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The bacteriological quality of water and fish of a pond system for the treatment of cattle feedlot effluent
Agricultural Wastes 9 (1984) 1-15
The Bacteriological Quality of Water and Fish of a Pond System for the Treatment of Cattle Feedlot Effluent T. E. Cloete, D. F. Toerien & A. J. H. Pieterse* Institute for Environmental Sciences, University of the Orange Free State, PO Box 339, Bloemfontein, South Africa 9300
ABSTRACT A cattle feedlot waste system consisting of a high-rate algal pond and a fish pond was regularly sampled for bacteriological analyses. High numbers of aerobic and anaerobic bacteria and potential pathogens (e.g. Salmonella spp.) were present in the waste water. The waste treatment system reduced all bacterial groups by more than 99"6 %. The skin, gills and intestines of the waste-grown fish housed large numbers of bacteria, including potential pathogens. However, similar bacterial numbers, including potential pathogens, were associated with the skins, gills and intestines of naturally-grown fish, which suggests that the health risk involved in the comsumption of waste-grown fish might not be substantially different to that of natural fish populations. In both cases the tissues and blood appeared to be sterile, which would contribute to a much reduced health risk.
INTRODUCTION Taiganides (1978) considered animal wastes to be resources out of place. Edwards (1980) pointed out the large potential o f such wastes for fish production. However, the microbial flora of fish directly reflect the microbiological condition of the water from which they are taken * Present address: Department of Botany, Universityof the Orange Free State, PO Box 339, Bloemfontein, South Africa 9300. 1
Agricultural Wastes 0141-4607/84/$03-00 © Elsevier Applied Science Publishers Ltd, England, 1984. Printed in Great Britain
2
T.E. Cloete, D. F. Toerien, A. J. H. Pieterse
(Guelin, 1962). Therefore the utilisation of animal or other wastes for fish production could result in a health risk because of the presence of pathogens in waste water (e.g. Janssen, 1970; Bryan, 1977; Hojovec, 1977; Wolfarth, 1978; Allen et al., 1979). In addition the high organic load, microbial activity and biological oxygen demand of waste water also create the risk of transmittance of fish diseases and parasites (Muttamara & Wattayakom, 1978; Schoonbee et al., 1979; Allen & Gearheart, 1980). Knowledge about the bacteriological quality of the water and fish of waste utilisation systems is needed to assess the associated health risks. The norm for assessing this health risk must be the bacteriological quality of naturally-grown or pond-grown (without waste addition) fish. An integrated cattle feedlot waste treatment system, which includes the production of fish, is operated by Soetvelde Farms Ltd of Vereeniging, South Africa. It consists of hydraulic removal of manure from concretefloored pens, the separation of the solids and slurry of the effluent, and the treatment of the slurry in a high-rate algal pond (HRAP) followed by passage through a fish pond. Water from the fish pond is used to move the manure (Cloete et al., 1982). To provide information on the bacteriological quality of the water in the different ponds of the waste treatment system, water samples were analysed at monthly intervals over the period March 1979 to June 1980. In addition, the bacteriological quality of different body parts of silver carp (Hypothalrnichthys molitrix), grass carp (Ctenopharyngodon idella) and common carp (Cyprinus carpio) harvested from the waste treatment system, was compared with that of common carp from a natural fish population.
