Document k6RNgRErVzyo1n5YN4O2m6ZOD

I V i-HOU A LOCATION) O ,: R.A. Lidgett - Ruabon OATC SUOJCCT ftFCftN'C 1 2?tli October, 1969* BIODEGRADATION OF AROCLORS ItALi/ineli TO W.It. RICHARDS . St . Louis v R.E. Keller, St. Louis R.A. Baxter, Ruabon J.H. Mainprize, Ruabon II.A. Vodden, Ruabon J.W. Barrett, London We are at present writing a detailed report on the work carried out to date on Aroclor 1242 biodegradation studies. This cannot be completed for a few more days so I have attempted to put the main observations and con clusions drawn, so far into this m. .:o. We will send the details later in the week. . Experimental Method , `- The samples of Aroclor 1242 hnv boon prepared at a standard 5 ppm level and submitted to biodegradation b\ J.U. Muinprize in the Effluent Research Group. This has involved a survey I . determine the most effective means, of dosing aqueous samples with both Vr 1242 and active bacteria and of methods for extracting the aqueo - sa'./les after degradation to obtain the - Arcelor 1242 in a form suitable for electron capture gas chromatography. The following method has be: '. o mblished A standard solution of 100 ppm .'i..- loi 1242 in ether is prepared. This is diluted to give 5} 2 and 1 ppm .-a,'in. i oos for gas chromatography. Aqueous samples are prepared by taking a 5 r.i.i aliquot of the standard solution, evaporating to dryness in the sample flask by evaporation at ambient teinpcraturi in a low air flow, then making up <0 100 mis with nutrient salt solution, inoculum etc. The inoculum used had previously been shown to be capable of complete degradation of biphenyl. These solutions are then used for the biodegradation tests. . The aqueous samples are shaken in closed vessels in the dark for specific periods, the flasks being vented evorj 3 days to ensure presence of sufficient oxygen, and are then extracted in a separating funnel with heptane (4 x 25 ml portions) to provide a 100 ml heptane solution for gas chromatography. Gas Chromatography % Conditions Instrument f *7 -- 11 P 402 equipped with Ni high temperature electron capture detector. Column . - 41 Silicone Oil 0V101 on 80-100 mesh Chromosorb W 11.P. (cx Supolco) Flow . '\ 40 ml s/min. through column plus 60 mls/min. purge flow in detector. Both of Argon/Mctham 95/5- * DSW 346669 - STLCOPCB4084675 -2- . Oven Temp. Flash Heater Detector Sample Size Palser - 170C - 250C . ' - 250C ` -- 4 jll. - 150 m. sec. . A calibration is prepared using a standard solution of Aroclor in heptane of 5, 2 and 1 ppm Aroclor 1242 plus an internal standard solution of 5*3 ppm 2 clilorobiphenyl (previously shown to be absent from the 1242 traces). Results-and Discussion In this work each series of experiments has led to a modification of the programme and this has meant that there has been little opportunity to carry out repeats or duplicates of critical experiments. As the pattern emerges, this will be done, but at present it should be understood that interpretation of results may be based, on a.single series of experiments. Results of the individual experiments are given in bar graph form on sheets attached to this memo and t!ie following observations can be made for each series. Figure No.l . From the bar chart it can be seen that 00 Peak No.l in the chromatogram is very easily lost, being reduced by 90$ . in three days and completely lost in the 8 day.and all subsequent samples. <n Q y 00 Samples 2 and 3 (3 and 8 days) appear to have undergone more severe degradation than 4, 5 and 6 (13 and 25 days). This may be an experimental factor with the former samples losing 1242 by a non degradative path or by physical loss. A further explanation could be that samples 4,5 and 6 have not been as efficiently biodegraded as samples 2 and 3* At the present time we have taken results on samples 4,5 and 6 as being more probably correct. ' - (c) Results for sample (l) are within experimental error and the dosing/ extraction/gas chromatographic methods arc