Phorochemrstrj and Photobiology. 1976. Val. 24. pp. 49-57.
Pergamon Press. Printed in Great Britain
THE ROLE OF SOLAR ULTRAVIOLET RADIATION IN ‘NATURAL’ WATER PURIFICATION JOHNCALKINS,JAMESD. BUCKLES and JOHNR. MOELLER*~ Departments of Radiation Medicine and tBiologica1 Sciences, University of Kentucky, Lexington, KY 40506, U.S.A. (Received 15 September 1975; accepted 3 February 1976)
Abstract-The conceqtration of Escherichiu coli in the ‘input and output of a tertiary wastewater system (4 lagoons) has been monitored over an 11 month period. The integrated flux of biologically active solar ultraviolet (UV) radiation was measured during this period. By also determining (1) the effective temperature in the system, (2) the growth rate of E . coli at the effective temperature, (3) the penetration of the solar U V into the lagoons, (4) the dose-response relation for killing of E. coli by U V and (5) the retention time of water in the system, it is possible to compare the ‘die off expected from
solar UV exposure to the actual ‘die OR observed for different batches of water. The observed killing of E . coli was quite close to the values calculated, considering the numerous factors involved. Solar UV light would thus seem to be a very important factor in the natural purification of water. Because each successful species must possess characteristics (physiological or behavioral) which provide adequate resistance to solar UV. the ecological role of solar UV radiation has not been widely appreciated.
INTRODUCTION
(Gloyna, 1971). Waste stabilization ponds are designed for water purification. Since in the pond the wastewater is not treated in any way, the purification obviously depends on the same processes which lead to ‘natural’ purification of water. A wide variety of organisms exist in the ponds in complex ecological relationships. Wastewater purification processes have a number of objectives; removal of toxic and undesirable organic materials, removal of nutrients which would support subsequent algal growth, and especially critical, the removal of pathogenic organisms. The processes which eliminate pathogenic organisms will be considered here. It will be considered that the observations made on a series of waste stabilization ponds (lagoons) typify the ‘natural’ purification process. As is common practice, it is assumed that the mildly pathogenic bacterium, E. coli, is a suitable ‘indicator organism’ and with appropriate extrapolations the behavior of the other pathogens can be deduced from observations of the responses of E. coli. Numerous factors have been suggested as contributing to the decline of pathogenic organism during the natural purification of water. We have been unable to locate any analysis of the role of a proposed factor which quantitatively determines the relative importance of the various purification agents in actual field situations. A number of studies have employed combinations of field and laboratory observations and qualitative reasoning to reach conclusions which seem convincing. Almost any agent or combination of agents observed to be bactericidal in the laboratory will from time to time occur in nature and will contribute to the natural purification of water. But for maximum
Human population growth and urbanization place increasingly heavy demands on the limited water resources of the earth. These demands make the rapid and efficient conversion of wastewater back into water suitable for human consumption and recreation a matter of great concern. Sufficient chlorination can make poor quality water pathogen free; however, there is recent evidence suggesting chlorination itself may generate carcinogenic substances (Bellar et al., 1974). If increasing fractions of the available water resources are polluted the quality of human life will of necessity be reduced. When the world population density was low, adequate water supplies were available through the simple ‘natural’ purification of water. It has been widely observed that the transmission of polluted waters down a few miles of stream or river or even holding the water in ponds o r lakes would often regenerate water free of pathogenic organisms and of high quality. The ‘natural’ purification of waters has been widely utilized as an inexpensive way of treating wastewaters. Wastewaters held in ‘waste stabilization ponds or ‘lagoons’ for an adequate time can then be released into a receiving stream without hazard. Waste stabilization ponds have particular economic advantages; they tend to be relatively inexpensive and do not increase greatly in cost per unit volume as the volume to be treated decreases, a problem encountered when using other forms of treatment in small communities
* Present address: Idaho State University, Department of Biology, Pocatello, ID 83209, U.S.A. PAP
24 I
49 D
50
J. CALKINS. J. D. BUCKLESand J. R. MOELLER
utilization of the natural purification processes the relative importance of the various purification factors should be known. The relation of the various factors might be expressed by stating the probabilities that a pathogenic organism entering a purification system would be killed by each of the various agents; the various mortality probabilities plus the probability of no mortality (surviving fraction) would naturally equal one. Clearly, data of this type are not presently available. The factors commonly cited as contributing to water purification include (1) biologically derived lethality, i.e. predation, parasites, toxins, nutrient depletion. pH. dissolved 0, and CO, levels, (2) chemical factors such as organic or heavy metal pollution, and (3) physical agents such as temperature and solar radiation. While complex multifactor interactions producing lethality are possible, it is only reasonable to expect that the major factors killing pathogenic organisms in nature would also be lethal in laboratory tests. Thus, in our particular situation, significant lethalities cannot be attributed to temperature, pH or 0, levels since measurement of these variables in the field showed they did not reach the levels required for lethality for E. coli in laboratory situations. Starvation alone seems unlikely to kill E. coli at a significant rate. E. coli shows a remarkably long life even under conditions where all possible nutrients were removed from the suspending medium (Gray, 1975). We have shown that there were, in fact, sufficient nutrients for growth of E. coli in both the input and output waters of the lagoon system. The system was not, to our knowledge, receiving toxic pollutants during this study. Phage may be responsible for reduction of bacteria in certain cases but are not regarded as a factor of established general significance (Chambers and Clark, 1966). The bacterial parasite Bdellouibro bacteriouorus has been proposed as a potential bactericidal agent, but the studies of Fry and Staples (1974) suggest that the actual conditions in a polluted river were unfavorable to the growth of this organism and tend to minimize its role in natural water purification. Parker (1962) considers microbial factors in an experimental lagoon system from a rather different viewpoint. He considers the growth of bacteria as a beneficial factor in the purification process, especially regarding BOD reduction. He observes that neither dissolved oxygen nor algal cell density appear to have an effect on the changes in bacteria count. He finds no indication that toxic substances released by the algae are significant in bacterial decline. Chambers and Clark (1966) likewise cite little support for antibiotic effects. Predation upon bacteria by the ciliates Paramecium or Tetrahymea can proceed to the point of rapid sterilization of dense bacterial cultures (unpublished observation). From time to time, numerous bacteriafeeding organisms such as Tetrahymea were observed in natural waters similar to the lagoon. Predation is well known to be a factor in the operation of the
activated sludge process. However, the role of predation in the situation such as a wastewater lagoon where the bacterial count is much reduced from the raw sewage situation is still equivocal. It is evident that there must be a relation between bacteria-feeding organisms and their food supply. However. Gray (1952) after a prolonged and detailed study of the microbial ecology of a chalk stream was unable to find a statistical correlation of numbers or fluctuations of numbers of ciliates with total bacteria counts. It is a common observation that bacterial predators will grow in stored natural water at the expense of the bacteria. Prior to the decline of bacterial numbers in stored waters, there is usually an increase in bacterial count (Camp, 1963). If the indigenous predators were constantly over-cropping the bacterial population in nature then the process should continue during storage rather than showing the transient bacterial increase upon storage. We are unable to estimate the relative importance of predation in water purification either from our own data or from the published materials available to us. T o test the hypothesis that solar UV radiation kills the pathoegens in the natural purification of water, we have assembled the data necessary to compute the extent of killing which would be expected during transit through a series of lagoons. The ‘die off calculated from these data has been compared with the actual ‘die off observed as batches of water traversed the lagoons system. It was found that the computed effects of solar UV radiation are in excellent agreement with the field observations. METHODS