MATERIALS A N D METHODS
Sampling procedures Water samples were collected at seven different sampling points in the system (Fig. 1). The samples were taken during monthly intervals during the period March 1979 to June 1980. The samples were collected in sterile 25 ml McCartney bottles and kept at 0 °C during transportation (360 km). Analyses were started within 24 h after sampling. In September 1979, fish (sized 400 to 500g each) were netted and transported live to the laboratory in a container holding 200 litres of fish
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Fig. 1. Schematicrepresentation of the Soetveldecattle feedlot system. A, confinement unit; B, concrete platform where particulate wastes are separated from slurried waste; C, storage pond; D, high-rate algal pond (HRAP); E, fish pond. Cage aerator is between sampling points 3 and 4. Bacteriologicalsamples were taken at points 1 (screen water), 2 (storage pond), 4 (below stream of cage aerator), 3 (upstream of cage aerator), 5 (outflow of HRAP), 6 (fish pond inflow) and 8 (fish pond outflow). pond water. On arrival the fish were killed, immediately dissected and bacterial enumerations performed. The same procedure was followed for fish collected from Wuras Dam, a freshwater impoundment. In September 1980, fish were again harvested from the waste treatment system and immediately dissected. The samples for bacteriological analyses were prepared on site and were then transported in an ice-box at 0 °C to the laboratory, where the analyses were completed.
Sample pretreatment Appropriate dilutions of the water samples were made using 9ml quantities of sterile water in test tubes. Analyses of the fish were done on different body parts, i.e. gill surfaces, skin, blood, tissue and intestinal contents. Sterile swabs were used to sample the gill surfaces and skin (2 cmZ). Sterile swabs were prepared in 10 ml sterile distilled water. Blood samples were obtained by separating the tail of the fish with a sterile scalpel, inserting the needle of a sterile 5 ml syringe into the caudal vein and withdrawing I ml of blood. Fish tissue samples were collected by either dissection with a sterile scalpel (September 1979) or by using a
4
T.E. Cloete, D. F. Toerien, A. J. H. Pieterse
sterile corkscrew after removing the scales with the aid of a sterile scalpel. The samples were transferred to sterile homogeniser tubes, weighed and homogenised. Intestinal samples were obtained by removing the intestines with a sterile scalpel and transferring them to a jar containing 50 ml sterile water. After shaking vigorously for 5 rain, a dilution series was prepared. Bacteriological analyses of intestinal contents were done only in September 1979 and analyses of blood in September 1980.
Bacteriological analyses Total viable aerobic and facultative anaerobic bacteria (hereafter referred to as total aerobic bacteria) were enumerated on Bacto Nutrient Agar by incubating at 37 °C for 48 h. Presumptive salmonellae were enumerated on Difco Bismuth Sulphite Agar by incubating at 37 °C for 48 h. Black colonies with a metallic sheen were counted. Coliform bacteria were enumerated with the MPN technique using MacConkey Broth (Oxoid) and incubating at 37°C for 24h. Presumptive clostridia (hereafter referred to as total anaerobic bacteria) were enumerated with the MPN technique using Reinforced Clostridial Medium (Oxoid) and incubating at 37 °C for 24 h. The basic procedures followed were as described by Orland (1965). Twenty randomly isolated presumptive Salmonella colonies on Bismuth Sulphite Agar were purified by repeated streaking on Nutrient Agar. These isolates were identified by the Gram stain and the use of the API-20E microtube system (e.g. Smith et al., 1972). RESULTS
Water quality The variations of the total aerobic bacteria, total anaerobic bacteria, coliforms and presumptive salmonellae for different stages of the system and with time are presented in Figs 2-5. Total aerobic bacteria (Fig. 2) The total aerobic bacterial numbers in the screen water did not show any distinct seasonal pattern and ranged from 1 x 10 6 t o 38 × 106m1-1 (Fig. 2(A)). In the storage pond these numbers were apparently higher (about 30 × 106 ml- 1) in the period August to January (spring-summer) than in autumn-winter (March to July) (Fig. 2(A)).
Bacteria in a feedlot effluent fish pond
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I I I J J A 1979
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1980
Fig. 2. Aerobic bacterial numbers in the waste treatment system during the study period. (A) H screen water, H storage pond; (B) HRAP, H below stream of cage aerator, O O upstream of cage aerator, [] [ ] outflow; (C) fish pond, ~ inflow, O O outflow.
