Environ. Sei. Technol. 1983, 17, 104-107
Effect of Nutrients on the Rates of Mineralization of Trace Concentrations of Phenol and p-Nitrophenol Howard E. Rubln and Martln Alexander" Laboratory of Soil Microbiology, Department of Agronomy, Cornell University, Ithaca, New York 14853
The rates of phenol mineralization in samples of three lakes were directly proportional to concentration at phenol levels of 2.0 pg/mL to 200 ng/mL. The rate at any one concentration was related to the trophic level of the water. In samples containing microbial cells collected from the eutrophic lake, reducing the nutrient supply decreased the mineralization rate. Inorganic nutrients, arginine, or yeast extract often enhanced but glucose reduced the rate of mineralization. Arginine was not stimulatory in stream water. Mineralization was slower than predicted at phenol concentrations of 1.5 pg/mL or less but occurred at predicted rates in the presence of arginine. Arginine also stimulated the mineralization of 19-197 pg of p-nitrophenol/mL by lake water microorganisms, but only at one p-nitrophenol concentration was it stimulatory in stream water. These data indicate that organic and inorganic nutrients in natural waters affect the rate of mineralization of organic compounds in trace concentrations. Introduction Because many organic chemicals present in inland waters are toxic to humans or aquatic animals or plants, their mineralization is of considerable importance. A variety of factors affect the rate of microbial destruction of such compounds, among which are the size and diversity of the microbial community (1, 2), sorption (3), and the structure of the chemicals ( 4 ) . Scant attention has been given to the effect of nutrient level on the mineralization of organic compounds in natural waters. However, a direct relationship has been reported between nutrient levels and microbial populations and diversity in eutrophic waters (1, 2). Moreover, an association has been noted between the rate of mineralization of high concentrations of 2,4-dichlorophenoxyacetate (2,4-D) and nutrient concentration (3, 5 ) . However, all studies of the effect of nutrient supply on mineralization have involved concentrations of organic compounds far higher than those that commonly exist in fresh water. Because the rates of mineralization of substrates at high concentrations differ from those occurring at concentrations characteristic of rivers and lakes and may follow different kinetics (6),a study was undertaken to determine the effect of nutrient supply on the mineralization of chemicals in trace concentrations. Experimental Methods To study mineralization rates, we passed freshwater samples through a glass fiber filter (Gelman Sciences, Inc., Ann Arbor, MI, No. 66085) and then amended the water with the 14C-labeledcompound to be tested. The samples were incubated in the dark at 29 "C without shaking. The volume of water varied from 100 mL to 6.0 L depending on the concentration and specific activity of the test chemical. At intervals, 5.0-250-mL samples were removed from the test flasks and acidified with concentrated HzSO4 to pH 2.0. Air was then bubbled through the samples for 15 min to remove 14C02or, for compounds at concentrations less than 20 pg/mL, the chemical was extracted with ethyl acetate. The radioactivity remaining was measured 104
Environ. Sci. Technol., Vol. 17, No. 2, 1983
with a Beckman liquid scintillation counter (Model LS7500). The rates of disappearance of each chemical at each concentration were determined from the linear portion of a plot of chemical disappearance vs. time. The entire procedure has been described in detail elsewhere To determine if the disappearance resulted from volatilization, sorption, or abiotic breakdown, we added the test compound to samples of lake water supplemented with 0.2% NaCN, but no disappearance was detected. When Beebe Lake water was incubated at 29 "C with 220 pg of radioactive phenol/mL for 18 h or 4.0 ng of radioactive p-nitrophenol/mL for 35 days under the test conditions, more than 99% of the radioactivity that had disappeared from solution was recovered as volatile products in an ethanolamine trap. At intervals, mineralization was determined in distilled water adjusted to pH 7.2 and supplemented with suspensions of cells of the microbial community of Beebe Lake, 52.8 mM tris(hydroxymethy1)aminomethane (Tris) buffer (pH 7.2), or distilled water augmented with inorganic salts, glucose, adenine, arginine, propionate, or yeast extract. The organic compounds were added to either distilled water or natural waters at the concentration of organic carbon that was present in Beebe Lake (38.5 pg/mL). Because Tris is slowly biodegradable, it was not considered to serve as a carbon or nitrogen source in these short-term experiments. [U-14C]Phenol(specific activity 87 mCi/mmol) was obtained from Amersham Corp., Arlington Heights, IL, and