Enchytraeus

Enchytraeus albidus
Enchytraeus buchholzi
Enchytraeus crypticus

1. Enchytraeus albidus

Enchytraeus albidus are important members of the soil fauna, being representative of the true soil layer. (ref. ID; 6816)

[ref. ID; 3555]

Test system

14-day acute toxicity test according to OECD (1999) (influence of soil characteristics)

Strains

Enchytraeus albidus (Henle, 1847) cultures were provided by J. Rombke. The cultures have been successfully maintained in our laboratory for five years.

Toxicants

CuCl2/2H2O, Pb(NO3)2.

Test design

Fractional Factorial Design (FFD), Central Composite Design (CCD).

Experimental conditions

Artificial soil (OECD, 1984: 70% dry wt sand, 20% dry wt kaolin clay, 10% dry wt Sphagnum peat, adjusted to pH 6 with CaCO3), 16L:8D. Temperature 20+/-1 degrees C.

Measurements/observations

Mortality.

Evaluations

LC50.

[ref. ID; 4447]

Test system

Acute and chronic toxicity assays

Strains

Provided by J. Rombke.

Toxicants

Arsenate

Test design/concentration

OECD Guideline 220 (1999) Artificial soil + rolled oats + Na2HAsO4 (0, 10, 18, 32, 56, and 100 mg As/kg dry wt) x 4 replications, 16L:8D photoperiod (400-800 lux). Temperature 20+/-1 degrees C.

Measurements/observations

Mortality, number of juvenile.

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; 5909]

Test system

Application method for aquatic toxicity test

Toxicants

Benomyl, PCP, Parathion, 2,4,5-Trichlorophenoxyacetic acid, Chloroacetamide, CdCl2, Tetrapropylenebenzolesulphonate, K2Cr2O7.

Test design

Aquatic Enchytraeid test: Test solution (CaCl2/2H2O 294 mg, MgSO4/7H2O 123 mg, NaHCO3 65 mg and KCl 5.8 mg per 1 liter deionized water with a conductivity of less than 10 uS cm-1, pH 7.8). Glass beaker (100 ml) containing test solution (50 ml) + 10 animals x 3 replications, without food, 96 hr.
Terrestrial Enchytraeid test: Artificial soil. Test boxes (8.4x8.4x4.5 cm) containing soil (10 g dry weight) + 10 animals + 20 mg of rolled oats (food), 4 weeks.

Measurements/observations

Aquatic Enchytraeid test: Mortality.
Terrestrial Enchytraeid test: Mortality, biomass production.

Evaluations

Aquatic Enchytraeid test: LC50 by the probit method or the geometrical median of the LC0 and LC100.
Terrestrial Enchytraeid test: LC50 by the probit method.

[ref. ID; 6016]

Test system

Uptake and elimination kinetics (28 day)

Strains

From J. Rombke.

Toxicants/concentrations

Zinc (10, and 100 mg/kg dry weight) and cadmium (10, and 100 mg/kg dry weight).

Test design

OECD standard artificial soil (70% sand, 20% kaolin clay, and 10% finely ground Sphagnum peat, adjusted to pH 6 with CaCO3). Temperature/light: 20 degrees C and a 16:8-hr light:dark cycle.

Measurements

The internal metal concentration.

[ref. ID; 6119]

Test system

The effect of ageing on the OECD-soil for zinc toxicity

Strains

From J. Rombke (ECT Oekotoxikologie GmbH, Germany).

Toxicants

ZnCl2

Temperature/light

20+/-1 degrees C. light:dark cycle of 16:8 at 400-800 lux.

Test design

Toxicity test were carried out according to OECD (1999). Per glass vessel, 10 adult worms with a fully developed clitellum were exposed in 20 g wet weight of soil. Four replicates were used per concentration and eight replicates were used as control treatments. Chronic toxicity test lasted for 6 weeks and weekly, ground rolled oates were put on the surface as a food source.
OECD-artificial soil (OECD guidline 207, 1984): 70% sand, 20% kaolinite clay and 10% finely ground Sphagnum peat, adjusted to pH 6 with CaCO3); OECD-soils were aged in four different ways: (1) storing for 8 weeks at 20 degrees C; (2) percolation (1 day of equilibration with a volume of deionized water corresponding to two times the water holding capacity) followed by storing at 20 degrees C; (3) alternately heating at 60 degrees C for 1 week and storing at 20 degrees C for 1 week; and (4) alternately freezing at -20 degrees C for 1 week and storing at 20 degrees C.

