Caenorhabditis elegans
Caenorhabditis elegans, a free-living soil nematode, is one of the best characterized animals at the genetic, physiological, molecular, and developmental levels. C. elegans has the properties of a short life cycle, small size, ease of cultivation, a simple cells lineage, and easily monitored behaviors. A standardized method for conducting laboratory soil toxicity tests using C. elegans was published in the American Society for Testing and Materials (ASTM 2002) Guide E2172-01. (ref. ID; 6700)
The fully sequenced genome of the nematode C. elegans contains two metallothionein isoforms (mtl-1 and mtl-2) both located on chromosome V. (ref. ID; 6738)
Adult worms are about 1 to 1.5 mm in length and can be distinguished into two sexes, hermaphrodites and males. Hermaphrodites usually reproduce by self-fertilization. After hatching, C. elegans reaches the adult stage by passing through four juvenile stages separated by molts. Under starvation a developmentally arrested stage, the dauer larva, can occur as an alternative third larval stage. The life cycle for worms grown on Escherichia coli is about 3 day at 20 degrees C. (ref. ID; 6855)
Caenorhabditis elegans, a free living soil nematode, was chosen as the test organism for several reasons: methods of testing C. elegans in an aquatic medium had previously been developed; and large numbers (60 animals per concentration) can be tested in a small volume of solution (6 ml). A 24 hr exposure period covers a significant percentage of the nematode's typical 10 day lifespan. C. elegans can be tested in a simple medium consisting of deionized water, Nacl and KCl, thus reducing interaction (i.e., precipitation) of metal ions with ligands in the testing matrix, and also C. elegans can survive over 24 hr without a food source. Elimination of a food source prevented the potential sorption of metals to food and consequent removal from the test media. (ref. ID; 7010)
[ref. ID; 495]
Test system
Chronically effect for Ultrastructure
Strains
Isogenic line of the Bristol, N2 strain. C. elegans was cultured on NG agar seeded with B strain of Escherichia coli.
Toxicants/concentrations
Mercuric chloride & Silver nitrate (3.25x10-2 M, 1.08x10-2 M, control)
Test design
0.4 ml of an aqueous solution of toxicants was added to the plates which E. coli were growing. Ten 3.5-day-old C. elegans were placed in each test plate. After 4 hr they were removed and the resulting progeny were left to develop for 5 days. Only chronically poisoned nematodes were subsequently removed from the dish and prepared for electron microscope examination.
Measurements/observations
Lesions in esophageal muscles and intestinal cells.
[ref. ID; 498]
Test system
Recovery method (extraction method) and soil type for soil toxicity test
Strains
var. Bristol (strain N2)
Toxicants
Copper chloride (Cucl2/2H2O) (Cu2+: 0, 100 and 200 mg/L).
Test design
Soil type: No soil (aquatic), Sand, Cecil (sandy loam), Worsham (sandy loam), Davidson (loam), Dyke (clay loam), Artificial soil.
Extraction method: Ludox HS-40, a 40% (w/w) colloidal silica suspension; Ludox AM, an aluminum-modified 30% (w/w) suspension.
Evaluation
LC50
[ref. ID; 506]
Test systems
Acute toxicity (4 days)
Strains
C. elegans var. Bristol (strain N2)
Toxicants
Water-soluble salts of metals: HgCl2, BeSO4/9H2O, Al(NO3)3/9H2O, CuCl2/2H2O, ZnCl2, Pb(NO3)2, CdCl2, Sr(NO3)2, NaAsO2, K2Cr2O7, AgNO3, SbCl3, NiCl2, Tl.
Test design
1.0 ml aliquot of a test solution of the metalic salt in K-Medium (NaCl 1.23 g/400 ml water, KCl 0.968 g/400 ml water) was placed into the 24-wells tissue culture plates. Four young adults (3- to 4-day old) from C. elegans synchronized cultures. Food (E. coli strains OP50). Temperatures 20 degrees C. The testing was usually repeated 25 times (100 individuals) for each concentrations.
Measurements/observations
Survival number.
Evaluations
24 hr-, 48 hr-, 72 hr-, 96 hr- LC50.
[ref. ID; 849]
Test system
168-hr growth & reproduction test
Strains
C. elegans (Maupas 1900), variety Bristol, is originally isolated from the soil in Great Britain (Briggs 1946). It has been successfully cultured since 1948. This species used in the test were originally obtained from Dr. Vanfleteren of the State University of Ghent, Belgium. Jevenile 1 stage (J-1), axenically.
Toxicants
CdCl2
Temperature
20+/-1 degrees C.
Test design/concentrations
Flat-bottom wells of microtiter plates (wells contained 100 ul medium (yeast extract 30 g/l, soy peptone (30 g/l, bacto-casitone 10 g/l, D-glucose 10 g/l, bovine hemoglobin) with CdCl2 concentrations ranging 0~320 uM) x 3 replicates.
Measurements/observations
Mean body length, number of offspring.
[ref. ID; 850]
Test system
Thermal stress and oxygen deprivation
Strains
Caenorhabditis elegans (var. Bristol (strain N2)). Adult worm (monoxenically, Escherichia coli strain OP50: 3-5 days old) and dauerlarvae.
