 specimens, 20 degrees C, 90% relative humidity, incubation time (30, 150, and 360 min).

Measurements

In vitro experiments: Cr(VI) concentration in the incubation mixture, oxygen consumption.
In vivo experiments: Total amount of Cr(VI) in the artificial soil.

[ref. ID; 6016]

Test system

Uptake and elimination kinetics (28 day)

Strains

From a commercial earthworm breeding plant.

Toxicants

Zinc (56, and 560 mg/kg dry weight) and Cadmium (56, and 560 mg/kg dry weight).

Temperature/light

20 degrees C and a constant illumination 400-800 lux.

Test design

OECD standard artificial soil (70% sand, 20% kaolin clay, and 10% finely ground Sphagnum peat, adjusted to pH 6 with CaCO3).

Measurements

The internal metal concentration.

[ref. ID; 6067]

Test system

Uptake of bound residues of Bentazon

Test design

20 individuals were held for 14 days in two soil types (sandy loam and loamy sand), both containing bound residues of [14]C-labelled (phenyl-u-[14]C) bentazon. The soil water content was adjusted to 17% (weight).

Measurements

Concentrations of [14]C-substances in different tissues of earthworm.

[ref. ID; 6069]

Test system

The effect of hydration, desiccation, cold stress, hypoxia, saline load, heavy metal (chromium and strontium) on chloragogenous tissue

Strains

Manure worms were obtained from a stock culture in our laboratory. Juvenile specimens (average live wt 178+/-32 mg).

Toxicants

Paraquat (250 ug/ml) for 2-4 weeks.

Test design

The culture medium consists of peaty marshland soil and horse manure in proportions 1:1 (w/w).

Measurements

Nuclear volume of the chloragocytes using by karyometric methods, elemental composition of chloragosomes using by electron microscopic X-ray microprobe analysis.

[ref. ID; 6074]

Test system

Correlation between data from Artificial Soil Test (OECD, 1984) & Field Test

Toxicants

Azinphos-methyl, Benomyl, Captafol, Captan, Cyfluthrin, Endosulfan, Ethiofencarb, Fenamiphos, Imidacloprid, mercaptodimethur, oxydemeton-methyl, Propoxur.

Field site (Sampling site)

Pastures near Leverkusen (Germany). The pastures have been covered by grass for more than 10 yr, and have a high abundance (up to 800 individuals m-2) and high diversity (up to 8 species) of earthworms. Plots of 10x10 m were treated in duplicate with the highest registered rate of each pesticide and with 4 times this rate.

Evaluation

LC50 and EEC (estimated environmental concentration).

[ref. ID; 6076]

Test system

24-hr acute toxicity

Toxicants

Carbaryl, Paraoxon.

Temperature

24-27 degrees C.

Test design

The standard OECD tests.

Measurements

Survival, Cholinesterase activity.

[ref. ID; 6100]

Test system

Acute toxicity (5-day)

Strains

Adult earthworms (300-500 mg), obtained from Carolina Biological Supply (Burlington, NC).

Toxicants/concentrations

PCB (Aroclor 1254) 0, 2.5, 5.0, 6.25, 10.0, 12.5, 25.0, 50.0, 75.0 and 100.0 ug cm-2.

Test design

Worms were exposed individually for 5 days to each of nine nominal PCB concentrations on Whatman #1 filter paper cylinders that completely lined the interior walls of 3x8 cm glass vials. PCB was applied evenly using a glass pipette to the filter paper in 1 ml acetone. After solvent evaporation, filter papers were moistened with 1 ml distilled-deionized water and earthworms introduced. Vials were sealed with plastic caps and placed into environmental chambers without light at 20+/-1 degrees C.

Measurements/observations

Mortality, PCB concentration of worm, the numbers and percentages of coelomocytes forming ERs (erythrocyte rosette) and SRs (secretory rosette), and phagocytosing RRBCs (rabbit red blood cells).

Evaluations

LC50 and LD50 by probit analysis.

[ref. ID; 6103]

Test system

Acute toxicity test

Strains

From a local vermiculturist. All worms were adult, at least 8 weeks old and fully clitellate.

Toxicants/concentration

Cadmium nitrate Cd(NO3)2/4H2O, Copper nitrate Cu(NO3)2/3H2O, Lead nitrate Pb(NO3)2, and Zinc nitrate Zn(NO3)2/6H2O. The concentrations of the four metals in the soils were (in ug/g dry weight of soil): Cadmium 5, 20, 80, 300, Copper 10, 40, 200, 1000, Lead 100, 400, 2000, 10000, Zinc 100, 400, 2000, 10000.

Test design

OECD guideline No. 207 (OECD, 1984), 10 worms per plastic box (275x155x95 mm), 4 replicates. The experiment was run for 56 days (8 weeks) at 20 degrees C in constant light.

Measurements

Mortality and growth were measured on a weekly basis by counting and weighting the surviving worms in each container. Cocoons were collected by sorting through the soil each week and were incubated on moist filter paper at 20 degrees C until all juveniles had emerged.

Evaluations

LC50 (14 day and 56 day) by probit analysis with the SAS software package.
EC50 cocoon production (56 day) were obtained from a logit model analysis in SAS.
NOEC mortalities (56 day) and NOEC cocoon production (56 day) were calculated using the Williams test.

[ref. ID; 6110]

Test system

Acute toxicity in different exposure systems (in solution, in artificial soil, and on filter paper), sperum deformity, and comet assay

Strains

Healthy earthworms of about 120 days old and weighting about 350 mg were used for all experiments.

Toxicants

Imidacloprid, RH-5849.

Test design

Acute toxicity

In solution: Earthworms were half immersed in distilled water in beakers and were incubated at 20+/-1 degrees C for 48 hr to make them purge their gut contents. Earthworms were put individually in glass tubes (3x8 cm) containing 1 ml of different concentrations of pesticide solution (Imidacloprid: 0.24, 0.48, 0.96 and 2.00 mg/l, RH-5849: 118.75, 237.50, 475.00, and 950.00 mg/l). 6 replicate, 20+/-1 degrees C, 48 hr incubation.
In artificial soil: Artificial soil prepared according to OECD (1984) with a water content of 35% on a wet weight basis. There are three 1-litre, open-mouth glass jars for each concentration and 10 worms were added to each jar. Earthworms were incubated in uncontaminated artificial soil for 24 hr and then exposed to different concentrations (Imidacloprid: 1, 2, 4, 8, and 16 mg/kg dry soil, RH-5849: 125, 250, 500, 1000, 2000, and 4000 mg/kg dry soil) of pesticides at 20+/-1 degrees C for 14 days in jars.
On filter paper: The standard test procedure recommended by the CEPA (1990). The compound was dissolved in acetone and 1 ml of solution was added to the paper in the glass tube (3x8 cm). After evaporating the acetone, 1 ml of distilled water was added to keep the paper moist and one earthworm was placed in the tube. 6 replicate. Exposure concentration (Imidacloprid: 0.004, 0.020, 0.100, and 0.500 ug/cm2, RH-5849: 160, 320, and 480 ug/cm2).

Sperm deformity
Earthworms were exposed to pesticides (Imidacloprid: 0.1, 0.2, and 0.5 mg/kg dry soil, RH-5849: 25, 50, and 100 mg/kg dry soil) in artificial soil. 6 earthworms were exposed at each concentration for 10 days.

Comet assay
Earthworms were exposed in solutions of pesticides (Imidacloprid: 0.05, 0.1, 0.2, and 0.5 mg/l, RH-5849: 5, 25, 50, and 100 mg/l) on ice for 2 hr. 3 earthworms were used for each concentration. The comet assay was performed as described by Singh et al. (1988). Tween-80 (1%) was used as negative control and 100 ug/l mitomycin C as positive control.

Measurements

Acute toxicity: Mortility.
Sperm deformity: Abnormalities in sperm morphology by using optical microscope (x 400).
Comet assay: Arbitrary units.

Evaluation

Acute toxicity: LC50 by the TSK (Trimmed Spearman-Karber Program, Version 1.5).
Comet assay: Statistical comparisons between the grade of DNA damage in control/exposed groups were made by using Kruskal-Wallis test.

[ref. ID; 6112]

Test system

The effect of acute toxicity test (48-hr LC50) on difference of generation

Strains

Juvenile worms with individual weights of between 120 and 260 mg (parent, F1, and F2).

Toxicants

Zn(NO3)2/6H2O

Test design

The contact filter paper test: In glass vials lined with strips of standard filter paper (Whatman No. 1), one worm, 20 degrees C, constant dark. Zn concentrations (48.3, 72.5, 84.6, 96.7, 109, 121, 133, 145, 169, 193, 216, 242, 266 and 290 ug Zn ml-1), 30 replicates.

Measurements

Mortality.

Evaluations

LC50, LC90, and LC99 and log-probit slope parameters were calculated by probit analysis using the MicroProbit 3.0 statistical software package.

[ref. ID; 6124]

Test system

OECD earthworm toxicity test

Strains

From Blades Biological (Cowden, Edenbridge, Kent, UK). Clitellate adults that were at least 3 months old and weighed 290-470 mg.

Toxicants

PbS (galena), PbCO3 (cerrusite) and Pb(NO3)2. Pb was added to the soil as solid. 625, 1200, 3125, 5000, 6250, 8000 and 12 500 ug Pb g-1 of soil.

Test design

OECD draft guideline for the testing of chemicals (OECD, 2000).
Sterilised Kettering Loam was dried at 40 degrees C and sieved to <2 mm. Total organic carbon 2.8+/-0.7% (n=24), cation exchange capacity 17.6+/-92 Meq 100 g-1 (n=24), pH 6.7+/-0.5 (n=24), water holding capacity was 48+/-16% (n=24). 4 replicates of each concentration and each of the Pb compounds, 8 replicates of Pb-free controls.

Measurements/observations

Number and weight of adult worm, cocoon production.

Evaluations

LC50 using Trimmed Spearman-Karber statistics, NOEC by ANOVA followed by Tukey's comparison. (after 7, 14, 28, 56 days).
EC50 using the Linear Interpolation Method, NOEC (28 days).