Measurements/observations

After 3 weeks of exposure, surviving animals were counted. After another 3 weeks of exposure, juveniles were counted. (The substrate was fixed with ethanol and a few drops of Bengal Red solution were added. The next day the substrate was washed through a 300-um sieve and the the juveniles were counted).

Evaluations

EC50s using the probit method.
LC50s using the binomial method.

[ref. ID; 6130]

Test system

Biotransformation in vivo and in vitro

Strains

From J. Rombke, Germany.

Toxicants

2,4,6-Trinitrotoluene (TNT) and TNT metabolites (2-hydroxyamino-4,6-dinitrotoluene (2-HADNT), 4-Hydroxyamino-2,6-dinitrotoluene (4-HADNT), 2-ADNT, 4-ADNT, 2,4-DANT, 2,6-DANT).

Test design

Run 1: Potworms were exposed to 1 ml of different concentrations of TNT in 0.2 um filtered tap water (measured prior to exposure: 0, 5.2, 10.2, 52.0 and 102.0 mg l-1 corresponding to 0, 22.7, 44.7, 228.9 and 449.0 nmoles ml-1, respectively) under static conditions at 20+/-1 degrees C in the dark for 3 hr and 20 hr.
Run 2: Potworms were exposed for 20 h, in triplicate test units (each containing 10 potworms), to 1 ml of different concentrations of TNT (0, 4.3, 20.9 and 42.1 mg l-1 corresponding to 0, 18.8, 92.2 and 185.4 nmoles ml-1, respectively) in the dark using 10-ml glass vials wrapped with aluminium foil.
Run 3: 10 Potworms exposed for 21 days, in triplicate to 20 g (dry amended artificial soil (a mixture of local garden soil and OECD artificial soil 1:1, w/w). Test TNT concentrations (nominal concentrations: 0, 25, 50, 100, 200, 300 and 1 000 mg TNT kg-1 dry soil). Food (lyophilized oats).

Measurements/observations

Chemical analyses of tissue extract.

[ref. ID; 6754]

Test system

Joint toxicity of chemical (CA model & IA model)

Strains

Toxicants

Atrazine, dimethoate, lindane, cadmium chloride anhydrous, zinc chloride.

Experimental design

Soil: Certified loamy sand soil LUFA2.2 (pH = 5.5, organic matter = 3.9%, texture = 6% clay; 17% silt; 77% sand). This soil type commercially available at the German institution LUFA Speyer.
Avoidance behavior tests: Using two-section vessels (ISO, 2007 (draft)). Circular plastic boxes (7.8 cm diameter x 4.3 cm highest). 20+/-degrees C. Photoperiod 16:8 hr (dark:light). 10 worms per replicate. Experimental period 48 h.

Test design

The single exposure bioassay.
The binary mixture exposure bioassay.

Measurements/observations

Evaluations

AC50

[ref. ID; 6755]

Test system

Avoidance response test

Strains

Adults with well-developed clitellum.

Toxicants

Carbendazim

Test conditions

Plastic Petri dishes (8.5 cm in diameter). The dishes were covered by their plastic lids and stored under constant conditions (20 degrees C, 16:8 h light:dark photoperiod). 5 replicates.
Soil: Natural standard soil LUFA 2.2 (50% WHC(max)).

Test design

1): Control-control test (with non-contaminated soil at the both sides of Petri dish).
2): The avoidance from six concentrations of carbendazim (0.25, 0.5, 2.5, 5, 25, 50 mg/kg) to non-contaminated soil was evaluated to determine EC50 value.
3): The avoidance from EC50 concentration at different exposure times was assessed to determine minimal test duration.
4): The avoidance from EC50 concentration was compared for fresh and 14 or 28 days aged contamination to see changes in carbendazim bioavailability.

Measurements/observations

Worms number.

Evaluations

Avoidance net response (NR). The non-parametric Mann-Whitney U test was used for the comparisons between groups of treatments. The concentrations causing 50% avoidance response (EC50 value) and time (ET50 value) necessary to reach the positive 50% NR by using this concentration (EC50 value) were calculated by probit regression.

[ref. ID; 6816]

Test system

The effect of aging on bioaccumulation and bioavailability

Strains

Toxicants

Lindane (1,2,3,4,5,6-gamma-HCH).

Test design

Two soil types.

Artificial OECD soil (OECD guideline no 207, OECD 1984): pH was adjusted to 4.7, similar to the pH of the natural soil. Although this was not the most suitable pH for these worms, which were not reproducing at this pH, the adults survival was not affected, and therefore no problems relative to the bioaccumulation test occurrence were noticed.
Natural agricultural soil: An agricultural alluvial soil from the Low Mondego Valley.