Toxicants
Thermal tolerance and anoxic tolerance.
Test design
Upper thermal tolerance limit: Dauerlarvae or adults which had been cultured at 20 degrees C were exposed to 37 or 40 degress C for up 26 hr.
Anoxic tolerance: Plates containing adult worms or dauerlarvae were transferred from a 20 degrees C incubator into anaerobic chambers (BBL GasPak anaerobic system). The anaerobic state is achieved 5-6 hr after sealing the chamber. Exposure period 1-16 days. After the end of the anoxic exposure, readmission of oxygen were had to measure of the worm's spontaneous locomotor activity.
Measurements/observations
Survival number, oxygen consumption and locomotor activity.
[ref. ID; 851]
Test system
Fecundity, growth and fine structure test
Strains
N2 strain + OP50 strain or B strain of Escherichia coli.
Toxicants
CdCl2/2.5H2O
Temperature
30 degrees C
Test design
Fecundity test: 60-mm petri dish (10 ml NG agar) + cadmium concentrations (0, 10E-8, 3.26x10E-8, 10E-7, 3.26x10E-7, 10E-6, and 3.26x10E-6 moles) x 6 replicates. Short term exposure (2, 6, and 24-hr) and long term exposure (3.5 days)
Growth test: Petri dish + cadmium concentrations (10E-7, 10E-6, 4x10E-7, and 4x10E-6 moles), 2-hr exposure
Measurements/observations
Body length, number of eggs and larvae, ultrastructure (esophagus, intestinal cells etc.).
[ref. ID; 852]
Test system
Strains
Caenorhabditis elegans var. Bristol, wild type N-2, J1 stage, axenically.
Toxicants/concentrations
Aldoxycarb (10, 100, 500, and 1000 ug/ml), Methylisothiocyanate (MITC) (1, 10, and 20 ug/ml), Methomyl (1, 10, and 100 ug/ml)
Temperature
20 degrees C.
Test design
300 ml Erlenmeyer flask containing 50 ml culture medium (3% soy peptone, 3% yeast extract, 1% casitone, 1% dextrose, and 500 ug/ml hemolobine), continuously shaking (70 rpm).
Measurements/observations
Body length and width, fecundity, survival.
[ref. ID; 853]
Test system
Genetics, ultrastructure, and tublins of wild-type and mutant (mebendazole-resistant) worms
Strains
Wild type: Strain N2 were originally obtained from Dr. Martin Samoiloff, Department of Zoology, University of Manitoba (Food: OP50 strains of Escherichia coli.
Mutant: Mebendazole-resistant mutants.
Toxicants
Mebendazole, Albendazole, Cambendazole, Fenbendazole, Methyl N-(2-benzimidazolyl)-carbamate, Thiabendazole
Measurements/observations
Body length, number of eggs.
[ref. ID; 857]
Test system
Strains
Strain N2 was provided by Dr. Martin Samoiloff, Department of Zoology, the University of Manitoba (food: Escherichia coli, strain OP50).
Toxicants/concentrations
Mebendazole (solvent: dimethyl sulphoxide (DMSO)), concentrations 0.05-6.4 ug/L.
Test design
Experimental cultures of C. elegans were grown in 60x15 mm or 35x10 mm plastic petri dishes containing 9.0 and 3.0 mL of NG agar, respectively.
Measurements/observations
Body length and width, reproductive capacity (egg-laying and egg hatching rate), motility.
[ref. ID; 1276]
Test system
Acute toxicity
Strains
Samples were taken near Wageningen, The Netherlands.
Toxicants
Cadmium chloride (CdCl2), Pentachlorophenol
Test design
LC50-tests were performed in water containing a defined mixture of minerals with concentrations resembling those found in interstitial water of sandy forest soils (K+: 0.1 mmol/L, Na+: 0.2 mmol/L, Ca2+: 0.35 mmol/L, Mg2+: 0.3 mmol/L, NH4+: 0.3 mmol/L, NO3-: 1.7 mmol/L, Cl-: 0.3 mmol/L) (Schouten and Van der Brugge 1989). pH 6.0+/-0.1.
Two replicates of the tests were carried out in multi-dishes (Greiner, 24 compartment plate, no. 662160) with lid and sealed with parafilm to minimize volatilization. Each compartment was filled with 0.9 ml of water containing toxicant. Samples of 0.1 ml of the multi-species suspension, containing 10-50 adult individuals of each species, were taken and suspended in each compartment. Temperature 20+/-0.1 degrees C in dark.
Measurements/observations
Mortality (24, 48, 72 and 96 hr).
Evaluations
LC50 according to the trimmed Spearmann-Karber method (Hamilton et al. 1977).
[ref. ID; 2165]
Test system
Stress response
Strains
Transgenic strains (PC71ubIn4, Pc72ubIn5, PC73ubIn6)
Toxicants
CdCl2, CuCl2, HgCl2, NaAsO2, Pb(NO3)2, ZnCl2 and paraquat.
Test design
Multiwell tissue culture dishes. Exposure to stress (8-96 hr).
Measurements/observations
Mortality, beta-galactosidase activity.