[ref. ID; 6125]

Test system

OECD earthworm toxicity test (the influence of time on lead mobility and bioaccumulation)

Strains

Toxicants

Lead nitrate

Test design

Experiment 1: OECD draft guideline for the testing of chemicals (OECD, 2000).
Sterilised Kettering Loam was dried at 40 degrees C and sieved to <2 mm. The pH was 6.8+/-0.4 and the mean water holding capacity was 48+/-16%. Lead nitrate was added in solution form to 500g subsamples of soil to give concentrations 100, 400, 1000, 2000, 3000, 5000, 7000, and 10,000 ug(Pb)/g(soil). Sufficient water was used to give a water content of 40% of water holding capacity. There were four replicates of each concentration and eight controls. The amended soil was left for 7 days before the addition of the worms (10 per replicate) and the experiments were left to run for 28 days. Adult, clitellate worms weighing 260-350 mg were acclimatised in uncontaminated test soil for 1 week before introduced to the lead amended soil. The mortality of the worms was assessed each week and the concentration of lead in the survivors measured after 28 days. The LC50 was determined by Trimmed Spearman-Karber and the NOEC was determined by ANOVA followed by Tukey's comparison. The EC50 for growth was determined by the Linear Interpolation Method.
Experiment 2: An experiment was also set up with concentrations of lead of 3000 and 5000 ug(Pb)/g(soil). Lead was added to the soil in the form of lead nitrate solution to give water contents of 40% of the soil water-holding capacity. Twenty-one 500 g samples of lead amended soil were established at each concentration. The amended soil was left for 7 days before the addition of 10 worms to each 500-g of soil. After 3, 7, 10, 14, 17, 24, 28 days worms from 3 of the 500 g soil samples at each Pb concentration were collected. Numbers of surviving worms were determined and tissue concentration of Pb in these worms was determined.
Experiment 3: Lead nitrate solution was mixed with 15 kg of soil to give a final concentration of 7000 ug(Pb)/g(soil) and a water contents of 40% water holding capacity. The soil was stored at 20 degrees C. At times 0, 7, 10, 14, 17, 21, 28 and 35 days after the addition of the lead, triplicate samples of 600 g of soil were taken and 10 worms added of each replicate. The time to death for all of the worms was determined.

[ref. ID; 6134]

Test system

Early-phase immunodetection of metallothionein and heat shock proteins

Strains

0.3-0.4 g b.w., were collected from the stockbreeding maintained in the Institute of Zoology of the Jagiellonian University.

Toxicants

Heavy metal (Zn, Cu, Pb, Cd) chlorides

Temprature

22 degrees C.

Test design

Filter paper protocols (Fitzpatrick et al., 1996), 3 days.

Measurements/observations

Heavy metal concentration in the body.
Coelomocytes number and viability (Tripan Blue exclusion test).
Expression of heat shock proteins (HSP70 and HSP72) and metallothionein 2 (MT2) in coelomycetes (The immunoperoxidase staining protocol was used on methanol-fixed cytospin preparation).

[ref. ID; 6135]

Test system

Bioavailability of Pb and Zn to earthworms after phosphorus amendments to soil

Strains

Adult (clitellum present) obtained from a local supplier (Timberline Fisheries Corp., Marion, IL, USA).

Toxicants

Soil obtained from southeast Kansans, southwest Missouri and northeast Oklahoma, collectively known as the tri-state mining area, contain elevated levels of Pb, Zn, and Cd from tailings and smelting activities.

Test design

Amendments: Amendments included five phosphorus forms, phosphoric acid (HPLC grade, Fisher Scientific, Fair Lawn, NJ, USA), triple super phosphate (TSP, Voluntary Purchasing Group, Bonham TX, USA), rock phosphate (RP, IMC Feed Ingredients, Lake Forest, IL, USA), monocalcium phosphate (MCP, BIOFOS, IMC Feed Ingredients, Lake Forest, IL, USA), or tricalcium phosphate (TCP, MULTIFOS, IMC Feed Ingredients, Lake Forest IL, USA) with pH adjustment with either HPLC grade phosphoric acid or certified ACS plus hydrochloric acid (Fisher Scientific, Fair Lawn, NJ, USA). After addition of the phosphorus amendment and deionized water to bring moisture content up to ~20%, the soils were mixed by rolling each day for 1 hr throughout a 13 day incubation period and stored at room temperature.
Bioaccumulation test: Approximately 80+/-2 g of soil (wet weight) and five reproductively mature E. fetida were added to each of four replicate 250-mL polypropylene containers for each tested combination and time point. Tests were conducted in a Precision Scientific 818 environmental chamber (Winchester, VA, USA) at 20 degrees C with continuous light. Soil was kept moist by the addition of deionzed water on a daily basis. Time points for these experiments included 0, 12, 24, 48, 96, 144 and 192 hr for a total of 28 containers or 140 worms in each soil treatment.

Measurements/observations

Metal analysis of soil and earthworm.

Evaluations

Bioaccumulation factors (BAFs) (analysis of variance was used PROC GLM of the SAS statistical packages).
LC50 using PROC PROBIT of the SAS statistical packages.

[ref. ID; 6683]

Test system

The separation of three molecular weight cadmium-binding protein

Strains

Eisenia foetida was collected from farmyard manure deposited behind a cowshed located in Akao, Saitama, and grown in a composed sewage sludge in wooden boxes (1 m long, 1 m wide, 0.5 m high), which were covered with rice straw to maintain the humidity and to minimize exposure to light.

Toxicants

Cadmium nitrate Cd(NO3)2/4H2O

Test design/concentrations

The earthworm was grown for 30 days in the composted sludge which contained different amounts of cadmium nitrate (41.6-511 ug/g dry compost).

Measurements/observations

Concentration of metal in earthworm, gel permeation-atomic absorption chromatography.

[ref. ID; 6688]

Test system

Metal uptake

Strains

From a synchronized culture, bred from stock cultures of the Stress Ecology Laboratory of the Department of Botany and Zoology at the University of Stellenboch, South Africa.

Toxicants

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%. 12 individuals per container. Worms did not receive food for the first 3 months. After that, worms were fed every second with fresh, urine free cattle manure for a total duration of 24 weeks. 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; 6702]

Test system

EDCs concentration in earthworms living in sewage treatment systems

Samples

Earthworms collected from five sewage percolating filter bed sites and five domestic gardens (no pesticides or herbicides had been routinely used in the last five years). Adult (0.4-0.6 g).

Toxicants

EDCs (Dibutylphthalate, Dioctylphthalate, Bisphenol A and 17 beta-Estradiol, 17beta-Estradiol-acetate, Estrone, 17alpha-Ethynylestradiol).

Measurements/observations

EDCs concentration in worms tissue.

[ref. ID; 6703]

Test system

The effect on heavy metal (Pb, Zn and Cd) fractionation of processing the soil through earthworm's digestive tracts, mobility, and oral-bioavailability in earthworms casts

Strains

Worms obtained from Regenwurmfarme Tacke GmbH, were used fully clitellated adult specimens and subadult specimens with developing tubercula pubertatis.

Toxicants

Soil was collected from the upper 30 cm layer of a managed vegetable garden near the abandoned lead smelter in the Mezica Valley in Slovenia. Pb mining and smelting activity in the Mezica Valley ceased in 1990, after more than 300 years of uninterrupted activity.

Test design

Pot experiment: Clean plastic pots (height 9 cm, diameter 12.5 cm) were filled with 250 g of air-dried non-leached and leached soil, in three replicates. 80% of soil field water capacity. 10 earthworms (average 0.55 g fresh weight) introduced into each pot and kept in the dark at 20 degrees C for 7 weeks.
Lead bioavailability in warm casts was determined as oral bioavailability in simulated stomach (pH 2.50+/-0.05) and intestinal (pH 7.00+/-0.05) phases of human gastrointestinal tract, using Ruby's physiologically based extraction test.

Measurements/observations

The concentration of Pb, Zn, Cd and pH in E. fetida casts. Fractionation (assessed using sequential extractions) of Pb, Zn and Cd in worm casts.

[ref. ID; 6706]

Test systems

Strains

Clitellid adults with a minimum weight of 300 mg.

Toxicants

Post-Katrina flooded soils. Surface soils was sampled from a location in the Chalmette, Saint Bernard Parish. (Metals and volatile organic compounds).

Test design

Procedures for E. fetida testing were based on guidelines of the American Society for Testing and Materials (ASTM, 1995) and the US EPA (1996). Tests consisted of three replicates of ten organisms for each treatment (seven flooded soils, soil from the background location, and positive (chemicals: 2-Chloroacetamide) and negative control). Exposure period 30 days. 21+/-2 degrees C.

Measurements/observations

Weight loss, burrowing time, bioaccumulation metal and mortality.

[ref. ID; 6737]

Test system

Comparison of techniques for estimating PAH bioavailability

Strains

Adult worms (average wet weight 0.3 g) with a clitellum.

Toxicants

PAH-contaminated soil was collected from a former gaswork site in Stockholm, Sweden.

Experimental design

(1): Exhaustive Soxhlet extraction to determine the total PAH concentration of the soil.
(2): Sequential leaching using solvents of decreasing polarity (methanol, n-butanol, acetone, and n-hexane, followed by toluene, which has high affinity for planar compounds) to assess the relative sorption strength of various PAH pools.
(3): Batch leaching experiments using mixtures of methanol/water, 50:50 (v/v), methanol/water 1:99 (v/v), n-butanol/water (1:99 v/v).
(4): Batch leaching experiments using aqueous solutions of two modifiers with different modes of action-a bioavailability-enhancing detergent (Tween-80, 3 mM; Volkering et al., 1995) and a complex-forming agent used in bioavailability assessments (HPCD (extraction using hydroxypropyl-beta-cyclodextrin), 50 mM; Reid et al., 2000).
(5): Passive sampling using soil-phase microextraction (SPME) and extraction using semi-permeable membrane devices (SPMDs).
(6): Uptake by earthworms (Eisenia fetida) to obtain a measure of the bioavailability of the PAHs. Six worms were exposed to 400 g of soil (water content raised to, and kept at, 15% to maintain a suitably moist environment for the worms) at 20 degrees C for 32 days.

[ref. ID; 6746]

Test system

Genotoxicity

Strains

About 350 mg with well-developed clitellum.

Toxicants

Soil (Farmland receiving wastewater (sewage and effluents from industrial plants) irrigation on the outskirts of Beijing, China) and soil extraction (PAHs and OCPs).

Test design

23+/-2 degrees C under continuous light.

In vivo bioassay: 100 g freeze-dried soil samples were blended with distilled water in a glass vessel, then 3 worms were laid. Exposure times 1, 3, 7, and 14 days.
In vitro bioassay: Coelomocytes cultures (with a final cell density 5-6x10E-5) were incubated in darkness for 18 hr in Hanks buffer mixed with DMSO-dissolved organic extracts (ratio 1:1) with a final concentration of DMSO of 0.1% in the culture solution.

Measurements/observations

Comet assay.

[ref. ID; 6748]

Test system

p,p'-DDE bioaccumulation from compost and soil

Strains

From uncontaminated soil.

Toxicants

Soil (56% sand, 36% silt, 8.0% clay, 1.4% organic carbon, and bulk density 1.14 g/cm3) containing p,p'-DDE residues as a result of historical application of DDT was collected from the Conneticut Agricultural Experiment Station's experimental farm. 474+/-59.3 ng p,p'-DDE/g of soil dry wt.
Compost aged approximately 6 months was collected from Lehigh Country, PA, USA. It contained 37.0% orgnaic carbon and had a bulk density of 0.61 g/cm3. The compost contained p,p'-DDE residures. 539+/-79.8 ng p,p'-DDE/g compost dry wt.

Test design

350 g each dried, seived medium (soil and compost) were mixed with 30 g of Perlite added to seven 1000-ml glass beakers (3 replicates + no-worm control). 30 individual (approximately 9 g biomass) added each beakers. 22+/-2 degrees C in dark.

Measurements/observations

Biomass of worm and p,p'-DDE concentration in the worm tissue.

