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Bacteriological, virological and chemical evaluation of a wastewater-aquaculture system
Water Res. Vol. 17, No. 12, pp. 1749-1755, 1983 Printed in Great Britain. All rights reserved
0043-1354/83 $3.00 +0.00 Copyright © 1983 Pergamon Press Ltd
BACTERIOLOGICAL, VIROLOGICAL AND CHEMICAL EVALUATION OF A WASTEWATER-AQUACULTURE SYSTEM THOMAS W. HEJKALI*, CHARLESP. GERBA2, SCOTT HENDERSON3 and MIKE FREEZE3 ~Department of Biological Sciences, Murray State University, Murray, KY 42071, -'Departments of Microbiology and Nutrition and Food Science, University of Arizona, Tucson, AZ 85721 and 3Arkansas Game and Fish Commission, Lonoke, AR 72086, U.S.A.
(Received February 1983) A~tract--Levels.of fecal coliforms (FC), fecal streptococci (FS), Salmonella spp and enteric viruses were monitored in the water, sediment and fish in experimental wastewater-fish ponds near Benton, Arkansas, U.S.A. Concentrations of five heavy metals were also monitored in the fish and wastewater. Concentrations of indicator bacteria were reduced by as much as 99.7~ through the series of six ponds which had a calculated total retention time of 72 days. Two filter-feeding species of Chinese carp, Hypophthalmichthys molitrix (silver carp) and Aristichthys nobilis (bighead carp), grown in the last three ponds accumulated FC and FS in their digestive tracts and skin at levels as great or greater than in the surrounding water and sediment. Only low levels of FC and FS were found in the fish muscle tissue (maximum of 25 FS per 100 g) even when concentrations of bacteria in the gut exceeded 105 per 100 g. Concentrations of bacteria in the water and sediment were not good predictors of concentrations in the fish. No Salmonella and no enteric viruses were isolated from the fish, but this lack of isolates was attributed to the extremely low levels which were present in the influent wastewater. Higher levels of copper and mercury were found in the fish flesh than in the surrounding water, with three of eleven fish samples containing higher than acceptable levels of mercury in the edible portion. Based on the efficiency of wastewater treatment, an aquaculture system using silver and bighead carp was judged to be a viable treatment system for domestic sewage resulting in a product suitable for animal or human consumption if proper precautions are taken in harvesting and processing the fish.
Key words--aquaculture, bacteria, fish, heavy metals, pesticides, sediment, wastewater, wastewater treatment, viruses
INTRODUCTION One alternative to conventional wastewater treatment systems is the use of aquaculture systems that convert the nutrients from domestic wastewater into plant, fish or shellfish biomass (Duffer, 1982). Since aquaculture-wastewater systems have the two-fold purpose of treating wastewater and producing a useful product, they must be capable of producing an acceptable effluent, and the aquaculture product must not contain unacceptable levels of harmful chemicals, pathogenic bacteria or viruses. Viruses and pathogenic bacteria which are present in domestic sewage present a potential health hazard to consumers of organisms grown in wastewater ponds. Vaughn and Ryther (1974) conducted studies in a model aquaculture system which used treated sewage as a nutrient supplement for primary production and found enhancement of bacteriophage survival by growing algae. Laboratory studies have shown that viruses may be accumulated by bottomfeeding fish which eat contaminated worms (Metcalf,
*To whom all correspondence should be addressed.
1975). Recent studies have been conducted by Buras (1980) which have shown that fish grown in ponds containing wastewater accumulate fecal bacteria and, that above a threshold concentration of about l 0 4 bacteria ml-~, detectable levels of bacteria penetrate into the muscle tissue. Other contaminants, such as pesticides and heavy metals also present a potential health hazard since some of these are known to accumulate in aquatic organisms (Mclntyre and Mills, 1975). The initial portion of this project demonstrated that an aquaculture system was at least as efficient in treating wastewater as a system of oxidation ponds without fish (Henderson, 1979). However, the effectiveness of an aquaculture system for removing bacteria, viruses and heavy metals has not been established. Knowledge of the levels of bacteria, viruses, heavy metals and other contaminants which accumulate in fish grown in wastewater ponds and the relationship to levels in wastewater, pond water and sediment is necessary to evaluate the public health risk. The present studies were performed to determine the efficiency of an experimental wastewater-aquaculture system in removing microbiological and chemical contaminants and to determine the
1749
THOMAS W. HE1KALet al.
1750
distribution o f these c o n t a m i n a n t s a m o n g the water, sediment a n d fish. MATERIALS AND M E T H O D S
Project site description Sewage that entered the test system came from Benton Services Center, Benton, Arkansas. The center provides mental rehabilitation programs, a nursing home facility and serves as a work release center. There are approx. 1000 fulltime residents and an additional 1000 employees contributing to the waste-load during working hours. A site plan for the system of oxidation ponds is shown in Fig. 1. The wastewater treatment facilities consisted of a bar screen and grinder for removing large debris, and a clarifier. Water from the clarifier entered the first of six oxidation ponds. The six ponds had a total surface area of 10.2 ha (24 acres). Individual ponds ranged in size from 1.55 to 1.8 ha. To prevent short-circuiting and provide maximum retention time, baffles were constructed diagonally, three-quarters of the distance across each pond. The average depth of each pond was 1.2-1.3 m. The average flow was 1711 m3day -I (0.45 MGD) with average loads of 444kg (9771b) of BOD 5 per day and 208.6 kg (459 lb) of total suspended solids (TSS) per day. This flow rate led to a calculated retention time of 12 days per pond or 72 days for the entire system. Ponds 1 and 2 served as stabilization ponds and were not stocked with fish. The remaining four ponds were stocked with fish as indicated below.
Sampling procedures Monthly samples of water, sediment and fish were taken for microbiological analyses from August to December 1980. Surface water samples were taken in sterile 20-1. containers as the influent entered Pond 1 and as the water Influent
/
/
•I I
,/ /
\
Pond I 1.83 ho Pond4 1.80ha
~ . ~
\
f
I
~"~'~" - Pond3 1.55 ha
------/I/I~ r - ' - " ' - ~ ~
Pond 6 1.56ha
/
1
\
Pond 5 1.67
Fish species Two species of Chinese carp Hypophthalmichthys molitrix (silver carp) and Aristichthys nobilis (bighead carp) were sampled for bacteriological, virological and chemical analyses. Both are filter-feeding fishes, capable of rapid growth and capable of withstanding fairly low levels of dissolved oxygen. The diet of the silver carp consists primarily of planktonic algae, and it is capable of removing particles as small as 4 pm in diameter (Omarov, 1970). These species are well suited to growth in eutrophic waters with dense algal populations. In January 1979 the Ponds 3, 4, 5 and 6 were stocked respectively with 20,270, 12,198, 12,070 and 8100 silver carp with an average weight of 41 g each. Bighead carp weighing 32 g each were added at levels of 4103, 2052, 2052 and 600 to Ponds 3, 4, 5 and 6 respectively. Six-hundred channel catfish, 100 buffalofish and 40 grass carp were also added to Pond 6 but were not collected for microbiological or chemical analysis. Due to a temporary malfunction in the system which necessitated bypassing Pond 1, there was a fish kill in Pond 3 due to oxygen depletion in July 1980. Therefore only fish from Ponds 4, 5 and 6 were collected for the microbiological studies which began in August 1980. At the time of sampling the fish had been in the ponds for 19-24 months and weighed 2-3 kg.