Evaluations
LC50
[ref. ID; 3301]
Test system
24-hr acute toxicity (by soil bioassay protocol)
Strains
Wild type N2 Bristol strain, age-synchronized adults
Toxicants
Nitrate salts of Cd, Cu, Ni, Pb, Zn.
Test design
The soil tests were conducted in 35-mm polystyrene culture dishes (including ASTM artificial soil, pH 7.21 (ASTM, 1997) 2.333 g + 1.5 ml of spiking solution).
Measurements/observations
Mortality.
Evaluation
LC50 (using TOXSTAT (Western Ecosystems Technologies).
[ref. ID; 3326]
Test system
24-hr & 72-hr sublethal toxicity test
Strains
Wild type strain N2, age synchronous adult worms.
Toxicants
Chloride salts of Cd2+, Cu2+, Pb2+.
Temperature
20 degrees C
Test design
12-well tissue culture plates including K-medium (0.032 M KCl, 0.051 M NaCl) + Escherichia coli (OP50 strain).
Measurements/observations
Rate of movement, feeding rate, growth rate (worm length), reproduction rate.
Evaluations
EC50, using probit analysis with Toxstat.
[ref. ID; 4474]
Test system
24-hr acute toxicity in three soils
Strains
The wild-type N2 strain was obtained from the Caenorhabditis Genetic Center (Minneapolis, MN, USA).
Toxicants/concentrations
Cd, Cu, Ni, Pb, Zn ([Cd] 0, 170, 600, and 1,050 mg/L; [Cu] 0, 150, 350, and 820 mg/L; [Ni] 0, 290, 780, and 1,600 mg/L; [Pb] 0, 240, 870, and 1,450 mg/L; [Zn] 0, 400, 350, and 1,230 mg/L) x 3 replications.
Test design
Soil
- ASTM (American Society for Testing and Materials artificial testing medium).
- Cecil soil (from the Piedmont region of Georgia, USA, containing higher fractions of clay and organic matter).
- Albany soil (from southern Georgia, USA, containing high sand content).
Measurements/observations
Mortality.
Evaluations
LC50s, using probit analysis with Toxstat.
[ref. ID; 4571]
Test system
Mixture toxicity
Strain
C. elegans var. Bristol, strain N2 was obtained from the Netherlands Cancer Institute (Amsterdam, The Netherlands). Age-synchronized, gravid young adults.
Toxicants
CdCl2/2H2O, ZnCl2, CdCl2, PbCl2, Carbendazim, Iprodione.
Test design
Petri dishes (diameter 6 cm) (5g of dry soil + 20 young, gravid adults + food (Escherichia coli 0.1 ml bacterial suspension/g dry wt soil) x 5 replications.
Soil used loamy sand (the Bezirks Verband Pfalz Landwirtschaftiche Untersuchungs- und Forschungsanstalt Speyer (LUFA; Speyer, Germany).
Evaluations
Toxic unit (TU) concept (TU=c/EC50, where EC50 is the median effective concentration and c is the toxicant concentration in the mixture).
[ref. ID; 6120]
Test system
72-hr (whole life cycle: completion of the self-fertilizing hermaphrodites) bioassay
Toxicants
4-Nonylphenol
Test design
Five age-synchronized juvenile worms of the first stage (J1; mean body length: 270+/-16 um standard deviation; n=50) were incubated in 1 ml of the respective NP solution (50-350 ug/l) in a phosphate buffer in polystyrene multiwells (12 wells). Food (Escherichia coli OP50, 10E9 cells/ml). 20 degrees C. 7 replicates.
Measurements/observations
Body length of the adult worms, the number of juvenile offspring.
Evaluations
Growth and reproduction by one-way ANOVA, post hoc: Dunnett; P<0.001.
[ref. ID; 6122]
Test system
Potential new method to study a behavioral toxicity parameter of nematodes in sediments -The Multispecies freshwater Biomonitor (MFB)
Strains
Caenorhabditis elegans var. Bristol, strain N2. All developmental stage (ca. 10 individuals) were placed in the test chamber.
Test design
The measurement principle of the MFB is based on the quadropole impedance conversion. Changes in an electrical field of high-frequency alternating current, that are caused by movements of organisms, can be automatically registered and analyzed with a Fast Fourier Transformation.
Test chambers are slots in a block made of polyacrylate with an inner diameter of 0.6 cm and a depth of 1.5 cm that were equipped with four vertically arranged platinum electrodes (diameter=1 mm, length=1.5 cm).
Measurements/observations
Measurements were performed at room temperature (20 degrees C) in three different types of medium: (1) Aqueous medium: Volvic water (300 ul), (2) natural freshwater sediment (Lake Ammersee, Germany) that was dried for 16 hr at 105 degrees C and again water saturated with Volvic water, and (3) NG agar with overlying Volvic water. Nematode movement was measured in six replicates and five successive measurements over a period of 1 hr (30 measurements).
[ref. ID; 6700]
Test system
Strains
Wild-type N2 and TJ356 were obtained from the Caenorhabditis Genetic Center. Transgenic line KC136 was from lab of Dr. King L. Chow. Age-synchronous populations (L4-larva stage).
