Document zoQ66nGzBd5r53n6G1n0wMGrg

t/\ ; , \ >V Vv T-:v^ <' A,.vt^*'^-'tj-'.^.. fit tf' yvV>,vv [Vi >j11 .Vu v. \-Dvix/ x* av*cUSU CH APT KK 25 Inhibition of Estuarine Microorganisms by Polychlorinated Biphenyls* A. W. Bourquin and L. A. Kiefer U.S. Environmental Protection Agency, Gulf Breeze Environmental Research Laboratory,** Sabine Island, Gulf Breeze, Florida 32501 N. H. Berner, S. Crow, and D. G. Ahearn Department of Biology, Georgia State University, Atlanta, Georgia 30303 Over 100 isolates of representative estuarine bacteria and fungi were screened for tbeir ability to grow in the presence of commercial preparations of polychlorinated biphenyls (1'CB). Super absorbant sensitivity discs impregnated with up to 0.5 mg of PCB were placed on the surface of freshly inoculated solid media. Twenty-six bacteria, representing both gram-positive and gram-negative strains of varying morphology, showed varying drgTe.es of sensitivity to PCB. In contrast to insensitive isolates, sensitive strains were mainly amylolytic and proteolytic. PCB had negligible effect on the growth of fungi. The sensitivity of select cultures of heterotrophic bacteria to PCB may be of considerable importance to nutrient turnover in estuarine ecosystems. Introduction Polychlorinated biphenyl formulations (PCB's) arc chemically and thermally stable and possess high dielectric constants. Because of these properties, PCB's have been important commercially as coolant-insulation fluids in capacitors and transformers, hydraulic fluids, plasticizers, lubricants, and fire retardants. Jensen et al. (1969) were among the first to note the magnification of PCB's in the food chain. Subsequent studies have shown their environmental effects to be similar to those of DDT. The chemistry and persistence of PCB's in the environment and their chronic toxicity for various animals have been reviewed by Peakall and Linccr (1970) and Gustafson (1970). One report (Kcil cl al. 1972) describes a commercial PCB formulation in concentrations of 0.1 jug/ml which stimulated the growth of Escherichia coli, Ahmed and Focht (1973) reported biodegradation of PCB isomers 2 to 5 chlorines by Achromobacter pCB. Littleinformation on the interactions of PCB's with heterotrophic microorganisms is available. Our investigation examines the effects of two commercial PCB formulations (Aroclor 1016 and 1242) on selected estuarine bacteria and fungi. *GBERL Contribution No. 230. Associate Laboratory of National Environmental Research Center. Corvallis, Oregon 97330. ^Registered Trademark* Monsanto Company, St. Ix>uis, Mo. Mention of commercial products or trade names does not constitute endorsement by the U.S. Environmental Protection Agency. 256 DSW 025322 STLCOPCB4009277 Of-'-!'&?*> ':* ' '' ` >Vx V,:/ -> V*><'- v*-1.. .y'.^yy- .?-* ?.-' *' *V; , **;/ ; y t'.W*Mta. ..w.' i,sirtn^Lbitha*..f/'XL-Cy''* C p IfftJrffj'Vi`liflft-Aiirfij -1 - CONTRIBUTED PAPERS 257 Materials and Methods Organisms. Bacterial isolates were obtained from estuarine waters and sediments of Pensacola Bay, near Gulf Breeze, Florida. Yeast isolates were obtained from waters and sediments of Bandana Bay, Louisiana. Biochemical analyses for bacteria were performed by the methods of Colwell and Wiebe (1970) and identifications by means of Breed et al. (1957). Media. The medium for bacteria contained 1.0 g yeast extract (Difeo) and 5.0 g peptone (Difco)/lilcr of aged artificial seawater (Rila Marine Mix, aged 1 mo in dark) al 20/oo salinity (adjusted with distilled water) and pH 7.4. For solid medium, 20 g agar (Fisher) were added per liter of medium. In growth curve studies, the above medium was diluted to one-half nutrient strength and the desired salinity. Mycological agar (Difeo) prepared with distilled water was used for the growth of fungi. For phosphatase