The total aerobic bacteria in the H R A P tended to be higher in the outflow than in the vicinity of the cage aerator (Fig. 2(B)). Higher numbers also occurred in the s p r i n g - s u m m e r - a u t u m n period than in winter. Maximum numbers were about 8 x 106m1-1 and minimum numbers were about 0.2 x 106 ml-1. The fish pond did not show any distinct seasonal pattern for total aerobic bacteria, but the numbers in the inflow were about ten thousand times higher than at the outflow (Fig. 2(C)).
Total anaerobic bacteria (Fig. 3) The total anaerobic bacterial numbers in the screen water and storage pond were lower in winter (June to August) than the rest of the year (Fig. 3(A)). The numbers varied from 10 x 106 to 150 x 106 ml-1. In the H R A P the total anaerobic numbers were appreciably lower (0.5 to 1 "8 x
6
T.E. Cloete, D. F. Toerien, A. J. H. Pieterse 15
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Fig. 3. Anaerobic bacterial numbers in the waste treatment system during the study period. (A) n screen water, H storage pond; (B)HRAP, H below stream of cage aerator, O O upstream of cage aerator, ~ outflow; (C)fish pond, H inflow, O O outflow. 10 6 ml - 1) than those of the screen water and storage pond and no distinct
seasonal pattern was observed, although the mid-winter ( J u n e - J u l y ) values were very low (0.5 to 1.5 x 10Sm1-1) (Fig. 3(B)). The total anaerobic bacterial numbers of the fish pond ranged from 1.5 x 105 to 10 x 105 ml-1 (Fig. 3(C)). The inflow values were somewhat higher than those of the outflow, but not as markedly so as for the total aerobic bacteria. Coliform bacteria (Fig. 4) N o seasonal pattern was evident for the coliforms in the screen water and the storage pond and the numbers varied from 0.3 × 10 6 to 6"5 × 10 6 ml - 1 (Fig. 4(A)). In the H R A P the numbers were appreciably lower than in the screen water and storage pond (0-2 x 104 to 8.3 × 104m1-1), and lower numbers occurred in winter (June to August) and higher numbers in
Bacteria in a feedlot effluent fish pond
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Fig. 4.
Numbers of coliforms in the waste treatment system during the study period. (A) screen water, I I - - - - U storage pond; (B) H R A P , H below stream of cage aerator, O O upstream of cage aerator, [ ] [] outflow; (C) fish pond, 4 - - - - - - 4 inflow, ~ outflow,
summer (November to December) (Fig. 4(B)). The coliform numbers in the fish pond were still lower (2.5 × 102 to 17 × 102 ml-1) and the inflow usually had somewhat higher numbers than the outflow (Fig. 4(C)).
Presumptive salmonellae (Fig. 5) In the storage pond and screen water the numbers were highest during the late winter (July-September 1979), but were lower in M a r c h - M a y 1979 and variable in M a r c h - M a y 1980 and ranged from 0.2 × 10 to 23 x 104 (Fig. 5(A)). In the H R A P the numbers ranged from 20 to 800 m l - ~ and showed no distinct seasonal pattern (Fig. 5(B)). There was also no distinct pattern in the fish pond where the numbers were from about 80 to 520 ml - 1 (Fig. 5(C)).
8
T . E . Cloete, D. F. Toerien, A. J. H. Pieterse
4 2 1 E
8
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Fig. 5, Numbers of salmonellae in the waste treatment system during the study period. (A) H screen water, H storage pond; (B) HRAP, H below stream of cage aerator, O O upstream of cage aerator, I-q[] outflow; (c) fish pond,
H
inflow, O
O outflow.