Toxicant/concentrations
BaCl2 (2.5 um, 75 uM, and 200 uM).
Test design
12-well sterile tissue culture plates. 20 temperature C. Exposure period 24 hr. No food.
Measurements/observations
Body size, brood size, generation time, head thrash, body bend fequency, changes of hsp-16.2 expression patterns, DAF-16:GFP localization, catalase activity, superoxide dismutase activity.
[ref. ID; 6706]
Test system
To simulate the exposure of nematodes to porewater following heavy rainfall or another flood event
Strain
Four-day-old, age synchronized adult nematode.
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
12-well cell culture plates with 1 ml of soil extract per well. Three replicates per sample and 9-11 test organisms per replicate. 20+/-1 degrees C in dark. Exposure period 48 hr.
Measurements/observations
Mortality.
[ref. ID; 6738]
Test system
Effect of metallothionein knockout for cadmium toxicity
Strains
Wild type and a double (mtl-1 and mtl-2) metallothionein deletion mutant.
Toxicants/concentrations
0, 2.5, 5 and 12 uM Cd.
Measurements/observations
Total brood size, number of worm.
Evaluations
Statistical differences between genotype and brood size, growth, lifespan and cadmium dose were identified using ANOVA, followed by the Tukey's post-hoc test.
[ref. ID; 6758]
Test system
Toxicity of binary mixtures
Strains
Stock cultures of C. elegans var. Bristol, strain N2, originally from the Caenorhabditis Genetics Center, Minnesota, USA.
Toxicants
Cadmium chloride, Chlorpyrifos, Diuron, Nickel chloride hexahydrate, Prochloraz.
Experimental design
Binary mixture experiments were carried out for all possible combinations of the five test compounds using a fixed-ratio design on fractions of the concentration required for a 50% effect (EC50) of the toxicants in the mixture. The fixed-ratio design provides sufficient coverage of the concentration-response surface to assess mixture toxicity effects while avoiding concentrations that show extreme toxicity and thus fail to provide meaningful data. The single-compound EC50 data required for the mixture test design were obtained from range-finding experiments using 12 concentrations and four replicates. The maximum single-chemicals exposure concentrations were set as four or six time the EC50, depending on the concentration-response curve slopes of the chemicals in each binary combination. The lower concentration to be used were determined by dividing the top concentration by a log factor of 1.5 to generate 15 decreasing concentrations spanning the concentration-response range of the single compouds. Top mixture concentrations of the test compounds needed to produce the mixtures at ratios of 1:1, 1:3, and 3:1 were generated in a similar manner to the single concentrations (i.e., 2/EC50(1):2/EC50(2), 1/EC50(1):3/EC50(2), and 3/EC50(1):1/EC50(2)), where 4/EC50 was used as the maximal concentration, and 3/EC50(1):3/EC50(2), 1.5/EC50(1):4.5EC50(2), and 4.5/EC50(1):1.5EC50(2), where 6/EC50 was used as the maximal concentrations. Single-compound and mixture exposures were run concurrently. Series were designed using a log factor of 1.5 to generate 15 treatments of decreasing concentrations from the maximum exposure concentration in each experiment. Thus, the design ultimately resulted in series of 15 concentrations for five ratios. In all cases except the controls (for which nine replicates were prepared to give a measurement of variation within the test system), no replicates were used. The design used thus focused effort on providing both a dense coverage of the entire concentration-response surface and maximum robustness of the design aganist drift in sensitivites between experiments. Since the data analysis framework of Jonker et al. (2005) for interpretation is based on regression analysis, these is no trade-off in statistical power using this design.
Toxicity test
Both 12-well plates (2.5 ml/well) and 60-mm Petri dishes (~10 ml/dish) were used for experiments, and each dish/well was inoculated with E. coli OP50 (10 ul/dish and 1 ul/well). Newly hatched nematodes from stock cultures were allowed to grow for 4 day to reach early maturation prior to being transferred to dosed 60-mm Petri dishes and incubated in the dark at 18 degrees C for the first 24-hr period. Four adult nematodes were then transferred to each corresponding dosed single 2.5-ml test well for a single-replicate test treatment. All plates were then incubated for a second 24-hr period under the same conditions. At the end of this second 24-hr period, the number of adult nematodes alive/dead in each test well was recorded, together with the total number of eggs and hatched juveniles (referred to hereafter as eggs) in each well.
Data analysis
Single-compound modeling: By fitting a log-logistic curve to the experimental data by minimizing the sum of the squared residuals using the Solver Function (Newton algorithm) in Microsoft Excel.
Mixture modelling: Independent action (IA) model. IA model is a measure of the joint probability of individual sensitivity to the compounds in the mixture assuming that the chemical mechanisms of action is fully independent.
[ref. ID; 6820]
Test system
Single and paired metal inputs in soil
Strains
The PC72 transgenic strain (carrying a C. elegans hsp16 promoter/E. coli lacZ fusion gene) was provided by Professor E.P.M. Candido. The lac-deleted E. coli strain P90C was obtained from Dr. A. Chisholm.
Toxicants
CdCl2/2.5H2O, ZnCl2, and CuCl2.