studies, the yeasts were grown on this medium plus 0.02% phcnolphthalcin diphosphate. The yeasts were also grown in a broth with this formulation. Test chemicals. Aroclors arc commercial PCB formulations containing many isomers. Two Aroclor formulations, 1242 and 1016, containing 42% chlorine, were examined in this study. Concentrations (w/v) of PCB's arc based on the Aroclor formulation as teemed from The Monsanto Company, considered as being 100% PCB. Sensitivity studies. Bacterial cells for inocula were grown in broth for 18 h at 28 C on a rotary shaker. The culture was diluted 1:1 with sterile 20%o seawater and 0.1 ml of the dilution was spread on the agar medium. Yeast cultures were grown for 48 h on mycological agar and colonics were suspended in distilled water to produce a cell suspension detectable by sight. The cell suspension was swabbed onto agar and prepared absorbant paper discs were positioned on the surface of the agar. f i'. r CY-.vY- " ` >.r ' " ti -i,- Vm ictwi w*.: 5' fi, MIREX HEPTACHLOR 1242 1016 0.5 mg 0.25 mg O.t mg PIG. 1. Growth inhibition of an estuarine bacterium by Aroclor 1242 and 1016 on solid marine medium. Min x and heptachlor (0.5 nig/disc), chlorinated hydrocarbon insecticides were included for screening ]>ui|X)scs only. Bacterial growth appears white on dark medium and the zones of inhibition appear dark surrounding the white disc due to the negative reproduction of the photographic plate. The culture dish served as the negative, placed directly on lire photographic paper. DSW 025323 STLCOPCB4009278 258 A. W. llOUHQUIN AND L. A. KItFF.R Tin' absorbanl paper tliscs (Schldclicr and Schucll, Inc., No. 7-10-E) were saturated with 0.1 ml of acetone solution containing ].0, 2.5, and 5.0 mg/ml of I'CB formulation. The discs were air-dried for 24 h at room temperature before use. I'CB-trcatcd discs and control discs treated only with acetone were placed on the agar plates within 2 h of inoculation. All tests were performed in duplicate and examined for possible inhibition after 24-48 h incubation at 24-28 C. Only isolates sensitive to 0,5 mg of cither l'CB formulation were tested further for response to PCB's. Sensitivity was defined as a zone of inhibition stirrounding the paper disc (Big. 1). Phosphatase studies. To test the effects oT PCB's on extracellular phosphatases, yeasts were grown in liquid medium and on PCB-impregnatcd membranes placed on agar plates. After 2-24 h incubation, the membranes were removed and the plates were exposed to vapors of concentrated NH^OIl. Occurrence of reddish zones on the plates demonstrated phosphatase activity. Growth curve studies. Cells for inocula were grown overnight in liquid medium and inoculated into 50-100 ml of the same medium in a 500-ml Ncphclo-culturc flask (Bcl)co, 515-A) to make a final cell concentration of 1%. Cell density was monitored as absorbance using a Bausch & Lomb Speclronic 20 at 660 nm or a Klctl-Surnmerson Photoelectric Colorimeter with a No. 66 red filter. Test chemicals were added in acetone to facilitate dispersion and medium plus acetone cultures were monitored as checks for acetone effects. Salinities were adjusted with distilled water prior to addition of nutrient and the pi I was adjusted to 7.4. Rj-isults and Discussion Of 106 bacterial isolates, growth of 28 was inhibited in varying degrees by the PCB formulations. Sensitive bacteria reacted similarly to both Aroclor formulations (Table 1). The PCB-scnsitivc bacteria included both gram-positive and gram-negative isolates. Of all strains tested, a slightly greater percentage of the sensitive bacteria were gram-positive (Table 2). These results differ from previous reports of sensitivity of gram-positive bacteria to other chlorinated compounds (Trudgill el al. 1971). Cyclodiene insecticides, shown to