Test vessels (7.5-cm diameter and 4 cm high), containing approximately 30 g dry weight of soil. 40% of the maximum water holding capacity. Temperature 20+/-degrees C, photoperiod 16:8 hr (light:dark). Exposure period 10 months.

Measurements

[14C]gamma-HCH concentration in the worm.

[ref. ID; 6826]

Test system

Avoidance test

Strains

Toxicants

Benomyl, Carbendazim, Phenmedipham.

Test design

Soils: The artificial OECD soil (OECD, 1984), the natural standard soil LUFA 2.2 and field soils (Euro-Soil).

Experimental conditions

Plastic boxes (8x5x10 cm) and a movable wall which divides the box in two halves. Previous to the introduction of the soils (25 g in each side), the wall is placed at the centre of the box and the control soil is introduced in one side of the vessel and the test soil on the other. After this, the wall is gently removed and ten adult worms are left on the contact line of the soils. The box is covered with a lid (containing small holes) and the test is running for 48 hr at 20 degrees C and a photoperiod of 16:8 hr. Five replicates per treatment are used. At the end of the test the movable wall is replaced in the centre and each side of the box is independently searched for worms.

Measurements/observations

Numbers.

Evaluations

The avoidance effect expresses the percentage of affected worms using the statistical software package SPSS 12.0. EC50.

[ref. ID; 6830]

Test system

Acute and chronic toxicity

Strains

From J. Rombke.

Toxicants

CdCl2/H2O

Test design

Toxicity tests were carried out according to draft OECD guideline 220 (1999). Grass vessels were filled with 30 g wet weight of soil and 10 adult worms. Vessels were kept at 20+/-1 degrees C and a light:dark cycle of 16:8 hr at 400-800 lux.

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; 6982]

Test system

Chronic toxicity assay

Strains

The strain was provided by J. Rombke. Adult worm with a fully developed clitellum.

Toxicants/concentrations

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

Test design

Chronic toxicity assays were carried out according to OECD Guideline 220 (1999). 10 adult worms. The glass vessel was filled with 20 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 a light:dark cycle of 16:8 hr at 400-800 lux. Exposure period six weeks. Rolled oats were put on the soil surface weekly as a food source.

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; 6984]

Test system

Influence of soil type for bioaccumulation and elimination

Strains

From a laboratory culture maintained under controlled conditions according to Rombke and Moser (1999).

Toxicants

Lindane

Test design

Artificial OECD soil: OECD guideline no. 207 (OECD, 1984, earthworm acute toxicity tests). pH was adjusted to 4.7 (similar to the natural soil) adding CaCO3.
Natural soil: An agricultural alluvial soil from the Low Mondego Valley in Central West Portugal.

Test vessels (7.5 by 4 cm plastic boxes) filled with 30 g DW (40% water holding capacity) of contaminated soil. Five animals placed into the test vessel with no food supply and the vessel was covered with a lid. Temperature of 20+/-2 degrees C and a photoperiod of 16/8 hr (light:dark). The experiment lasted for a period of 20 days, 10 for the uptake phase plus 10 for the elimination phase.

Measurements/observations

Lindane concentration in worm.

Evaluations

Bioaccumulation factor (BAF).

[ref. ID; 7034]

Test system

The influence of soil characteristics on the toxicty

Strains

The strains were provided by J. Rombke.

Toxicants

CdCl2/H2O, ZnCl2.

Test design

Toxicity tests were carried out according to the OECD (1999, Test Guideline 220). Immediately after spiking, 10 adult worms were exposed in 20 g of soil in covered glass vessels. During exposure, vessels were kept at 20+/-1 degrees C at a 16:8-hr light:dark cycle. Exposure period 14-days.
The influence of soil characteristics on the toxicty: A fractional factorial design (FFD). Soil parameter (pH, clay, organic matter, FeO2, H2O, K+, Mg2+).

Measurements/observations

Number of surviving worms.

Evaluations

LC50s

[ref. ID; 7135]

Test system

Reproduction and bioaccumulation

Strains

Toxicant

Phenanthrene

Test design

Test soil (The natural standard soil LUFA 2.2, originating from Speyer, Germany. The test soil has an organic matter content 4.4%, organic carbon content of 2.5%, pH (0.1 M CaCl2) approximately 5.8, 6% clay, 17% silt, and 77% sand, cation exchange capacity of 11 cmol/kg, and a maximum water holding capacity of approximately 55%.