Test design
Exposure to soil: One-gram soil (Lufa 2.2, pH 6.1) samples were prepared by adding solution of toxicants, plus E. coli P90C at approximately 10E8 CFU/g-1 soil. The soil:metal:bacterial mixtures were incubated in 15-ml centrifuge tubes for 24 hr at 25 degrees C to allow equilibration. Worms were added and exposed for a 24-hr incubation period at 25 degrees C, then killed by freezing for at least 2 h. Moisture holding capacity 50%.
Recovery of worms from soil: The worms were recovered from soil using a silica suspension (Ludox-HS40, Dupont) as described by Donkin and Dusenbery (1993).
Exposure to soil water: Exposure of PC72 worms to 100 ul of the soil water sample was conducted in 24-well tissue culture plates.
Exposure to metal solutions:
Measurements/observations
Metal concentration in worm tissue and beta-galactosidase activity.
[ref. ID; 6821]
Test system
Biomarkers (stress-inducible transgenic nematode) of heavy metal pollution in water samples
Strains
Transgenic strains have been engineered to carry bacterial (E. coli strain P90C: lac-operon-deleted strain) stress-inducible lacZ reporter genes.
- CB4027 transgenic strain of C. elegans (carrying a Drosophila hsp70/lacZ fusion gene; Fire 1986) was obtained from Dr A. Chisholm (MRC, Cambridge).
- The transgenic PC72 strains of C. elegans carrying a C. elegans hsp16/lacZ fusion gene.
Toxicants
Water were taken from two sites on the Carnon river and from two tributary streams, the relatively unpolluted Helstone stream and th Clemows stream which drains the tailigns lagoon (where Wheal Jane minewater is treated with lime and flocculant to remove heavy metals and raise the pH). Minewater.
Test design
Worm exposured for 7 hr to metal solutions or water samples (normally about 10E3 worms in 1 ml of sample contained in replicate wells of a 24-well Coring CellWell plate), as described by Guven et al. (1994). PC72 worms were exposed at 25 degrees C whereas CB4027 worms were exposed at 31 degrees C.
Measurements/observations
In situ staining and calculation of stain scores and beta-galactosidase enzyme activities.
[ref. ID; 6823]
Test system
Predictive models for relative toxicity of mono-, di- and trivalent metal ions
Strains
A wild-type (N2) strain.
Toxicants
Metals (nitrate salts): Li+, Na+, Mg2+, K+, Ca2+, Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Sr2+, Cd2+, Cs+, Ba2+, La3+, Pb2+.
Test design
Costar-3512 12-well tissue culture plates (Corning Costar, Kennebunk, ME) were contained 1 ml of test solution per well. Test solutions consisted of six metal concentrations and a control with each replicated six times. 9-11 (average ten) nematodes per well. Temperature 20 degrees C. Exposure period 24+/-1 hr.
Measurements/observations
Number of dead.
Evaluations
LC50s (expressed as total and free ion concentrations) using the probit procedure of Toxstat 3.4 (WEST, Inc. and Gulley, 1994).
[ref. ID; 6824]
Test system
Single metal and multiple metals, normal and transgenic strain
Strains
Gravid C. elegans hermaphrodites and Synchronized L3 stage of the transgenic derivative (KC125, KC136 carring hsp16-41 and hsp16-2 promoter driven reporter genes).
Toxicants
AlCl3, CoCl2/6H2O, CdCl2, CuSO4/5H2O, HgCl2, K2Cr2O7, MnCl2/4H2O, NiSO4/6H2O, PbCl2, and ZnSO4/6H2O.
Test design
Lethality test: Assays were performed in disposable borosilicate tubes (final volume 2 ml) with constant shaking for 48 hr at room temperature (22 degrees C). 1000 nematodes per tube. E. coli (OP50) added.
Stress test of transgenic strain: Tests were performed in disposable borosilicate tubes (final volume 1 ml) with constant shaking for 5 hr at room temperature (22 degrees C). 500 nematodes per tube. Six replication.
Measurements/observations
Lethality test: The inactive individuals number.
Stress test: The induced fluorescnet signal at the pharyngeal bulb.
Evalutations
LC10, LC15, LC20, LC30, LC50.
[ref. ID; 6854]
Test system
Influence of developmental stage, salts and food presence for aquatic toxicity testing
Strains
Wild-type N2 strain, va. Bristol.
Toxicants
CdCl2, Pb(NO3)2, CuCl2/2H2O, HgCl2, and Sodium pentachlorophenate (PCP).
Test design
A two-factor randomized complete block design was used in which the factors were development stage of the nematodes tested (L2-L3, L4, adults from egg, and adults from dauer) and test medium (K-medium with and without bacteria, and deionized water, with and without bacteria). 24-well tissue culture plates (Falcon 3047). 20 degrees C at dark.
Measurements/observations
24-hr and 96-hr mortality, development of larval stage nematodes to adulthood after 96 hr, and evidence of reproduction determined by the presence of off-spring after 96 hr.
[ref. ID; 6855]
Test system
Bioassay
Strains
C. elegans var. Bristol, strain N2. Synchronous worms (length 270+/-16 um).
Toxicants
CdCl2 (0, 0.071, 0.142, 0.282, 0.563, and 1.124 mg/L).