[ref. ID; 6749]

Test system

The effect on heavy metal (Pb and Zn) fractionation of processing the soil through earthworm's digestive tracts, mobility, and oral-bioavailability in earthworms casts

Strains

Worms obtained from Regenwurmfarme Tacke GmbH (Borken, Germany), were used fully clitellated adult specimens and subadult specimens with clear signs of developing tubercula pubertatis.

Toxicants

Soil was collected from the 0-30 cm surface layer of an abandoned vegetable garden in the vicinity of a former industrial site in the Mezica Valley in Slovenia. The Mezica Valley has been exposed to more than 300 years of active Pb mining and smelting.

Test design

Pot experiment: Clean plastic pots (height 9 cm, diameter 12.5 cm) were filled with 250 g of air-dried non-remediated and remediated soil, in three replicates. 80% of soil field water capacity. 10 earthworms (0.16-1.42 g fresh weight) introduced into each pot and kept in the dark at 20 degrees C for 4 weeks.
Lead bioavailability in warm casts was determined as oral bioavailability in simulated stomach (pH 2.50+/-0.05) and intestinal (pH 7.00+/-0.05) phases of human gastrointestinal tract, using Ruby's physiologically based extraction test.

Measurements/observations

Fractionation (assessed using sequential extractions) of Pb and Zn in worm casts.

[ref. ID; 6750]

Test system

Effect of metal on life cycle parameters

Strains

From Blades Biological, Cowden, Edenbridge, Kent, UK.

Toxicants

Contaminated soils (0-5 cm depth) were collected from eight metalliferous sites in the United Kingdom. Seven sites are disused mines: Shipham, Cwmystwyth, Weymyss, Llatrisant, Charterhouse and Glenridding; one was on the perimeter of a lead-zinc smelter, Avonmouth.
Uncontaminated soils were collected from Dinas Powys and Sonning.

Test design

Plastic box (18 cm x 13 cm x 17 cm) were filled with 500 g of air-dried soil. Soil moisture content 60%. 12 earthworms introduced into each box. No food. 18 replicates for each soil. 20 degrees C. Experiment period 42 days.

Measurements/observations

Mortality, body weight, cocoon production and hathing rate of worm. Metal concentration in worm tissue.

Evaluations

LC50 was calculated using probit analysis on SPSS version 10.1 for those metals for which there was a significant relationship between life cycle parameters and soil, soil pore water and earthworm tissue concentrations.
EC50s for weight change were calculated by Linear Interpolation analysis using the USEPA Inhibition Concentration Approach software version 2.0.
A CoInertia analysis was carried out using ADE-4 statistical software to test the covariation between biotic (life cycle parameters and earthworm tissue concentration) and abiotic (metal in soil, soil properties) parameters.

[ref. ID; 6781]

Test system

Strains

Worms(cd): Forty clitellate worms were placed in an artificial soil medium prepared according to OECD Guideline No. 207 (OECD, 1984), in a 5-liter container. The worms were fed on a weekly basis with fresh cow manure contaminated with 0.01% cadmium sulfate (m/m). 25 degrees C, moisture content 70-75%. The worms had been kept in this container for more than 3 years and were fed regularly with cadmium-contaminated food. A control, treated in the same manner but fed uncontaminated cow manure was maintained. After 32 months worms were used.
Worms(abs): No preexposure.

Toxicants

Cadmium sulfate

Test design

Growth, Cocoon production, and Hatching success: The substrate used was urine-free cattle manure. Cadmium sulfate concentrations 0, 600 and 1200 ug/g. 400 g wet wt (moisture 70-75%) of the specific substrate was placed into a plastic container with perforated lid and 20 clitellate worms were added. Worms(cd) series, and Worms(abs) series, respectively. Experimental period 35 days.
Survival: According to the OECD Guideline No. 207. Cadium sulfate concentrations 0, 1500, 2000, 2500, 3000, 3500, and 4000 ug/g. 750 g wet wt (moisture content 35%) of the test substrate was placed into a plastic container with perforated lid and 10 cilitellate worms were added. Worms(cd) series, and Worms(abs) series, respectively. Experimental period 14 days.

Measurements

Cadmium contents of the worms, worm weight and number, cocoon production.

[ref. ID; 6788]

Test system

Effect of two test substrates on Ni toxicity

Strains

Pre-cilitellate specimens (297-378 mg wet weight). The species had a history of long-term pre-exposure in stock cultures maintained in the ecotoxicology laboratory of the Department of Botany and Zoology at the University of Stellenbosch, Western Cape, South Africa.
Pre-exposed cultures were fed bi-weekly with urine-free cattle manure containing 0.02% nickel as NiCl2/6H2O and 0.8% manganese as MnSO4/4H2O.

Toxicants

NiCl2/6H2O

Test design

Test substrate

1. Artificial ground water: 100 mg NaHCO3, 20 mg KHCO3, 200 mg CaCl2/2H2O and 180 mg MgSO4 per litre distilled water.
2. Agar medium: Artificial ground water was heated up to 80 degrees C, 1.5% of normal melting agarose was dissolved in it and the different concentrations of nickel were added. The agarose medium was then poured into 500 ml plastic containers. After cooling down and solidifying, the agar gel was cut into small cubes. 3 replicates per metal concentration and 6 worms per replicate were exposed for 96 hr in the artificial ground water gel and for 48 hr in 500 ml of artificial ground water to different concentrations of nickel by addition of the specific concentrations of NiCl2/6H2O.

Both exposure media were kept in a dark, climate-controlled (20 degrees C) room and covered with black plastic sheet. The plastic containers containing agar were closed with a perforated lid for aeration and the artificial ground water was continuously aerated with an aquarium pump. Experimental period: groud water 48 hr, agar medium 96 hr.

Measurements

Mortality, body weight.

Evaluations

LC50 using the Probit Program Version 1.5 published by the Environment Protection Agency.

[ref. ID; 6802]

Test system

The effects of competing cations (Ca2+, Mg2+, and H+) on Cd uptake by earthworms

Strains

From a farming factory in the Dachang district of Nanjing, China. Mature earthworms weighing 190-385 mg.

Toxicants

CdCl2

Test design

1. Ca set: Ca2+ concentration 0.2-1.0 mM + Cd concentration 0.2-0.9 mM (Test media: MOPS (3-[N-morpholino]propane sulfonic acid, 0.75 g/L) buffer).
2. Mg set: Mg2+ concentration 0.1-4.0 mM + Cd concentration 0.2-1.0 mM (Test media: MOPS (3-[N-morpholino]propane sulfonic acid, 0.75 g/L) buffer).
3. H set: H+ concentration 5.5-7.1 mM + Cd concentration 0.1-0.5 mM (Test media: pH 6.0-7.1 MOPS buffer, pH 5.5-6.0 MES (2-[N-morpholino]ethane sulfonic acid, 2.0 mM) buffer).

Tests were conducted in an environmental chamber (20+/-1 degrees C) in darkness. Eight worms were exposed indiviually for 48 hr in 100 ml of exposure solution. The test solutions were renewed after 24 hr of exposure.

Measurements

Mortality, Cd concentration in test media and worm.

[ref. ID; 6818]

Test system

Bioassay

Strains

From a vermiculturist (Granny's Hillside Farms, Gore, OK). Adult worm.

Toxicants

Remediation of heavy metal-contaminated soils using phosphorus; Two different site soils were collected from Joplin, Mo, which is situated in the Tri-State mining area. The area were conducted large-scale mining operations from the mid-1800s to the 1950s. The soils were treated with two different forms of phosphorus fertilizers, KH2PO4 (potassium phosphate monobasic) and P2O5 (Super Triple Phosphate fertilizer). Two levels phosphorus, which a high phosphorus (5,000 mg/kg dry weight) and low phosphorus treatment (600 mg/kg dw) were used.

Test design

Bioaccumulation experiments: Approximately 80+/-2 g of soil (wet weight) and four E. fetida were added to each replicate 250-ml polypropylene container. A total of five treatments were used for each metal (Pb, Zn, and Cd) and included nonamended and high and low levels of KH2PO4 and P2O5. Temperature 20 degrees C under constant light. Time points for the bioaccumulation experiment included: 0, 12, 24, 48, 144, and 192 hr.
Depuration experiments; Earthworms were exposed to approximately 400 g (wet weight) of site soil during the contaminant-loading phase for each treatment. After a 192-h contaminant-loading phase, earthworms were then transferred to clean artificial soil. A modified OECD standard artificial test soil (74% medium sand, 20% kaolinite clay, 5% Canadian peat moss, and 1% CaCO3. pH 8.2+/-0.14) was used for all tests. Sampling time points were 0, 12, 24, 48, 96, 144, and 192 hr.

Measurements/observations

Lead, zinc, cadmium concentrations in worm.

Evaluations

BAFs.

[ref. ID; 6827]

Test system

Strains

Mature (clitellate) earthworms (weight: approx. 0.2-0.4 g) were obtained from in-house cultures.

Toxicants

Cd(NO3)2

Test design

Standard protocol (ASTM, 1997).
Glass jar contained 200 g (dry weight) artificial soil, consisting of 69.5% silica sand, 20% kaolin clay, 10% 2-mm sieved Sphagnum peat moss, and approximately 0.5% CaCO3. pH 6.5+/-0.5. water holding capacity 35% (w/w). Temperature 20+/-1 degrees C under constant light. 10 worms per each replicates. Experimental period 14 days.

Measurements/observations

Cd concentration in tissue (tissue fraction: supernatant (metallothioneien-bound) and pellet (toxicologically active)) of worm.

[ref. ID; 6830]

Test system

Acute and chronic toxicity

Strains

From a commercial earthworm breeding plant.

Toxicants

CdCl2/H2O

Test design

Acute toxicity was assessed as described in OECD guideline 207 (1984), and chronic toxicity as suggested by van Gestel et al. (1989). Grass vessels were filled with 750 g wet weight of soil and 10 adult worms. Vessels were kept at 20+/-1 degrees C under constant illumination.

Soil type

Standard artificial soil: OECD guideline 207 (1984) 70% sand, 20% kaolin clay and 10% finely ground Sphagnum peat. pH 6.
Sandy field soil: Kalmthout (Antwerp, Belgium).
Loamy field soil: Termunck (Flemish Brabant, Belgium).

Measurements/observations

Mortality, number of cocoons.

Evaluations

EC10s and EC50s using the probit method.
LC50s using the moving average method.

[ref. ID; 6832]

Test system

Comparison of exposure methods

Strains

Mature earthworms displayed a prominent clitellum band and weighed 370-450 mg.

Toxicants

CdCl2/2.5H2O and [109]CdCl2

Test design

Acute toxicity
Earthworms were exposed via filter paper exposure or artificial soil exposure essentially as described in OECD guideline for testing chemicals, Method 207: Earthworms, acute toxicity test (1984). All exposures were conducted at 20+/-3 degrees C.