acceptable. The reduction in peak (l) is probably caused by evaporation loss in the dosing step. From these results it is possible to infer that ' (a) Loss of 1242 at the 5 ppm level can reach 40$ of initial concentration for the majority of the measured peaks after 13 days. Further loss appears to be inhibited: this may be caused by poisoning of the bacteria by 1242 or its degradation products at the relatively high concentration of 1242 usdd. * . `` ' DSW 346670 STLCOPCB4084676 00 The first peak in the chromatogram is rapidly lost under the conditions of the experiment. Results given la(,cr with sterile samples indicate that this loss is mainly caused by biodegradation. FIgure No.2 To ensure that the observed disap,,e.-trance of 1242 was caused by biodegradation and not other factors J.H.M. also ran a series of control samples which contained copper sulphate solution at a level known to sterilise the media. These samples gave the surprising result that the 1242 was still lost in significant amounts and this can be seen in the bar chart. This can 1 be interpreted as follows . (a) The dosing/extraction/gas chromatographic methods have an acceptable accuracy (however, see discussion later concerning electron capture detection of p.c.b's.) 00 In the presence of- CuSO^ (known to be o bacteriostat) Aroclor 1242 is . lost by a chemical or catalytic process and this process is time dependent. It is thought to be non hydrolytic as a similar situation does not occur . when the CuSO^ is replaced by'llgPln (see later). _ Figure No.3 . The two 25 day samples have been compared here with a similar sample in which 10 ppm CUSO4 has been used as a bacteriostat. . It is interesting to see that peak (l) is lost with the inoculated samples but in the presence of CUSO4 the rate of loss is much less. This is almost certainly loss through chemical reacti03; in the sterile sample. .* Figure No.4 - - As there appeared to be a possible chemical reaction between 1242 and CuSO^ a series of flasks was prepared in which the sterilising agent was IIgCl2 and not CuS04. ' , From the bar chart it can be seen that (a) When 5 ppm of 1242 (without additives and with ether removed) is shaken for 14 days there is a loss of approximate]}' 20-30$ of the original concentration added to the flask. Or, if the concentration present before shaking but after ether evaporation is used, there is a loss of approx. 10-15$ caused by the shaking procedure. 00 Samples 4 and 5 show that shaking 12'i2 (5 ppm) in a sterile aqueous medium for 14 days causes a loss of approximately 20$ of the 1242. It is noticeable that in these samples, peak No.l behaves in .an equivalent manner to the later peaks, i.e. biodegradation is not occurring. (c) Samples 5, 6 and 7 give equivalent results. DSW 346671 STLCOPCB4084677 -4- The results can he interpreted as follows (a) Shaking Aroclor 1242 in a sterile aqueous medium causes a loss of 1242, but this is relatively small. The loss may be flue to adsorption of the Aroclor on glass surfaces or free, other as yet unknown causes. 00 The IIgCl2 is acting as a baci or i osta (. at all levels used. This is important for future work where sterile controls must be used. From the foregoing results, the following tentative conclusions may be made ` (a) Loss of Aroclor 1242 on shaking for 14 days in a sterile medium (HgC^) is approximately 20 - 30$ based on measurement of the major peaks in the chromatogram. 