Polluted sediment samples: The River Elbe near Hamburg, Germany.
Unpolluted sediment samples: Mesotrophic Lake Klostersee.
Test design
The test vessel (polystyrene cylinders with lids, diameter 29 mm, height 36 mm).
Liquid medium: Five first-stage juveniles were transferred to the test vessels containing 1 ml of the test medium. The samples were placed on shaking desks at 20+/-1 degrees C for 72 hr. 10 replicates for each concentration.
Sediment test: 10 first-stage juveniles were transferred to the test vessels containing 1 g of sediment (wet weight) and 1 ml of bacterial suspension (Escherichia coli). The samples were placed on shaking desks at 20+/-1 degrees C for 96 hr. 5 replicates for each concentration.
Measurements/observations
Liquid medium: Living number, body length, number of eggs inside body, and offspring per worm.
Sediment test: The percentage of gravid worms, body length, number of eggs inside gravid worms.
Evaluations
LOEC.
[ref. ID; 7002]
Test system
Tolerance of pH, salinity, and hardness in aquatic media
Strains
N2, wild type strain. Synchronized adult worm. Food: Escherichia coli (OP50).
Toxicants
pH 3.0-12.3 (0.1 unit increments, 0.1M HCl and 1M NaCl), NaCl and KCl (as approximations of salinity), and NaHCO3 (as hardness).
Test design
Tests were performed in two media: K-medium (2.36 g KCl + 3.0 g NaCl per L distilled water) and moderately hard reconstituted water (MHRW, 96 mg NaHCO3 + 60 mg CaSO4/2H2O + 60 mg MgSO4 + 4 mg KCl per L distilled water).
Aquatic toxicity tests were performed in 12-well tissue culture plates (Costar, 3512). Ten (+/- 1) adult worms were transferred into each well containing 1 ml of test solution. 3 replicate weels per concentration. Temperature 20 degrees C. Exposure period 24 hr (without food) and 96 hr (with food).
Measurements/observations
Mortality.
[ref. ID; 7003]
Test system
72-hr toxicity test
Strains
Synchronized adult worm.
Toxicants
The Washington municipal wastewater treatment plant (WWTP). The wastewater treatment facility located in the city of Washington in Wikes Country, Georgia, USA, an area known for high diversity of industrial activity. The effluents of all three industries (a yarn dyeing facility (dyehouse), a fiberglass manufacturer, and a pulp processing facility) combine prior to entrance into the WWTP, where this influent subsequently mixes with town sewage waste, undergoes biological treatment, and is discharged into a stream known as Rocky Creek.
Test design
One milliliter of each sample/food (Escherichia coli OP50 strain) mixture was pipetted into each of six wells of a Costar 3512 well dish plate (Corning Co., Kennebunk, ME). 8 to 12 worms were located into each of the six wells. The worms were incubated at 20 degrees C for 72 hr.
Measurements/observations
Surivival counts.
[ref. ID; 7005]
Test system
24- and 72-hr toxicity test
Strains
Synchronized adult worms.
Toxicants
Three synthetic azo dye wastewaters (two reactive dyes and one acid-based dye) and byproducts after ozonation (t=0, 8, 32, 64 min).
Test design
The toxicity test consisted of 10 (+/-2) nematodes in four wells (Costar 3512 well dishes, Corning, Kennebunk, ME). For 24-hr test without food, for 72-hr test with food (Escherichia coli OP-50 strains). Well dishes were covered and placed in a 20 degrees C incubator.
Measurements/observations
Number of surviving worms.
Evaluations
LC50s by probit analysis using ToxStat Software (WEST, Inc).
Duncan's multiple range test.
[ref. ID; 7009]
Test system
Rapid aquatic toxicity test
Strains
Transgenic strain CB4027 and the lac-operon deleted E. coli strain P90C (lacZ gene deleted) were obtained from Dr. J. Hodgkin and Dr A. Chisholm of the MRC Molecular Biology Laboratory, Cambridge, UK.
Toxicants
AgNO3, CdCl2/2.5H2O, HgCl2, MnCl2/4H2O, SnCl2/2H2O, ZnSO4/7H2O, Lindane, Tributyltin-cl.
Test design
Exposure of worms to toxicants was carried out by adding appropriate volumes of 50x to 500x concentrated solutions to each well (multiwell plates, Corning Cell-Wells) or Petri dishes containing the worms suspsended in 1 or 10 ml of K-medium. Exposure was continued for 7 hr at 32 degrees C.
Measurements/observations
Metal concentrations in worm, beta-galactosidase enzyme activity, and histochemical staining of beta-galactosidase.
[ref. ID; 7010]
Test system
Predicting relative toxicity
Strains
A wild type (N2), synchronized adult worms.
Toxicants
Metal chlorides (Ca2+, Cd2+, Cu2+, Hg2+, Mg2+, Mn2+, Ni2+, Pb2+, Zn2+).