Filter paper exposure: Shell vials (23x85 mm) were lined with Whatman No.1 filter paper strips (8.3x6.4 cm; surface area, 53.12 cm2) and wetted with 1 ml of test solution. Earthworms were placed individually into the vials, which are covered with Parafilm M and placed on their side in the dark during the exposure. Exposure concentrations 80, 40, 20, 10, 5 and 2.5 ug/cm2 in duplicate with 10 earthworms per concentration.
Artificial soil exposure: Artificial soil was prepared consisting of 70% industrial sand, 20% kaolin clay, and 10% sphagnum peat moss with the pH adjusted to 6.5+/-0.5 with CaCO3. The soil was spiked by adding the appropriate amount of cadmium and adjusting the moisture content to 35% of the final weight. Five kilogram lots of the soil were prepared and mixed using a Hobart mixer. The soils had to be mixed at least 30 min to obtain homogeneously spiked soils. All artificial soil exposure were conducted under continuous lighted conditions to keep the earthworms burrowed in the soil. 10 earthworms were placed in each beaker. Duplicate 600-g aliquots of 10, 100, and 100 ug Cd/g artificial soil dry weight.

Measurements/observations

Filter paper exposure: Mortality at 24 and 48 hr.
Artificial soil exposure: Mortality at 1, 7, 14 and 28 days.

Evaluations

LC50s using the method of moving averages (Thompson and Weil, 1952).

Cd Uptake and elimination
For uptake studies, earthworms were exposed to Cd at exposure concentrations of 0.625, 1.25, or 6.25 ug/cm2 for filter paper exposures and 5, 10, 50, 100, 500, or 1000 ug Cd/g artificial soil dry weight for artificial soil exposures. For elimination studies, earthworms exposed for 48 hr to 1.25 ug/cm2 were transferred to noncontaminated vials and sampled at 0.25, 0.5, 1, 2, 4, 8, 24, or 72 hr postexposure, and 28 days to 10 ug/g were transferred to noncontaminated artificial soil and sampled at 3, 7, 14, or 28 days postexposure.

Cd distribution
Exposure concentrations were 6.25 ug Cd/cm2, sp act 4x10E6 dpm/mg Cd, for filter paper exposures and 50 mg Cd/kg dry wt, sp act 4x10E6 dpm/mg Cd, for artificial soil exposures. Artificial soil-exposed earthworms were placed in groups of 14 earthworms in 200 g of [109]Cd-spiked artificial soil in 1-liter polyethylene beakers covered with plastic wrap to prevent moisture evaporation.

Measurement/observations

Tissue localization, subcellular distribution and autoradiography.

[ref. ID; 6834]

Test system

Effects of Metal-contaminated soils on juvenile worm

Strains

Juvenile.

Soil sampling site

Contaminated soils (Cadmium, Copper, Lead, Zinc) were collected from seven sites in the vicinity of the Avonmouth smelting works. Uncontaminated soil was collected from the Reading University campus.

Test design

Containers (plastic boxes with dimensions 175x120x60 mm, 4 replicate) were filled with 500 g soil. Ten individuals were weighed and placed ino each test replicate. All containers were covered to prevent water loss and maintained at 20 degrees C under constant light for 20 weeks. Worms were supplied with uncontaminated horse manure as a source of food.

Measurements/observations

Survival, growth (= weight after 5 weeks exposure), time to sexual maturation (= percentages of adults present after 8 weeks), reproduction (= number of cocoons produced by the worms).

[ref. ID; 6850]

Test system

The use of acid insoluble residue as a marker fraction in the soil

Strains

Lumbricus terrestris, Allolobophora longa, Allolobophora caliginosa, Allolobophora chlorotica were collected from the soil in Rothamsted Park. Eisenia foetida was collected from cattle manure. Mature, clitellate individuals for experimental use.

Toxicants

Zn, Cu, Cd, Pb
Soil sampling site: 4 soils (Frongoch, Ystwyth, Shipham, Broek Polder).

Test design

Groups of each species of earthworm were placed on separate subsamples of each soil. A ratio of approximately 5 g (live weight) Of earthworms to 600 g (air dry weight) of soil. Experimental period 15 days. Temperature 15 degrees C.

Whole worm: Immediately after removal from the soil, earthworms were rinsed in distilled water, killed by oven drying at 85 degrees C to constant weight. These 'whole worm' samples comprised both earthworm tissue and soil within the alimentary canal.
Dissected worm: Earthworms were dissected along the entire length of the alimentary canal which was rinsed free of soil with distilled water before being oven dried at 85 degrees C to constant weight.
Starved worm: Earthworms were placed in clean petri dishes at 100% humidity (a few millilitres of distilled water) for 48 hr (transferred to clean petri dishes after the first 24 hr). All soil egested by the earthworms during the 48 hr period was collected, dried and analysed for acid insoluble residue. Earthworms which had been starved for 48 hr were rinsed in distilled water, killed by oven drying at 85 degrees C to constant weight.

Measurements/observations

Heavy metals and AIR concentration in earthworm tissue.

[ref. ID; 6858]

Test system

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

Strains

Eisenia fetida were reared in a synchronous laboratory culture maintained on uncontaminated horse manure. Fully clitellate individuals with an average wet weight of 422 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%. 10 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 day. 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/observations

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; 6863]

Test system

Strains

Adult with clitella weighing 0.25 to 0.35 g.

Toxicants

Diesel

Test design

Experimental system for oxidation of diesel-contaminated sand: The systems was composed of a liquid oxygen reservoir, an ozone generator a flow meter, a pressure meter, a fiber-optic transflection dip probe system for the measurement of ozone concentration, and the 3-D test cell.
Three-dimensional (3-D) test cell with a length of 3 m, width of 2 m, and height of 2 m was constructed with stainless steel. A total 15,330 kg of sand (available commercially: Doyang Sience, Chumunjin, Korea) was packed in the 3-D test cell, and commercially available diesel fuel was weathered, and then gaseous ozone was introduced continuously for 20 days.
Toxicity test: Contaminated soil samples were collected at several points in the 3-D test cell before and after the ozonation process was performed. OECD guideline no. 207.

Measurements/observations

Mortality, growth, and the behavioral response.

[ref. ID; 6864]

Test system

Strains

Toxicants

Chemicals: Cadmium (as nitrate) 0, 5, 20, 80 and 300 ug g-1 dry wet soil. Copper (as nitrate) 0, 10, 40, 200 and 1000 ug g-1 dry wet soil. Lead (as nitrate) 0, 100, 400, 2000 and 10000 ug g-1 dry wet soil. Zinc (as nitrate) 0, 100, 400, 2000 and 10000 ug g-1 dry wet soil.
Contaminated soil: Contaminated soils collected from sites at different distances from a smelting works situated at Avonmoutn, south-west England.

Test design

Using the modified OECD (1984) toxicity test, described by Van Gestel et al. (1989). Plastic boxes (dimensions 175 mm x 120 mm x 60 mm) were filled with soil and 10 worms. 20 degrees C in constant light.

Run 1. Worms were exposed to single metals in standard artificial soil (70% sand, 20% kaolin clay and 10% coarse ground Sphagnum peat, pH 6.1).
Run 2. Worms were exposed to contaminated soil.
Run 3. Worms were exposed to mixtures of metals in artificial soil.

Measurements/observations

Survival, growth, cocoon production and cocoon viability.
Earthworm population density and species diversity in the contaminated field.

Evaluations

LC50s by probit analysis.
EC50s by logit analysis, and NOEC using the derivation of the Williams (1971, 1972) test.

[ref. ID; 6866]

Test system

Accumulation of heavy metals

Strains

Laboratory studies: From a stock culture maintained in the College laboratory.
Field study: From Cecoms, Inc., Santa Clara, Calif., where the field study was conducted.

Toxicants

CdSO4, CuSO4, NiSO4, Pb acetate, and Zn acetate.

Laboratory studies: Waste-activated sludge were obtained from drying beds at the Meadowbrook-Limestone Wastewater Treatment Plant, Onondaga County, New York.
Field study: A sludge previously sun-dried for 1 year in a 8-cm layer in a lagoon was obtained from San Jose, Calif.

Test design

Laboratory studies

1. The effect of E. foetida on extractability of metals from sludge with 0.1N HCl, five replicates of 50 g of earthworms were placed in 400-g samples of aerobic sludge at 15 degrees C in glass dishes 6 cm high and 20 cm diameter. Control was without worm. Exposure time 0, 2, 4, 7, 11 days.
2. Uptake of heavy metals into E. foetida was determined following separate addition of CdSO4 (10, 50, 100ppm Cd), CuSO4, NiSO4 (100, 500, 1000 ppm Ni), Pb acetate (2500, 5000 ppm Pb), and Zn acetate (2500, 5000, 10000 ppm Zn) to samples of the aerobic sludge. Experimental period 5 weeks.

Field study
Approximately 1 m3 of the sludge was distributed over a sheet of plywood, 1.2 by 2.4 m. Samples of worms, about 1.8 kg live weight, were removed every 2 weeks, as were pooled, random samples of sludges weighing about 9 kg wet weight each.

Measurements

Heavy metal concentrations in the worm tissue.

[ref. ID; 6868]

Test system

Bioavailability

Strains

Sexaully mature (developed clitellum) worm.

Toxicants

Individual metal test: Pb 1000 mg/kg, Zn 400 mg/kg, 100 Cd mg/kg (nitrate salts).
Mixture metals test: Pb 333 mg/kg, Zn 133 mg/kg, 33 Cd mg/kg (nitrate salts).

Test design

OECD artificial soil was modified by decreasing the proportion of peat moss from 100 to 50 g kg-1 and increasing the amount of sand from 690 to 740 g kg-1.

Soil amending method: Phosphorus (P: Monobasic potassium phosphate (KH2PO4)) or Organic matter (OM: composted leaves obtained from a local recycling plant).
Treatments or soil amendments for the individual experiments consisted of the following: nonamended (NA), low P, high P, OM, low P + OM, and high P + OM.
Treatments or soil amendments for the mixture experiments consisted of the following: nonamended (NA), high P, OM, and high P + OM.

Experiments for all soils were conducted in 250-ml polypropylene containers with approximately 100 g wet weight, metal-spiked soil. Four adult worms added. All tests were conducted in a Sherer 710 Dual Jet environmental chamber at 20 degrees C with continuous photoperiod.

Measurements

Metal concentrations in worm tissues.

Evaluations

BAFs.

[ref. ID; 6872]

Test system

Bioavailability

Strains

From a local angling shop. Average mass of 1.13 (+/-0.22) g.

Toxicants

Dredge spoil: The dredge disposal site is located on the north bank of the R Yare opposite the Whitlingham sewage treatment works just outside the city of Norwich. The river is cutting through chalk and thus the sediment is composed primarily of calcite with minor quartz and kaolinite. The primary contaminants within the sediment are Hg and copper which were discharged to the river until 1986.

Test design

The bioassay was conducted at 17+/-1 degrees C under neon fluorescent lighting for 28 days, essentially following the methodology described by Rhett et al. (1988).

Measurements/observations

Metal (Fe, Mn, Cu, and Hg) concentrations in worm.

[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

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%. 10 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; 6895]

Test system

Sublethal effect

Strains

From our own laboratory.