For comparison of the observed killing of pathogenic bacteria (E. coli being the model organism) with effects expected from solar UV light, a number of data are required. These include: (1) the number of E. coli present at the beginning and at the end of the natural purification process, (2) the amount of growth which would occur during transit, (3) the dose of ultraviolet radiation an E . coli would receive during its transit through the system and (4) the dose-response relation for killing of E . coli by solar radiation. The methods of determining these factors are described in detail below. The West Hickman sewage lagoon system, a system located just outside Lexington, KY, is a series of four rectangle ponds in sequence, each 5 acres in surface area, 5 feet (1.52 m) deep and containing 8.25 million gallons for a total capacity of 33 million gallons. The design of the system was such that ‘channeling’ was considered to be of little significance. Thus, the average transit time of a batch of water through the system will be the time for 33 million gallons of wastewater to enter the system after the batch in question. The system is analagous to a long pipe. Water entering the system displaces water at the output. There is vertical mixing in the ponds but the configuration virtually eliminates input-output mixing. Flow of wastewater is constantly recorded and transit times have been computed from these records. Samples were taken at the input to the system (lagoon 1) and at the output (lagoon 4) and at intermediate points as well. These measurements extended from September, 1973, to June, 1974; sixty-four sets of data were collected during this time. By culturing these samples for both fecal
51
Solar UV in water purification and total coliforms it was possible to determine the net killing of E. coli as various batches traversed the system. The equipment. culture media, laboratory technique and analysis of the plated samples were as recommended by the Millipore Corporation (1974). Media were prepared from M-FC broth dehydrate (fecal coliforms) and M-Endo broth dehydrate (total coliforms). All sampling was done according to procedures recommended in American Public Health Association ‘Standard Methods’ (1971). This included the addition of sodium thiosulfate to each sample as a means of neutralizing the sanitizing effect of any residual chlorine. Although sample dates were randomly selected, all collections were taken at approximately 1O:OO a.m. A composite sample, consisting of equal volumes of effluent from the seven overflow pipes interconnecting each lagoon, was taken from each lagoon. Coliform tests were run on each of the seven overflows of lagoon one in order to validate statistically the use of a composite sample. The composite sample did not significantly alter the laboratory results. The growth rate of E. coli in the nutrients available in lagoon water was estimated by purifying samples of lagoon water (input and output) by filtration and innoculating with a clone of E . coli previously isolated from lagoon samples. Samples were grown at different temperatures and growth monitored by optical density at 420 nm. The temperature of lagoon three was measured from 2 to 5 times during the transit of each batch and the average of these measurements is considered to be the effective growth temperature. The dose of biologically active solar UV (UV-B) an organism would receive during transit depends on two factors, the amount of UV-B incident on the lagoon system and the absorption of the water above the E. coli. A major complication in evaluating the biological action of UV-B is the great variation in biological efficiency within a short wavelength range. Luckiesh (1946) has measured the relative efficiency of different wavelengths of UV radiation. He finds a photon of 280 nm to be about 150 times as efficient at killing of E . coli as a 320 nm photon. The action spectrum for human erythema is quite similar to the E. coli killing action spectrum (Luckiesh, 1946). Dr. D. F. Robertson (1969, 1975) has developed a sensor system using the flourescence of MgW04 and optical filters which closely simulate the action spectrum for production of human erythema. This device termed the Robertson sensor has been widely used to measure the amount of biologically effective UV-B incident on various parts (Berger et al., 1975). A Robertson sensor was used in conjunction with an integrating recorder to record the amount of biologically active UV-B incident in Lexington, KY. The integrated output of Robertson sensors is expressed in an arbitrary unit termed the Sunburn Unit (SU). One SU is approximately one minimum erythema dose of UV-B. The solar UV-B dose rate at Lexington, KY at summer noon is typically 2.5 Su/h. (we have once recorded 1.5 Su/half h); the maximum daily dose we have measured was 18.84 SU. Since the Robertson sensor was occasionally used for other purposes, the UV-B dose for some days was estimated from data on cloud cover and UV-B incident on clear days near the same time of year. The attenuation of UV-B in the lagoons has been measured by lowering the Robertson sensor into the lagoons and reading the level of UV-B as a function of depth of the sensor. In all cases it was observed that attenuation of UV-B was exponential i.e. I , ~= , IOe-KZ (1) where Iz is the intensity at depth Z, lois the intensity measured immediately below the surface, and K is the broad beam attenuation coefficient.
It is assumed that there was in effect complete vertical mixing in the system, i.e. elements of water would spend equal time at all depths from top to bottom, a reasonable assumption since transit normally takes about 6 to 12 days; there is little or no channeling and in passing through each of the 4 lagoons, water enters at the bottom and leaves at the top of each lagoon. The average dose (D,) per unit area A will be the dose to each element of volume divided by the volume or z=B
AJZ=, l o e - K Z d Z
D,
(2)
=
AJdZ which integrating and inserting limits simplifies to the Morowitz formula (1950)
(3) where the symbols are defined as noted above and ZR is the depth to the bottom (B). The ability of UV-B to kill E. coli has been determined using simulated solar UV-B. The simulator and its characteristics are described in detail in Calkins (1975). In brief, it is a bank of 10 closely spaced Westinghouse FS-20 lamps filtered by a Pyrex plate of nominal thickness of 1/4 inch. The spectral output of this system closely resembles the solar spectrum in the UV-B region with a slightly reduced ozone thickness; the dose rate was adjusted to equal summer noontime rate, i.e. 2.5 SU/h, and maintained at this level throughout irradiations by controlling the voltage applied to the FS-20 lamps. ‘ Bacteria were irradiated either on an agar surface or in thin layers of non-absorbent balanced salt solution and subsequently plated on agar. These results and direct irradiation of agar plates before plating indicated that there were no toxic substances formed from the medium in the agar plates when exposed to simulated solar UV-B. All E . coli results were obtained by irradiating E . coli directly on the agar plates and subsequently incubating at 25°C for 24-48 h. The dose-response relations for UV-B letality were determined using a locally isolated strain of E. coli which has been used in this laboratory for some time.
RESULTS AND DISCUSSION
Observations Figure 1 shows the growth rate (two experiments) of a strain of E. coli recently isolated from the lagoon system; growth was at 2 different temperatures. It is evident that in the absence of injurious agents there could be net growth of E. coli as it passes through the lagoon system in summer. The E. coli were grown in filtered samples of the input and output water of the lagoon system. The determination of the low growth rates of E. coli in media which are relatively nutrient poor such as the secondary effluent of an efficient treatment plant is not simple. We have obtained a rough estimate of growth rate by determination of optical density over a short period of time. We encountered the same problems noted by Hendricks (1972) in determining E. coli growth in river water. Part of the growth is in the form of a slime layer on the glass, the slime layer is unstable
J. CALKINS, J. D. BUCKLES and J. R. MOELLER
52
I
.o I
I
26.
L c,
2 8
E
,005
:
I .
.
.02
8 .o I ,005 7
v 0
,001
Input Wotor Output Water
J
0
10 20 TIME IN HOURS
30
Figure I . The growth of E. coli (two experiments) in filtered lagoon water at two temperatures.
DOSE I N SU
Figure 2. The dose-response relation for killing of pathogenic bacteria by simulated solar UV-B.
Table 1 No. of Days UV-B est.
Temp. of No. 3 Lagoon "C
Sample date
Retention dates
Survival FC TC
Incident UV-B dose in SU
1
2
3
4
5
77.4
2
23,23,22
75.4 97.2 91.7 77.2 40 17.1 18.2 6.7 5.9 6.01 7.1 4.3 27.5
4 4 3 2 0 1
23,23,22,21 23,22,21,20 21,20.18.18 18,18,17,14 13.11,12,9 18.12.12 12,12,10 10,11,4,5
44.8 29.5 63.1 73.1 76.5 89.7 101.5 87.2 95.0 108.5
4 3 1 0 2
10/9/73 10/11 10/16 10/22 10/30 11/12 12/4 12/6 1/3 1/9 1/15 1/30 2/5 2/27 3/11 4/4 4/9 4/11 4/17 4/23 5/20 5/28 6/13 7/17
9/28-10/8 10/1 -10/10 10/4 -10/15 10/11-10/21 10/18-10/29 1111 -11/11 11/27-12/3 11/28-12/5 12/27-1/2 1/2 - 1/8 1/9 -1/14 1/24-1/29 1/30-2/4 2/19-2/26 314 -3/10 3/28-4/3 4/2 -4/8 4/4 -4/10 4/9 -4/16 4/11 -4/22 5/9 -5119 5/20-5/27 6/5 -6/12 7/9 -7116
.032 .0045 .0028 .021 .01 .21 .121 .00445 .0108 .031 .0024 .00325 .00637 .0049 .07 .0035 .0037
.0013 .00052
.014 .014 .0138 .0053 .015 .00445 * 021 ,275 .00645 .125 .114 .062 .0067 .0085 .02 95 .056 .046
.0042 .010 .016 0017 .0014 .000244
.