p-nitr0[2,6-~~C]phenol (26.6 mCi/mmol) was purchased from Tracerlab, Waltham, MA. When inorganic nutrients were added to the solutions, the supplement contained (per liter of distilled water) the following: MgSO4.7Hz0,0.10 g; MgC12,0.066 g; (NH4)2S04, 0.50 g; CaCl2.2Hz0,0.02 g; FeCl,, 1.0 mg; Na2Mo04.2H20,0.062 g; MnS04.H20,0.023 g. A natural aquatic microbial community was added to distilled water in the following manner. After fresh samples of Beebe Lake water were passed through the glass fiber filter to remove large particulates, the cells were removed by centrifugation at 41000g at 4 "C, washed twice with 0.3 mM phosphate buffer, pH 7.2, and concentrated 100-fold. A suspension of these cells was added to distilled water to give a cell density equal to that originally found in Beebe Lake water. Samples of fresh water were taken from Beebe Lake, Cayuga Lake, and Enfield Creek near Ithaca, NY, and from White Lake near Old Forge, NY. Beebe Lake is eutrophic, Cayuga Lake is mesotrophic, and the stream and White lake are oligotrophic. Total organic carbon analysis was performed by using a Beckman (Beckman Instruments, Inc., Fullerton, CA) Model 915A organic carbon analyzer fitted with a Beckman Model 865 infrared analayzer. Sodium oxalate was used as the standard. The flow rate of the compressed air was 150 mL/min, and the temperature of the column was 970 "C. Each experiment was conducted in triplicate, and a single set of analyses was performed on each sample. Statistical analysis was either by the least-squares me-
(n.
0013-936X/83/0917-0104$01.50/0
0 1983 American Chemical Society
CI
I 10 ng
Table I. Mineralization of Phenol a t Different Dilutions of Beebe Lake Water with a Constant Natural Microbial Community
-
#
i
a W n Y
ing-
W I-
PO
-
10 PO
-
gloo
initial phenol concn, ng/mL
CAYUGA LAKE (
z
P c a
i
TREAM WATER (A)
d
a
K W
3 5
1po-
fg
mineralization rate, pg/(mL h ) Beebe Lake
diluted
lox
diluted
looox
t1 A i o 0 pg
1 ng
10 ng
H2O
1.0 0.89 1.1 1.4 0.019 13Aa 13A 14 14 0.197 121 93 134 144 1.98 lllOB 1140B 1230 1620 19.9 10800 9980 14200 16800 198 a Values with a letter in common are not significantly different, All other differences in rates among the various dilutions of lake water and in distilled water and for all phenol concentrations are statistically significant a t the 95% confidence level.
F
loo
i o pg
diluted
100 ng 1.2
PHENOL CONCENTRATION (PER ML)
Figure 1. Rates of phenol mineralization in samples of natural waters.
thod or by the Newman-Keuls multiple range test at the 95% confidence level.
Results The rates of mineralization of different concentrations of phenol were determined in filtered samples from Beebe Lake, Cayuga Lake, stream water, and White Lake. The total organic carbon concentrations of these waters were 38.5, 27.0, 7.9, and 5.7 pg/mL, respectively. The data indicate that the rate of mineralization of phenol at any one concentration in the range of 2.0 p g / d to 200 n g / d tested increased as the concentration of organic carbon in these waters increased (Figure 1). However, differences in the carbon concentrations in these waters also reflect differences in trophic levels and in the composition of the indigenous microbial communities. It is also evident that mineralization rates in these waters were directly proportional to phenol concentration over a range of 5 or more orders of magnitude. As an aid in establishing why the rates of phenol mineralization were different in the several waters, Beebe Lake water was diluted with distilled water, and the dilutions were then amended with a suspension of the normal lake microflora. The data show that the rate of mineralization decreased as the extent of dilution increased (Table I). Such findings indicate that a dilutable factor is present that affects the rate of mineralization. Because species diversity and the size of the microbial community were the same at each dilution, it is likely that some other factor such as nutrients was influencing the mineralization. To determine if organic nutrients affected phenol mineralization, we added arginine, adenine, glucose, propionate, or yeast extract (at concentrations of 38.5 pg of carbon/mL) to distilled water containing 2.0 ng of phenol/ mL. The liquid was inoculated with a portion of the microbial community from Beebe Lake. Under these conditions, phenol mineralization was enhanced by arginine (Figure 2). On the other hand, adenine and glucose did not shorten the apparent lag phase and reduced the subsequent rate of phenol mineralization. Propionate almost totally suppressed mineralization during the test period. To determine the effect of various carbon sources at 38.5 pg of carbon/mL on the rates of mineralization of various
t -
-1 0
0.B
z n W
i -I a K W
z
5 0.6 P W
E 0.3
I 4
8
12
HOURS
Figure 2. Effect of organic compounds on phenol mineralization in distilled water inoculated with cells from Beebe Lake.