inhibit a range of gram-positive bacteria, had no effect on gram-negative bacteria tested (Widdus et al. 1971; Trudgill ct al. 1971). Differences in toxicity of PCB's and cyclodiene pesticides to gram-negative bacteria may be related to. type of molecule rather than to degree of chlorination. Biochemical activities of sensitive and nonsensitive bacteria arc compared in Table 2. The majority of sensitive strains produced both amylase (75%) and gclatinasc (89%), whereas of all strains tested, only 37% were amylase-producers and only 457o were gclatinasc-produccrs. The significance of these results in relation to total nutrient catabolism must await further investigation. Fig. 2 shows the effect of Aroclor 1242 on the growth of four estuarine bacteria in liquid culture. Two bacteria were completely inhibited for 18 h. Since tire PCB's were added in acetone solution, we believe that after volatilization (or degradation) of the acetone, PCB's were adsorbed to the cells and glass, allowing cells with no adsorbed PCB to attain logarithmic growth after 18-20 h incubation (not shown). However, in nature, if the PCB's were adsorbed to the microbial substrate at inhibitory concentrations, no growth would occur. DSW 025324 CONTRIBUTED PAPERS 259 Table 1. Inhibition of growth of estuarine bacteria on nutrient seawater medium by PCB's GBLRL Culture No. Gram Reaction Sc Morphology Genus Aroclor 1242 (mg) 0.1 0.25 0.5 Aroclor 1016 ( mg) 0.1 0.25 0.5 3 + ROD Unknown . ++ + 4- + + 4- + + ++ 21 - HOD Unknown ++ ++ 4-4-4- ++ ++ 35 -ROD Flavobactcrium sp. ++ ++ + 4- + +++ 39 -COCCOID Unknown . ++ +++ 4-44- ++ +++ 53 -ROD Unknown ++ ++ + 4-4-4- +4+ +++ 54 + ROD Bacillus sp. +++ ++ + 4-4-+ + +++ 7 + ROD Bacillus sp. + ++ X + 9 + ROD Bacillus sp. + ++ 4-4 + + ++ 51 -ROD Unknown + + +++ + + 60 + ROD Bacillus sp. + + + + + ++ 86 - ROD Flavobacterium sp- + + + X + 100 -ROD Pseudomonas sp. + 4 + ++ + + + 8 + ROD Corynebacterium sp. X 4- ++ X + 11 - ROD Achromobacter sp. X + ++ X + 42 +COCCUS Micrococcus sp. X 4- ++ X + 44 +COCCUS Micrococcus sp. X 4- + -- X 93 -t ROD Unknown X 4- + X 43 t COCCUS Micrococcus sp. - 4- + + + 5 -COCCOID Serralia sp. -- _ 4-4- -- _ 13 - ROD Achromobacter sp. -- -- ++ -- -- 28 -ROD Achromobacter sp. -- -- ++ -- 32 -t ROD Corynebacterium sp. _ 4- + _ X 41 -COCCOID Unknown -- ++ -- -- 67 - ROD Achromobacter sp. -- 4- + _ -- 69 - ROD Unknown - - ++ - - Degree o F sensitivity: +++ (18-20 mm zone), ++ (16-18 mm), + (14-16 mm), x (slightly). -- (not sensitive). +++ +++ +++ +++ +++ +4+ +4- +++ +++ ++ + ++ ++ ++ ++ + + ++ ++ ++ ++ + ++ + ++ TABLE 2. Biochemical activities of PCB-test bacteria (percent of cultures showing positive reaction) Bacteria Tested Urease Production of Amylase Lipase Gelatinasc HjS Citrate Gram Utilization Reaction Sensitive cultures 25 75 29 89 4 43 54 Total test cultures 14 37 19 45 5 37 40 us rD.` l-J i f. - ; f . i; L*V I FlCi.2. Growth of estuarine bacteria in liquid marine medium (20%o) containing 10 fig/ml Aroclor 1242 of cultures no. 31 (unknown, gram-negative) and no. 60 {Bacillus sp.) were sensitive to PCB's and cultures no. 12 (unknown, gram-negative) and 47 [Pseudomonas sp.) were not sensitive. Average data points given for the latter two bacteria, two curves (experimental ------- , and control------- ), are not sig nificantly different. ..... (-- ~ir ' " ----- H ` tIMt [Ktvti) DSW 025325 STLCOPCB4009280 " A ., 'f '` " I V ' ' \ V.' ; v.. - ' " ' ' . . - : - 260 A. W. BOUKQUIN AND I.. A. KIEFER IFFKT Of VARYING CONCINTR ATFONJ OF AftOCLOR 1242 ON GROWTH OF ISTUARINi BACTERIA WsVl*-!