Qualifying and quantifying linuron degradation capacity with colorimetric reaction? Test case on a linuron treated agricultural soil

The phenylurea herbicides (PUH), such as diuron, isoproturon (IPU) and linuron, are one of the main categories of crop protection products and they kill weeds and other plants that grow where they are not wanted. In recent years researchers are paying greater attention to this family of herbicides because of their high biotoxicity and possible carcinogenic properties and because they require several weeks to months for their removal from the environment. Biodegradation is recognized as the primary force in transformation and mineralization of phenylurea herbicides. Degradation products of phenylureas such as 3,4-dichloroaniline (3,4-DCA) may be even more persistent and bind strongly to soil constituents. The 3,4-DCA (possible metabolite from linuron or diuron) displayed a much higher toxicity than their mother compounds. Several tests can be used to see if there is degradation capacity for herbicides present in soil. The most important techniques are mineralization and degradation experiments. In this study, a facile and sensitive method (diazotization-coupling colorimetric test) was tested to see if it is capable to detect and / or quantify the metabolites of isoproturon, linuron and diuron. If this is possible the researchers can replace the time consuming and costly experiments (like mineralization experiments and detection of metabolites in degradation experiments by HPLC) by a colorimetric test to detect aniline compound metabolites of PUH. This was tested on aniline compounds by testing the colorimetric reaction introduced by Pease et al (1962) on this metabolite. In the first part of this study we wanted to test the possibility to create the metabolite by chemical hydrolysis of the herbicide or to create it by means of microbial degradation , so we tried to detect and quantify the aniline compound with color reaction and to compare the results with those obtained by HPLC.
Then in the second part a case study on a linuron treated agricultural soil was performed. Nine soil microcosms were set up in glass columns and subjected to different irrigation treatments during 4 weeks: (1) a discontinuous irrigation with sterile tap water (this treatment is designated as H2O), (2) a discontinuous irrigation with sterile tap water containing linuron (50 mgL-1) (this treatment is designated as ? x LIN), (3) a single addition of 1mL of 50 mgL-1 linuron in sterile tap water at the start of different treatments, followed by discontinuous irrigation with sterile tap water (this treatment is designated as 1x LIN). Each treatment was performed in triplicate. Just before the start of the different irrigation treatments and after 4 weeks of incubation small amounts of soil were taken from the surface of the soil column and mineralization and degradation experiments (evaluated with both HPLC and the colorimetric test) were started. After four weeks of different linuron treatments soil samples were taken for DNA-extraction to perform a molecular analysis of the Variovorax population and the linuron degradation genes under different exposures. The results obtained shows an effect of the exposure to linuron on the soil microbial community. Although we could not detect a significant increase in the suspected linuron degrading genus (Variovorax), we could observe a shift in the Variovorax community. Under the influence of the discontinuous linuron supply a Variovorax population (S) not part of the dominant Variovorax populations in soils without linuron supply, seems to have become more dominant. This implies that this strain plays an important role in situ linuron degradation and thus supports the hypothesis of Breugelmans et al. (2007) that the genus Variovorax plays a dominant role in in situ linuron degradation. Based on the results obtained in this study, significant differences in the qualification of linuron degradation and the quantification of 3,4-DCA as main metabolite of linuron between HPLC and the diazotization-coupling reaction was observed. We can therefore conclude that the colorimetric reaction is not trustworthy for the quantification of 3,4-DCA and thus for the detection of linuron degradation.