Test design
Nematodes were tested in Costar-3512 12-well tissue culture plates containing 1 ml of test solution per well. Test solutions consisted of six metal concentrations and a control with each replicated six times. Using a dissecting microscope, 9-11 (average of 10) nematodes were transferred into each test well with a 10 ul pipette. Worms were incubated at 20 degrees C for 24+/-1 hr.
Measurements/observations
Number of dead worms.
Evaluations
Three LC50 values for each metal were calculated with the probit procedure of Toxstat 3.4 using the measured metal concentrations and a log transformation of concentration. The LC50 values obtained by the probit analysis were converted to percentage of free ion or percentage of free ion + neutral chloro-complexes with values obtained from MINTEQA2 Version 3.10.
[ref. ID; 7011]
Test system
The influence of non-ionic surfactant on metal toxicity
Strains
Transgenic hsp16/lacZ strain of C. elegans PC72ubln5 (here abbreviated to PC72) was provided by Dr E.P.M. Candido (Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada). The lac-deleted E. coli strain P90C was obtained from Drs. A. Chisholm and J. Hodgkin (MRC Molecular Biology Laboratory, Cambridge, UK).
Wild-type N2
Toxicants
Metal: CdCl2/2.5H2O, CuSO4/5H2O, HgCl2, MnCl2/4H2O, and ZnSO4/7H2O.
Non-ionic surfactant: Pluronic F-127.
Test design
Worms were exposed for 7 hr at 25 degrees C to 0-50 ppm of various metal ions in K-medium, in the presence or absence of freshly dissolved Pluronic F-127 at a final concentration of 10 ppm.
Measurements/observations
Beta-galactosidase activity and staining.
[ref. ID; 7033]
Test system
Biomonitor
Strains
Transgenic lines C. elegans that contains mtl-2:lacZ transgene were generated by microinjecting the pMTL2ILACZ plasmid (100 ug/ml) into the gonadal syncytium of adult C. elegans. Food (Escherichia coli strain OP50). All exposure studies were performed using mixed populations (L1-L4 larvae and adults).
Toxicants
CdCl2/2.5H2O, HgCl2, NiSO4/6H2O, and ZnSO4/7H2O.
Test design
Equal volumes of the C. elegans suspension were transferred into 50-ml (final volume 10 ml), capped centrifuge tubes, and various concentrations of metal were added. Liquid cultures of C. elegans were exposed at 20 degrees C with constant agitation. 3 replicates.
Measurements/observations
Histochemical staining of beta-galactosidase (absorption at 615 nm).
Evaluations
EC50 using the probit procedure.
[ref. ID; 7035]
Test system
The influence of particulate organic matter (POM) and dissolved organic matter (DOM) on Cd toxicity
Strains
var. Bristol, strain N2.
Toxicants
CdCl2. Stock solutions were prepared with K-medium (3.1 g/L NaCl, 2.4 g/L KCl).
Test design
Sediment (uppermost 2 cm): From Lake Starnberg, Germany, a mesotrophic lake that only has groundwater input (no stream input) and no industrial impact.
POM spiking: The sediment was divided into three equal parts (each ~1.2 kg). One part was used without further treatment. Second part was mixed with low amounts of peat (1-mm sieved). Third part was mixed with high amounts of peat.
Cd spiking: Each type sediments was divided into six fractions (each 180 g). Cd sediment concentrations of 10, 100, 400, 800, and 1200 mg/kg wet weight. Sediments were incubated for 72 hr at 20 degrees C.
Pore-water extraction: Portions of the spiked sediment were centrifuged for 20 min at 2000 g in a swing-out centrifuge. The supernatant was filtered through a glass fiber filter (pore size: 1 um), and then was filtered through a 0.45-um membrane filter. The filtrate (pore water) was used in the bioassay.
Bioassay: Sediment (0.5 g wet wt) or pore water (0.5 ml) were transferred into polystyrene multiwells (12 wells; Nunc, Wiesbaden, Germany) and then mixed with 0.5 ml of a suspension of E. coli (~10E10 cells/ml) in K-medium. Five first-stage juvenile worms (270+/-16 um body length) were transferred to each test vial. Six replicates. The samples were incubated on a shaker at 20 degrees C, for 72 hr.
Measurements/observations
Body length.
Evaluations
Inhibition of growth %.
[ref. ID; 7045]
Test system
Effect of intact soil on acute toxicity test
Strains
C. elegans wild type var. Bristol, strain N2. E. coli strain OP50 for food source.
Toxicants
CdCl2, CuCl2/2H2O, Pb(NO3)2, ZnCl2.
Test design
Four soil types native to the southeastern United States.
- Cecil sandy loam (66% sand, 18% silt, 16% clay, 1.7% organic matter, pH in water = 6.2)
- Worsham sandy loam (55% sand, 29% silt, 16% clay, 3.0% organic matter, pH in water = 5.1)
- Davidson loam (51% sand, 29% silt, 20% clay, 3.4% organic matter, pH in water = 6.1)
- Dyke clay loam (28% sand, 33% silt, 39% clay, 2.2% organic matter, pH in water = 6.2)
The test samples consisted of 7 g soil in 60x15 mm Pyrex petri dishes with lids, spiked with 3 mL volume of test solution. 20 adult nematodes were added. 5 replicates. Temperature 20 degrees C. Exposure period 24 hr. Control is no soil.
