Folsomia candida (Collembola) mortality test for ecotoxicity assessment of chemical substances

The testing methodology in case of chemical substances is identical with the methodology used for soil and ground water (https://enfo.hu/en/node/13265). The only difference is that for toxicity assessment of chemical substances the sample is suspended in distilled water and then the dilution series is prepared.

 

Summary:

 

Type of the test: one species laboratory, animal, acute toxicity and chronic test. It can be used in a microcosm test.

Feasible for: testing the soil directly, or the soil extract should be poured onto standard soil.

Testorganism: Folsomia candida, springtail Collembola, sensitive to organic contaminants, mainly to the volatile contaminants and to chemicals absorbed through the skin. Not sensitive enough for heavy metals.

Endpoint: number of animals; lethality; from dilution: EC20, EC50, and ED20, ED50; based on reproductivity test: NOEC.

Necessary instrument: cytoplast microscope or visual.

Duration of the test: acute: 5-10 days, reproductivity test: 20 days.

Standard protocol: ISO 11267:1999, Soil quality -- Inhibition of reproduction of Collembola (Folsomia candida) by soil pollutants; OECD/OCDE 232, OECD Guidelines for testing chemicals: Collembolan reproduction test in soil (2009)

Comment: highly reproducible, easily performed

 

Testorganism:

The Folsomia candida species belongs to the subclass Collembola (Springtails).

Tiny (max. 3-4 mm) white animals with a furcula (tail-like appendage, that is folded beneath the body to be used for jumping. It is held under tension by a small structure called the retinaculum and when released, snaps against the substrate, flinging the springtail into the air. All of this takes place in as little as 18 milliseconds.). They live in the soil, in forests, there can be 100,000 Collembola in 1 m2 soil. They respirate with a ventral tube (collophore), that is why they are sensitive to soil vapours. Collembolas reproduce by epimorphosis. In case of suitable humidity environment of 20 °C the eggs hatch within 10–15 days and the animals mature within 10–15 days.

Test protocol:

The Folsomia candida species is feasible for acute and chronic tests, the duration of the acute test is 5–10 days, the measuring endpoint of the test is the percentage of the surviving animal. With this test the EC20, EC50 or the ED20 and ED50 can be determined from the dilution of the soil sample.

 

Necessary tools:

370 ml volume glasses

portable balance with tare button

automatic pipette and tips (5 ml)

 

Necessary materials:

tapwater

granulated yeast

standard OECD soil.

 

The composition of the OECD soil: peat (10%), kaolinite clay, min. 30% kaolinite content (20%), quartz sand (70%).

 

The test:

The test is carried out with 20-20 g of air-dried soil sample in 370 ml jars. The samples are prepared by diluting with OECD soil. OECD soil is used as a control. The dilutions used:

Sample, g	20	10	5	2.5	1.25
OECD, g	0	10	15	17.5	18.75

 

The samples are moistened with 5-5 ml of water and 2-2 mg yeast is added.  

10 test animals are given into the sample jar.

The test containers are kept in dark at 20-25 ° C for 7 days.

 

 

Evaluation of  the test:

• The soil in test jars is suspended in water.  
• The suspended material is gently mixed for the soil clots should disintegrate. This way the surviving animals can swim up onto the surface of water.
• Surviving animals are counted on the surface.
• From the number of the surviving and dead animals toxicity can be counted comparing to the toxicity of the control OECD soil, given in mortality percentage. The mortality percentages of the different dilutions are plotted according to the amount of the contaminated soil by Software Origin. From the resulting sigmoid curves ED20 and ED50 values can be determined.

Based on the ED20 and ED50 values the examined samples are compared for toxicity.

 

Table 1 Characterization of the toxicity of the soil samples based on the Folsomia candida mortality test.

ED20 [g]	ED50 [g]	Characterization
> 20	> 20	Non toxic
12-20	16-20	Slightly toxic
2-12	4-16	Toxic
< 2	< 4	Very toxic