Bacteriological assays
Pond 2 1.76 ha
I
exited Ponds 2, 4 and 6. Grab samples of sediment were also taken near the outfalls from Ponds 2, 4 and 6 as indicated in Fig. 1. Fish were captured from Ponds 4, 5 and 6 with seine nets. They were killed, placed individually in plastic bags and packed in ice for shipment back to the laboratory. One fish from each pond was used for bacteriological tests and a second fish for virological analysis each month. A total of three samples of fish from each pond were collected intermittently between March 1979 and September 1980 to determine levels of metals and pesticides. For these tests, fish were gutted but not skinned, wrapped in aluminum foil and placed on ice for shipment to the laboratory.
~ t'- ,--
-
Effluent
Fig. 1. Site plan for wastewater-fish pond system. Ponds 3~5 were stocked with fish as listed in Table 1. A fish kill in Pond 3 eliminated these fish from most of the study. Sampling locations for water and sediment are indicated by the symbol *
Samples of fish gut and skin were blended in a Stomacher 400 (Dynatech Laboratories, Inc.) (Emswiler et al., 1977) with 10ml of 0.1M phosphate-buffered saline (PBS), pH 7.5, per g of fish tissue. Due to the low levels of bacteria in the flesh, samples offish muscle were blended in only 5 ml of PBS per g. Water samples were tested for fecal coliforms (FC) and fecal streptococci (FS) using the membrane filter technique (APHA, 1980). Sediment samples were mixed with 0.01 M PBS for 5 min and immediately assayed for fecal coliforms, fecal streptococci and Salmonella spp using multiple tube techniques. Dulcitol selenite broth was used for initial enrichment of Salmonella followed by streaking on brilliant green agar and identification by biochemical (API 20E) and serological tests (APHA, 1980). Lactose broth (Difco) was used as the initial enrichment medium for fecal coliforms. Cultures showing gas production within 48 h were transferred to EC broth (Difco) and incubated at 44.5°C. Gas production in EC broth within 24 h was considered a positive test for fecal coliforms. Azide dextrose broth was used to enrich for fecal streptococci. Inocula from tubes showing turbidity within 48 h were streaked onto PSE agar plates (APHA, 1980). Formation of black colonies indicative of esculin hydrolysis was interpreted as a positive test for fecal streptococci.
Virological assays Samples of fish flesh, skin or guts were blended in 0.05 M glycine, pH 9.5, using a Stomacher. Each 100ml of homogenate was mixed with 10 ml of a 1~o solution of Cat-Floc (Calgon Corp., Pittsburgh, PA) and centrifuged at 2500 revmin ~ for 30min to clarify the suspension. The
Evaluation of a wastewater-aquaculture system supernatant was decanted into a dialysis bag and hydroextracted overnight with polyethylene glycol flakes at 4°C. The contents of the dialysis bag were resuspended in 37O beef extract, pH 10. All concentrates were clarified by centrifugation at 10,000 rev rain -~ for 30 min and by filtration through positively charged Zeta-plus type 50S filters (AMF/CUNO, Meriden, CT) (Hejkal et al., 1982). They were then treated with 0.2ml of an antibiotic solution containing 100U penicillin ml-~ and 100/~g streptomycin ml 1. Samples were inoculated onto monolayers of buffalo green monkey kidney (BGM) cells in 75 cm 2 tissue culture flasks. After a 1.5 h adsorption period, the sample was withdrawn and saved, and the bottle was rinsed with PBS to reduce cytotoxicity. An agar overlay containing neutral red was applied, and the bottles were incubated at 37°C. Plaques that appeared on the monolayer were harvested and inoculated into bottles containing BGM monolayers under a liquid medium (Eagles minimum essential medium containing 2% fetal calf serum) to confirm them as viral. Twenty-liter samples of pond water were collected in a stainless steel pressure vessel. The samples were prefiltered, if necessary, through a 142mm diameter 3.0/~m nominal pore size fiberglass Filterite filter (Filterite Corp., Timmonium, MD). The sample was adjusted to pH 3.5 with 1 N HCI, and A1CI3 was added to a final concentration of 0.005M. The sample was then filtered through a 3.0-0.45 # m Filterite series (Farrah et al., 1976). Both filter and prefilter were eluted with 50 ml of 37o beef extract at pH 10. This eluate was concentrated to a final volume of 8-12 ml by hydroextraction as above. Sediment samples were mixed with 300ml of 3% beef extract for each 100 g of sediment. This mixture was centrifuged at 1500revmin -~ for 10min. The supernatant was adjusted to pH 3.5 with 1 M glycine, pH 1.5. The floc that formed was sedimented by centrifugation and the sedimented material was eluted with 0.05 M glycine at pH 9.5 (Katzenelson et al., 1976).
Chemical analyses Concentrations of five heavy metals (lead, copper, cadmium, mercury and arsenic) in the water and fish tissue were determined by flame atomic adsorption (APHA, 1980). Fish flesh and water were also monitored for the pesticides aldrin, dieldron, endrin, mires, DDT, toxaphene and kepone, and for potychlorinated biphenyls (PCB) using solvent extraction concentration followed by electron capture gas chromatography (APHA, 1980). Em'ironmental conditions in ponds Measurement of other water quality parameters was done on the influent wastewater and on the effluent from each pond. These measurements were performed according to standard methods (APHA, 1980). The water temperature in the ponds during the microbiological sampling period ranged from 30°C in August to 9 C in December. Average values for other selected variables are listed in Table 1. Statistical procedures For statistical analysis, concentrations of indicator bac-
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6
[ ] Fecal coliforms
5
[ ] Fecal streptococci
0
o
I I nfl uent
Pond 2
Pond 4
Fig. 2. Concentrations of indicator bacteria in pond water. teria in fish, water and sediment were transformed by taking the logarithm of the number per 100 g. Paired t-tests were used to detect significant differences between bacterial levels in the fish and in the surrounding water and sediment and between fish from Pond 4 and Pond 6. Correlation coefficients and significance levels were calculated using the Statistical Package for the Social Sciences (SPSS). The SPSS was also used to perform a multiple linear regression analysis to look for good predictors of bacterial levels in fish. Other confidence intervals were calculated using the t-distribution. RESULTS
Bacteriological results Levels o f indicator bacteria decreased steadily t h r o u g h the system o f ponds. Figure 2 illustrates the decrease in c o n c e n t r a t i o n s o f indicator bacteria in water going from influent to P o n d 6. There was a total decrease of at least 2.5 log~0 (99.7%) for F C a n d a total decrease of 2.3 logt0 (99.5%) for FS, based o n the average c o n c e n t r a t i o n in each pond. The decrease was not substantially different from p o n d to pond. There was a n average 62% decrease per p o n d for F C a n d a n average 58% decrease per p o n d for FS. Based on a calculated retention time of 12 days per p o n d this represented inactivation rates of 0.034 a n d 0.032 log~0 day ~, respectively. Bacterial c o n c e n t r a t i o n s in the sediments followed a different p a t t e r n t h a n in the overlying waters (Fig. 3). There was a substantial decrease in F C from P o n d 2 sediments to P o n d 6 sediments. A cumulative decrease of 2.71og10 was observed for FC. This represents a n average 79% decrease per p o n d for F C in the sediments of the last four ponds. The concentration of FS in the p o n d sediments decreased by only 0.4 log~0 from P o n d 2 to P o n d 6. The decrease of FS
Table 1. Environmental conditions in ponds
Pond
pH
Influent 1 2 3 4 5 6
6.5 7.6 7.9 8.4 8.2 8.2 8.3
*Dissolved oxygen. ?Total suspend solids.