0
0 0 0 0 0 6
2
3 0 5 1
6
495,797 7.7 11.12.11 11,6 10,6,6 14,13 13,16,18 16,18,11 18,11.15 11.15.16 15,16,17 17,22,22.22 22,22.22 24.24 29,28
Solar U V in water purification
53
relation of lethality and solar UV, we feel some important corrections must be applied to reveal the actual relation of solar UV to natural water purification. Analysis
o o
v A
Lapoon I
Lagoon 2
Logoon 3 Logoon 4
d
'PO
'40 DEPTH (CM)
Figure 3. Two measurements of the penetration of solar UV-B into each of the lagoons using the Robertson sensor as a UV-B detector. and abrupt drops in optical density can occur when the layer peels off. The survival of four strains of pathogenic bacteria (or closely related strains) irradiated with UV-B is shown in Fig. 2. The killing of E. coli by sunlight observed by Luckiesh (1946) is indicated assuming Cleveland, OH and Lexington, K Y have essentially the same UV-B intensity in midsummer. The attenuation of the UV-B component of natural sunlight as a function of depth for different lagoons and time is shown in Fig. 3. From these measurements the value of K , the broad beam attenuation coefficient was computed as 1/0.0625 m = 16/m. Figure 4 shows the observed die-off of E. coli during transit through the system plotted as a function of the amount of solar UV-B incident at the surface of the lagoon system. Observations on 23 separate batches of water covering a 9-month span are plotted. Table 1 lists the data plotted in Fig. 4 and additional information which will be used for further interpretation of the primary data. It should be noted that the UV dosage ranged from less than 5 SU to over 100 SU. Temporarily disregarding those dosages below 50 SU, it can be seen that larger doses of UV seemingly correspond to lower fractions of surviving coliforms. Regression analyses were run on samples taken between midApril and early November, all samples of which exceeded 50 SU. Analysis of the regression statistics indicates that a significant portion ( p < 0.02) of the variation in both fecal and total coliform survival (above a dosage of 50 SU) may be explained by linear regression. While Fig. 4 shows there is a general cor-
Phelps (1944) notes that E. coli appear to multiply immediately after discharge of sewage into a receiving stream and that seasonal variations in coliforms suggest growth of E. coli in either the receiving streams or the sewage system. The observations of Hendricks (1972) directly demostrate growth of a number of strains of enteric bacteria in samples of river water taken below a sewage outfall. Both Phelps and Hendricks note that growth rate will depend on temperature and nutrients. The rate of growth of E. coli in complete medium as a function of temperature as determined by Barber (1908) and by Ng et al. (1962) is plotted in Fig. 5 along with the much lower growth rate in nutrient-poor lagoon water (our observations) and river water (Hendricks 1972). The general trend of these observations is indicated by the solid line. The retention time, lagoon 3 temperature, and the corresponding growth rate (solid line in Fig. 5 ) have been used to compute the total growth of E. coli which would have occurred during transit in the absence of any lethal agent. These factors are shown in Table 2. The growth-corrected survival curve is plotted in Fig. 6. Dose is expressed as average dose and the E. coli dose-response relation is indicated by the solid line. The 8 data points where the average temperature in lagoon 3 was below 10" have been omitted from Fig. 6 and will be considered separately. Although there is a considerable scatter of points, the lethality during transit through the lagoon system is quite close to that expected from UV-B exposure. There are doubtless random errors which contribute to the large scatter of points including the necessity of estimating part of the UV-B dose for some sample dates and estimating the effective growth temperature from a limited number of measurements of lagoon 3 temperature. There are, of course, errors of sampling and counting and in determination of effective transit time. Two factors may also introduce systematic errors into the results. Only a limited number of determinations of penetration of UV-B into the lagoons have been made; penetration was quite similar on two different occasions. However, in other natural waters, the transparency to UV-B relative to transparency to visible light increases considerably in summer and decreases in the winter (Calkins, 1975). Also, the computed growth factors (55-2700) for summer (warm weather) might not be attained due to depletion of nutrients before the organisms traverse the system, a complication which would not occur a t lower temperatures. These two possible systematic errors are difficult to evaluate quantitatively but both would tend to bring the high dose data points into better
J. CALKINS, J. D. BUCKLESand J. R. MOELLER
54
Table 2.
Days
Growth corrected survival Fecal Total
Average UV dose
Effective temperature "C
Growth Factor
1
2
3
4
5
6
10/09/73 10111 10/16 10122 10130 11/12 12/4 12/6 113 1 19 1/15 1/30 21 5 2/27 3/11 414 4/ 9 4/11 4/17 4/23 5/20 5/28 6/13 7/17
11 11
3.1 3.0 3.9 3.7 3.1 1.6 .69 .73 .27 .24 .24 .2a .172 1.1 1.8 1 .2 2.4 3.9 3.1 3.6 4.0 3.5 3.8 4.3
23 22 22 19 17 11 14 11 7 6 7 11 9 7 13.5 16 15 15 14 16 21 22 24 28.5
Sample date
.000071 .00000564 .000035 .000263 .0055
12
11 12
11 6 7 8 7 6 4 6 8 6 6 6 6 8 11 11 9 8 8
1
.15 .097 .0041 .009 .024 .00185 .00254 .0035 ,00111 .021 .00185 .0000169 .0000087 .00000019
.0000212 .000031 .GO00173 .000066 .000188 .00243 01 05 .197
.
.00585 .lo4 .088
.046 .00525 .00472 .0067 .017 .014 .0022 .00066 .000073 .000014 .000056 .00000009
660 450 800 80 80 1 .8 2 1.4 1.25 1.1 1.2 1.3 1.3 1.28 1 .8 4.4 3.3 3.3 1.9 15 220 150 250 2,700
0 Foe01 Callfarms 0 Total Collfarmi
50 loo
.
5
1,000
Borbdr 1908 v Np e l a l 1962 Hendricks 1972 Input woter 0 Output water 0
so0
~
0 0
V
0
re
200 00
t
00
5
0
.
0
.os
-
.02
-
.OI
-
2
0
00
n
50
0
a .O
B0
On
V
o @
I00
0
P
20
0
0 I.o .5
.001
T 0
20
40
60
80
100
DOSE INCIDENT IN LAGOONS IN
I20
SU
.I
0
10
I5
20
TEMPERATURE
Figure 4. The 'die off (ratio of viable E. coli/m/ leaving the system to viable E. coZi/mt when the same batch of water entered the system) as a function of solar UV-B dose incident on the surface of the system during the transit time.
1
1
S
25
30
35
'C
Figure 5. The growth of E. coli under various conditions of nutritients and temperature. The solid curve is assumed to indicate growth in the lagoons as a function of temperature.
Solar UV in water purification Fecal Colilormr
0
Total Coilfarms
.OOOl .oooos ,0002
no&
I
_
I
Figure 6. The doseeresponse relation for killing E. coli by solar UV-B during transit through the lagoon system. Doses have been corrected to average dose and a growth correction has been made as indicated in the text. Survival data when the effective lagoon temperature was less than 10°C have been omitted. The solid line indicates observed dose-response relation for killing of E. coli by simulated
measurements. Hendricks (1972) was able to demonstrate a slight growth of E. coli in sewage enriched river water at 5°C (although some enteric bacteria were killed). Barber (1908) notes no or very slight growth at 6-10°C but does not indicate killing. Cultures are routinely stored in refrigerators at these temperatures and the short term viability of stored cultures can be quite good. It is possible that some unsuspected factor might explain the low-temperature results. There is, however, a possible explanation of the observed lethality without recourse to 'unknown' factors. It is well known that biological processes have very high temperature coefficients. Photoreactivation, the most completely elucidated repair process, is much more efficient in the normal temperature range than at low temperatures (Jagger, 1958). The low efficiency of photoreactivation at low temperatures is associated with a low rate of complex formation between the repair enzyme and the lesions. Similar behavior might occur with other repair enzymes leading to a very low rate of repair of DNA damage during the cold treatment. Stapleton et al. (1953) observed that holding E. coli for 24 h at different temperatures following X-irradiation modified survival substantially. The rate of recovery was measured by a variable holding period before incubation at 37°C. Optimum recovery temperature for three strains of E. coli was found to range from about 15°C to about 25°C. The rate of recovery during low temperature holding of strain B/r was found to decrease progressively from a maximum at 18" to a value about half maximum at 12" and recovery was completely eliminated at 6°C. While the mechanisms of 100
suv.
agreement with the survival curve. Increase UV-B penetration would increase the average dose in summer and nutrient depletion would, through the growth factor, lead to 'higher actual survival levels than those computed using the laboratory growth factor. In summary, Fig. 6 demonstrates that using laboratory observations of UV-B sensitivity and growth rate in lagoon water when combined with field observation of temperature, incident UV-B intensity, and transmission of UV-B into lagoons, leads to predictions of E. coli survival in good agreement with the observed survival in the lagoons. The low temperature (below 10°C) response was separated from the other data. It is evident (note Fig. 4) that the very low doses of UV-B in winter are associated with much greater lethality than might be expected from the W - B dose-response curve. The 8 points omitted in Fig, 6 are plotted in Fig. 7. There is no correlation of killing with UV-B dose. It might be suggested that low temperatures in the lagoons are themselves lethal and UV-B exposure is immaterial. This hypothesis is not borne out by laboratory
55
so 20
10
ie $ 5 cr, .t
,Response of Repolr Defective $ 2
E.
from Horm 1969
$ I
T*7
0
0
.S
eT-6
.2
T-9
T-7
.2
.4
.6
i
ISU
Figure 7. The dose-response relation for killing E. coli at temperatures below 10°C. The killing expected from simulation experiments and if response were that of the repair deficient E . coli strain (Harm, 1969).