--v FIG. S. Dose-response curve showing growth (O.D.) response of estuarine bacterial isolates no. 9 (Haciltus p.) and no. 100 (Pseudomonas sp.) to varied concentrations of Aroclor 1242 (0-5 ug/ml). The PCB was added in acetone to facilitate dispersion. Check, cultures contained marine broth at 207,,<,salinity only and with acetone (0.5 ml/flask). Due to the insoluble nature of PCB's, the minimum inhibitory level of PCB formulation could not be determined by the paper disc method. The lowest inhibitory concentration of PCB's for two selected sensitive organisms is demonstrated in Fig. 3. Growth of the gram-positive isolate (culture No. 9, Bacillus sp.) was inhibited at 1.0 pg/ml, whereas the gram-negative isolate (culture No. 100, Pseudomonas sp.) was sensitive at 1.0 pg/ml and completely inhibited at 5.0 pg/ml. Inhibition of growth of fungi (Cladosporium sp., Cephalosporivm sp., Sacchnromyces sp., Candida lipolytica, C. subiropicalis, Pichia spartinae, and Kluyveromyccs drosophitarum) by PCB's on paper discs was negligible. Although yeasts failed to grow on membranes completely saturated with PCB's, they grew on membrane areas that were free of PCB's and demonstrated phosphatase activity. In liquid media with PCB's added in petroleum ether (which was evaporated), yeast growth and phosphatase activity were similar to those of controls. Sensitivity of estuarine bacteria to PCB formulations was greater in liquid than on solid medium. No inhibition of growth of estuarine fungi or phosphatase activity was noted except on PCB-saturated membranes. Inhibition of bacterial growth by PCB formulations adsorbed onto paper discs is a simple technique which may be used to detect, in the laboratory, the possibility of inhibit ion in the environment. Although PCB's arc almost insoluble in water, they are DSW 025326 STLCOPCB4009281 CONTRIBUTED PAPERS 261 readily adsorbed to solid surfaces (Rizwanul ci al. 1974). In nature, if the solid surface to which I'CB's arc adsorbed is a potential microbial substrate, inhibition of microbial catabolism could occur. Such inhibition could account for unexplained increases in BOD of effluents of sewage treatment facilities that receive industrial wastes containing large amounts of PCB's. The nature of the inhibiting substance in the PCB formulation that acts to interfere with the rate of nutrient turnover is uncertain. Literature Cited Ahmed, M., and D. D. Focht. 1973. Oxidation of polychlorinated biphenyls by Achromobacter pCB. Bull. Environ. Contam. Toxicol. 10:70-72. Dreed, R. S,, E. G. D. Murray, and N, R. Smith. 1957. Bergey's Manual of Determinative Bacteriology. - Williams & Wilkins Co., Baltimore, Md. " Colwell, R. R., and W.J. Wicbe. 1970. "Core" characteristics for use in classifying aerobic, hetcrotrophic bacteria by numerical taxonomy. Bull. Georgia Acad. Set. 28:165-185. Gustafson, C. G. 1970. PCB's--prevalent and persistant. Environ. Sci. Technol. 10:814-819. Jensen, S., A. G. Johneis, S. Olson, and G. Ouerlind. 1969. DDT and PCB in marine animals from Swedish waters, Nature 224:247-250. Keil, J. E., S. H. Sandifer, C. D. Grabcr, and L. E. Pricster. 1972. DDT and polychlorinated biphenyl (Aroclor 1242) effects of uptake on E. coli growth. Water Res. 6:83 7-841. Peakall, D. II., and J. E. Lincer. 1970. Polychlorinated biphenyls. Another long-life widespread chemical in tite environment. BioScience 20:958-964. Rirwanul, 11., D. W. Schmcdding, and N. H. Freed. 1974. Aqueous solubility, adsorption, and vapor behavior of polychlorinated biphenyl Aroclor 1254. Environ. Sci. Technol. 8:139-142. Trudgill, P. W., R. Widdus, and J. S. Rees. 1971. Effects of organochlorjne insecticides on bacterial growtli, respiration and viability. J. Gen. Microbiol. 69:1-13. Widdus, R,, P. W. Trudgill, and D. C. Turncl). 1971. The effects of technical chlordanc on growth and energy metabolism of Streptococcus faecalis and Mycobacterium phlci: a comparison with Bacillus subtilis.J. Gen. Microbiol. 69:23-31. -A * -V mm ''V' f.. ' * 'Vv'V V'wwW.; T * ITn mi. uwvjm* ' r f c t fi STLCOPCB4009282