Average of monthly samples Jan. 1979-Jan. 1981 DO* TSSt 5-day BOD Ammonia N (mgl 1) (mg i - i ) (mgl -I) [mgl i) 0 2.1 2.6 6.9 5.7 6.9 7.4
97 81 47 47 25 21 20
Pond 6
251 63 30 24 15 11 10
24 6.2 5.0 3.2 2.4 1.1 1.2
PO 4 P (mgl -I)
NO 3 N (mgl i)
22 3.9 3,0 2.6 2.3 2.0 2.0
0 0.018 0.026 0.071 0.14 0.38 0.33
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THOMASW. HEJKALet al. 6 5
[]
-
a n d 10 4 of each type of bacteria per 100 g in the digestive tract and skin. The concentrations of FC in the gut and of FS in the gut and skin were correlated with the concentration of FS in the surrounding water (Table 3). FS levels in sediment also showed a weak positive correlation with FS levels in fish flesh. Other than this, concentrations of FC in the water and sediment and FS in the sediment were not significantly correlated with bacterial levels in the fish. Concentrations in the three types of fish tissue were generally correlated with each other, and concentrations of FC showed a strong positive correlation with concentrations of FS in most types of samples (correlation coefficients not shown). Table 4 shows the levels of bacteria which were detected in the fish flesh. Two methods were used for sampling the fish muscle. The samples in August and September were taken by a normal filet procedure using a decontaminated filet knife. Samples taken during these two months yielded sporadically high levels of FC and FS in the muscle tissue, probably due to contamination by bacteria from the fish skin. Beginning in October, all muscle samples were taken aseptically to avoid contamination from the skin. Three of nine samples of muscle tissue obtained from October through December were positive for either FC or FS at low levels. The highest level of indicator bacteria in fish muscle sampled aseptically was 25 MPN (most probable number) of FS/100 g from a fish which had a concentration of 1.4 × 105 MPN of FS/100 g in the digestive tract. Using a normal filet procedure, contamination occurred in 8 of 12 samples with levels up to 22,000 MPN of FS/100 g. Salmonella spp was detected in 2 of 4 influent samples at levels of 0.4 and 2.3 MPN/100 ml using dulcitol selenite enrichment. Salmonella spp was also isolated from a single water sample from Pond 2 in December. No Salmonella was isolated from any of the other pond water, sediment or fish samples.
Fecal coliforms
[ ] F e c a l streptococci
4.o
3
,/,
-
g ..J
fL
7/, ~"L "//, f,,'/, "//, F/, sis
I --
Pond2
Pond4
Pond6
Fig. 3. Concentrations of indictor bacteria in pond sediments.
in the sediments from pond to pond was substantially less than the decrease of FS in the water. Inactivation rates could not be calculated for the sediments since retention time in the sediments was unknown. The concentrations of FC and FS in the fish guts were on the average greater than in the surrounding water and sediment (Table 2; Figs 2 and 3). Mean concentrations of FC and FS on the fish skin were lower than in the gut. Mean concentrations of both FC and FS in the gut were correlated with concentrations of FS on the skin with r = 0.607 and 0.825, respectively. On the average, fish in Pond 6 had levels of F C and FS 54~ lower than fish from Pond 4. However, due to the variability between samples, this decrease was not considered significant (P < 0.14), and fish in the last pond still averaged between 102 Table 2. Levels of fecal coliforms (FC) and fecal streptococci (FS) in fish gut and skin samples Average concentration (Iog~0/100g)* FC Gut Skin Out Pond 4 Pond 5 Pond 6
3.07 3.01 2.73
2.00 2.43 2.21
FS Skin
4.36 4.03 3.75
3.57 3.47 2.95
*Averages of five monthly samples. Table 3. Correlation coefficients between levels of fecal coliforms (FC) and fecal streptococci (FS) in fish and in water and sediment*
Virological results
Variable
FC water
FC gut FC skin FC flesh
0.559 0.019 0.357
0.097 -0.423 0.003
0.712t -0.029 0.330
FS gut FS skin FS flesh
0.307 0.363 0.455
0.280 0.525 0.537
0.646]" 0.691t 0.375
Six of the 90 samples tested for enteric viruses yielded at least 1 PFU (plaque-forming unit) on the BGM monolayers (Table 5). Three of five influent samples were positive at low levels. Only a portion of each influent sample was tested for virus because of the necessity of dilution to reduce toxicity of these concentrates. The concentrations based on the
FC sediment
FS water
FS sediment -0.251 -0.217 0.099 -0.236 0.370 0.630t
*Data from five monthly samples from Ponds 4 and 6. tSignificant at P < 0.05.
Table 4. Concentrations of fecal coliforms (FC) and fecal streptococci (FS) in fish flesh M P N / 1 0 0 g fish flesh Pond 5
Pond 4
Pond 6
Month
FC
FS
FC
FS
August* September* October November December
<30 230 < 11 II <11
140 80 25 <11 <15
<30 430 < 11 <11 <11
140 22,000 < 11 <11 <15
FC
FS
40 < 30 < 6.6 <11 <11
860 < 60 15 <11 <15
*August and September samples were taken by a normal filet procedure with possible contamination from the skin. All other flesh samples were taken aseptically. Data from pooled samples taken from several locations from single fish.
Evaluation of a wastewater-aquaculture system Table 5. Samples yielding plaques on BGM monolayers Sample Influent lnfluent Influent Pond 2 sediment Pond 4 sediment Pond 2 water
September
2
Estimated concentrationt 20PFU1 -I 15PFUI-' 7.5PFU1-I 2VFU/50Og
November
1
1 PFU]500 g
December
1
Month
Total PFU counted*
August November December
5 3 2
0.05PFUI-'
*Plaques could not be confirmed as viral. ~Dilution factors varied for influent samples.
number of plaques counted ranged from 7.5 to 20 P F U 1-~. Two of 15 sediment samples and one water sample from Pond 2 also yielded 1-2 P F U per sample of 500 g or 201. A single P F U was detected in the water concentrate from Pond 2 in December. All other pond water samples were negative for virus. No viruses were detected in any of the 45 fish samples processed. Attempts were made to isolate and identify the virus from each plaque. The plaques recorded in Table 5 produced CPE when inoculated onto BGM monolayers under liquid overlay. However, attempts at additional passages and identification were unsuccessful. Thus, although the original plaques were virus-like, it is possible that they were caused by nonviral agents,
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higher than in the composite sample of fish prior to stocking the ponds (0.063 ppm) and was 70-fold higher than the average concentration of mercury in pond water from which the fish were taken ( 0 . 0 0 5 ppm). A considerable bioconcentration of copper was also observed with an average of 3.58 ppm in the fish flesh and <0.01 ppm in the pond water. All fish and water samples, including influent, tested for PCB and pesticides were negative. Other water quality parameters The system substantially improved many of the usual indicators of wastewater quality. Through the series of six ponds, the 5-day BOD decreased by 96~, total suspended solids by 79~, ammonia by 95~ and reactive phosphorous by 91~ (Table 1).