56
J. CALKINS.J. D. BUCKLES and J. R. MOELLER
dark repair of solar UV injury are not totally understood, dark repair systems have been found to be relatively nonselective in substrate. Mutants of E. coli deficient in either excision or recombination capacity tend to be sensitive to a wide range of injurious agents such as UV, ionizing radiation and chemicals. If the rate of repair were depressed during a cold transit through the lagoon system, then obviously there would be more lesions to be repaired (in the time available for repair) when the E. coli were tested for growth capacity. If it were assumed that temperatures below 10°C eliminated repair processes in E. coli then the doseresponse relation should be more like the response Harm (1969) observed with repair-deficient E. coli (in which photoreactivation was also suppressed by low temperatures and short exposure to visible light). The UV-B killing of a totally repair-deficient E. coli deduced from Harm (1969) is plotted in Fig. 7. The lethality under ‘no repair’ conditions is clearly more than adequate to explain the high mortality at low temperature conditions. There is, however, no direct evidence that suppression of repair processes is the explanation of the low temperature observation. Implications The quantitative agreement of UV-B exposure and killing of E. coli makes it probable that solar UV-B is an important factor in the natural purification of water. Gameson and Saxon (1967) reached a similar conclusion from studies of the killing of E. coli in marine sewage disposal. They demonstrated sunlight killing of E. coli to depths of 4 m in sea water. When the significant parameters such as the solar UV effects are recognized, then it may be possible to use them more effectively. Our findings would suggest that larger surface area for a given volume should make wastewater stabilization ponds more efficient. Likewise, clarification of the secondary effluent, before release into the ponds, which would increase UV-B penetration, might improve efficiency. Our findings are also relevant to aquatic ecology in general. Because of the lethal potential of UV-B, each species of light-exposed organism must have sufficient resistance to solar exposure to maintain its particular life cycle. The normal habitat of E. coil, the digestive system of higher organisms, is well protected from UV-B exposure. Yet E. coli must from timt to time move to a new host. The overall viability of E. coli in exposed natural waters indicates that, even in an exposed situation such as the lagoon system, about 1% of the coli survive for about 1 week (Fig. 4). Such a survival level could be expected to allow sufficient time for transfer of this organism to a new host. There are numerous strains of E. coli which have lost one or more of the repair systems found in the wild type. Repair-defective E. coli are quite viable in laboratory culture where they are not exposed to solar UV-B. The lethal effect of sunlight on the repair-
defective E. coli strain AB2480 used by Harm (1969) can be calculated (Fig. 7). A 1-h exposure to solar UV-B on a sunny winter day with the average dosage in the water columns would reduce survival to 17;; similarly a clear summer day would reduce survival by a factor of lo6’! Radiation repair systems in microorganisms clearly represent a utilization of cellular resources, any expenditure of which would soon be eliminated by mutation if not needed. The life cycle of E. coli demands a certain level of resistance to solar radiation, a resistance known to arise from the action of at least three different repair processes (Harm, 1969). E. coli represents an intermediate level of resistance. It can exist in natural waters for a few days, but without continuing input natural waters become free of E. coli. The repair-deficient strains would be much less able to transfer from host to host. Microorganisms which spend their entire life cycle in exposed habitats, for instance surface-film bacteria, would have to possess more UV-B resistance than wild-type E. coli. Bacteria which are clearly long-term residents of natural waters are indeed much more resistant to UV-B than the pathogenic group reported here (Calkins, 1975). Mycobacteriurn phlei, an airborn pathogen which would not be protected by the attenuation of the water column, was observed to be more resistant than E. coli, while Neisseria species, an organism transmitted by personal contact, was found to be much more UV-B-sensitive than E. coli. Although our observations of response of pathogenic bacteria strongly suggest that importance of solar UV-B as an environmental factor, it might be argued that UV resistance is merely a ‘spin off from some other vital function. There are other examples of loss of resistance to solar UV in protected situations. Organisms isolated from caves are remarkably sensitive to the shorter wavelengths in solar radiation (T. Barr, personal communication). A strain of protozoan isolated from Mammoth Cave was found to lack a functional photoreactivation capacity (Calkins and Griggs, 1969 a, b). Considering the observations noted above from the more general viewpoint, it is clear that solar UV-B is an important environmental factor. Solar UV-B is highly lethal. Recent calculations (Calkins, manuscript in preparation) indicate that a variety of even fully repair-competent organisms must attenuate (by epidermis, skin, hair, or by position in an absorbing water column for unicellular organisms) the incident solar UV-B by a factor of about 10,OOO to reach a level of exposure which can be tolerated on a long-term basis. Because each successful species must possess adequate UV-B resistance for its particular ecological niche, the injurious aspects of solar radiation and the multiple physiological and behavioral adaptations required to live in various environments exposed to solar UV have been little appreciated. Our observations quantify the role of solar UV-B in the sterilization of domestic water supplies. Comprehension of
Solar UV in water purification the effects of sunlight will permit a more effective use of this natural resource. Acknowledgements-This work has been supported in part by the Climatic Impact Assessment Program, Office of the Secretary, U.S. Department of Transportation.
57
The work on which this report was based was supported in part by the Office of Water Research and Technology, U.S. Department of Interior under the provisions of Public L~~ 88-379,
REFERENCES
American Public Health Association (1971) Standard Methods for the Examination of Water and Wastewater, 13th Edition, New York. Barber, M. A. (1908) J . Infect. Disease, 379-396. Bellar, T. A., J. J. Lichtenberg and R. C. Kroner (1974) J. Am. Water Works Assoc. 66. 703-706. Berger, D., D. F. Robertson, R. E. Davies and F. Urbach (1975) CIAP Monograph V, Chapter 2, Appendix D. pp. 235-264. Camp, T. R. (1971) Water and its Impurities, p. 231. Reinhold Publishing Co., New York. Calkins, J. (1975) CIAP Monograph V., p. 231. Calkins, J., and G. Griggs (1969 a) Photochem. Photobiol. 10. 61-66. Calkins, J., and G. Griggs (1969 b) Photochem. Photobiol. 10. 445-449. Chambers, C. W., and N. A. Clarke (1966) Ado. Appl. Microbiol. 8. 105-143. Fry, J. C., and D. G. Staples (1974) Water Res. 8. 1029-1035. Gameson, A. L. H., and J. R. Saxon (1967) Water Res. 1. 279-295. Gloyna, E. F. (1971) Waste Stabilization Ponds; World Health Organization, Geneva. Harm, W. (1969) Radiation Res. 40, 63-69. Hendricks, C. W. (1972) Appl. Microbiol. 24, 168-174. Jagger, J. (1958) Bacteriol. Rev. 22. 99-142. Luckiesh, M. (1946) Applications of Gernicidal, Erythemal and Infrared Energy, D. Van Nostrand, New York. Millipore Corporation (1973) Application Manual AM 302 Biological Analysis of Water and Wustewater, Bedford, Massachusetts. Morowitz, H. J. (1950) Science 111. 229-230. Ng, H., J. L. Ingraham and A. G. Marr (1962) J. Bacteriol. 84. 331-339. Parker, C. D. (1962) J . Water Pollut. Control Fed. 34, 149-161. Phelps. E. B. (1944) Stream Sanitation, Wiley, New York. Robertson, D. F. (1969) Biological Effrcts of Ultraviolet Radiation (Edited by F. Urbach), p. 433. Pergamon Press, Oxford. Robertson, D. F. (1975) C U P Monograph V., Chapter 2, Appendix B, pp. 203211. Stapleton. G. E., D. Billen and A. Hollaender (1953) J . Cell. Comp. Physiol. 41. 345-357.