concentrations of phenol, we added the microbial community from Beebe Lake to distilled water amended with the various organic materials. The rate of phenol mineralization was often enhanced in the presence of yeast extract or arginine as compared to the water without a second carbon source (Table 11). In contrast, glucose was inhibitory, and phenol at a concentration of 2 pg/mL was not mineralized in the presence of glucose. The effect of arginine on the rate of phenol mineralization was then assessed by using a wider range of phenol concentrations. In this test, distilled water was supplemented with the normal flora from Beebe Lake (Figure 3). Arginine stimulated the rate of mineralization at very low as well as high concentrations of phenol. At concentrations of phenol from 0.39 to 1.5 pg/mL, the rate of mineralization was less than predicted from a linear extrapolation of the plot of rate vs. substrate concentration at higher phenol levels. A line for the linear regression was calculated from the data for phenol concentrations from Environ. Sci. Technol., Vol. 17, No. 2, 1983
105
Table 11. Rates of Phenol Mineralization in Distilled Water Amended with Organic Compounds and Cells from Beebe Lake initial phenol concn, ng/mL
mineralization rate, pg/(mL h ) distilled water
yeast extract
arginine
adenine
glucose
0.0196 1.4 1.8 2.6 0.20 0.19 1.8 185 195 118 76.4 95.1 19.8 74 7 1 250Aa 1530 1230A 1030 200 21 700B 19 200 2 1 300B 1 9 800 12 300 2000 1 7 1 000 233 000 196 000 220 000 Ob a Values with a letter in common are not significantly different. All other rates are significantly different at the 95% level. b N o mineralization. 0
Table 111. Mineralization of Phenol in Stream Water or in Distilled Water Amended with Buffer, Inorganic Salts, or Arginine
water
distilled
stream
/
0
1 PO
0.33Ba 0.31B 0.47 0.36C 0.36C
46.7B 45.8B 74.5 57.5C 53.5C
5640B 5080B 7750 6810C 6320C
0.49
90.8
8830
0.041A 0.042A
3.49A 3.68A
286A 307A
a Values in a single column with a letter in common are not significantly different. All other rates are significantly different at the 95% confidence level.
PHENOL(*)
10 PO
100 PO
1
no
PHENOL CONCENTRATION (PER ML)
Figure 3. Effect of arginine on rates of phenol mineralization in distilled water inoculated with cells from Beebe Lake.