DISCUSSION Indicator bacteria
The consistent decline of FC and FS levels in pond water indicated that these bacteria were being removed by a combination of natural inactivation processes, adsorption to sediments and uptake by fish. The sediment may serve as a sink for some microorganisms, for example, FS which showed little decrease in concentration in the sediments going from Pond 2 to Pond 6. This is most reasonably explained Chemical contaminants by extended survival of these bacteria in sediments, Measurable levels of lead, copper and mercury although other factors such as growth of FS in the were found in one or more fish grown in the waste- sediments or more efficient adsorption to sediments water ponds (Table 6). Influent concentrations in the downstream ponds may be involved. averaged < 0.053 mg Cu 1-1, <0.067 mg Pb I-~ and The levels of bacteria in the fish also showed little < 0.0036 mg Hg 1 i. Cadmium and arsenic were not decrease through the final 3 ponds. Thus the fish may detected in any of the fish flesh samples (detection also serve as sinks for bacteria in the wastewater limit = 0.02 ppm) although arsenic was detected in 7 ponds. The concentrations of bacteria in the gut were of 19 water samples (detection limit = 0.002 ppm) highly correlated with the concentrations on the skin, from the influent and ponds at concentrations up to whereas correlations between bacterial levels in fish 0.013 ppm. and in water or sediment were generally weak. The Three of 11 fish contained higher than acceptable bacterial levels in water and sediment, therefore, were levels of mercury in the edible portion. The average not good predictors of bacterial levels in fish in the level of mercury in fish taken from the wastewater range found in our study. The lack of good corponds was 0.350 +__0.274 (95~o C.I.) ppm which was relation between bacterial levels in the water and fish may be explained if the fish accumulated bacteria to a saturation level which may have been reached even Table 6. Concentrations of selected heavy metals in fish flesh* in the final pond. However, if this were true, fish from Copper Lead Mercury each pond would be expected to contain approxiSamplesite (ppm) + ~ppm> tppm> mately equal concentrations of indicator bacteria Before stocking 0.77 0.57 0.063 which was not the case. The apparent cause of the 0.45 Pond 3 3.0 <0.5 0.54 <0.5 0.079 lack of correlation was the large variability between 0.60 <0.5 0.64~bacterial levels in individual fish. This could be 0.099 Pond 4 30.0 1.7t
1754
THOMAS W. HEJKAL et al.
The lack of a good correlation between bacteria in the water and bacteria in the fish does not imply that monitoring of indicator bacteria in water would be useless. Based on these results, we expect that if indicator bacteria and presumably pathogens were present in the water, silver and bighead carp would accumulate them in their digestive tracts at levels as high or higher than the levels in the water. Since the muscle tissue is the critical portion of the fish if it is to be used for human consumption, bacterial levels in the fish muscle were a major concern. Even when levels of bacteria exceeded 105/100 g in the fish guts, very little penetrated into the fish muscle tissue. However, when the muscle tissue was sampled using normal filet procedures, contamination of the muscle occurred in 8 of 12 samples. The conclusion is that while the fish do not accumulate bacteria in the muscle tissue, contamination of the muscle tissue during processing is difficult to avoid. Viruses The sewage entering the Benton fish ponds was atypical from a virological standpoint. The levels of virus in the sewage were much lower than would be expected for untreated sewage from a larger and more diverse community. For example, concentrations in raw sewage from treatment plants in St Petersburg, Florida, averaged 90 P F U 1-~ and, at a larger treatment plant in Tampa, Florida, concentrations of over 2000 P F U 1-~ were found (Hejkal et al., 1981). The sewage entering Benton ponds had an average concentration of < 9 P F U I ~ for the 5 samples tested. The low levels of virus in the sewage in this study can be attributed to the population from which the sewage is derived. The population consists of approx. 1000 persons residing fulltime at the Benton Services Center with an additional 1000 fulltime employees. Infants and young children contribute most of the enteric viruses to the wastewater of any given community, since they are the most susceptible age group to infection by these viruses (Fox and Hall, 1980). The lack of infants and young children in the population at the Benton Services Center explains the low levels of enteric viruses in the sewage from the center. Additionally, half of the population contributing to the waste load are employees who do not live at the Center. These persons would be less likely to come to work if they had an enteric viral infection and therefore would not be likely to contribute to the viral contamination of the watewater. Because of the low levels of virus found in the influent, the results cannot be extrapolated to make conclusions or predictions about the survival and transport of viruses in other fish pond systems that may have a much higher input of viruses. The lack of virus isolated from the fish and pond water in this study does not preclude the possibility of viruses surviving in the fish ponds and being accumulated by
the fish if the initial levels of virus were higher. Since relatively high levels of FC and FS were found in the ponds and fish, it is likely that viruses would also be present if the input rate were higher because viruses generally survive inactivation processes better than do indicator bacteria (Scarpino et al., 1972; Shuval et al., 1971). Chemical contaminants The accumulation of persistent chemical contaminants in the fish poses an even greater public health threat than microbiological contaminants since decontamination is not feasible. Bioaccumulation of mercury and other contaminants is a known hazard (Mclntyre and Mills, 1975). The levels of mercury present in the fish taken from the Benton ponds demonstrate the necessity to be extremely cautious in the utilization of fish grown in wastewater. Although the levels found in the fish flesh were not excessive in this study, the fact that concentrations of mercury were much higher in the fish than in the water reemphasizes the need for controls on the quality of wastewater used for aquaculture purposes. Efficiency o f treatment and environmental factors The efficiency of the system in treating the wastewater is also important. Previous studies have shown the fish pond system to be at least as efficient as a system of oxidation ponds without fish (Henderson, 1979). The system used in the present study was effective in producing an effluent which met standards for secondary wastewater treatment. The data were insufficient to assess seasonal variations or to draw conclusions about the effects of variables such as temperature, pH and ammonia concentrations in this system. These factors influence the survival of pathogenic bacteria and viruses in water and can also affect adsorption to sediments. There would probably be seasonal and short-term fluctuations in the survival of pathogens and in rates of adsorption to sediments and uptake by fish. For example, inactivation of pathogens may be enhanced in warmer months since high temperatures are detrimental to pathogens. However, in this study there was no evident relationship between temperature and bacterial or viral survival. Interactions between temperature and other variables will need to be determined before the overall effect can be assessed. CONCLUSIONS
Aquaculture-wastewater treatment systems are potentially valuable alternatives to conventional sewage treatment plants. This study shows that while concentrations of indicator microorganisms are reduced by as much as 99.7% they are not eliminated by the fish ponds. Significantly, neither do conventional activated sludge or trickling filter processes eliminate indicator organisms, pathogens or toxic chemicals. It is clear that fish or other organisms
Evaluation of a wastewater-aquaculture system raised in wastewater have a high probability of becoming contaminated with bacteria, viruses and toxic chemicals and appropriate cautions need to be taken when these organisms are harvested and utilized for human or animal consumption. However, the public health risk may be no greater under these carefully controlled conditions than the risk from the uncontrolled harvesting of fish from waters that are contaminated by effluents from conventional sewage treatment plants. Acknowledgements--This research was supported by a grant from the U.S. Environmental Protection Agency (R8054301) to the Arkansas Game and Fish Commission. The assistance of Catherine Castefiada in the bacteriological and virological analyses is gratefully acknowledged. REFERENCES
APHA (1980) Standard Methods for the Examination of Water and Wastewater, 15th Edition. American Public Health Association, Washington, DC. Buras N. (1980) Personal communication. Coleman M. S., Henderson J. P., Chichester H. G. and Carpenter R. L. (1974) Aquaculture as a means to achieve effluent standards. In Wastewater Use in the Production of Food and Fiber, Proceedings, EPA Technology Series, EPA-660/2-74-041, pp. 199-212. Duffer W. R. (1982) Assessment of aquaculture for reclamation of wastewater. Water Reuse (Edited by Middlebrooks E. J.), pp. 349-367. Ann Arbor Science, Ann Arbor, MI. Emswiler B. S., Pierson C. J. and Kotula A. W. (1977) Stomaching vs blending. Food Technol. 31, 40-42.