Pergamon Press. Printed in Great Britain
THE ROLE OF SOLAR ULTRAVIOLET RADIATION IN ‘NATURAL’ WATER PURIFICATION JOHNCALKINS,JAMESD. BUCKLES and JOHNR. MOELLER*~ Departments of Radiation Medicine and tBiologica1 Sciences, University of Kentucky, Lexington, KY 40506, U.S.A. (Received 15 September 1975; accepted 3 February 1976)
Abstract-The conceqtration of Escherichiu coli in the ‘input and output of a tertiary wastewater system (4 lagoons) has been monitored over an 11 month period. The integrated flux of biologically active solar ultraviolet (UV) radiation was measured during this period. By also determining (1) the effective temperature in the system, (2) the growth rate of E . coli at the effective temperature, (3) the penetration of the solar U V into the lagoons, (4) the dose-response relation for killing of E. coli by U V and (5) the retention time of water in the system, it is possible to compare the ‘die off expected from
solar UV exposure to the actual ‘die OR observed for different batches of water. The observed killing of E . coli was quite close to the values calculated, considering the numerous factors involved. Solar UV light would thus seem to be a very important factor in the natural purification of water. Because each successful species must possess characteristics (physiological or behavioral) which provide adequate resistance to solar UV. the ecological role of solar UV radiation has not been widely appreciated.
INTRODUCTION
(Gloyna, 1971). Waste stabilization ponds are designed for water purification. Since in the pond the wastewater is not treated in any way, the purification obviously depends on the same processes which lead to ‘natural’ purification of water. A wide variety of organisms exist in the ponds in complex ecological relationships. Wastewater purification processes have a number of objectives; removal of toxic and undesirable organic materials, removal of nutrients which would support subsequent algal growth, and especially critical, the removal of pathogenic organisms. The processes which eliminate pathogenic organisms will be considered here. It will be considered that the observations made on a series of waste stabilization ponds (lagoons) typify the ‘natural’ purification process. As is common practice, it is assumed that the mildly pathogenic bacterium, E. coli, is a suitable ‘indicator organism’ and with appropriate extrapolations the behavior of the other pathogens can be deduced from observations of the responses of E. coli. Numerous factors have been suggested as contributing to the decline of pathogenic organism during the natural purification of water. We have been unable to locate any analysis of the role of a proposed factor which quantitatively determines the relative importance of the various purification agents in actual field situations. A number of studies have employed combinations of field and laboratory observations and qualitative reasoning to reach conclusions which seem convincing. Almost any agent or combination of agents observed to be bactericidal in the laboratory will from time to time occur in nature and will contribute to the natural purification of water. But for maximum
Human population growth and urbanization place increasingly heavy demands on the limited water resources of the earth. These demands make the rapid and efficient conversion of wastewater back into water suitable for human consumption and recreation a matter of great concern. Sufficient chlorination can make poor quality water pathogen free; however, there is recent evidence suggesting chlorination itself may generate carcinogenic substances (Bellar et al., 1974). If increasing fractions of the available water resources are polluted the quality of human life will of necessity be reduced. When the world population density was low, adequate water supplies were available through the simple ‘natural’ purification of water. It has been widely observed that the transmission of polluted waters down a few miles of stream or river or even holding the water in ponds o r lakes would often regenerate water free of pathogenic organisms and of high quality. The ‘natural’ purification of waters has been widely utilized as an inexpensive way of treating wastewaters. Wastewaters held in ‘waste stabilization ponds or ‘lagoons’ for an adequate time can then be released into a receiving stream without hazard. Waste stabilization ponds have particular economic advantages; they tend to be relatively inexpensive and do not increase greatly in cost per unit volume as the volume to be treated decreases, a problem encountered when using other forms of treatment in small communities
* Present address: Idaho State University, Department of Biology, Pocatello, ID 83209, U.S.A. PAP
24 I
49 D
50
J. CALKINS. J. D. BUCKLESand J. R. MOELLER
utilization of the natural purification processes the relative importance of the various purification factors should be known. The relation of the various factors might be expressed by stating the probabilities that a pathogenic organism entering a purification system would be killed by each of the various agents; the various mortality probabilities plus the probability of no mortality (surviving fraction) would naturally equal one. Clearly, data of this type are not presently available. The factors commonly cited as contributing to water purification include (1) biologically derived lethality, i.e. predation, parasites, toxins, nutrient depletion. pH. dissolved 0, and CO, levels, (2) chemical factors such as organic or heavy metal pollution, and (3) physical agents such as temperature and solar radiation. While complex multifactor interactions producing lethality are possible, it is only reasonable to expect that the major factors killing pathogenic organisms in nature would also be lethal in laboratory tests. Thus, in our particular situation, significant lethalities cannot be attributed to temperature, pH or 0, levels since measurement of these variables in the field showed they did not reach the levels required for lethality for E. coli in laboratory situations. Starvation alone seems unlikely to kill E. coli at a significant rate. E. coli shows a remarkably long life even under conditions where all possible nutrients were removed from the suspending medium (Gray, 1975). We have shown that there were, in fact, sufficient nutrients for growth of E. coli in both the input and output waters of the lagoon system. The system was not, to our knowledge, receiving toxic pollutants during this study. Phage may be responsible for reduction of bacteria in certain cases but are not regarded as a factor of established general significance (Chambers and Clark, 1966). The bacterial parasite Bdellouibro bacteriouorus has been proposed as a potential bactericidal agent, but the studies of Fry and Staples (1974) suggest that the actual conditions in a polluted river were unfavorable to the growth of this organism and tend to minimize its role in natural water purification. Parker (1962) considers microbial factors in an experimental lagoon system from a rather different viewpoint. He considers the growth of bacteria as a beneficial factor in the purification process, especially regarding BOD reduction. He observes that neither dissolved oxygen nor algal cell density appear to have an effect on the changes in bacteria count. He finds no indication that toxic substances released by the algae are significant in bacterial decline. Chambers and Clark (1966) likewise cite little support for antibiotic effects. Predation upon bacteria by the ciliates Paramecium or Tetrahymea can proceed to the point of rapid sterilization of dense bacterial cultures (unpublished observation). From time to time, numerous bacteriafeeding organisms such as Tetrahymea were observed in natural waters similar to the lagoon. Predation is well known to be a factor in the operation of the
activated sludge process. However, the role of predation in the situation such as a wastewater lagoon where the bacterial count is much reduced from the raw sewage situation is still equivocal. It is evident that there must be a relation between bacteria-feeding organisms and their food supply. However. Gray (1952) after a prolonged and detailed study of the microbial ecology of a chalk stream was unable to find a statistical correlation of numbers or fluctuations of numbers of ciliates with total bacteria counts. It is a common observation that bacterial predators will grow in stored natural water at the expense of the bacteria. Prior to the decline of bacterial numbers in stored waters, there is usually an increase in bacterial count (Camp, 1963). If the indigenous predators were constantly over-cropping the bacterial population in nature then the process should continue during storage rather than showing the transient bacterial increase upon storage. We are unable to estimate the relative importance of predation in water purification either from our own data or from the published materials available to us. T o test the hypothesis that solar UV radiation kills the pathoegens in the natural purification of water, we have assembled the data necessary to compute the extent of killing which would be expected during transit through a series of lagoons. The ‘die off calculated from these data has been compared with the actual ‘die off observed as batches of water traversed the lagoons system. It was found that the computed effects of solar UV radiation are in excellent agreement with the field observations. METHODS
