MATERIALS

All materials were of Analar grade where possible. Picolinic acid was supplied by Aldrich Chemical Co. (Gillingham, Dorset, UK) 3,6-dich- loropicolinic acid, analytical and technical grades, Methyl 3,6-dich- loropicolinate, 3,6-dichloropyridinol, [1l|C] 3,6-dichloropicolinic acid were kindly donated by The Dow Chemical Co., Kings Lynn, Norfolk, UK.

CHAPTER 3

MINERALISATION OF 3,6 DICHLOROPICOLINIC ACID (36DCPA)

3.1 RESULTS

To test the feasibility of screening soil micro-organisms for any activity against recalcitrant molecules, the compounds first need to be tested for mineralisation in soil and an examination made for any metabolised breakdown products. It has been demonstrated that micro­ organisms capable of the degradation of organic pollutants can be enriched for by application of biodegradable analogues to the microbial ecosystem (Focht & Alexander, 1970) and this phenomenon has been termed "analogue enrichment" (see section 5.1).

It is not necessary, when examining mineralisation alone, to use the high concentrations that are normally necessary for obtaining measurable growth in liquid media, so field application rates may be used. This gives a more accurate indication of the fate of the herbicide in the natural environment.

Four soils were used to study mineralisation. Two of these had shown high rates of 36DCPA disappearance in field trials (Dow Chemical Co., personal communication) and were thus termed high activity soils and the other two had shown long residence times of 36DCPA during field studies and so were termed low activity soils. All the soils were treated identically (see section 2.5) and were inoculated with 0.25 ppm (ug g_1 soil) 2,6 [ 1**C]-3,6DCPA as the sole source of carbon.

Table 3*1 shows the amount of ^ C O g absorbed from the soil culture effluent air, expressed as disintegration per minute, for each soil. Because the nature of the soils (i.e. their activity towards 36DCPA) was specified as high or low by the Dow Chemical Co., the times of sampling were varied for the two activities of soil.

CHAPTER 3

MINERALISATION OF 3,6 DICHLOROPICOLINIC ACID (36DCPA)

3.1 RESULTS

To test the feasibility of screening soil micro-organisms for any activity against recalcitrant molecules, the compounds first need to be tested for mineralisation in soil and an examination made for any metabolised breakdown products. It has been demonstrated that micro­ organisms capable of the degradation of organic pollutants can be enriched for by application of biodegradable analogues to the microbial ecosystem (Focht & Alexander, 1970) and this phenomenon has been termed "analogue enrichment" (see section 5.1).

It is not necessary, when examining mineralisation alone, to use the high concentrations that are normally necessary for obtaining measurable growth in liquid media, so field application rates may be used. This gives a more accurate indication of the fate of the herbicide in the natural environment.

Four soils were used to study mineralisation. Two of these had shown high rates of 36DCPA disappearance in field trials (Dow Chemical Co., personal communication) and were thus termed high activity soils and the other two had shown long residence times of 36DCPA during field studies and so were termed low activity soils. All the soils were treated identically (see section 2.5) and were inoculated with 0.25 ppm (pg g_1 soil) 2,6 [1**C]-3,6DCPA as the sole source of carbon.

Table 3.1 shows the amount of ^ C O g absorbed from the soil culture effluent air, expressed as disintegration per minute, for each soil. Because the nature of the soils (i.e. their activity towards 36DCPA) was specified as high or low by the Dow Chemical Co., the times of sampling were varied for the two activities of soil.

Table 3-1 Amount of absorbed by the absorbing reagent when field application rates of 2,6 [ 1l,C]-36DCPA were to the soils specified. Values have been adjusted for blank and sterile control.

TABLE 3.1

I Activity of absorption reagent (D.P.M) i i___________________________________________ Time (days) 1 1 1 ! M46 j Soils M47 M51 M53 1 1 1 6798 ND 8022 ND 2 ! 12288 ND 11170 ND 3 i 19152 ND 25713 ND 4 ! 33675 ND 36761 ND 5 ! 56241 ND 48479 ND 7 I 104448 ND 71121 ND 10 ! 268572 43956 144239 64026 14 ! 357675 ND 181302 ND 21 ! 644235 106743 310806 136242 28 ! 941039 ND 491061 ND 30 ! ND 215558 ND 232221 42 i ND 339876 ND 360179 55 I ND 543484 ND 531408 67 ! ND i_i 1113202 ND 1243759 4 7

Figure 3.1(a) shows the percentage of 36DCPA remaining in the soil at the various times tested. Control samples of heat sterilised soil plus identical quantities of 2,6 [^C]-36DCPA and COg absorption reagent plus scintillant were allowed for in the calculation of the percentage of 36DCPA remaining. After 28 days it can be seen that approximately 30J of the radio-active 36DCPA has disappeared from the culture vessel.

Figure 3.1(b) shows the disappearance of 36DCPA from the same soil but on a logarithmic scale. From this it can be seen that up to day 14 the rate of disappearance of 36DCPA was relatively slow (8% in 14 days). At day 14 there appeared to be a sharp rise in the degradation rate. The new rate of degradation was approximately 3 times that of the old rate. From this figure, the half life of 36DCPA in soil M46 was calculated to be 43 days (calculated from the steeper half of the graph).

Figures 3.2(a) and (b), 3.3(a) and (b) and figures 3.4(a) and (b) show similar results for soils M51, M47 and M53 respectively. Soils M47 and M53 were the biologically less active soils. The half lives have been calculated to be 71 days, 181 days and 152 days respectively (calculated from the shallower portion of the graph). In soil M51 there appeared to be the same increase in the rate of degradation that was apparent in soil M46 but it appeared to be absent in the two other soils. This shows, to some extent, that the larger organic component of these soils was more able to produce a more rapid mechanism of degradation after a suitable induction period.