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Farrah S. R., Gerba C. P., Wallis C. and Melnick J. L. (1976) Concentration of viruses from large volumes of tapwater using pleated membrane filters. Appl. envir. Microbiol. 31, 221-226. Fox J. P. and Hall C. E. (1980) Viruses in Families. PSG Publishing Co., Littleton, MA. Hejkal T. W., Wellings F. M., Lewis A. L. and LaRock P. A. (1981) Distribution of viruses associated with particles in wastewater. Appl. envir. Microbiol. 41, 628-634. Henderson S. (1979) Utilization of Silver and Bighead Carp for Water Quality Improvement. Proceedings of Symposium on Aquaculture for Wastewater Treatment, EPA430/9-80-006, pp. 309-350. Katzenelson E., Fattal B. and Hostovesky T. (1976) Organic flocculation: an efficient second-step concentration method for the detection of viruses in tapwater. Appl. envir. Microbiol. 32, 638-639. McIntyre A. D. and Mills C. F. (1975) Ecological Toxicology Research. Plenum Press, New York. Metcalf T. G. (1975) Final Report, GI 38976: RANN Program, National Science Foundation. Omarov M. O. (1970) The daily food consumption of the Silver Carp. J. Ichthyol. 10, 425-426. Roberts H. R. (1981) Food Safety. Wiley, New York. Scarpino P. V., Berg G., Chang S. L., Dahling D. and Lucas M. (1972) A comparative study of the inactivation of viruses in water by chlorine. Water Res. 6, 959-965. Shuval H. I., Thompson A., Fattal B., Cymbalista S. and Wiener Y. (1971) Natural virus inactivation processes in seawater. Proceedings of the National Specialty Conference on Disinfection, pp. 429-452. American Society of Civil Engineers, New York. Vaughn J. M. and Ryther J. H. (1974) Bacteriophage survival patterns in a tertiary sewage treatment-aquaculture model system. Aquaculture 4, 399-406.
0043-1354/83 $3.00 +0.00 Copyright © 1983 Pergamon Press Ltd
BACTERIOLOGICAL, VIROLOGICAL AND CHEMICAL EVALUATION OF A WASTEWATER-AQUACULTURE SYSTEM THOMAS W. HEJKALI*, CHARLESP. GERBA2, SCOTT HENDERSON3 and MIKE FREEZE3 ~Department of Biological Sciences, Murray State University, Murray, KY 42071, -'Departments of Microbiology and Nutrition and Food Science, University of Arizona, Tucson, AZ 85721 and 3Arkansas Game and Fish Commission, Lonoke, AR 72086, U.S.A.
(Received February 1983) A~tract--Levels.of fecal coliforms (FC), fecal streptococci (FS), Salmonella spp and enteric viruses were monitored in the water, sediment and fish in experimental wastewater-fish ponds near Benton, Arkansas, U.S.A. Concentrations of five heavy metals were also monitored in the fish and wastewater. Concentrations of indicator bacteria were reduced by as much as 99.7~ through the series of six ponds which had a calculated total retention time of 72 days. Two filter-feeding species of Chinese carp, Hypophthalmichthys molitrix (silver carp) and Aristichthys nobilis (bighead carp), grown in the last three ponds accumulated FC and FS in their digestive tracts and skin at levels as great or greater than in the surrounding water and sediment. Only low levels of FC and FS were found in the fish muscle tissue (maximum of 25 FS per 100 g) even when concentrations of bacteria in the gut exceeded 105 per 100 g. Concentrations of bacteria in the water and sediment were not good predictors of concentrations in the fish. No Salmonella and no enteric viruses were isolated from the fish, but this lack of isolates was attributed to the extremely low levels which were present in the influent wastewater. Higher levels of copper and mercury were found in the fish flesh than in the surrounding water, with three of eleven fish samples containing higher than acceptable levels of mercury in the edible portion. Based on the efficiency of wastewater treatment, an aquaculture system using silver and bighead carp was judged to be a viable treatment system for domestic sewage resulting in a product suitable for animal or human consumption if proper precautions are taken in harvesting and processing the fish.
Key words--aquaculture, bacteria, fish, heavy metals, pesticides, sediment, wastewater, wastewater treatment, viruses
INTRODUCTION One alternative to conventional wastewater treatment systems is the use of aquaculture systems that convert the nutrients from domestic wastewater into plant, fish or shellfish biomass (Duffer, 1982). Since aquaculture-wastewater systems have the two-fold purpose of treating wastewater and producing a useful product, they must be capable of producing an acceptable effluent, and the aquaculture product must not contain unacceptable levels of harmful chemicals, pathogenic bacteria or viruses. Viruses and pathogenic bacteria which are present in domestic sewage present a potential health hazard to consumers of organisms grown in wastewater ponds. Vaughn and Ryther (1974) conducted studies in a model aquaculture system which used treated sewage as a nutrient supplement for primary production and found enhancement of bacteriophage survival by growing algae. Laboratory studies have shown that viruses may be accumulated by bottomfeeding fish which eat contaminated worms (Metcalf,
*To whom all correspondence should be addressed.
1975). Recent studies have been conducted by Buras (1980) which have shown that fish grown in ponds containing wastewater accumulate fecal bacteria and, that above a threshold concentration of about l 0 4 bacteria ml-~, detectable levels of bacteria penetrate into the muscle tissue. Other contaminants, such as pesticides and heavy metals also present a potential health hazard since some of these are known to accumulate in aquatic organisms (Mclntyre and Mills, 1975). The initial portion of this project demonstrated that an aquaculture system was at least as efficient in treating wastewater as a system of oxidation ponds without fish (Henderson, 1979). However, the effectiveness of an aquaculture system for removing bacteria, viruses and heavy metals has not been established. Knowledge of the levels of bacteria, viruses, heavy metals and other contaminants which accumulate in fish grown in wastewater ponds and the relationship to levels in wastewater, pond water and sediment is necessary to evaluate the public health risk. The present studies were performed to determine the efficiency of an experimental wastewater-aquaculture system in removing microbiological and chemical contaminants and to determine the
1749
THOMAS W. HE1KALet al.
1750
distribution o f these c o n t a m i n a n t s a m o n g the water, sediment a n d fish. MATERIALS AND M E T H O D S
Project site description Sewage that entered the test system came from Benton Services Center, Benton, Arkansas. The center provides mental rehabilitation programs, a nursing home facility and serves as a work release center. There are approx. 1000 fulltime residents and an additional 1000 employees contributing to the waste-load during working hours. A site plan for the system of oxidation ponds is shown in Fig. 1. The wastewater treatment facilities consisted of a bar screen and grinder for removing large debris, and a clarifier. Water from the clarifier entered the first of six oxidation ponds. The six ponds had a total surface area of 10.2 ha (24 acres). Individual ponds ranged in size from 1.55 to 1.8 ha. To prevent short-circuiting and provide maximum retention time, baffles were constructed diagonally, three-quarters of the distance across each pond. The average depth of each pond was 1.2-1.3 m. The average flow was 1711 m3day -I (0.45 MGD) with average loads of 444kg (9771b) of BOD 5 per day and 208.6 kg (459 lb) of total suspended solids (TSS) per day. This flow rate led to a calculated retention time of 12 days per pond or 72 days for the entire system. Ponds 1 and 2 served as stabilization ponds and were not stocked with fish. The remaining four ponds were stocked with fish as indicated below.