For comparison of the observed killing of pathogenic bacteria (E. coli being the model organism) with effects expected from solar UV light, a number of data are required. These include: (1) the number of E. coli present at the beginning and at the end of the natural purification process, (2) the amount of growth which would occur during transit, (3) the dose of ultraviolet radiation an E . coli would receive during its transit through the system and (4) the dose-response relation for killing of E . coli by solar radiation. The methods of determining these factors are described in detail below. The West Hickman sewage lagoon system, a system located just outside Lexington, KY, is a series of four rectangle ponds in sequence, each 5 acres in surface area, 5 feet (1.52 m) deep and containing 8.25 million gallons for a total capacity of 33 million gallons. The design of the system was such that ‘channeling’ was considered to be of little significance. Thus, the average transit time of a batch of water through the system will be the time for 33 million gallons of wastewater to enter the system after the batch in question. The system is analagous to a long pipe. Water entering the system displaces water at the output. There is vertical mixing in the ponds but the configuration virtually eliminates input-output mixing. Flow of wastewater is constantly recorded and transit times have been computed from these records. Samples were taken at the input to the system (lagoon 1) and at the output (lagoon 4) and at intermediate points as well. These measurements extended from September, 1973, to June, 1974; sixty-four sets of data were collected during this time. By culturing these samples for both fecal
51
Solar UV in water purification and total coliforms it was possible to determine the net killing of E. coli as various batches traversed the system. The equipment. culture media, laboratory technique and analysis of the plated samples were as recommended by the Millipore Corporation (1974). Media were prepared from M-FC broth dehydrate (fecal coliforms) and M-Endo broth dehydrate (total coliforms). All sampling was done according to procedures recommended in American Public Health Association ‘Standard Methods’ (1971). This included the addition of sodium thiosulfate to each sample as a means of neutralizing the sanitizing effect of any residual chlorine. Although sample dates were randomly selected, all collections were taken at approximately 1O:OO a.m. A composite sample, consisting of equal volumes of effluent from the seven overflow pipes interconnecting each lagoon, was taken from each lagoon. Coliform tests were run on each of the seven overflows of lagoon one in order to validate statistically the use of a composite sample. The composite sample did not significantly alter the laboratory results. The growth rate of E. coli in the nutrients available in lagoon water was estimated by purifying samples of lagoon water (input and output) by filtration and innoculating with a clone of E . coli previously isolated from lagoon samples. Samples were grown at different temperatures and growth monitored by optical density at 420 nm. The temperature of lagoon three was measured from 2 to 5 times during the transit of each batch and the average of these measurements is considered to be the effective growth temperature. The dose of biologically active solar UV (UV-B) an organism would receive during transit depends on two factors, the amount of UV-B incident on the lagoon system and the absorption of the water above the E. coli. A major complication in evaluating the biological action of UV-B is the great variation in biological efficiency within a short wavelength range. Luckiesh (1946) has measured the relative efficiency of different wavelengths of UV radiation. He finds a photon of 280 nm to be about 150 times as efficient at killing of E . coli as a 320 nm photon. The action spectrum for human erythema is quite similar to the E. coli killing action spectrum (Luckiesh, 1946). Dr. D. F. Robertson (1969, 1975) has developed a sensor system using the flourescence of MgW04 and optical filters which closely simulate the action spectrum for production of human erythema. This device termed the Robertson sensor has been widely used to measure the amount of biologically effective UV-B incident on various parts (Berger et al., 1975). A Robertson sensor was used in conjunction with an integrating recorder to record the amount of biologically active UV-B incident in Lexington, KY. The integrated output of Robertson sensors is expressed in an arbitrary unit termed the Sunburn Unit (SU). One SU is approximately one minimum erythema dose of UV-B. The solar UV-B dose rate at Lexington, KY at summer noon is typically 2.5 Su/h. (we have once recorded 1.5 Su/half h); the maximum daily dose we have measured was 18.84 SU. Since the Robertson sensor was occasionally used for other purposes, the UV-B dose for some days was estimated from data on cloud cover and UV-B incident on clear days near the same time of year. The attenuation of UV-B in the lagoons has been measured by lowering the Robertson sensor into the lagoons and reading the level of UV-B as a function of depth of the sensor. In all cases it was observed that attenuation of UV-B was exponential i.e. I , ~= , IOe-KZ (1) where Iz is the intensity at depth Z, lois the intensity measured immediately below the surface, and K is the broad beam attenuation coefficient.
It is assumed that there was in effect complete vertical mixing in the system, i.e. elements of water would spend equal time at all depths from top to bottom, a reasonable assumption since transit normally takes about 6 to 12 days; there is little or no channeling and in passing through each of the 4 lagoons, water enters at the bottom and leaves at the top of each lagoon. The average dose (D,) per unit area A will be the dose to each element of volume divided by the volume or z=B
AJZ=, l o e - K Z d Z
D,
(2)
=
AJdZ which integrating and inserting limits simplifies to the Morowitz formula (1950)
(3) where the symbols are defined as noted above and ZR is the depth to the bottom (B). The ability of UV-B to kill E. coli has been determined using simulated solar UV-B. The simulator and its characteristics are described in detail in Calkins (1975). In brief, it is a bank of 10 closely spaced Westinghouse FS-20 lamps filtered by a Pyrex plate of nominal thickness of 1/4 inch. The spectral output of this system closely resembles the solar spectrum in the UV-B region with a slightly reduced ozone thickness; the dose rate was adjusted to equal summer noontime rate, i.e. 2.5 SU/h, and maintained at this level throughout irradiations by controlling the voltage applied to the FS-20 lamps. ‘ Bacteria were irradiated either on an agar surface or in thin layers of non-absorbent balanced salt solution and subsequently plated on agar. These results and direct irradiation of agar plates before plating indicated that there were no toxic substances formed from the medium in the agar plates when exposed to simulated solar UV-B. All E . coli results were obtained by irradiating E . coli directly on the agar plates and subsequently incubating at 25°C for 24-48 h. The dose-response relations for UV-B letality were determined using a locally isolated strain of E. coli which has been used in this laboratory for some time.
RESULTS AND DISCUSSION
Observations Figure 1 shows the growth rate (two experiments) of a strain of E. coli recently isolated from the lagoon system; growth was at 2 different temperatures. It is evident that in the absence of injurious agents there could be net growth of E. coli as it passes through the lagoon system in summer. The E. coli were grown in filtered samples of the input and output water of the lagoon system. The determination of the low growth rates of E. coli in media which are relatively nutrient poor such as the secondary effluent of an efficient treatment plant is not simple. We have obtained a rough estimate of growth rate by determination of optical density over a short period of time. We encountered the same problems noted by Hendricks (1972) in determining E. coli growth in river water. Part of the growth is in the form of a slime layer on the glass, the slime layer is unstable
J. CALKINS, J. D. BUCKLES and J. R. MOELLER
52
I
.o I
I
26.
L c,
2 8
E
,005
:
I .
.
.02
8 .o I ,005 7
v 0
,001
Input Wotor Output Water
J
0
10 20 TIME IN HOURS
30
Figure I . The growth of E. coli (two experiments) in filtered lagoon water at two temperatures.
DOSE I N SU
Figure 2. The dose-response relation for killing of pathogenic bacteria by simulated solar UV-B.
Table 1 No. of Days UV-B est.
Temp. of No. 3 Lagoon "C
Sample date
Retention dates
Survival FC TC
Incident UV-B dose in SU
1
2
3
4
5
77.4
2
23,23,22
75.4 97.2 91.7 77.2 40 17.1 18.2 6.7 5.9 6.01 7.1 4.3 27.5
4 4 3 2 0 1
23,23,22,21 23,22,21,20 21,20.18.18 18,18,17,14 13.11,12,9 18.12.12 12,12,10 10,11,4,5
44.8 29.5 63.1 73.1 76.5 89.7 101.5 87.2 95.0 108.5
4 3 1 0 2
10/9/73 10/11 10/16 10/22 10/30 11/12 12/4 12/6 1/3 1/9 1/15 1/30 2/5 2/27 3/11 4/4 4/9 4/11 4/17 4/23 5/20 5/28 6/13 7/17
9/28-10/8 10/1 -10/10 10/4 -10/15 10/11-10/21 10/18-10/29 1111 -11/11 11/27-12/3 11/28-12/5 12/27-1/2 1/2 - 1/8 1/9 -1/14 1/24-1/29 1/30-2/4 2/19-2/26 314 -3/10 3/28-4/3 4/2 -4/8 4/4 -4/10 4/9 -4/16 4/11 -4/22 5/9 -5119 5/20-5/27 6/5 -6/12 7/9 -7116
.032 .0045 .0028 .021 .01 .21 .121 .00445 .0108 .031 .0024 .00325 .00637 .0049 .07 .0035 .0037
.0013 .00052
.014 .014 .0138 .0053 .015 .00445 * 021 ,275 .00645 .125 .114 .062 .0067 .0085 .02 95 .056 .046
.0042 .010 .016 0017 .0014 .000244
.