Toxicant/concentrations

Metal chloride: NaCl, KCl, CaCl2, MgCl2, CoCl2, CuCl2, BaCl2, MnCl2, NiCl2, SnCl2, SrCl2, AlCl3 and FeCl3 (concentration of introduced salts in the wetted substratum were 20, 40, 60, 80, 100, 120 or 180 mM kg-1), LiCl and TlCl (concentration of introduced salts were 1, 5 and 10 mM kg-1).

Test design

Plastic bag containing 500 g wet mass of the medium (peaty marshland soil and horse manure in proportions 1:1 (m/m)) were used, 10 worms (mean initial mass 177+/-32 mg) per each bag. Experiments were carried out at room temperature (20-30 degrees C) over 10 weeks.

Measurements/observations

Number and the live mass of the worm, cocoon number.

Evaluations

Results of each treatment were one by one compared statistically with those of the control.

[ref. ID; 6898]

Test system

Critical body residues (CBRs) in assessment the toxicity

Strains

Adult worms (0.25-0.35 g) were purchased from a commercial grower (Early Bird Ecology and Bait Ltd, Smithville, ON, Canada).

Toxicants

Pentachlorophenol.

Test design

Toxicity tests by using standard procedures (OECD, 1984; ISO, 1991). Testing was conducted in Conviron Environmental Chambers (Model E-7, Winnipeg, MN, canada) with constant humidity, light and a temperature of 24+/-0.25 degrees C.

Measurements/observations

Motality and PCP residues in worm.

Evaluations

LC50s using the Trimmed Spearman-Karber method (Hamilton et al., 1977).
The 1CFOK (one compartment first-order kinetics) model was applied to the LC50-time data, using non-linear regression, to estimate the ILLs (incipient lethal levels) as the asymptonic value of the exponential curve of the inverse of the LC50 vs time data. All residues were analyzed by ANOVA and means were compared using Tukey's Honestly Significant Difference test (alpha = 0.05).

[ref. ID; 6901]

Test system

Development of the laboratory chronic test conditions

Strains

Test design

Improvements for producing a synthetic food made by microorganisms grown under standardized conditions.

[ref. ID; 6902]

Test system

Strains

From our own laboratory.

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). 500 g (dry wt) soil and 10 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; 6906]

Test system

DNA adducts

Strains

From the laboratories of the Ministry of the Environment of the Province of Quebec.

Toxicants

The study site (a coal gasification plant located in Quebec city, Canada) was heavily contaminated with polycyclic aromatic hydrocarbons (PAHs), with maximum concentrations of 3500 ug g-1 of total PAHs and 209 ug g-1 of benzo(a)pyrene.

Test design

Twenty five earthworms were put in a 1 L plastic container with approximately 500 g (wet weight) of contaminated soil. Room temperature. Exposure period 1 and 2 weeks.

Measurements/observations

The DNA isolation procedure developed for E. fetida and the nuclease P1 modification of the [32]P-postlabelling method are described respectively by El Adlouni et al. (1994) and Walsh et al. (1995).

[ref. ID; 6907]

Test system

Effects on the spermatozoan ultrastructure

Strains

From a stock maintained in our laboratory over several years, originating from garden compost heaps. Pre-clitellate worms.

Toxicants

Pb(NO3)2, MnSO4/4H2O.

Test design

Four plastic containers with a surface area of 140 cm2 and a perforated lid, covered with fine gauze, were used for the exposures with 200 g of the wetted substrate (dried, urine-free, groud and sieved cattle manure with a particle size of 500 < /_ 1000 um moisture content of 75%) used for each container. 10 worms per container. The fresh food was spread over the upper layer of the substrate. Temperature 25 degrees C. Exposure period 8 weeks.

Measurements/observations

Spermatozoan ultrastructure and metal concentration in worm tissue.

[ref. ID; 6924]

Test system

Subcellular partitioning in the worm

Strains

From a commercial supplier.

Toxicants

CdCl2

Test design

Soil (Udeic Ferrosols, the top 20-cm layer) was sampled from Yingtan, Jiangxi Province, China. Cd solution was sprayed into the soil to 1 mg/kg dry weight. Soil (70% water-holding capacity) was put into plastic jars (1000 ml) with a perforated lid, and four worms were placed to the surface soil in each jar. No food. Temperature 20 degrees C under constant illumination. Exposure period 0, 2, 4, 7, 14, and 21 days.

Measurements/observations

Metal concentration in subcellular fraction (the granular fraction, the tissue and membranes and cell organelle fraction, the cytosolic fraction, and the organelles fraction).

[ref. ID; 6955]

Test system

Strains

From a local composting farm. Adult specimens with a well-developed clitellum and body weight of 400-700 mg were used.

Toxicants

Imazalil, imazalil sulfate, R 14821 (transformation product), chloroacetamide, copper sulfate, pentachlorophenol, trichloroacetic acid.

Test design

Temperature 20+/-2 degrees C, light/dark cycle of 12/12 hr.

Contact toxicity test: Brown glass vials (8x3.2 cm; height x diameter), contact area: 50 cm2. Strips of filter paper (Whatman No. 604; 83 g/m2 - 6.25x8 cm; height x width). 10 replicates for each concentration. Exposure period 48 hr.
Artificial soil toxicity test: According to OECD guideline 207. 1-liter glass beakers were filled with 750 g of soil and 10 worms. Moisture content 35-40 g water/100 g dry soil equivalent. 4 replicates for each concentration. Exposure period 7 and 14 days.

Measurements/observations

Mortlity, imazalil concentration of earthworm tissues.

Evaluations

LC50 using the Probit analysis (the method of Finney, 1962).

[ref. ID; 6959]

Test system

Strains

Toxicants

44 chemicals. 2,4-Dinitrophenol, 4-Nitrophenol, 2-Chlorophenol, 2,4-Dimethylphenol, Pentachlorophenol, 2,4-Dichlorophenol, 2,4,6-Trichlorophenol, Phenol, 2-Nitrophenol, Chloroacetamine, N-Nitrosodiphenylamine, N-Nitroso-N-dipropylamine, carbaryl, Nitrobenzene, 1,2-Dichlorobenzene, 1,2,4-Trichlorobenzene, Chlorobenzene, Ethylbenzene, Toluene, Benzene, Hexachlorobenzene, Acenaphthene, Fluorene, Fluoranthene, Naphthalene, n-Butylphthalate, Diethylphthalate, Dimethylphthalate, Dioctylphthalate, Bis(2-ethylhexyl)phthalate, 1,2-Dichloropropane, Hexachloro-1,3-butadiene, Chloroform, 1,2-Dichloroethane, 1.1.1-Trichloroethane, 1.1.2-Trichloroethane, Tetrachloroethane, Hexachloroethane, Bis(2-chloroethyl) ether, 2-Chloroethylvinyl ether, Methylene chloride, Carbon tetrachloride, 1,2-Trans-dichloroethylene, Trichloroethylene.

Test design

Contact filter paper test: The glass vials used (Wheaton, 90 mL snap cap) were 8-cm long and 3-cm in diameter. Strips of filter paper 12 by 6.7 cm, (Whatman no. 1) lined and completely covered the sides of the vial. One adult earthworm (300-500 mg) was added per vial and the vials were kept on their side at 20 degrees C in darkened incubator for 48 hr.
Artificial soil test: The EEC tentative "artificial soil" test method (Edwards, 1983). Medium (finely ground sphagnum peat 100 g/kg, kaolinite clay 200 g/kg, fine sand 690 g/kg, pulverized calcium carbonate 10 g/kg; pH 6.0+/-0.5, water content 35%). 10 adult worms (300-500 mg) were added to 400 g (dry weight) of the test mixture in covered glass dishes 6.5 cm high and 12.5 cm in diameter. The dishes were incubated at 20 degrees C. Exposure period 2 weeks.

Measurements/observations

Mortality.

Evaluations

LC50 using the method of Litchfield and Wilcoxon (1949).

[ref. ID; 6980]

Test system

Strains

Mature (clitellate) worms (weight: approx. 0.2-0.4 g) were obtained from in house-cultures.

Toxicants

CdSO4, Pb(NO3)2, ZnSO4, Cd-Pb-Zn mixture.

Test design

Soil toxicity test: Standard protocol (ASTM, 1997).

Single-metal tests
Mixture-metal tests

All replicates contained 200 g (dry weight) artificial soil, consisting of 69.5% silica sand, 20% kaolin clay, 10% 2-mm sieved Sphagnum peat moss, and approximately 0.5% CaCO3. pH 6.5+/-0.5. Water content 38% (w/w). 10 worms per each replicate.

Measurements/observations

Mortality.

Evaluations

LC50 using the Trimmed Spearman-Karber method (Hamilton et al., 1977).
ILL (Incipient Lethal Levels).

[ref. ID; 6982]

Test system

Chronic toxicity assay

Strains

From a commercial earthworm breeding farm. Adult worm with a fully developed clitellum.

Toxicants/concentration

NiCl2 100, 180, 320, 560, and 1000 mg Ni/kg dry wt.

Test design

Chronic toxicity assays were performed as suggested by van Gestel et al. (1989). 10 adult worms. The glass vessel was filled with 750 g wet weight of the artificial soil (OECD Guideline 207, 1984). Soil moisture content of 55% of the water holding capacity. All test vessels were kept at 20+/-1 degrees C and constant illumination. Exposure period three weeks. At the start of the test, 2% (dry weight) finely ground cow dung was supplied in a shallow depression in the test soil.

Measuremetns/observations

Number of juveniles, cocoons and adult.

Evaluations

EC50s using the probit method.
LC50s using the moving average method (Stephen, 1977).
NOECs were calculated by Kruskal-Wallis ANOVA followed by post-hoc multiple comparisons (Conover, 1980).

[ref. ID; 7012]

Test system

48-hr Contact toxicity test

Strains

From Bert's Bait Farm, Irvine, KY. Mature worms showing a developed clitellum and weighing 370 to 450 mg.

Toxicants

90 chemicals. Acephate, 2-Acetamidofluorene, Acetone, Acetylsalicylic acid, Agrinine, Aldicarb, Ammonium nitrate, Asparagine, Benomyl, Benzaldehyde, Benzene, Benzo(a)pyrene, Cadmium chloride, Caffeine, Canavanine, Captan, Carbaryl, Carbendazim, Carbofuran, Carbon tetrachloride, p-Chlorophenyl-N-methylcarbamate, Chlorpyrifos, Copper sulfate, Cyclohexane, Cypermethrin, Cysteine, 2,4-D, DDT, Dichloromethane, 2,4-Dichlorophenol, Dicrotophos, Dicyanamide (DCD), O,O-Diethyl-O-naphthyl-phosphorothioate, 2,3-Dihydro-2,2-dimethyl-7-hydroxyobenzofuran, Dimethylacetamide, Dimethylformamide, Dimethyl sulfoxide, Diesel fuel, Diuron, Endosulfan, Eserine salicylate, Ethyl alcohol, Ethyl carbamate, Fenvalerate, Formaldehyde, Fonophos, Gasoline (unleaded), Guthion, Histidine, Kepone, Lead nitrate, Lysine, Malathion, Methanol, Methionine, Methomyl, Methylamine-HCl, Methyl iodide, Methyl methanesulfonate, N-Methyl-N'-nitro-N-nitrosoguanidine, Methylurea, Metribuzin, Mirex, Myosmine, 1-Naphthol, 2-Naphthylamine, 1-Naphthyl acetate, 1-Naphthyl glucoside, 1-Naphthyl sulfate, Nicotine, p-Nitrophenol, p-Nitrophenyl glucoside, p-Nitrophenyl-N-methylcarbamate, Nornicotine, Paraquat chloride, Parathion, Permethrin, Piperonyl butoxide, Phenmetrazine, Phenylalanine, Proline, Propoxur, Sodium chloride, Sodium nitrite, 2,4,5-T, Taurine, Theophylline, Thiophanate-methyl, 2,4,5-Trichlorophenol, Trifluralin, Tryptophan.