Sampling procedures Monthly samples of water, sediment and fish were taken for microbiological analyses from August to December 1980. Surface water samples were taken in sterile 20-1. containers as the influent entered Pond 1 and as the water Influent
/
/
•I I
,/ /
\
Pond I 1.83 ho Pond4 1.80ha
~ . ~
\
f
I
~"~'~" - Pond3 1.55 ha
------/I/I~ r - ' - " ' - ~ ~
Pond 6 1.56ha
/
1
\
Pond 5 1.67
Fish species Two species of Chinese carp Hypophthalmichthys molitrix (silver carp) and Aristichthys nobilis (bighead carp) were sampled for bacteriological, virological and chemical analyses. Both are filter-feeding fishes, capable of rapid growth and capable of withstanding fairly low levels of dissolved oxygen. The diet of the silver carp consists primarily of planktonic algae, and it is capable of removing particles as small as 4 pm in diameter (Omarov, 1970). These species are well suited to growth in eutrophic waters with dense algal populations. In January 1979 the Ponds 3, 4, 5 and 6 were stocked respectively with 20,270, 12,198, 12,070 and 8100 silver carp with an average weight of 41 g each. Bighead carp weighing 32 g each were added at levels of 4103, 2052, 2052 and 600 to Ponds 3, 4, 5 and 6 respectively. Six-hundred channel catfish, 100 buffalofish and 40 grass carp were also added to Pond 6 but were not collected for microbiological or chemical analysis. Due to a temporary malfunction in the system which necessitated bypassing Pond 1, there was a fish kill in Pond 3 due to oxygen depletion in July 1980. Therefore only fish from Ponds 4, 5 and 6 were collected for the microbiological studies which began in August 1980. At the time of sampling the fish had been in the ponds for 19-24 months and weighed 2-3 kg.
Bacteriological assays
Pond 2 1.76 ha
I
exited Ponds 2, 4 and 6. Grab samples of sediment were also taken near the outfalls from Ponds 2, 4 and 6 as indicated in Fig. 1. Fish were captured from Ponds 4, 5 and 6 with seine nets. They were killed, placed individually in plastic bags and packed in ice for shipment back to the laboratory. One fish from each pond was used for bacteriological tests and a second fish for virological analysis each month. A total of three samples of fish from each pond were collected intermittently between March 1979 and September 1980 to determine levels of metals and pesticides. For these tests, fish were gutted but not skinned, wrapped in aluminum foil and placed on ice for shipment to the laboratory.
~ t'- ,--
-
Effluent
Fig. 1. Site plan for wastewater-fish pond system. Ponds 3~5 were stocked with fish as listed in Table 1. A fish kill in Pond 3 eliminated these fish from most of the study. Sampling locations for water and sediment are indicated by the symbol *
Samples of fish gut and skin were blended in a Stomacher 400 (Dynatech Laboratories, Inc.) (Emswiler et al., 1977) with 10ml of 0.1M phosphate-buffered saline (PBS), pH 7.5, per g of fish tissue. Due to the low levels of bacteria in the flesh, samples offish muscle were blended in only 5 ml of PBS per g. Water samples were tested for fecal coliforms (FC) and fecal streptococci (FS) using the membrane filter technique (APHA, 1980). Sediment samples were mixed with 0.01 M PBS for 5 min and immediately assayed for fecal coliforms, fecal streptococci and Salmonella spp using multiple tube techniques. Dulcitol selenite broth was used for initial enrichment of Salmonella followed by streaking on brilliant green agar and identification by biochemical (API 20E) and serological tests (APHA, 1980). Lactose broth (Difco) was used as the initial enrichment medium for fecal coliforms. Cultures showing gas production within 48 h were transferred to EC broth (Difco) and incubated at 44.5°C. Gas production in EC broth within 24 h was considered a positive test for fecal coliforms. Azide dextrose broth was used to enrich for fecal streptococci. Inocula from tubes showing turbidity within 48 h were streaked onto PSE agar plates (APHA, 1980). Formation of black colonies indicative of esculin hydrolysis was interpreted as a positive test for fecal streptococci.
Virological assays Samples of fish flesh, skin or guts were blended in 0.05 M glycine, pH 9.5, using a Stomacher. Each 100ml of homogenate was mixed with 10 ml of a 1~o solution of Cat-Floc (Calgon Corp., Pittsburgh, PA) and centrifuged at 2500 revmin ~ for 30min to clarify the suspension. The
Evaluation of a wastewater-aquaculture system supernatant was decanted into a dialysis bag and hydroextracted overnight with polyethylene glycol flakes at 4°C. The contents of the dialysis bag were resuspended in 37O beef extract, pH 10. All concentrates were clarified by centrifugation at 10,000 rev rain -~ for 30 min and by filtration through positively charged Zeta-plus type 50S filters (AMF/CUNO, Meriden, CT) (Hejkal et al., 1982). They were then treated with 0.2ml of an antibiotic solution containing 100U penicillin ml-~ and 100/~g streptomycin ml 1. Samples were inoculated onto monolayers of buffalo green monkey kidney (BGM) cells in 75 cm 2 tissue culture flasks. After a 1.5 h adsorption period, the sample was withdrawn and saved, and the bottle was rinsed with PBS to reduce cytotoxicity. An agar overlay containing neutral red was applied, and the bottles were incubated at 37°C. Plaques that appeared on the monolayer were harvested and inoculated into bottles containing BGM monolayers under a liquid medium (Eagles minimum essential medium containing 2% fetal calf serum) to confirm them as viral. Twenty-liter samples of pond water were collected in a stainless steel pressure vessel. The samples were prefiltered, if necessary, through a 142mm diameter 3.0/~m nominal pore size fiberglass Filterite filter (Filterite Corp., Timmonium, MD). The sample was adjusted to pH 3.5 with 1 N HCI, and A1CI3 was added to a final concentration of 0.005M. The sample was then filtered through a 3.0-0.45 # m Filterite series (Farrah et al., 1976). Both filter and prefilter were eluted with 50 ml of 37o beef extract at pH 10. This eluate was concentrated to a final volume of 8-12 ml by hydroextraction as above. Sediment samples were mixed with 300ml of 3% beef extract for each 100 g of sediment. This mixture was centrifuged at 1500revmin -~ for 10min. The supernatant was adjusted to pH 3.5 with 1 M glycine, pH 1.5. The floc that formed was sedimented by centrifugation and the sedimented material was eluted with 0.05 M glycine at pH 9.5 (Katzenelson et al., 1976).
Chemical analyses Concentrations of five heavy metals (lead, copper, cadmium, mercury and arsenic) in the water and fish tissue were determined by flame atomic adsorption (APHA, 1980). Fish flesh and water were also monitored for the pesticides aldrin, dieldron, endrin, mires, DDT, toxaphene and kepone, and for potychlorinated biphenyls (PCB) using solvent extraction concentration followed by electron capture gas chromatography (APHA, 1980). Em'ironmental conditions in ponds Measurement of other water quality parameters was done on the influent wastewater and on the effluent from each pond. These measurements were performed according to standard methods (APHA, 1980). The water temperature in the ponds during the microbiological sampling period ranged from 30°C in August to 9 C in December. Average values for other selected variables are listed in Table 1. Statistical procedures For statistical analysis, concentrations of indicator bac-
1751
6
[ ] Fecal coliforms
5
[ ] Fecal streptococci
0
o
I I nfl uent
Pond 2
Pond 4
Fig. 2. Concentrations of indicator bacteria in pond water. teria in fish, water and sediment were transformed by taking the logarithm of the number per 100 g. Paired t-tests were used to detect significant differences between bacterial levels in the fish and in the surrounding water and sediment and between fish from Pond 4 and Pond 6. Correlation coefficients and significance levels were calculated using the Statistical Package for the Social Sciences (SPSS). The SPSS was also used to perform a multiple linear regression analysis to look for good predictors of bacterial levels in fish. Other confidence intervals were calculated using the t-distribution. RESULTS
Bacteriological results Levels o f indicator bacteria decreased steadily t h r o u g h the system o f ponds. Figure 2 illustrates the decrease in c o n c e n t r a t i o n s o f indicator bacteria in water going from influent to P o n d 6. There was a total decrease of at least 2.5 log~0 (99.7%) for F C a n d a total decrease of 2.3 logt0 (99.5%) for FS, based o n the average c o n c e n t r a t i o n in each pond. The decrease was not substantially different from p o n d to pond. There was a n average 62% decrease per p o n d for F C a n d a n average 58% decrease per p o n d for FS. Based on a calculated retention time of 12 days per p o n d this represented inactivation rates of 0.034 a n d 0.032 log~0 day ~, respectively. Bacterial c o n c e n t r a t i o n s in the sediments followed a different p a t t e r n t h a n in the overlying waters (Fig. 3). There was a substantial decrease in F C from P o n d 2 sediments to P o n d 6 sediments. A cumulative decrease of 2.71og10 was observed for FC. This represents a n average 79% decrease per p o n d for F C in the sediments of the last four ponds. The concentration of FS in the p o n d sediments decreased by only 0.4 log~0 from P o n d 2 to P o n d 6. The decrease of FS
Table 1. Environmental conditions in ponds
Pond
pH
Influent 1 2 3 4 5 6
6.5 7.6 7.9 8.4 8.2 8.2 8.3
*Dissolved oxygen. ?Total suspend solids.