0
0 0 0 0 0 6
2
3 0 5 1
6
495,797 7.7 11.12.11 11,6 10,6,6 14,13 13,16,18 16,18,11 18,11.15 11.15.16 15,16,17 17,22,22.22 22,22.22 24.24 29,28
Solar U V in water purification
53
relation of lethality and solar UV, we feel some important corrections must be applied to reveal the actual relation of solar UV to natural water purification. Analysis
o o
v A
Lapoon I
Lagoon 2
Logoon 3 Logoon 4
d
'PO
'40 DEPTH (CM)
Figure 3. Two measurements of the penetration of solar UV-B into each of the lagoons using the Robertson sensor as a UV-B detector. and abrupt drops in optical density can occur when the layer peels off. The survival of four strains of pathogenic bacteria (or closely related strains) irradiated with UV-B is shown in Fig. 2. The killing of E. coli by sunlight observed by Luckiesh (1946) is indicated assuming Cleveland, OH and Lexington, K Y have essentially the same UV-B intensity in midsummer. The attenuation of the UV-B component of natural sunlight as a function of depth for different lagoons and time is shown in Fig. 3. From these measurements the value of K , the broad beam attenuation coefficient was computed as 1/0.0625 m = 16/m. Figure 4 shows the observed die-off of E. coli during transit through the system plotted as a function of the amount of solar UV-B incident at the surface of the lagoon system. Observations on 23 separate batches of water covering a 9-month span are plotted. Table 1 lists the data plotted in Fig. 4 and additional information which will be used for further interpretation of the primary data. It should be noted that the UV dosage ranged from less than 5 SU to over 100 SU. Temporarily disregarding those dosages below 50 SU, it can be seen that larger doses of UV seemingly correspond to lower fractions of surviving coliforms. Regression analyses were run on samples taken between midApril and early November, all samples of which exceeded 50 SU. Analysis of the regression statistics indicates that a significant portion ( p < 0.02) of the variation in both fecal and total coliform survival (above a dosage of 50 SU) may be explained by linear regression. While Fig. 4 shows there is a general cor-
Phelps (1944) notes that E. coli appear to multiply immediately after discharge of sewage into a receiving stream and that seasonal variations in coliforms suggest growth of E. coli in either the receiving streams or the sewage system. The observations of Hendricks (1972) directly demostrate growth of a number of strains of enteric bacteria in samples of river water taken below a sewage outfall. Both Phelps and Hendricks note that growth rate will depend on temperature and nutrients. The rate of growth of E. coli in complete medium as a function of temperature as determined by Barber (1908) and by Ng et al. (1962) is plotted in Fig. 5 along with the much lower growth rate in nutrient-poor lagoon water (our observations) and river water (Hendricks 1972). The general trend of these observations is indicated by the solid line. The retention time, lagoon 3 temperature, and the corresponding growth rate (solid line in Fig. 5 ) have been used to compute the total growth of E. coli which would have occurred during transit in the absence of any lethal agent. These factors are shown in Table 2. The growth-corrected survival curve is plotted in Fig. 6. Dose is expressed as average dose and the E. coli dose-response relation is indicated by the solid line. The 8 data points where the average temperature in lagoon 3 was below 10" have been omitted from Fig. 6 and will be considered separately. Although there is a considerable scatter of points, the lethality during transit through the lagoon system is quite close to that expected from UV-B exposure. There are doubtless random errors which contribute to the large scatter of points including the necessity of estimating part of the UV-B dose for some sample dates and estimating the effective growth temperature from a limited number of measurements of lagoon 3 temperature. There are, of course, errors of sampling and counting and in determination of effective transit time. Two factors may also introduce systematic errors into the results. Only a limited number of determinations of penetration of UV-B into the lagoons have been made; penetration was quite similar on two different occasions. However, in other natural waters, the transparency to UV-B relative to transparency to visible light increases considerably in summer and decreases in the winter (Calkins, 1975). Also, the computed growth factors (55-2700) for summer (warm weather) might not be attained due to depletion of nutrients before the organisms traverse the system, a complication which would not occur a t lower temperatures. These two possible systematic errors are difficult to evaluate quantitatively but both would tend to bring the high dose data points into better
J. CALKINS, J. D. BUCKLESand J. R. MOELLER
54
Table 2.
Days
Growth corrected survival Fecal Total
Average UV dose
Effective temperature "C
Growth Factor
1
2
3
4
5
6
10/09/73 10111 10/16 10122 10130 11/12 12/4 12/6 113 1 19 1/15 1/30 21 5 2/27 3/11 414 4/ 9 4/11 4/17 4/23 5/20 5/28 6/13 7/17
11 11
3.1 3.0 3.9 3.7 3.1 1.6 .69 .73 .27 .24 .24 .2a .172 1.1 1.8 1 .2 2.4 3.9 3.1 3.6 4.0 3.5 3.8 4.3
23 22 22 19 17 11 14 11 7 6 7 11 9 7 13.5 16 15 15 14 16 21 22 24 28.5
Sample date
.000071 .00000564 .000035 .000263 .0055
12
11 12
11 6 7 8 7 6 4 6 8 6 6 6 6 8 11 11 9 8 8
1
.15 .097 .0041 .009 .024 .00185 .00254 .0035 ,00111 .021 .00185 .0000169 .0000087 .00000019
.0000212 .000031 .GO00173 .000066 .000188 .00243 01 05 .197
.
.00585 .lo4 .088
.046 .00525 .00472 .0067 .017 .014 .0022 .00066 .000073 .000014 .000056 .00000009
660 450 800 80 80 1 .8 2 1.4 1.25 1.1 1.2 1.3 1.3 1.28 1 .8 4.4 3.3 3.3 1.9 15 220 150 250 2,700
0 Foe01 Callfarms 0 Total Collfarmi
50 loo
.
5
1,000
Borbdr 1908 v Np e l a l 1962 Hendricks 1972 Input woter 0 Output water 0
so0
~
0 0
V
0
re
200 00
t
00
5
0
.
0
.os
-
.02
-
.OI
-
2
0
00
n
50
0
a .O
B0
On
V
o @
I00
0
P
20
0
0 I.o .5
.001
T 0
20
40
60
80
100
DOSE INCIDENT IN LAGOONS IN
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Figure 4. The 'die off (ratio of viable E. coli/m/ leaving the system to viable E. coZi/mt when the same batch of water entered the system) as a function of solar UV-B dose incident on the surface of the system during the transit time.
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Figure 5. The growth of E. coli under various conditions of nutritients and temperature. The solid curve is assumed to indicate growth in the lagoons as a function of temperature.
Solar UV in water purification Fecal Colilormr
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Figure 6. The doseeresponse relation for killing E. coli by solar UV-B during transit through the lagoon system. Doses have been corrected to average dose and a growth correction has been made as indicated in the text. Survival data when the effective lagoon temperature was less than 10°C have been omitted. The solid line indicates observed dose-response relation for killing of E. coli by simulated
measurements. Hendricks (1972) was able to demonstrate a slight growth of E. coli in sewage enriched river water at 5°C (although some enteric bacteria were killed). Barber (1908) notes no or very slight growth at 6-10°C but does not indicate killing. Cultures are routinely stored in refrigerators at these temperatures and the short term viability of stored cultures can be quite good. It is possible that some unsuspected factor might explain the low-temperature results. There is, however, a possible explanation of the observed lethality without recourse to 'unknown' factors. It is well known that biological processes have very high temperature coefficients. Photoreactivation, the most completely elucidated repair process, is much more efficient in the normal temperature range than at low temperatures (Jagger, 1958). The low efficiency of photoreactivation at low temperatures is associated with a low rate of complex formation between the repair enzyme and the lesions. Similar behavior might occur with other repair enzymes leading to a very low rate of repair of DNA damage during the cold treatment. Stapleton et al. (1953) observed that holding E. coli for 24 h at different temperatures following X-irradiation modified survival substantially. The rate of recovery was measured by a variable holding period before incubation at 37°C. Optimum recovery temperature for three strains of E. coli was found to range from about 15°C to about 25°C. The rate of recovery during low temperature holding of strain B/r was found to decrease progressively from a maximum at 18" to a value about half maximum at 12" and recovery was completely eliminated at 6°C. While the mechanisms of 100
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agreement with the survival curve. Increase UV-B penetration would increase the average dose in summer and nutrient depletion would, through the growth factor, lead to 'higher actual survival levels than those computed using the laboratory growth factor. In summary, Fig. 6 demonstrates that using laboratory observations of UV-B sensitivity and growth rate in lagoon water when combined with field observation of temperature, incident UV-B intensity, and transmission of UV-B into lagoons, leads to predictions of E. coli survival in good agreement with the observed survival in the lagoons. The low temperature (below 10°C) response was separated from the other data. It is evident (note Fig. 4) that the very low doses of UV-B in winter are associated with much greater lethality than might be expected from the W - B dose-response curve. The 8 points omitted in Fig, 6 are plotted in Fig. 7. There is no correlation of killing with UV-B dose. It might be suggested that low temperatures in the lagoons are themselves lethal and UV-B exposure is immaterial. This hypothesis is not borne out by laboratory
55
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Figure 7. The dose-response relation for killing E. coli at temperatures below 10°C. The killing expected from simulation experiments and if response were that of the repair deficient E . coli strain (Harm, 1969).