Measurements/observations
Mortality.
Evaluations
LC50s and confidence intervals were determined using the probit regression line of survival data (Finney, 1971) and the SAS microcomputer software.
[ref. ID; 7086]
Test system
Effect of metal exposure on associative learning behavior
Strains
Wild-type N2, originally obtained from the Caenorhabditis Genetics Center (CGC, University of Minnesota, Minneapolis, USA). Age-synchronous popluation of larva (L4 stage).
Toxicants
AgNO3, CuSO4, HgCl2,Pb(NO3)2, and ZnSO4. Metal Concetrations 2.5, 10, and 50 uM.
Test design
Exposures were performed in 12-well sterile tissue culture plates. All exposures were 6 hr long and carried out in 20 degrees C incubator in the presence of food (Escherichia coli OP50).
Measurements/observations
Body bend frequency/20sec: Control or metal-exposed nematodes were pick onto a NGM plate and scored for the number of body bends in an interval of 20sec. Thirty nematodes were examined per treatment.
Thermotaxis Assay: Approximately 50 control or metal-exposed nematodes were examined.
Associative Learning Assay: Approximately 50 control or metal-exposed nematodes were grown at 25 or 17 degrees C for at least 12 h in the presence of a fresh lawn of the bacteria on a 9-cm Petri dish, and then shifted individually to a seeded plate at 20 degrees C for different time intervals (0, 0.5, 2, 5, and 18 hr). Upon removal of the nematodes from the plate, tracks left were photographed and analyzed.
[ref. ID; 7113]
Test system
Acute and chronic toxicity
Strains
Worm (Wild-type (N2) and NW1229) and E. coli (NA22 and OP50) were provided by the Caenorhabditis Genetics Center, which is funded by the Nathional Institutes of Health National Center for Reseach Resources. NW1229 expresses a pan-neuronal green fluorescent pattern due to the integration of Ex[F25B3.3::GFP;dpy-20(+)] in dpy-20(-) backgroud.
Synchronous L2 C. elegans.
Toxicants
Touchdown (52.3% glyphosate), Mancozeb with Zinc Flowable (37% Mn/Zn-EBDC).
Test design
Acute toxicity: Worms were exposed to the respective pesticide for 30 min, washed at least 3x with sterile dH2O, then placed on fresh nematode growth media (NGM; 51,3 mM NaCl, 17.0 g bactoagar/L, 2.5 g bactopepton/L, 1 mM CaCl2, 1 mM MgSO4, 0.5 mM potassium phosphate buffer (pH 6.0), 12.9 mM cholesterol in 95% ethanol, 1.25 ml nystatin/L, 0.2 g streptomycin/L) plates, with a lawn of OP50 E. coli (grown in 25 g Luria broth/L, 200 mg streptomycin/L). Following a 24-hr incubation at 20 degrees C.
Chronic toxicity: Worms were exposed to the respective pesticide for 30 min, then without additional washes, placed on NGM plates seeded with a lawn of OP50 E. coli and counted or photographed after a 24-hr incubation period at 20 degrees C.
Dual pesticide treatment: Worms were treated with 2% glyphosate (recommended application concentration) followed by varying concentration of MZ.
Measurements/observations
Number of live worms. Fluorescence observations (green pixels) for NW1229.
Evaluations
LC50s using GraphPad Prism (v5.03 for Windows, GraphPad Software, San Diego, CA).
The Bliss additivism model was used to classify the effect of combining TD and MZ as additive, synergistic or antagonistic. Using the equation E(Bliss)=E(TD)+E(MZ)-(E(TD) x E(MZ)), where E(TD) and E(MZ) are the fractional inhibitions obtained by TD alone and MZ alone at specific concentrations of each of the pesticides used in the dual treatment protocol. E(Bliss) is defined as the fractional inhibition expected if the combination of the two pesticides was exactly additive.
The potential contribution of acute treatments of either TD or MZ to the dual treatment was determined by calculating the percent change in pixel numbers compared to control ([total pixel number/average pixel number of control]x100), for the LC25, LC50 or LC75 for either MZ or the TD/MZ concentrations.
[ref. ID; 7249]
Test system
Inhibition of growth and reproduction
Strains
Strain N2, was provided by Dr. Martin Samoiloff, Department of Zoology, University of Manitoba. Synchronized L1 larvae.
Toxicants
Amidantel (ADT or BAY d 8815) and its deacylated derivative (dADT or BAY d 9216).
Test design
Growth test: Synchronized L1 larvae were inoculated onto 3.5-cm plates of NG agar, with (0.02-1.0 mM) or without drug supplementation, and cultured for 96 hr.
Egg-laying rates: Synchronized L1 larvae were allowed to grow for 59 hr on NG medium or MG medium supplemented with ADT (100 ug/ml-1) or dADT (50 ug/ml-1). The worms were transferred to fresh plates of the same medium at intervals until 155 hr after synchronization. Egg counts were made at 0.5 and 1.5 hr after transfer on three replicate plates of 10 worms.
Measurements/observations
Growth test: Worm length.
Egg-laying rates: Egg-laying per worm.