1.5 pg/mL to 27 ng/mL, and it was then determined that the values for the three lowest phenol levels in the figure differed from this line by 3.8-4.9 standard deviations of y. In the presence of arginine, on the other hand, the rates of mineralization at the lowest two phenol concentrations were similar to those predicted from the linear regression. The influence of inorganic salts, Tris buffer (pH 7 . 2 ) , or arginine at a concentration of 38.5 pg C/mL on phenol mineralization in distilled water was determined. The solution was inoculated with cells from Beebe Lake water. The addition of the buffer did not increase the rate in water alone (Table 111). Addition of the inorganic salts or arginine increased the rate at which phenol was mineralized. The highest rate was obtained in solutions amended with both arginine and inorganic salts. In stream water, in contrast, arginine did not enhance phenol mineralization, and the rates with and without arginine at any one concentration were not statistically different. To determine if nutrients might affect the mineralization of a more slowly mineralized compound, we added p nitrophenol at various concentrations to distilled water containing cells derived from Beebe Lake and to stream water. Arginine (38.5 pg of carbon/mL) was added to half the samples. The mineralization rate was slower in distilled water than in stream water with no supplemental carbon (Table IV). Arginine stimulated the rate of p nitrophenol mineralization in distilled water, but only at one p-nitrophenol level did it have a stimulatory effect in samples of stream water. 108
amendment none Tris salts arginine arginine t Tris arginine + salts none arginine
mineralization rate, pg/(mL h ) 20 pg 2.0ng 200ng of of of phenol/ phenol/ phenol/ mL mL mL
Environ. Sci. Technol., Vol. 17, No. 2, 1983
Table IV. Mineralization of p-Nitrophenol in Distilled Water Inoculated with Cells from Beebe Lake and in Stream Water initial p-nitroconcn, phenol
mineralization rate, pg/(mL h ) stream water
distilled water
unamended + arginine unamended + arginine 0.18 0.58 0.20A 0.25Aa 0.019 2.42B 2.97A 2.49B 3.04A 0.199 37.8 48.9A 51.8B 48.3AB 1.95 391 495 577A 438A 19.8 1260 6850 5810A 5580A 197 Values in each row with a letter in common are not significantly different, All other rates are significantly different at the 95% confidence level. ng/mL
Discussion Nutrient content of natural waters has been shown to increase the rate of decomposition of glycine, glucose, malate, phenylacetate (Z),and 2,4-D (3,5). However, the effect of naturally occurring nutrients on the rates of mineralization of trace concentrations of compounds has not previously been investigated. The present data suggest that nutrient levels stimulate the mineralization rate of two phenols independent of their concentrations. The greater rates of mineralization in waters of increasing trophic levels could be a result of either the greater nutrient concentrations or the larger and possibly more diverse microbial community with increasing amounts of nutrients in the waters. Nevertheless, the finding that the mineralization rates by an introduced microflora declined as the lake water was diluted indicates a role for natural
Environ. Sci. Technol, 1983, 17, 107-112
nutrients, either inorganic or organic. Mineralization of low concentrations of organic chemicals may be a result of activities of oligotrophic populations. The finding that propionate or glucose at 38.5 pg of carbon/mL markedly inhibited the mineralization of phenol is consistent with the view that these organisms are sensitive to organic chemicals at concentrations often used to test for biodegradation; that is, at levels that would sustain eutrophic microorganisms. Alternatively, the cells may have selectively utilized glucose and propionate rather than the small amount of'phenol. It has been shown previously that mineralization of 2,4-D in fresh waters sometimes ceased at concentrations as low as 200 ng of 2,4-D/mL, presumably because of the sensitivity of the active organisms (8). The significance of the oligotrophs, which have been reported in natural waters (9-11), thus requires further inquiry. Concentrations of substrates below which no mineralization occurs have been demonstrated in some natural waters (6)and in pure culture (12). The failure to observe such thresholds in other waters (8) may result from the presence in these waters of nutrients that influence the indigenous community. In support of this view is the finding of Law and Button (13) that the threshold concentration for glucose in culture was lowered in the presence of arginine and was lowered beyond the limits of detection in the presence of a mixture of amino acids. The especially marked enhancement by arginine of the mineralization of less than 2 pg of phenol/mL by the community from lake water may reflect a similar type of microbial response. A threshold may be expected in environments in which the indigenous populations are obligate eutrophs that are not able to metabolize the test chemicals at low levels regardless of the presence of other nutrients.
Acknowledgments We thank Deidre M. Brophy for technical assistance. Registry No. p-Nitrophenol, 100-02-7;glucose, 50-99-7; adenine, 73-24-5; arginine, 74-79-3; propanoic acid, 79-09-4; Mg, 7439-95-4; NH4, 14798-03-9; Ca, 7440-70-2; Fe, 7439-89-6; Mo, 7439-98-7; Mn, 7439-96-5; phenol, 108-95-2.