Test design

Glass shell vials (22 mm x 85 mm) were lined with Whatman No. 1 filter paper strips (9.5 cm x 6.8 cm; surface area, 65 cm2) and placed in cardboad scintillation vial trays. 10 worms were placed in the vials, the containers were capped and kept stored in the dark in the horizontal position for 48 hr.

Measurements/observations

Mortality.

Evaluations

LC50 by the Litchfield-Wilcoxon log dose-effect probit transformation method.

[ref. ID; 7019]

Test system

Reference toxicants for use of the 14-d earthworm toxicity test

Strains

From Carolina Biological Supply (Burlington, NC).

Reference toxicants

2-Chloroacetamide (C2H2ClNO), Potassium chloride (KCl) and Ammonium chloride (NH4Cl). 2-Chloroacetamide (C2H2ClNO) is the U.S.Environmental Protection Agency's (EPA) reference toxicant of choice for the earthworm toxicity test in artificial and site soil.

Test design

Test chambers were screw-top glass jars with one small nail hole punched in the lid for air exchange. A weight of hydrated soil (40 ml water to every 100 g dry weight artificial soil) that contained 200 g dry weight was added to each of four replicate chambers per treatment group. 10 adult clitellate worms (300-600 mg) were added. Tests were conducted in an incubator kept at 22+/-2 degrees C under continuous light intensity of approximately 400 lux. Exposure period 7 and 14 days.

Measurements/observations

Mortality.

Evaluations

LC50 using the graphical method.

[ref. ID; 7022]

Test system

Method for assessing sublethal effect

Strains

From P.R.Sheuerman (East Tennessee State University, Johnson City, TN, USA), derived originally from E. foetida cultures at the EPA laboratory in Athens, Georgia.

Toxicants/concentrations

2-chloroacetamide (1, 2, 5, 10, 15, 25, or 35 mg/kg of dry soil).

Test design

Approximately 100 g of soil aliquots were placed into 6.5 cm x 15 cm (1.15-ml-thick) transparent resealable polyethylene bags. The bags were punctured on identically with a template of 10 thumbtacks (10 punctures on each side) before the test soil was added to allow aeration on the soil. Two adult E. foetida were added to each bags. The bags and their contents were incubated in an environmental chamber maintained at 20+/-2 degrees C on a 12:12-hr day:night lighting regime. Incubation period 21 days.

Experiment A: Food supplement.
Experiment B: Compost-remediated soils.
Experiment C: Application for ecological risk assessment.
Experiment D: Reference toxicants in dilution series.

Measurements/observations

Number and weight of adult, juveniles, and cocoons.

Evaluations

Survival(%), reproduction(%), growth(mg).

[ref. ID; 7024]

Test system

Avoidance test

Strains

From Carolina Biological Supply.

Toxicants/concentrations

KCl (5,000 and 6,000 ppm), NH4Cl (150 ppm), 2-Chloroacetamide (20 and 80 ppm), hazadous waste site soil.

Test design

This test can be run with the same artificial soil (AS) as used in the U.S.Environmental Protection Agency (U.S.EPA), Organization for Economic Co-operation and Development (OECD), and American Society for Testing and Materials (ASTM) acute toxicity protocols, and with conditions of soil hydration, pH, light, temperature, and test organisms that are compatible with these test protocols.
Test chambers were circular glass evaporating dishes (150x75 mm) with a line drawn on the outside to divide the chamber into halves. 10 adult clitellate worms were placed on the soil surface, along the center line (control/test soils) of test chamber. 22+/-2 degrees C, approximately 600 lux continuous illumination. Test period 1, 2, 3, 4, 7 days.

Measurements/observations

Number of worms in each soils.

[ref. ID; 7031]

Test system

Lethal and sublethal toxicity (the relationship between laboratory experiments using soluble metal salts for spiking) and real exposure conditions in the field)

Strains

Adult (visible clitellum) 300-600 mg live weight.

Toxicants

Cu.

Test design

The experiments were conducted in containers containing 600 g moist sandy clay soil, i.e. 500 g dry soil and 100 ml demineralized water. 4 replicates, each with 10 earthworms per replicate, were used at each Cu concentration. The experiments were continued for 21 day at a temperature of 20+/-1 degrees C and with a 12:12 hr light:dark. 3 g horse manure (wetted with 10 ml glass-distilled water) was added for food to the soil surface every 7 day.

Cu-spiked soil: CuCl2/H2O spiked in the laboratory with Cu concentrations ranging from 0, 50, 100, 300, 700, and 1400 mg Cu/kg. pH 6.5-7.0.
Field contaminated soil: Worms were exposed in the laboratory to soil collected from various points along gradient in a Cu-contaminated field in Hygum, Denmark. The Cu gradient: 15, 67, 211, 421, 829, and 1369 mg Cu/kg. The soil pH 6.5-7.0.

Measurements/observations

Adult survival, growth (wet weight), cocoon production, cocoon wet weight, neutral-red retention time, and Cu concentration in worm.

Evaluations

NOEC and LOEC by multiple comparison Dunnett's procedure.
EC10 and EC50 by fitting a logistic model.

[ref. ID; 7090]

Test system

Subchronic bioaccumulation/toxicity test and avoidance bioassay

Strains

Mature with clitellate. 0.3-0.6 g.

Toxicants

Micron-sized Al2O3 (type WN-3, size range 50-200 um). Nano-sized Al2O3 (nominal diameter=11 um).

Test design

Subchronic bioaccumulation/toxicity test: The test were conducted over a period 28 d following the American Society for Testing and Materials E 1676-04 method. Nano- and micron-sized Al2O3 was dry-spiked into Grenada-Loring soil (the Brown Loam Experimental Station, Learned, MS, USA) to create five nominal concentrations plus control (100, 300, 1000, 3000, and 10000 mg/kg), plus an additional treatment (13000 mg/kg) for micron-sized Al2O3. Spiked soil were divided into three replicates of 300 g in glass 500 ml exposure chambers with porous screw-top lids. 85% water holding capacity. All exposure were conducted in an environmental chamber set to 22+/-1 degrees C under continuous light and 80% humidity. 10 worms added, no food.
Avoidance bioassay: A modified version of a method published by Environment Canada (2004). Plexiglas avoidance wheels containing six pie-shaped compartments and separated by removable partitions. The six compartments were connected by a circular, center chamber that contains openings to each treatment. The GL soil was amended with micron-sized and nano Al2O3 to create nominal concentrations of 625, 1250, 2500, 5000, and 10000 mg/kg. All exposure were conducted in an environmental chamber set to 22+/-1 degrees C under continuous darkness and 80% humidity. 10 worms added. Exposure period was 48-hr.

Measurements/observations

Subchronic bioaccumulation/toxicity test: Survival number and weight. Cocoon number. Al concentration in worm tissues.
Avoidance bioassay: Location of worms.

[ref. ID; 7132]

Test system

Bioaccumulation test

Strains

From Carolina Biological Supply.

Toxicants

Biosolids (sewage sludge) including Triclocarban (TCC) and Triclosan (TCS).

Test design

The Metropolitan Water Reclamation District of Greater Chicago initiated a three-yaer field scale research experiment at two farms in Illinois, USA, with Ashkum silty clay loam (fine, mixed, mesic Typic Haplaquolls, W soil) and Chelsea fine sand (mixed, mesic, Alfic Udipsamments, K soil).
Biosolids application rates/time:

W soil (heavy textured clay soil) 0, 5.0, 7.5, 10, 12.5, and 20 dry tons of biosolids per acre per year. In fall of 2004, 2005, and 2006.
K soil (light textured clay soil) 0, 2.5, 5.0, 7.5, 10, and 15 dry tons of biosolids per acre per year. In spring of 2005, 2006, and 2007.

Approximately 120 g soil was placed in 150-ml beaker. The beakers were kept in a water bath under constant temperature (22-24 degrees C) and lighting. Worm aliquots (approximately 1-1.5 g wet weight of worms per aliquot, four individuals) were added. Triplicates. Exposure period 28 days.

Measurements/observations

Worm weight. TCC and TCS concentations in worm.

[ref. ID; 7137]

Test system

Effects of aging and mixed nonaqueous-phase liquid sources (NAPLs) in soil system

Strains

From the Carolina Biological Supply.

Toxicants

Unlabeled and radiolabeled (9-[14]C) pyrene.

Test design

Two sandy loam soil 'North Campus soil (organic carbon 2.47%)' and 'Chelsea soil (organic carbon 5.95%)' were employed. The radiation-sterilized soils were spiked aseptically in sterilized 250-ml wide-mouth glass jars with pyrene in unlabeled and [14]C-labeled forms dissolved in NAPLs (hexadecane, 2,2,4,4,6,8,8-heptamethylnonane (HMN), toluene, and dimethyl phthalate (DMP), and dichloromethane) to yield a final pyrene soil concentration of 100 ug/g and a radioactivity of 1.7 kBq/g soil. Soil aging period were 0, 30, 110, 167 days.
Bioavailability of pyrene: Three adult worms were placed to moist (22% water) soil samples (75 g) in 250-ml glass jars. The jars were loosely closed with a cap to prevent worm escape and allow air exchange, then were held in the dark for 14 days at 21+/-1 degrees C. Three replicates.

Measurements/observations

Pyrene concentration in worm.

[ref. ID; 7145]

Test system

Growth test

Strains

Toxicants/concentrations

Activated Sludge (The Meadowbrook-Limestone Wastewater Treatment Plant, Onondaga County, N.Y.) + CaCO3, Ca(CH3COO)2/H2O, CaCl2/2H2O, CaSO4/2H2O, CdO, 3CdSO4/8H2O, CoCl2/6H2O, CoCO3/6H2O, Co(No3)2/6H2O, Cr2O3, Cu(CH3COO)2/H2O, CuCl2/2H2O, CuSO4/5H2O, FeSO4/7H2O, Hg2(CH3COO)2, HgCl2, KCl, KNO3, K2HPO4, Mg(CO3)4/Mg(OH)2/5H2O, MgCl2/6H2O, MgSO4/7H2O, Mg(CH3COO)2/4H2O, Mn(CH3COO)2/4H2O, MnSO4/H2O, NaCl, NaNO3, NH4Cl, NH4(CH3COO), NH4H2PO4, NiCO3/2Ni(OH)4/4H2O, NiSO4/6H2O, Pb(CH3COO)2/3H2O, PbCO3, 3Zn(OH)2/2ZnCO3, ZnSO4/7H2O Five concentrations (0, 0.01, 0.1, 0.5, and 1.0%) of the test salts and oxides.