Reproduction test: According to OECD Guideline 220 and International Organization (ISO) Guideline 16387. Test vessel (glass containers of 250 ml with 25 g (dry wt equivalents) moist soil, plus food). Ten adult worms, clitellate and with eggs. Phenanthrene concentrations 5, 10, 20, 40, 80, and 160 mg/kg dry soil, 4 replicates per concentration. Containers were covered with parafilm with a few holes for aeration and incubated in a climate room at 20 degrees C, with a 16:8 hr light:dark cycle.
Bioaccumulation test: According to OECD Guidlines for testing of chemicals: Bioaccumulation: soil test using terrestrial oligochaetes (2010).

The experiment included a 14-days uptake phase followed by a 14-days elimination phase. Test vessels (glass containers of 250 ml) with 25 g (dry wt equivalents) of moist soil spiked with 8 mg PHE/kg dry soil. 15 worms with a well-developed clitellum. Containers were covered with parafilm with a few holes for aeration and incubated in a climate room at 20 degrees C, with a 16:8 hr light:dark cycle. After 14-days exposure, the remaining animals were transferred into clean soil.

Measurements/observations

Reproduction test: Adult and juvenile number.
Bioaccumulation test: Phenanthrene concentrations in worms.

Evaluations

Reproduction test: LC50 using the trimmed Spearman-Karber method. EC50 applying a four-parameter logistic model according to Van Ewijk and Hoekstra and a generalized likelihood ratio test.
Bioaccumulation test: BAF.

[ref. ID; 7140]

Test system

DNA microarray

Strains

Toxicants/concentrations

CuCl2/2H2O 320 mg/kg soil dry wt. and Phenmedipham 10 and 32 mg a.i./kg soil dry wt (157 g/L active ingredient [a.i.]).

Test design

Two different soil were used the LUFA2.2 and the OECD artificial soil. The OECD soil was manipulated to obtain three different soils in terms of constituents' composition. 15 adult worms with well-developed clitellum were introduced into a glass vessel containg 25 g of most soil (40-60% of the maximum water holding capacity). Each vessel was covered with a lid having small holes. Test were maintained at 20 degrees C for 48 hr with a photoperiod of 16:8 hr light:dark. Six replicates per treatment.

Measurements/observations

Total RNA was extracted from all replicates of each exposure treatment. For the constructed of the normalized cDNA library, the protocol of Shagin was used.

2. Enchytraeus buchholzi

[ref. ID; 1320]

Test system

4-days mortality test and 16-days reproduction test & enzyme assay

Strains

Mature, 3-4 mm; juveniles >1 mm.

Toxicants

CdCl2.

Test design

1% Agar medium (for reproduction test) and solution (for mortality test), both media composition (Ca(NO3)2 1g/l, MgSO4/7H2O 0.25 g/l, KNO3 0.25 g/l, KH2PO4 0.25 g/l, a trace of FeSO4), pH 5.0.

Measurements/observations

Number, mRNA encoding a nonmetallothionein 33-kDa protein.

Evaluations

LC50, accumulation.

3. Enchytraeus crypticus

[ref. ID; 5974]

Test system

The 28-days enchytraeid reproduction test

Strains

Toxicants

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

Test design

International Standard Organization [ISO 11267].

Measurements/observations

Adult survival and cocoon production.

Evaluations

NOEC, LOEC, EC20, EC50.

[ref. ID; 6783]

Test system

Prediction of metal bioavailability

Strains

Toxicants

Twenty soils were collected at moderately contaminated (Cd, Cu, Pb, and Zn) sites in The Netherlands. For site codes see Janssen et al. (1997) (Environ. TOxicol. Chem. 16, 2470-2478).

Test design

Plastic jars (15 ml) filled with 10 g fresh soil at field humidity, and then, adult specimens of E. crypticus added. 4 replicates. Once a week a spatula tip of rolled oats was added to feed the worms. Exposure period 0, 1, 2, 3, 4, 7, 14, 21, 28, and 35 days.

Measurements/observations

Metal concentrations in worms.

Evaluations

Uptake rate constants and equilibrium concentrations were estimated using compartment modeling.

[ref. ID; 6836]

Test system

Joint toxicity of a binary metal mixture of copper and zinc

Strains

From Dr. J. Rombke from a culture at Osnabruck.

Toxicants

Cucl2/H2O, ZnCl2.

Test design

OECD artificial soil. Exposure duration 4 weeks. Copper singly, zinc singly, and equitoxic mixtures of copper and zinc. Six replicates were prepared for each treatment, four for observations on reproduction, and two for body concentration measurements. Adult enchytraeids were randomly distributed in groups of 15 individuals and put in plastic containers (15 ml) filled with 10 g soil to start exposure. During exposure, the containers were kept at 17+/-1 degrees C in the dark. Animals were fed weekly with oat meal.

Measurements

Juvenile production.

Evaluations

EC50, Toxic Unit.