Average of monthly samples Jan. 1979-Jan. 1981 DO* TSSt 5-day BOD Ammonia N (mgl 1) (mg i - i ) (mgl -I) [mgl i) 0 2.1 2.6 6.9 5.7 6.9 7.4
97 81 47 47 25 21 20
Pond 6
251 63 30 24 15 11 10
24 6.2 5.0 3.2 2.4 1.1 1.2
PO 4 P (mgl -I)
NO 3 N (mgl i)
22 3.9 3,0 2.6 2.3 2.0 2.0
0 0.018 0.026 0.071 0.14 0.38 0.33
1752
THOMASW. HEJKALet al. 6 5
[]
-
a n d 10 4 of each type of bacteria per 100 g in the digestive tract and skin. The concentrations of FC in the gut and of FS in the gut and skin were correlated with the concentration of FS in the surrounding water (Table 3). FS levels in sediment also showed a weak positive correlation with FS levels in fish flesh. Other than this, concentrations of FC in the water and sediment and FS in the sediment were not significantly correlated with bacterial levels in the fish. Concentrations in the three types of fish tissue were generally correlated with each other, and concentrations of FC showed a strong positive correlation with concentrations of FS in most types of samples (correlation coefficients not shown). Table 4 shows the levels of bacteria which were detected in the fish flesh. Two methods were used for sampling the fish muscle. The samples in August and September were taken by a normal filet procedure using a decontaminated filet knife. Samples taken during these two months yielded sporadically high levels of FC and FS in the muscle tissue, probably due to contamination by bacteria from the fish skin. Beginning in October, all muscle samples were taken aseptically to avoid contamination from the skin. Three of nine samples of muscle tissue obtained from October through December were positive for either FC or FS at low levels. The highest level of indicator bacteria in fish muscle sampled aseptically was 25 MPN (most probable number) of FS/100 g from a fish which had a concentration of 1.4 × 105 MPN of FS/100 g in the digestive tract. Using a normal filet procedure, contamination occurred in 8 of 12 samples with levels up to 22,000 MPN of FS/100 g. Salmonella spp was detected in 2 of 4 influent samples at levels of 0.4 and 2.3 MPN/100 ml using dulcitol selenite enrichment. Salmonella spp was also isolated from a single water sample from Pond 2 in December. No Salmonella was isolated from any of the other pond water, sediment or fish samples.
Fecal coliforms
[ ] F e c a l streptococci
4.o
3
,/,
-
g ..J
fL
7/, ~"L "//, f,,'/, "//, F/, sis
I --
Pond2
Pond4
Pond6
Fig. 3. Concentrations of indictor bacteria in pond sediments.
in the sediments from pond to pond was substantially less than the decrease of FS in the water. Inactivation rates could not be calculated for the sediments since retention time in the sediments was unknown. The concentrations of FC and FS in the fish guts were on the average greater than in the surrounding water and sediment (Table 2; Figs 2 and 3). Mean concentrations of FC and FS on the fish skin were lower than in the gut. Mean concentrations of both FC and FS in the gut were correlated with concentrations of FS on the skin with r = 0.607 and 0.825, respectively. On the average, fish in Pond 6 had levels of F C and FS 54~ lower than fish from Pond 4. However, due to the variability between samples, this decrease was not considered significant (P < 0.14), and fish in the last pond still averaged between 102 Table 2. Levels of fecal coliforms (FC) and fecal streptococci (FS) in fish gut and skin samples Average concentration (Iog~0/100g)* FC Gut Skin Out Pond 4 Pond 5 Pond 6
3.07 3.01 2.73
2.00 2.43 2.21
FS Skin
4.36 4.03 3.75
3.57 3.47 2.95
*Averages of five monthly samples. Table 3. Correlation coefficients between levels of fecal coliforms (FC) and fecal streptococci (FS) in fish and in water and sediment*
Virological results
Variable
FC water
FC gut FC skin FC flesh
0.559 0.019 0.357
0.097 -0.423 0.003
0.712t -0.029 0.330
FS gut FS skin FS flesh
0.307 0.363 0.455
0.280 0.525 0.537
0.646]" 0.691t 0.375
Six of the 90 samples tested for enteric viruses yielded at least 1 PFU (plaque-forming unit) on the BGM monolayers (Table 5). Three of five influent samples were positive at low levels. Only a portion of each influent sample was tested for virus because of the necessity of dilution to reduce toxicity of these concentrates. The concentrations based on the
FC sediment
FS water
FS sediment -0.251 -0.217 0.099 -0.236 0.370 0.630t
*Data from five monthly samples from Ponds 4 and 6. tSignificant at P < 0.05.
Table 4. Concentrations of fecal coliforms (FC) and fecal streptococci (FS) in fish flesh M P N / 1 0 0 g fish flesh Pond 5
Pond 4
Pond 6
Month
FC
FS
FC
FS
August* September* October November December
<30 230 < 11 II <11
140 80 25 <11 <15
<30 430 < 11 <11 <11
140 22,000 < 11 <11 <15
FC
FS
40 < 30 < 6.6 <11 <11
860 < 60 15 <11 <15
*August and September samples were taken by a normal filet procedure with possible contamination from the skin. All other flesh samples were taken aseptically. Data from pooled samples taken from several locations from single fish.
Evaluation of a wastewater-aquaculture system Table 5. Samples yielding plaques on BGM monolayers Sample Influent lnfluent Influent Pond 2 sediment Pond 4 sediment Pond 2 water
September
2
Estimated concentrationt 20PFU1 -I 15PFUI-' 7.5PFU1-I 2VFU/50Og
November
1
1 PFU]500 g
December
1
Month
Total PFU counted*
August November December
5 3 2
0.05PFUI-'
*Plaques could not be confirmed as viral. ~Dilution factors varied for influent samples.
number of plaques counted ranged from 7.5 to 20 P F U 1-~. Two of 15 sediment samples and one water sample from Pond 2 also yielded 1-2 P F U per sample of 500 g or 201. A single P F U was detected in the water concentrate from Pond 2 in December. All other pond water samples were negative for virus. No viruses were detected in any of the 45 fish samples processed. Attempts were made to isolate and identify the virus from each plaque. The plaques recorded in Table 5 produced CPE when inoculated onto BGM monolayers under liquid overlay. However, attempts at additional passages and identification were unsuccessful. Thus, although the original plaques were virus-like, it is possible that they were caused by nonviral agents,
1753
higher than in the composite sample of fish prior to stocking the ponds (0.063 ppm) and was 70-fold higher than the average concentration of mercury in pond water from which the fish were taken ( 0 . 0 0 5 ppm). A considerable bioconcentration of copper was also observed with an average of 3.58 ppm in the fish flesh and <0.01 ppm in the pond water. All fish and water samples, including influent, tested for PCB and pesticides were negative. Other water quality parameters The system substantially improved many of the usual indicators of wastewater quality. Through the series of six ponds, the 5-day BOD decreased by 96~, total suspended solids by 79~, ammonia by 95~ and reactive phosphorous by 91~ (Table 1).
