Common biodegradation test procedures allow the classification of chemicals in either non-, inherently- or ready biodegradable. Only rough assessments of the biodegradation kinetics in environmental compartments can be made on the basis of this classification scheme. During the last years, improvement and availability of computers and mathematical models resulted in a growing interest to assess the fate of chemicals in the different compartments on the basis of, among others, biodegradation kinetics, measured in test environmentally relevant test systems.

To develop a new laboratory test system for the simulation of biodegradation in surface waters, the German Federal Environmental Agency, Berlin initiated a research project (UBA-F+E 106 03 120). The new system was partly based on an existing guideline for a sediment/water system (BBA 1990). Two test chemicals, Lindane and 4-nitrophenol were chosen and their degradation and distribution in systems with sediment and water from two locations was measured for up to 91 days. Radiolabelled (14C) test chemicals were used to achieve testing at very low concentrations (< 1 µg/L). The effects of the aeration method, sediment type, feeding, illumination, temperature, test substance application method on the fate of the test chemicals were determined.

The research project ended in 1997. Based on the experiences with the new system a test guideline proposal was written (Kalsch et al. 1997). Two publications describe the results from experiments with lindane (Kalsch et al. 1998) and 4-nitrophenol (Kalsch et al. 1999).

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Step 1: Draw aerobic sediment and water samples from an unpolluted surface water.

Step 2: Fill test reactors with sieved sediments and water (1:10). Add radiolabelled test chemical.

Step 3: Incubate under controlled conditions:

• weak illumination (approx. 300 lx)

• Temperature (e.g. 5 °C or 15 °C)

• aeration (e.g. 150 mL)

• water rotation (by use of "aerators")

• nutrient status (either not corrected or add organic material)

Step 4: Measure the release of 14C-carbon dioxide (mineralisation), volatile, water soluble and sediment bound radioactivity.

Step 5: Analyse mineralisation kinetics and calculate half-lives (if first order kinetics) or DTx values (degradation time until x % is mineralised).

Figure 1: Water and sediments were collected at the Fraenkische Saale, a small unpolluted river in Northern Bavaria, Germany.

Figure 2: The "surface water" in the laboratory. Flat flange beakers (reactors, 1) are filled with 2 cm of sediment and 20 cm of water. They are tightly closed with a lid, that has four openings, two for the aeration tubes and a larger one that allows the introduction of a pipette and pH or redox electrodes. The fourth opening in the center of the lid is optional and is used to safely attach the aerator (2) described below. A suction pump (3) is used to draw 100 - 200 mL/min of premoistened ambient air (4) through the reactors. After leaving the reactors the air is led through traps for volatile compounds and for carbon dioxide (6). An empty bottle prevents the flow-back of the trapping liquids into the reactor when the traps are opened.

Figure 3: How aerators work. An air inlet tube is fixed at the lower end of the aerators inner tube. The ascending bubbles force water to rise within this tube. This water flows into the outer tube which surrounds the inner one causing a slight hydrostatic overpressure. The outer tube has three openings at its lower end which form tangential jets. The force of the water leaving the jets causes the whole water body in the reaction vessels to rotate.

Figure 4: Fate of ¹⁴C-lindane in a sediment/water test (example).

Different approaches have been proposed to study biodegradation under simulated conditions. Among them, shake flask assays (1), water/sediment systems, with undisturbed but mixed and sieved sediment (2), continuos flow river models (3) as well as microcosms with undisturbed sediment cores (4) provide a variety of methods. However, a guideline for a the second type has not been accepted by all experts. To discuss the guideline proposal based on the UBA reserach project, an international workshop took place in March 1999 at Hochheim, Germany. Based on the results of this workshop the guideline draft was revised (Kalsch and Knacker 1999). Future steps may include the further discussion of the draft to achieve acceptance as an international guideline (e.g. ISO). Validation of the test method, e.g. by ring testing may also be required.

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Dr. Th. Knacker
th-knacker@ect.de
++6145 956411

The financial support for the research project (UBA-F+E 106 03 120) and for the workshop (UBA 298 67 429) is gratefully acknowledged.

BBA 1990. Biologische Bundesanstalt für Land- und Forstwirtschaft (Federal Biological Research Centre for Agriculture and Forestry), Braunschweig, Germany. Richtlinien zur Prüfung von Pflanzenschutzmitteln im Zulassungsverfahren Teil IV. No. 5-1: Abbaubarkeit und Verbleib von Pflanzenschutzmitteln im Wasser/Sediment-System (Degradability and persistence of plant protection products in the water/sediment-system).

Kalsch W., Knacker T., Robertz, M. and Schallnass H.-J. 1997. Entwicklung eines Testsystems für die Prüfung des biologischen Abbaus in Oberflächengewässern. Final report to the German Federal Environmental Agency, Berlin. In German, including a first draft for a test guideline in english. UBA-Tetxe 13-97.

Kalsch W., Knacker T., Robertz, M, Studinger G. and Franke C. 1998. Partitioning and mineralization of [¹⁴C]lindane in a laboratory sediment-water system- Environ. Toxicol. Chem 17, 662-669.

Kalsch W., Knacker T., Danneberg G., Studinger G. and Franke C. (1999). Biodegradation of [¹⁴C]-4-nitrophenol in a sediment-water simulation test. Accepted for publication to Int. Biodet. Biodeg.

Kalsch W. and Knacker T. 1999. Workshop on "Biodegradation: Simulation test using surface water and sediment". Final report submitted to the German Federal Environmental Agency, Berlin.
Copies available from Th. Knacker (th-knacker@ect.de).

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