Test design

About 50 g of a Teel silt loam was placed into a dish (10-cm-diam, 5-cm deep). Distilled water, 30 ml, was added. At the beginning experiment, a test salt or oxide was added as a solid to 50 g of sludge (13-15% solid), mixed thoroughly by hand, and placed on the soil together with two hatchlings. Exposure period 8 weeks. At four intervals of 2 weeks each the worms were removed, rinsed, blotted, weighted, and returned to their dishes. Four weeks after an experiment had begun, a fresh supply of sludge with test salt was placed into each dish, and the bulk of old sludge material was removed and discarded. Temperature 24+/-1 degrees C.

Measurements/observations

Weight of worms.

Evaluations

One-way analysis of variance of weights achieved at 4 and 8 weeks.

[ref. ID; 7155]

Test system

The modification of the initial defined medium

Strains

Test design

All experiments were carried out at 25 degrees C, in darkness, with an RH of about 75%, using wide-mouthed 1 L round glass jars (115 mm x 110 mm), with a copper screw lid with a single hole. MCU grade commercial vermiculite from Micronised Products (South Africa), with a water holding capacity of 0.48 ml g-1 was used as a matrix throughout. Three 20-day development-stage-synchronised worms (0.037 g). Experimental period 90 days.
Base medium (MCU 193 g, H2O 86 g, Cellulose 14 g, Casein 0.7 g, DNA from salmon 0.8 g, Humic acid 2.0 g) + Stearic acid, Palmitic acid, Lecithin, Glycerophosphate, Phytin, Adenosine triphosphate, Oxy-humic acid. Control medium was cow manure (200 g at 80% water capacity).

Measurements/observations

Weight, and number of cocoon and clitelate worms.

Evaluations

Data was analysed with one-way analysed of variance (ANOVA), with Tukey's multiple comparison test for comparisons between treatments (P=0.05) using GraphPad Prism (Version 4.03).

[ref. ID; 7156]

Test system

Effect of different cations (Ca2+, Mg2+, Na+, K+, and H+) on the acute toxicity (48-hr LC50)

Strains

From the Dachang Earthworm Cultivation Farm in Nanjing. Adult worm (approximately 200-400 mg).

Toxicants/concentrations

CdCl2, 2.0x10E-4 - 4.0x10E-3 Cd mol/L.

Test design

Each treatment was performed by exposing eight worms singly into a plastic cup containing 100 mL test media (simulated soil solution was prepared by adding different volumes of stock solutions of Ca(NO3)2, MgSO4, NaNO3, and KNO3 to deioninzed water). Test were carried out in an environmental chamber (20+/-1 degrees C) in darkness for 48 hr.

Measurements/observations

Number of surviving.

Evaluations

LC50 using the trimmed Spearman-Karber method.

[ref. ID; 7159]

Test system

Contact test (48 hr) and aritifical soil test (2 weeks)

Strains

Toxicants

Carbaryl, 1,2-Dichloropropane, Dimethyl phthalate, Fluorene, Nitrobenzene, N-nitrosodiphenylamine, 4-Nitrophenol, Phenol, 1,2,4-Trichlorobenzene, 2,4,6-Trichlorophenol.

Test design

Contact test: The glass vials used (Wheaton, 3 oz. snap cap) were 8 cm long and 3 cm in diameter. Strips of filter paper 12x6.7 cm (Whatman No.1) lined and completely covered the sides of the vial. One milliliter of water was added to the filter paper to provide enough moisture for earthworm survival. One adult earthworm was added per vial and the vials were kept on their side at 20 degrees C in a darkened incubator for 48 hr. At least five concentrations were evaluated in the definitive test, with 10 or more replicates used for each concentration tested.
EEC aritifical soil test: The medium consists by weight of 10% finely ground sphagnum peat, 20% kaolinite clay, 69% fine sand and 1% pulverized calcium carbonate. pH 6.0+/-0.5. water content 35% of the dry wt of the four components. Ten adult earthworms were added to 400 g (dry wt) of the test mixture in covered glass dishes 6.5 cm high and 12.5 cm in diameter. The dishes were incubated at 20 degrees C and mortality was assessed after 2 weeks. This test had at least five chemical concentration and four control dishes, all containing 10 worms per dish.

Measurements/observations

Mortality.

Evaluations

Contact test: LC50 using the method of Litchfield & Wilcoxon (1949). The LC50 values are reported as ug of test chemical cm-2 of filter paper.
EEC aritifical soil test: LC50 using the method of Litchfield & Wilcoxon (1949). The LC50 values are reported as mg chemical/kg dry wt of artificial soil.

[ref. ID; 7162]

Test system

Bioavailability

Strains

From Carolina Biological Supply (Burlington, NC).

Toxicants

DDT (75% p,p'-isomer, 18% o,p'-isomer), DDE, DDD, and Dieldrin.

Test design

Field study
Worm and soil samples: Samples of Chester loam (pH 5.5, 6.5% organic matter) and Sassafras silt loam (pH 5.2, 4.4% organic matter) that had been treated with DDT and dieldrin in 1949 or that did not receive the insecticides were obtained at depths of 0-25 cm from experimental plots at the Beltsville Agricultural Research Center (U.S.Department of Agriculture, Beltsville, MD).

Samples of Kendaia loam (pH 6.6, 12.5% organic matter) were obtained from Aurora, NY.
Samples of a sandy loam (pH 6.6, 6.0% organic matter) that had been contaminated with DDT and diledrin approximately 30 years earlier and an adjacent uncontaminated silt loam (pH 6.4, 5.4% organic matter) from a remediation site at the U.S. Navy Surface Weapons Testing Center in Dahlgren, VA.

Laboratory study
Chester loam (uncontaminated soil): DDT (final concentration 13.6 mg/kg of soil), DDE (5.28 mg/kg), (3.26 mg/kg), and dieldrin (9.56 mg/kg) in hexanes were added to 2 kg of Chester loam, respectively. The soil was thoroughly mixed with a Teflon-coated spatula and stored in an EPA-cetrified ultraclean 1-L glass jar with a Teflon-lined screwcap. The height of the headspace above the soil was 5 cm. The soil was stored in the dark for 90 days at approximately 22 degrees C.

Kendaia loam (uncontaminated soil): DDT (final concentration 46.6 mg/kg of soil), DDE (19.8 mg/kg), and DDD (11.2 mg/kg) in hexanes were added to 2 kg of Kendaia loam, respectively. The soil was stored 190 days.
The remediation site in Dahlgren and in adjacent soil (uncontaminated soil): DDT (final concentration 45.9 mg/kg of soil), DDE (11.0 mg/kg), DDD (15.6 mg/kg), and dieldrin (13.6 mg/kg) in hexanes were added to 2 kg, respectively. Unaged.

Six worms were placed in 60 g (dry wt) of pesticide-amended soil contained in 250-ml glass jars (90% of field capacity with water). The jars were covered with Saran wrap bearing holes for air entry, and then kept under constant room lighting. Exposure period 8 days.

Measurements/observations

Field study: Toxicants concentration in worm tissue.
Laboratory study: Weight. DDT, DDE, DDD, dieldrin concentrations in worms.

Evaluations

Earthworm uptake (ug/g).

[ref. ID; 7163]

Test system

Contact test (2-d) and Soil test (14-d)

Strains

Toxicants

Acenaphthene, Benzene, n-Butyl phthalate, Cadmium acetate, Cadmium chloride, Cadmium nitrate, Cadmium sulfate, Carbaryl, Chloroacetamide, Chlorobenzene, 2-Chloroethyl vinyl ether, 2-Chlorophenol, Copper chloride, Copper nitrate, Copper sulfate, 1,2-Dichlorobenzene, 1,2-Dichloroethane, 1,2-trans-Dichloroethylene, Dichloromethane, 2,4-Dichlorophenol, 1,2-Dichloropropane, Dieldrin, Diethyl phthalate, Dimethyl phthalate, 2,4-Dimethyphenol, Dioctyl phthalate, Diphenylnitrosamine, Di-n-propylnitrosamine, Ethylbenzene, Fluoranthene, Fluorene, Hexachlorobutadiene, Hexachloroethane, Lead nitrate, Lead sulfate, Naphthalene, Nickel acetate, Nickel chloride, Nickel nitrate, Nickel sulfate, Nitrobenzene, 2-Nitrophenol, 4-Nitrophenol, Pentachlorophenol, Phenol, Tetrachloroethane, Tetrachloroethylene, Tetrachloromethane, Toluene, 1,1,2-Trichloroethane, Trichloroethylene, Trichloromethane, 2,4,6-Trichlorophenol, Zinc acetate, Zinc chloride, Zinc nitrate, Zinc sulfate.

Test design

Standard protocols.

Measurements/observations

Mortality.

Evaluations

LC50 and Weibull function.

[ref. ID; 7171]

Test system

Lethal toxicity (7-day and 14-day), chronic toxicity (28-day) and molecular endpoints

Strains

From the Earthworm Breeder Company in Tianjin, China. Adult animals with well-developed clitellum (400+/-500 mg weight).

Toxicants

Polycyclic musks (HHCB, AHTN).

Test design

Natural (uncultivated and unpolluted) field soils were collected from the surface layer (0-20 cm in depth) of arboretum area in Tianjin, China.

Lethal toxicity test: OECD guideline. AHTN and HHCB concentration 100.0, 140.0, 196.0, 274.4, 384.2, 537.8, 752.9, 1054.1, and 1475.8 ug g-1 air-dried soil. Three replicates of 10 worms were used for each container containing 500 g soil and placed in an incubation chamber (20+/-1 degrees C, light/dark cycle 12/12 hr). Dry cow manure 2 g.
Chronic toxicity test: AHTN and HHCB concentration 0 (solvent control), 3, 10, 30, 50 and 100 ug g-1 air-dried soil. Four replications of 10 worms. Dry cow manure 2 g.

Three earthworms per replicate of different groups were pooled together and were snap-frozen in liquid nitrogen and stored at -80 degrees C until required for gene expression analysis.

Measurements/observations

Lethal toxicity test: Mortality.
Chronic toxicity test: Body weight, cocoon number.
Molecular analysis: beta-actin, SOD, CAT, HSP70, ANN, TCTP, CRT.

Evaluations

Lethal toxicity test: 7 day- and 14 day-LC50.
Chronic toxicity test: Growth rate and reproduction rate using one-way anlaysis of variance (ANOVA) followed by Dunnett's comparison.