00- Loss of Aroclor 1242 on shaking for 13 days (or longer) in an active medium causes a loss of approximately 40$ for later peaks and 100$ for an early peak which may or may not be composed of more than one p.c.b. (this is being examined). . (c) . Control samples containing CuSO;, (10 ppm or greater) as a bacteriostat lose Aroclor 1242 at a significant rate via an at present unknown process (possibly catalytic or direct chemical). This appears to cause an overall loss of material but there is evidence that production of other compounds also electron capturing may he occurring. ' The above conclusions are based on the assumption that the 60 - 65$ loss of Aroclor 1242 (samples 2 and 3 Figure T) is an erroneous resultj hut this will be re-examined. ' While the above tentative interpretations of.the results can be made, there * is an increasing need to treat the quantitative measurement of Aroclor chromat ograms obtained with the electron capture detector with more caution. This was also expressed by Widmark at his last lecture and at the present time there is < ' no experimental evidence to suggest that all the Aroclor components have the same or similar electron capturing powers (it is not known even whether p.c.b. isomers have similar responses to the e.e. detector). Thus the loss of one peak cr the appearance of a second peak in the 1242 chromatograms discussed above, leads us to believe that this behaviour could well be occurring beneath the envelope of later peaks which we know to be composed of many components. The complexity has been shoivn by comparison of a normal packed column electron capture detector chromatogram of 1242, and a wide bore tubular column chromatogram of the same _ product obtained with a flame ionisation detector and copies of these chromatogram are included with this memo. : DSW 346672 STLCOPCB4084678 1 * 5 For this reason our future work on degradation will proceed along the following lines . (1) Submission of individual p.c.b.'s (supplied earlier hy Applied Sciences Section) to biodegradation procedures and quantitative measurement of component loss. (2) Development of a wide bore tubular column for quantitative analysis of Aroclor 1242. Such a column can be used without an injection splitter, it will provide high resolution chromatograms suitable for quantitative measurement. RAL/mch 28th October, 1969* R.A. LIDGETT % ; i | . . '** * DSW 346673 I ' : ! . - ! '' .. . i* STLCOPCB4084679 0 KEY Figure No.1 1. 5 ppm 1242 in ether added to flask. Ether removed by low air flow Inoculum plus water (10 nil) and minerals (90 mis.) added, total 100 mis. Immediately extracted with heptane (4 x 25 mis). 2. As 1 - extracted after 3 days. - \^ 3. " " " " 8 days. ' 11 13 days. . " 25 days. 6. ' Duplicate of 5* ' . Figure No.2 . 1. 5 ppm of 1242 in heptane. ' 2. 5 ppm 1242 in ether added to flask. Ether removed by low air flow. 100 mis. heptane added. 3* 5 ppm 1242 in ether added to flask. Ether removed by low air flow. Mineral soln. (90 mis), water (10 mis) plus CuSO^ (10 ppm) added. Extracted immediately with 4 x 25. ml portions heptane. 4. As 3 extracted after 3 days.- . 5. " -" " 13 days. . . .` * _ .` ` - ' Note ?ealc No.4, sample No.5 was not measured because it was off scale under the attenuation conditions used in this particular g.c. run. DSW 346674 STLCOPCB4084680 KEY (Continued) Figure No.3 1. 5 ppm 1242 in ether added to flask. Ether removed hy low air flow then mineral solution (90 mis.), water (jO mis) and inoculum added. Flask shaken 25 days before e.\i iv.eLi on with heptane (k x 25 ml). 2. As 1. .. 5. As 1 but with addition of CuSh.^ (H) ppm). Samples 1 and 2 in this chari mj , fh. .-a ho as 5 and 6 in Figure 1. Figure No.4 ` 1. 5 ppm 1242 in 100 mis heptane. 2. 5 ppm 1242 in ether added to flask. Ether removed by low air flow. Hade up to 100 mis. with heptane. . 5* 5 ppm 1242 in ether added to flask. Ether removed by low air flow. Flask shaken (1242 only in'flash) for 14 days. Extracted with heptane 100 mis. . 4. 5 ppm 1242 in ether added to f!nsk. Ether removed by low air flow. Minerals (90 ml), water (10 ml) and llgi'lo (50 ppm) added. Solution .extracted immediately with 4 x 25 ml. heptane. . 5` .6. 7* As 4 but shaken for 14 days before extradion with heptane (4 x 25 ml). . ` * As 5 but 10 ppm HgClg added in pi arc of 50 ppm. As 5 but 25 ppm n m it if n * DSW 346675 STLCOPCB4084681 IV, L-/ Peak dumber7 ro co STLCOPCB4084682 Number ja q iu n M ^V3 e d Peak dumber Q __ p-p.ra..Aroclor_____ Q (\o co ^Fs. cn STLCOPCB4084685 3