56
J. CALKINS.J. D. BUCKLES and J. R. MOELLER
dark repair of solar UV injury are not totally understood, dark repair systems have been found to be relatively nonselective in substrate. Mutants of E. coli deficient in either excision or recombination capacity tend to be sensitive to a wide range of injurious agents such as UV, ionizing radiation and chemicals. If the rate of repair were depressed during a cold transit through the lagoon system, then obviously there would be more lesions to be repaired (in the time available for repair) when the E. coli were tested for growth capacity. If it were assumed that temperatures below 10°C eliminated repair processes in E. coli then the doseresponse relation should be more like the response Harm (1969) observed with repair-deficient E. coli (in which photoreactivation was also suppressed by low temperatures and short exposure to visible light). The UV-B killing of a totally repair-deficient E. coli deduced from Harm (1969) is plotted in Fig. 7. The lethality under ‘no repair’ conditions is clearly more than adequate to explain the high mortality at low temperature conditions. There is, however, no direct evidence that suppression of repair processes is the explanation of the low temperature observation. Implications The quantitative agreement of UV-B exposure and killing of E. coli makes it probable that solar UV-B is an important factor in the natural purification of water. Gameson and Saxon (1967) reached a similar conclusion from studies of the killing of E. coli in marine sewage disposal. They demonstrated sunlight killing of E. coli to depths of 4 m in sea water. When the significant parameters such as the solar UV effects are recognized, then it may be possible to use them more effectively. Our findings would suggest that larger surface area for a given volume should make wastewater stabilization ponds more efficient. Likewise, clarification of the secondary effluent, before release into the ponds, which would increase UV-B penetration, might improve efficiency. Our findings are also relevant to aquatic ecology in general. Because of the lethal potential of UV-B, each species of light-exposed organism must have sufficient resistance to solar exposure to maintain its particular life cycle. The normal habitat of E. coil, the digestive system of higher organisms, is well protected from UV-B exposure. Yet E. coli must from timt to time move to a new host. The overall viability of E. coli in exposed natural waters indicates that, even in an exposed situation such as the lagoon system, about 1% of the coli survive for about 1 week (Fig. 4). Such a survival level could be expected to allow sufficient time for transfer of this organism to a new host. There are numerous strains of E. coli which have lost one or more of the repair systems found in the wild type. Repair-defective E. coli are quite viable in laboratory culture where they are not exposed to solar UV-B. The lethal effect of sunlight on the repair-
defective E. coli strain AB2480 used by Harm (1969) can be calculated (Fig. 7). A 1-h exposure to solar UV-B on a sunny winter day with the average dosage in the water columns would reduce survival to 17;; similarly a clear summer day would reduce survival by a factor of lo6’! Radiation repair systems in microorganisms clearly represent a utilization of cellular resources, any expenditure of which would soon be eliminated by mutation if not needed. The life cycle of E. coli demands a certain level of resistance to solar radiation, a resistance known to arise from the action of at least three different repair processes (Harm, 1969). E. coli represents an intermediate level of resistance. It can exist in natural waters for a few days, but without continuing input natural waters become free of E. coli. The repair-deficient strains would be much less able to transfer from host to host. Microorganisms which spend their entire life cycle in exposed habitats, for instance surface-film bacteria, would have to possess more UV-B resistance than wild-type E. coli. Bacteria which are clearly long-term residents of natural waters are indeed much more resistant to UV-B than the pathogenic group reported here (Calkins, 1975). Mycobacteriurn phlei, an airborn pathogen which would not be protected by the attenuation of the water column, was observed to be more resistant than E. coli, while Neisseria species, an organism transmitted by personal contact, was found to be much more UV-B-sensitive than E. coli. Although our observations of response of pathogenic bacteria strongly suggest that importance of solar UV-B as an environmental factor, it might be argued that UV resistance is merely a ‘spin off from some other vital function. There are other examples of loss of resistance to solar UV in protected situations. Organisms isolated from caves are remarkably sensitive to the shorter wavelengths in solar radiation (T. Barr, personal communication). A strain of protozoan isolated from Mammoth Cave was found to lack a functional photoreactivation capacity (Calkins and Griggs, 1969 a, b). Considering the observations noted above from the more general viewpoint, it is clear that solar UV-B is an important environmental factor. Solar UV-B is highly lethal. Recent calculations (Calkins, manuscript in preparation) indicate that a variety of even fully repair-competent organisms must attenuate (by epidermis, skin, hair, or by position in an absorbing water column for unicellular organisms) the incident solar UV-B by a factor of about 10,OOO to reach a level of exposure which can be tolerated on a long-term basis. Because each successful species must possess adequate UV-B resistance for its particular ecological niche, the injurious aspects of solar radiation and the multiple physiological and behavioral adaptations required to live in various environments exposed to solar UV have been little appreciated. Our observations quantify the role of solar UV-B in the sterilization of domestic water supplies. Comprehension of
Solar UV in water purification the effects of sunlight will permit a more effective use of this natural resource. Acknowledgements-This work has been supported in part by the Climatic Impact Assessment Program, Office of the Secretary, U.S. Department of Transportation.
57
The work on which this report was based was supported in part by the Office of Water Research and Technology, U.S. Department of Interior under the provisions of Public L~~ 88-379,
REFERENCES
American Public Health Association (1971) Standard Methods for the Examination of Water and Wastewater, 13th Edition, New York. Barber, M. A. (1908) J . Infect. Disease, 379-396. Bellar, T. A., J. J. Lichtenberg and R. C. Kroner (1974) J. Am. Water Works Assoc. 66. 703-706. Berger, D., D. F. Robertson, R. E. Davies and F. Urbach (1975) CIAP Monograph V, Chapter 2, Appendix D. pp. 235-264. Camp, T. R. (1971) Water and its Impurities, p. 231. Reinhold Publishing Co., New York. Calkins, J. (1975) CIAP Monograph V., p. 231. Calkins, J., and G. Griggs (1969 a) Photochem. Photobiol. 10. 61-66. Calkins, J., and G. Griggs (1969 b) Photochem. Photobiol. 10. 445-449. Chambers, C. W., and N. A. Clarke (1966) Ado. Appl. Microbiol. 8. 105-143. Fry, J. C., and D. G. Staples (1974) Water Res. 8. 1029-1035. Gameson, A. L. H., and J. R. Saxon (1967) Water Res. 1. 279-295. Gloyna, E. F. (1971) Waste Stabilization Ponds; World Health Organization, Geneva. Harm, W. (1969) Radiation Res. 40, 63-69. Hendricks, C. W. (1972) Appl. Microbiol. 24, 168-174. Jagger, J. (1958) Bacteriol. Rev. 22. 99-142. Luckiesh, M. (1946) Applications of Gernicidal, Erythemal and Infrared Energy, D. Van Nostrand, New York. Millipore Corporation (1973) Application Manual AM 302 Biological Analysis of Water and Wustewater, Bedford, Massachusetts. Morowitz, H. J. (1950) Science 111. 229-230. Ng, H., J. L. Ingraham and A. G. Marr (1962) J. Bacteriol. 84. 331-339. Parker, C. D. (1962) J . Water Pollut. Control Fed. 34, 149-161. Phelps. E. B. (1944) Stream Sanitation, Wiley, New York. Robertson, D. F. (1969) Biological Effrcts of Ultraviolet Radiation (Edited by F. Urbach), p. 433. Pergamon Press, Oxford. Robertson, D. F. (1975) C U P Monograph V., Chapter 2, Appendix B, pp. 203211. Stapleton. G. E., D. Billen and A. Hollaender (1953) J . Cell. Comp. Physiol. 41. 345-357.