[ref. ID; 7182]

Test system

Growth and survival

Strains

Toxicants/concentrations

Acetovanillone, Acetylsalicylic acid, p-Anisic acid, Anisole, Apocynol (alpha-Methylguaiacylalcohol), Benzaldehyde, Benzene, Benzoic acid, Catechol, 3,5-Dihydroxybenzoic acid, 3,4-Dimethylphenol, 3,5-Dimethylphenol, o-Dimethoxybenzene, m-Dimethoxybenzene, p-Dimethoxybenzene, Ferulic acid, Gallic acid, Gentistic acid, Guaiacol (o-Methoxyphenol), Humic acids (From activated sludge, From anaerobic sludge), Hydroquinone, p-Hydroxybenzoic acid, Lignins (Cherry, Indulin AT, Marasperse, Orzan A, Reax 31), Melilotic acid, 1-Methyl 3,5-dihydroxybenzene, p-Methoxyacetophenone, m-Methoxyphenol, p-Methoxyphenol, Phenol, Phloroglucinol, Protocatechuic acid, Pyrogallol, Resorcinol, Salicylic acid, Salicylic alcohol, Salicylic aldehyde, Tannic acid, Toluene, Vanillic acid, Vanillin.
The test substances, except lignins and humic acids, were processed for assay by mixing 0.01, 0.1, 0.5 or 1.0 g or ml into 100 g sludge which contained 11-15% solids. The humic acids and lignins were tested at concentrations of 0, 1, 4, 8 and 16%.

Test design

About 30 g sludge (ca. 13% solids) with test substance were placed over a ca. 4 mm depth of silt loam in a 20x100 mm Petri dish. Two hatchlings, each under 100 mg live, wt, were added. The set of 25 dishes with 5 replicates of 5 concentrations was then stored at 24+/-1 degrees C.

Measurements/observations

Mortality. Growth (body weight: 2, 4, and 6 weeks).

Evaluations

Significant differences by the Neuman-Keul test.

[ref. ID; 7218]

Test system

Uptake and elimination

Strain

Without a clitellum, individual weight 0.352+/-0.09 g.

Toxicants

PAC-contaminated soil from an old gaswork site in Stockholm, Sweden.

Test design

The fraction with particles less than 5 mm was divided among a total of 15 vessels with 300 g (wet wt) soil in each (water content 10%, organic matter (OM) content 6.2% of the dry matter (DM)). The glass test vessels were covered with aluminum foil but were not completely dark inside. Samples were protected from ultraviolet radiation by ultraviolet filters on hoods. Six worms were added to each vessel, and each vessel represented one sample. The vessels were regularly weighed to check that the moisture contents remained constant. Room temperature (~22 degrees C). No food.

Uptake study: Duplicate samples of worms were taken after 0, 2, 5, 8, 11, and 19 days of exposure.
Elimination study: Worms were exposed for 19 d to contaminated soil transferred from five vessels to five new vessels with artificial soil (according to the Organization for Economic Cooperation and Development guidelines for testing chemicals). Worms were sampled after 0, 1, 3, 6, 10, and 14 days of elimination.

Measurements/observations

Concentrations of Anthracene, 9,10-Anthraquinone, Benzo[a]anthracene, Benzo[a]pyrene, Benzo[b]fluoranthene, Benzo[k]fluoranthene, Benzo[ghi]perylene, 1,2-Benzoanthraquinone, Carbazole, Chrysene, Dibenzo[a]anthracene, Dibenzotiophene, Fluoranthene, Indeno[cd]pyrene, Phenantrene, Pyrene in worm extracts and soil extracts.

3. Eisenia fetida andrei

[ref. ID; 831]

Test system

Acute toxicity

Strains

Adult with a well-developed clitellum, more than 6 weeks old, with an average weight of 300-420 mg.

Toxicants

Cadmium chloride, Chloroacetamide, 3,4-Dichloroaniline, Pentachlorophenol.

Temperature

23+/-2 degrees C.

Test design/concentration

A modification of OECD (1984) filter paper contact test: the influence of soil characteristics (artificial soil (10% <0.5 mm ground sphagnum peat, 20% kaolin clay, 69% fine sand, 1% calcium carbonate) & Gilze soil (low-organic-matter sand)), darkness, toxicants concentration: 0, 0.1, 1.0, 10 and 100 ug/cm2 of the test substance x 5 replicates.

Measurements/observations

24-hr and 48-hr mortality.

Evaluations

LC50.

[ref. ID; 3582]

Test system

Biochemical response

Strains

Purchased from 'Ver'Humus' (Cahors, France). Adults with well-developed clitellae.

Toxicants

Carbaryl

Test design

The earthworms were exposed to carbaryl in artificial soil as described by OECD (1984). To obtain media containing 12, 25 and 50 mg carbaryl kg-1 soil, 600 g of soil was contaminated by adding the appropriate amount of carbaryl to 180 ml acetone. The soil was then mixed for at least 30 min in a Rotavapor with heat applied to obtain homogeneously spiked soil and the evaporate the acetone. The moisture content was adjusted to 35% of the final wieght. All exposures were conducted at 20+/-1 degrees C under continuous light to ensure that the earthworms remained burrowed in the soil. Three exposure period 2, 7, and 14 days.

Measurements/observations

Biochemical measurements (MROD activity, NADPH Red activity, NADH Red activity, GST activity, AChE activity, LP, LPI, total GSH, %GSSG, CAT activity, GR activity).

[ref. ID; 3584]

Test system

Biochemical response

Strains

Purchased from 'Ver'Humus' (Cahors, France). Adults with well-developed clitellae.

Toxicants

Lead acetate

Test design

The earthworms were exposed to lead acetate in artificial soil method described by OECD (1984). To obtain media containing 30, 60, 120 and 250 mg Pb kg-1 soil, 200 g of soil was contaminated by adding the approriate amount of Pb to 70 ml distiled water. The soil was mixed carefully. The moisture content was adjusted to 35% of the final weight. The pH not modified (5.80-6.20). All exposures were conducted at 20+/-1 degrees C under continuous light to ensure that the earthworms remained burrowed in the soil. Four exposured period: 2, 7, 14 and 28 days, and for each duration and dose condition, three replicates, consisting of five worms pooled together, were anaylsed.

Measurements/observations

Biochemical measurements (MROD activity, NADPH Red activity, NADH Red activity, GST activity, AChE activity, LP, LPI, total GSH, %GSSG, CAT activity, GR activity).

Evaluations

Multivariate analyses (MANOVA), Univariate analyses (ANOVA), post-hoc comparison (LSD test) and Discriminant analyses (DA) were carried out using Statistica software (release 5.1H, Statsoft Inc, Ed., 1997).

[ref. ID; 6822]

Test system

Immuno-modulator effects

Strains

From "ver'Humus" (Cahors, France). Adults with a well-developed clitellum.

Toxicants

Carbaryl, 2,4-dichlorophenoxy acetic acid (2,4-D).

Test design

Contact test method (EEC, 1985). Exposure period 48 hr at 20 degrees C.
Preparation of Coelomic Fluid (CF) and Cytosol (CL): Earthworm released about 50 ul of liquid through the dorsal pores. The supernatant of a 5 min centrifugation at 11,000 g, 4 degrees C corresponded to the coelomic fluid (CF). The pellet of coelomocytes was resuspended in a volume of 0.1 M TRIS buffer, pH 8, equal to the CF volume, then sonicated. The supernatant of 5 min centrifugation at 11,000 g, 4 degrees C reffered to the cytosol.

Lysozyme activity:
Cytolytic activity:
Protease activity:
Serpin activity (Activity of serine protease inhibitor):
Phagocytic activity:

Evaluations

LC50 using the Log-Probit method (Mantel and Bryan 1961).

[ref. ID; 6908]

Test system

Biochemical response

Strains

From 'Ver'Humus' (Cahors, France). Adults with well-developed clitellae.

Toxicants/concentrations

Benzo(a)pyrene 50 ug, 1mg, 100 mg and 1 g per 1 kg artificial soil.

Test design

The earthworms were exposed to Benzo(a)pyrene in artificial soil as described by OECD (1984). 20+/-1 degrees C under continuous light. Duration period 1, 2, 7 and 14 days.

Measurements/observations

Catalase, acetylcholinesterase, glutathione-S-transferase, methoxyresorufin-O-deethylase, NADH (NADH Red) and NADPH (NADPH Red) cytochrome reductase activites, lipid peroxides, peroxidizable lipids, total glutathione concentrations and percentage of oxidized glutathione in worm.

[ref. ID; 6953]

Test system

Toxicity and bioaccumulation

Strains

Adults with a well-developed clitellum (more than 6 weeks old) and average weight 324-486 mg.

Toxicants

3-Chlorophenol, 3,4-Dichlorophenol, 2,4,5-Trichlorophenol, 2,3,4,5-Tetrachlorophenol, Pentachlorophenol.

Test design

Soils: Soils was collected from the top 20 cm of agricultural fields. Holten soil (very humic sand) and Kooyenburg soil (moderately humic sand).
Glass jar filled with about 0.5 kg soil (dry wt) and 10 worm. The jars were placed in an incubator at 23 degrees C. Experimental period 7 and 14 days.

Measurements

Mortality.

Evaluations

LC50 values based on 14 days'mortality data were calculated according to a logit model.

[ref. ID; 6954]

Test system

Reproduction toxicity

Strains

Adults with a well-developed clitellum (8-13 weeks old) and average weight 248-425 mg.

Toxicants

Pentachlorophenol (PCP) (0, 1.0, 3.2, 10, 32, 100 mg/kg dry soil), copper(II)chloride/2H2O (0, 60, 120, 180, 240, 300 mg/kg dry soil), 2,4-dichloroaniline (DCA) (0, 18, 32, 56, 100, 180 mg/kg dry soil).

Test design

Artificial Soil (10% sphagnum peat, 20% kaolin clay, 69% fine sand; pH 6.0+/-0.5, water holding capacity 55% (w/w)). Food: Finely ground (<1.0 mm) cow dung. Earthworms are exposed to the chemical substances for 3 weeks. The number of cocoons produced is determined, and cocoons are incubated in untreated artificial soil for 5 weeks to assess hatchability. 0.5 kg dry soil were placed in 1-liter glass jars. In the middle of the soil a hole was made and filled with 2% (dry weight) food. Ten adult earthworms were added to each jar. Jars were loosely covered with a glass petri dish to prevent moisture loss by evaporation and incubated at 20+/-2 degrees C in an illuminated climatic chamber (ca. 400 lux). 4 replicates.

Measurements/observations

Number of cocoon produced and cocoon hatchability.

Evaluations

EC50 according to a logit model. No-effect levels (NEL) using Student's t test.

4. Eisenia veneta

[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

Lethality test (14d-LC50): Ten worms were placed in glass containers with 500 g dry mass of a soil-like substrate composed of a mixture of sphagnum peat, kaolinite clay, industrial quartz sand in a dry-weight ratio of 1:2:7. The pH (1N KCl) was adjusted to 6.0+/-0.5 with calcium carbonate. The water content was kept at 55% on a dry mass basis. Temperature 15 degrees C. Forty worms were tested at each test concentration.
Reproduction test (14d-EC50): Similar above experimental conditions, however, some modification following; a) the replacement of the artificial soil with a natural sandy soil (Kooyenburg) containing 3.7% organic matter, 1.4% clay, and pH (1N KCl) 4.8, and b) the supply of coarsely groud air-dried leaves of alder, Alnus glutinosa, for food.

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; 6076]

Test system

24-hr acute toxicity

Toxicants

Carbaryl, paraoxon.

Temperature

24-27 degrees C.

Test design

The standard OECD tests.

Measurements

Survival, Cholinesterase activity.