Literature Cited (1) Tanaka, N.; Ueda, Y.; Onizawa, M.; Kadota, H. Jpn. J. Limnol. 1977,38, 41. (2) Vaccaro, R. F. Limnol. Oceanogr. 1969, 14, 726. (3) Nesbitt, H. J.; Watson, J. R. Water Res. 1980, 14, 1689. (4) Alexander, M. In "Microbiology 1980"; Schlesinger, D., Ed.; American Society for Microbiology: Washington, DC, 1980; p 328. (5) Nesbitt, H. J.; Watson, J. R. Water Res. 1980, 14, 1683. (6) Boethling, R. S.; Alexander, M. Appl. Environ. Microbiol. 1979,37, 1211. (7) Subba-Rao, R. V.; Rubin, H. E.; Alexander, M. Appl. Environ. Microbiol. 1982, 43, 1139. (8) Rubin, H. E.; Subba-Rao, R. V.; Alexander, M. Appl. Environ. Microbiol. 1982, 43, 1133. (9) Akagi, Y.; Taga, N.; Simidu, U. Can. J . Microbiol. 1977, 23, 981. (10) Kuznetsov, S. I.; Dubinina, G. A.; Lapteva, N. A. Annu. Rev. Microbiol. 1979, 33, 377. (11) Moaledj, K.; Overbeck, J. Arch. Hydrobiol. 1980,89, 303. (12) Jannasch, H. W. Limnol. Oceanogr. 1967,12, 264. (13) Law, A. T.; Button, D. K. J. Bacteriol. 1977, 129, 115.
Received for review December 28, 1981. Revised manuscript received August 9, 1982. Accepted October 26, 1982. This research was supported by the Environmental Protection Agency cooperative agreement CR806887. The statements do not necessarily reflect the views and policies of the Environmental Protection Agency.
Environmental Fate and Effects of Ethylene Oxide Richard A. Conway," Gene 1.Waggy, Milton H. Splegel, and Ronald L. Bergiund Research and Development Department, Solvents and Coating Materials Division, Union Carbide Corporation, South Charleston, West Virginia 25303
rn A study has been completed to learn more about the fate and effects of ethylene oxide (EO) in the environment and especially to fill gaps revealed in a recently published in-depth survey of available literature. Key findings on this large-volume product (3 X loe kg/year) include (1) desorption coefficient of 0.36 times that of oxygen, (2) hydrolysis half-life of 14 days in natural fresh water at 25 "C, (3) hydrolysis/hydrochlorination half-life of 9 days in 3% salt water at 25 "C and a chlorohydrin/glycol product ratio one-tenth of literature estimate, (4) biooxidation of EO and derivatives at a rate indicating nonpersistence, and (5) aquatic toxicity at moderate levels (e.g., 96-h LCmwith fathead minnows of 84 mg/L, 48-h LC50 with Daphnia magna of 212 mg/L, and 48-h LC50 with brine shrimp of 745 mg/L). Also, literature cited indicates that in air ethylene oxide is nonpersistent due to washout by rain and degradation by free-radical processes.
Introduction The production of ethylene oxide (EO) in the United States is about 3 X lo9 kg/year, ranking it among the top 15 chemicals in volume. After various governmental committees identified a need to know more about the environmental fate and effects of this material (1-3), a com0013-936X/83/0917-0107$01.50/0
prehensive paper study toward this end was conducted by Syracuse Research Corp. (SRC) ( 4 ) . Union Carbide Corp. now has completed environmental chemistry and ecotoxicity studies to supplement and extend the SRC findings. Studies were conducted in the areas of volatilization, hydrolysis, hydrochlorination, biodegradation, and aquatic toxicity, As a result, risk analyses can be made for various ecological situations.
Methods EO has a molecular weight of 44.053, freezing point of -112.44 "C, boiling point of 10.5 "C at 760 torr, vapor pressure of 1305 torr at 25 "C, and complete solubility in water (4). The high solubility indicates a low potential to accumulate in fatty tissue or to partition onto clays. The relative desorption coefficient for use in estimating volatilization rate was determined experimentally for EO by using an unbaffled 2-L vessel, 10 cm deep. EO was added to water (22 "C) and rapidly mixed for a 4-h period by using a 3-cm, 45" propellor at 450 rpm. Prior to introducing EO, the oxygen-transferrate was determined for this system. Tests were conducted both with a 5 m/s wind flow over the liquid surface and with no induced wind flow. After addition of EO, the dissolved EO concentration was
0 1983 Amerlcan Chemical Society
Environ. Sci. Technol., Vol. 17,No. 2, 1983 107
