Manual for Soil Analysis-Monitoring and Assessing Soil Bioremediation Phần 10 doc

18 Assessment of Ecotoxicity of Contaminated Soil Using Bioassays 329

■ Procedure

In the guideline ISO 10381–6 (1993) collection, handling, and storage of

soil for the assessment of aerobic microbial processes in the laboratory is

described. For testing contaminated soils it has to be considered that some

contaminants may interact with vessel material (see Sect. 18.1). Moreover,

alteration of the redox potential during storage should be minimized for

anaerobic soils for which only investigation by aquatic ecotoxicological and

genotoxicological tests is relevant.

Sieving (According to ISO 10381–6 1993)

If the soil is too wet for sieving, it should be spread out, where possible in

a gentle air stream, to facilitate uniform drying. The soil should be finger

crumbled and turned over frequently to avoid excessive surface drying.

Normally this procedure should be performed at ambient temperature.

The soil should not be dried more than necessary to facilitate sieving.

Water Extraction (According to ISO/DIS 21268–2 2004)

The soil samples are extracted by a ratio of 1 part soil dry mass to 2 parts of

water with a minimum amount of 100 g soil dry mass. The water content in

the soil has to be considered. The samples are shaken intensively to simulate

worst-case conditions for 24 h and then centrifuged. The supernatant is

filtered with a glass microfiber filter and stored at 4 ◦C in Duran (Schott

AG, Mainz) glass bottles in the dark. The pH of the elutriates is adjusted

to 7 ± 1 with conc. HCl or NaOH. Ecotoxicological and genotoxicological

testing should be performed within 8 days.

Preparation of Solid-Phase Extracts from the Water Extracts

for Genotoxicological Testing

The solid-phase extraction of the water extract is performed with Serdo￾lit PAD-1 resin, an ethylstyrene-DVB-copolymer with a particle size of

0.3−1.0 mm and a pore diameter of ca. 25 nm with a specific surface of

ca. 250 m2/g. The PAD-1 beads are pretreated by rinsing for 2 h in warm

10% (v/v) HCl, Millipore water, 10% (v/v) NaOH, and Millipore water

successively followed by 8 h Soxhlet extraction with pentane/acetone in

a ratio of 1:2. The beads are dried at a temperature of 110 ◦C. Shortly before

solid phase extraction 10 g PAD-1 beads are preconditioned by shaking

them with 25 mL methanol.

The water extract should be concentrated by a factor of 15 by mixing

375 mL with 10 g Serdolit PAD-1 beads. This suspension is placed on an

overhead shaker for 2.5 h. The beads are removed from the water extract

and dried under nitrogen atmosphere in a Baker-spe-10 system (J.T. Baker,

330 A. Eisentraeger et al.

Phillipsburg, New Jersey, USA). The dried beads are then extracted with

a mixture of 9 parts dichloromethane and 1 part methanol. One mL of

DMSO is added to the solvent, which is then evaporated under nitrogen

atmosphere to a final volume of 1 mL. The concentrated sample is stored

for less than 8 days at 4 ◦C. The sample is adjusted with distilled water to

a volume of 25 mL for the genotoxicity tests. The final DMSO concentration

is 4%. Therefore, the concentration factor for the water soil extract is 15.

■ Notes and Points to Watch

• As already mentioned in Sect. 18.1, localized drying of the soil has to be

avoided.

• The soil should be processed as soon as possible after sampling. Any

delays due to transportation should be minimized.

• Microbial tests:if storageis unavoidable, this should not exceed 3 months,

unless evidence of continued microbial activity is provided. Even at low

temperatures the active soil microflora decreases with increasing storage

time; the rate of decrease depends on the composition of the soil and the

microflora.

• Soil fauna tests and tests using higher plants: there are no specific recom￾mendations for soil storage with respect to soil fauna and higher plants

in ISO standards. Therefore it is recommended to store the soil sam￾ples under the same conditions as for testing of microbes and microbial

processes.

• Aquatic tests: for testing the leaching potential, water extracts for aquatic

tests should be prepared immediately after sieving. If the tests cannot be

performed within 8 days (storage of the extracts at 4 ± 2 ◦C in the dark),

extracts should be stored at −20 ◦C.

• An ISO guidance paper on the long and short term storage of soil samples

is in process.

18.3

Water-Extractable Ecotoxicity

18.3.1

Vibrio fischeri Luminescence-Inhibition Assay

■ Introduction

Objectives. This test is an acute toxicity test with the marine lumines￾cent bacterium Vibrio fischeri NRRL B-11177 (formerly known as Photo-

18 Assessment of Ecotoxicity of Contaminated Soil Using Bioassays 331

bacterium phosphoreum). It is standardized for the determination of the

inhibitory effect of water samples in the ISO guideline 11348 parts 1-3

(1998). In the strategy presented here, it is used to determine whether toxic

substances are present in the aqueous soil extracts.

Principle. The test system measures the light output of the luminescent

bacteria after they have been challenged by a sample and compares it to

the light output of a blank control sample. The difference in light output

(between the sample and the control) is attributed to the effect of the sample

on the organisms. The test is based on the fact that the light output of the

bacteria is reduced when it is introduced to toxic chemicals.

Theory. V. fischeri emits a part of its metabolic energy as blue-green light

(490 nm). Biochemically luminescence is a byway of the respiratory chain.

Reduction equivalents are separated and transmitted to a special acceptor

(flavinmononucleotide, FMN; Engebrecht et al. 1983). During the oxidation

of substrates by dehydrogenase hydrogen is transferred to nicotinamide

adenine dinucleotide (NAD). The reduced NAD (NADH2) transfers the hy￾drogen normally to the electron transport chain. To get bacterial lumines￾cence, a part of the NADH2 is used to build reduced flavin mononucleotide

(FMNH2). FMNH2 builds a complex with luciferase which involves the

oxidation of a long-chain aliphatic aldehyde, developing an excited energy

state. The complex decomposes and emits a photon. The oxidation prod￾ucts FMN and the long chain fatty acid are reduced in the next reaction

cycle by NADPH2.

FMNH2 + RCHO + O2 → Luciferase → FMN + RCOOH + H2O + hν

This luminescence is inhibited in the presence of hazardous substances.

Since it is dependent on reduction equivalents, the luminescence inhibitory

test is a physiological test belonging to the electron-transport-chain-activi￾ty group.

■ Procedure

Equipment, reagents, sample preparation, procedure, and calculations are

described in detail in ISO 11348 (1998).

18.3.2

Desmodesmus subspicatus Growth-Inhibition Assay

■ Introduction

Objectives. This fresh water algal growth inhibition assay is performed

according to the standard ISO 8692 (1989). It is applicable both for the