Biodegradation rates of chemicals in surface water and groundwater assessed in batch simulation tests.

The aim of this PhD project was to evaluate some of the factors and mechanisms that are responsible for the biodegradation in batch simulation tests with specific focus on adaptation phenomenon and degradation rates. In Manuscript I the aim was to compare degradation rates of aniline in laboratory shake flask simulation tests with field rates in the river Rhine. The combined events of a low flow situation in the Rhine and residual aniline concentrations in the effluent from the BASF treatment plant in Ludwigshafen temporarily higher than normal, made it possible to monitor aniline at trace concentrations in the river water downstream the wastewater outlet by means of a sensitive GC headspace analytical method. Aniline was analyzed along a downstream gradient and the dilution along the gradient was calculated from measurements of conductivity, sulfate and a non-readily biodegradable substance, 1,4-dioxane. Compensating dilution, field first-order degradation rate constants downstream the discharge of BASF were estimated at 1.8 day^-1 for two different dates with water temperatures of 21.9°C and 14.7°C, respectively. This field rate estimate was compared with results from 38 laboratory shake flask batch tests with Rhine water which averaged 1.5 day^-1 at 15°C and 2.0 day^-1 at 20°C. These results indicate that laboratory shake flask batch tests with low concentrations of test substance can be good predictors of degradation rates in natural water bodies - at least as ascertained here for short duration tests with readily degradable compounds among which aniline is a commonly used reference.

Manuscript II describes a semi-continuous preexposure procedure (SCEP) for surface water batch simulation biodegradability tests at low chemical concentrations (0.1-100 µg/L). This type of tests are normally used for determining "initial rates" characteristic of the water as sampled, while the aim of the SCEP is the determination of reproducible "adapted rates". The SCEP maintains the microbial diversity and a supply of test substance and natural substrates, and thereby facilitates the process of adaptation. Conducting a SCEP involves regular renewal of a part (e.g. one third) of the test suspension (e.g. every two weeks) using freshly collected natural water with test compound added to the initial concentration. A study prototype was performed with aniline, 4-nitrophenol, 2,4-dichlorophenoxyacetic acid, 4-chloroaniline, and water from the urban river Mølleå. Following preexposure considerably reduced and much more reproducible lag phases resulted, whereas adapted rates of degradation were roughly the same as final rates in batch tests. The adapted rate constant is perceived as an inherent characteristic of the test compound at a specific concentration and under environmental influence (temperature, natural substrates, etc.) but with no simple links back to the original microbial population. By contrast, the initial rates in batch tests are determined also by the microbial population initially present.
In Manuscript III it was shown that increasing the total volume of test medium resulted in decreased lag phases in biodegradability shake flask batch tests conducted with either surface water or with synthetic mineral medium inoculated with supernatant from settled activated sludge. Experiments were performed with test volumes ranging from 1.8 ml to 100 L using two ¹⁴C-labeled model chemicals, 2,4-D and p-nitrophenol both of which are known to be readily degradable after variable lag phases. Lag phases ranged from 2.1 to 30.4 days for PNP and from 16 to 37 days for 2,4-D. Decreasing the test volume tended to increase the lag phase even when a single test batch was redistributed into smaller flasks. At small volumes of 10 ml or less, degradation failed randomly. Our findings are partly explained by the hypotheses that a sufficient total amount as well as a sufficient concentration of specifically degrading microorganisms or consortia of bacteria must be present initially for biodegradation to get started, from which follows that with too small inoculations or with too small test volumes, biodegradation may fail randomly. A straight forward practical implication of the findings is that the test volume in biodegradability tests can significantly influence the lag time and thus sometimes be decisive for the outcome in biodegradation studies.
The knowledge on degradation kinetic at naturally relevant low concentration is important in order to give qualified estimates for the time needed in order to decrease contaminant concentrations to below the accepted limit of 0.1 µg/L for pesticides. In Manuscript IV the biodegradation kinetics of two phenoxy acid herbicides, MCPP and 2,4-D were studied in laboratory batch microcosms at low concentrations (0.025 to 100 µg/L) using ¹⁴C-technique with sediments and groundwater from a shallow aerobic sandy aquifer. Below a certain threshold concentration of approximately 1 µg/L for 2,4-D and 10 µg/L for MCPP, the biodegradation followed first order non-growth kinetics and no adaptation was observed within the experimental period of 341 days. Half-lives for ultimate degradation were 500 days for 2,4-D and 1100 days for MCPP at 10°C in unpolluted aquifer sediment, in this environmentally relevant concentration regime. Above the threshold concentrations the biodegradation rate accelerated gradually due to selective growth of specific biomass, which was ascertained from ¹⁴C-MPN (most probable number) enumerations of specific phenoxy acid degraders. At the highest concentration tested (100 µg/L), specific degraders increased from 10^-1 to 10⁵ cells/g during the experiment, and half-lives after adaptation decreased to approximately 5 days. The enhanced rate of degradation by adapted systems was maintained during degradation of the last residuals measured to less than 0.1 µg/L. In situ long-term preexposure of the aquifer sediment also resulted in significant higher degradation rates of the phenoxy acids.
Adaptation phenomena and biodegradation kinetics of the phenoxy acid herbicide 2,4-D was studied in laboratory microcosms at low concentrations (0.1 to 100 µg/L) using ¹⁴C-technique in Manuscript V. Experiments were carried out with groundwater amended with 4.3 gSS/L of sediment fines from a shallow aerobic sandy aquifer. Tests were either batch tests or as semi-continuous preexposed tests (SCEP). Conducting a SCEP one third of the test suspension was renewed monthly using freshly collected aquifer material and groundwater with test compound added to the initial concentration. Below a certain threshold concentration of approximately 1 µg/L for 2,4-D, the biodegradation followed first order nongrowth kinetics and no adaptation was observed within the experimental period of 335 days. Reproducible half-lives for ultimate degradation were approximately 90 days for 2,4-D at 15°C in unadapted batch test with low spatial variability. At the highest concentration tested (100 µg/L), specific degraders increased by up to a factor of 1000 during the experiment, and half-lives after adaptation decreased to 1-2 days. Adaptation was still prominent 162 d after the batch test and the decay rate was estimated to 0.007 d^-1.
In general, the experiments conducted and the literature reviewed suggests that the uses of the standardized laboratory batch simulation tests are necessary and feasible for estimation of biodegradation rates in the environments. However, more knowledge about the complex assessment of biodegradation of organic chemicals at low concentrations in the natural environment is still needed.