DISCUSSION Indicator bacteria
The consistent decline of FC and FS levels in pond water indicated that these bacteria were being removed by a combination of natural inactivation processes, adsorption to sediments and uptake by fish. The sediment may serve as a sink for some microorganisms, for example, FS which showed little decrease in concentration in the sediments going from Pond 2 to Pond 6. This is most reasonably explained Chemical contaminants by extended survival of these bacteria in sediments, Measurable levels of lead, copper and mercury although other factors such as growth of FS in the were found in one or more fish grown in the waste- sediments or more efficient adsorption to sediments water ponds (Table 6). Influent concentrations in the downstream ponds may be involved. averaged < 0.053 mg Cu 1-1, <0.067 mg Pb I-~ and The levels of bacteria in the fish also showed little < 0.0036 mg Hg 1 i. Cadmium and arsenic were not decrease through the final 3 ponds. Thus the fish may detected in any of the fish flesh samples (detection also serve as sinks for bacteria in the wastewater limit = 0.02 ppm) although arsenic was detected in 7 ponds. The concentrations of bacteria in the gut were of 19 water samples (detection limit = 0.002 ppm) highly correlated with the concentrations on the skin, from the influent and ponds at concentrations up to whereas correlations between bacterial levels in fish 0.013 ppm. and in water or sediment were generally weak. The Three of 11 fish contained higher than acceptable bacterial levels in water and sediment, therefore, were levels of mercury in the edible portion. The average not good predictors of bacterial levels in fish in the level of mercury in fish taken from the wastewater range found in our study. The lack of good corponds was 0.350 +__0.274 (95~o C.I.) ppm which was relation between bacterial levels in the water and fish may be explained if the fish accumulated bacteria to a saturation level which may have been reached even Table 6. Concentrations of selected heavy metals in fish flesh* in the final pond. However, if this were true, fish from Copper Lead Mercury each pond would be expected to contain approxiSamplesite (ppm) + ~ppm> tppm> mately equal concentrations of indicator bacteria Before stocking 0.77 0.57 0.063 which was not the case. The apparent cause of the 0.45 Pond 3 3.0 <0.5 0.54 <0.5 0.079 lack of correlation was the large variability between 0.60 <0.5 0.64~bacterial levels in individual fish. This could be 0.099 Pond 4 30.0 1.7t
1754
THOMAS W. HEJKAL et al.
The lack of a good correlation between bacteria in the water and bacteria in the fish does not imply that monitoring of indicator bacteria in water would be useless. Based on these results, we expect that if indicator bacteria and presumably pathogens were present in the water, silver and bighead carp would accumulate them in their digestive tracts at levels as high or higher than the levels in the water. Since the muscle tissue is the critical portion of the fish if it is to be used for human consumption, bacterial levels in the fish muscle were a major concern. Even when levels of bacteria exceeded 105/100 g in the fish guts, very little penetrated into the fish muscle tissue. However, when the muscle tissue was sampled using normal filet procedures, contamination of the muscle occurred in 8 of 12 samples. The conclusion is that while the fish do not accumulate bacteria in the muscle tissue, contamination of the muscle tissue during processing is difficult to avoid. Viruses The sewage entering the Benton fish ponds was atypical from a virological standpoint. The levels of virus in the sewage were much lower than would be expected for untreated sewage from a larger and more diverse community. For example, concentrations in raw sewage from treatment plants in St Petersburg, Florida, averaged 90 P F U 1-~ and, at a larger treatment plant in Tampa, Florida, concentrations of over 2000 P F U 1-~ were found (Hejkal et al., 1981). The sewage entering Benton ponds had an average concentration of < 9 P F U I ~ for the 5 samples tested. The low levels of virus in the sewage in this study can be attributed to the population from which the sewage is derived. The population consists of approx. 1000 persons residing fulltime at the Benton Services Center with an additional 1000 fulltime employees. Infants and young children contribute most of the enteric viruses to the wastewater of any given community, since they are the most susceptible age group to infection by these viruses (Fox and Hall, 1980). The lack of infants and young children in the population at the Benton Services Center explains the low levels of enteric viruses in the sewage from the center. Additionally, half of the population contributing to the waste load are employees who do not live at the Center. These persons would be less likely to come to work if they had an enteric viral infection and therefore would not be likely to contribute to the viral contamination of the watewater. Because of the low levels of virus found in the influent, the results cannot be extrapolated to make conclusions or predictions about the survival and transport of viruses in other fish pond systems that may have a much higher input of viruses. The lack of virus isolated from the fish and pond water in this study does not preclude the possibility of viruses surviving in the fish ponds and being accumulated by
the fish if the initial levels of virus were higher. Since relatively high levels of FC and FS were found in the ponds and fish, it is likely that viruses would also be present if the input rate were higher because viruses generally survive inactivation processes better than do indicator bacteria (Scarpino et al., 1972; Shuval et al., 1971). Chemical contaminants The accumulation of persistent chemical contaminants in the fish poses an even greater public health threat than microbiological contaminants since decontamination is not feasible. Bioaccumulation of mercury and other contaminants is a known hazard (Mclntyre and Mills, 1975). The levels of mercury present in the fish taken from the Benton ponds demonstrate the necessity to be extremely cautious in the utilization of fish grown in wastewater. Although the levels found in the fish flesh were not excessive in this study, the fact that concentrations of mercury were much higher in the fish than in the water reemphasizes the need for controls on the quality of wastewater used for aquaculture purposes. Efficiency o f treatment and environmental factors The efficiency of the system in treating the wastewater is also important. Previous studies have shown the fish pond system to be at least as efficient as a system of oxidation ponds without fish (Henderson, 1979). The system used in the present study was effective in producing an effluent which met standards for secondary wastewater treatment. The data were insufficient to assess seasonal variations or to draw conclusions about the effects of variables such as temperature, pH and ammonia concentrations in this system. These factors influence the survival of pathogenic bacteria and viruses in water and can also affect adsorption to sediments. There would probably be seasonal and short-term fluctuations in the survival of pathogens and in rates of adsorption to sediments and uptake by fish. For example, inactivation of pathogens may be enhanced in warmer months since high temperatures are detrimental to pathogens. However, in this study there was no evident relationship between temperature and bacterial or viral survival. Interactions between temperature and other variables will need to be determined before the overall effect can be assessed. CONCLUSIONS
Aquaculture-wastewater treatment systems are potentially valuable alternatives to conventional sewage treatment plants. This study shows that while concentrations of indicator microorganisms are reduced by as much as 99.7% they are not eliminated by the fish ponds. Significantly, neither do conventional activated sludge or trickling filter processes eliminate indicator organisms, pathogens or toxic chemicals. It is clear that fish or other organisms
Evaluation of a wastewater-aquaculture system raised in wastewater have a high probability of becoming contaminated with bacteria, viruses and toxic chemicals and appropriate cautions need to be taken when these organisms are harvested and utilized for human or animal consumption. However, the public health risk may be no greater under these carefully controlled conditions than the risk from the uncontrolled harvesting of fish from waters that are contaminated by effluents from conventional sewage treatment plants. Acknowledgements--This research was supported by a grant from the U.S. Environmental Protection Agency (R8054301) to the Arkansas Game and Fish Commission. The assistance of Catherine Castefiada in the bacteriological and virological analyses is gratefully acknowledged. REFERENCES
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