Reduction in bacterial numbers An examination of the average bacterial numbers at the various stages of the treatment system (Table l) showed that an overall reduction exceeding 99.6 % was achieved in the waste treatment system. Whereas the total aerobic bacteria and the coliforms increased in numbers between the screen water and the storage pond, the total anaerobic bacteria and presumptive salmonellae decreased throughout the waste treatment system. A comparison of the ratios between the different bacterial groups in the system (Table 2) reflects the rates at which the groups were either stimulated or eliminated by conditions in the different stages of the waste treatment system. High ratios of aerobic bacteria to the other bacterial groups in the HRAP indicate a more rapid elimination of anaerobes,
7x 70 x 1× 1-8 x
106 106 106 10 s
Screen water
12 × 106 55 x 106 4 x 106 1.8 x 104
Storage pond
2.1 3.3 2 2
x x x x
106 105 104 102
at cage aerator x 106 x l0 s x 104 × 103
outflow 4 6 3-5 2
HRA P
3.5 3,4 2 2.2
x 106 x l0 s × 103 x 102
inflow 2-5 2.8 1.8 2
x 104 x l0 s x 103 × 102
outflow
Fish pond
Coliforms
Total anaerobic
0.10 0-22 6,36 6.67 1.03 0.09
Screen water Storage pond HRAP inflow HRAP outflow Fish pond inflow Fish pond outflow
7.0 3-0 105.0 114.3 175,0 13.9
Total aerobic
Total aerobic
Stage of treatment system 38.9 666.7 10 500.0 20 000.0 1 590.9 125-0
Salmonellae
Total aerobic
70-0 13.8 16.5 17.1 170.0 155.6
Coliforms
Total anaerobic
388.8 3 055.6 165.0 300.0 154.5 1400.0
Salmonellae
Total anaerobic
99.64 99.60 99.98 99.89
% Overall reduction
5.6 222,2 100.0 17,5 9-1 9.0
Salmonellae
Coliforms
TABLE 2 Ratios of the Different Bacterial Groups in the Treatment System (Based on Average Bacterial Numbers)
Total aerobic bacteria Total anaerobic bacteria Coliforms Salmonellae
Bac~r~
TABLEI ComparisonoftheAverageBacterialNum~rsattheVariousSta~sofTreatment
10
T. E. Cloete, D. F. Toerien, A. J. H. Pieterse
coliforms and salmonellae by the conditions in the HRAP than aerobes (since there was a decrease in aerobes between storage pond and the HRAP, the high ratios of aerobes to the other groups are not a result of increased numbers of aerobic bacteria). The reduction in the ratios of aerobes to the other groups in the fish pond (except for the inflow of the fish pond) suggests a more rapid rate of elimination of aerobes other than coliforms and salmonellae in the fish pond. This could indicate a higher grazing pressure by the organisms living in the fish pond on these aerobes. The anaerobic bacterial numbers were drastically reduced between the storage pond and the HRAP, suggesting that the oxygenated conditions which occurred in the H R A P (Cloete et al., 1982) could have been the cause of an elimination of obligate anaerobes. On the other hand fairly high numbers of apparently anaerobic bacteria were recorded in the H R A P and fish pond (Table 1). If the reduction in numbers between the storage pond and the HRAP was due to the elimination of obligate anaerobes, then the anaerobes detected in the HRAP must have been facultative anaerobes or bacterial spores able to survive oxygenated conditions. The ratios of anaerobes to coliforms were relatively constant in the screen water, the storage pond and the HRAP, but increased in the fish pond. A more rapid rate of coliform elimination in the fish pond was the reason for this. The anaerobe/salmonellae ratio varied widely and did not show a distinct pattern. Salmonellae were eliminated more rapidly than coliforms in the storage pond and the HRAP (higher coliform/salmonellae ratios), but less rapidly in the fish pond (lower coliform/salmonellae ratios). Selective grazing by bacterivorous organisms and differential behaviour towards environmental conditions in the different ponds probably contributed to this phenomenon. The above illustrates that the dynamics of the bacterial population and groups in the system are complex and probably influenced by factors such as state of oxygenation, selective grazing, etc. Quality of the fish Some significant facts emerged from a comparison of the bacterial numbers of different body parts of waste-grown and natural fish (Table 3). The first is that potential pathogens in the form of salmonellae were present in all fish, including those derived from a natural population.
N.D.
ND.
8"5 x 108
Number/ml blood
Number/g tissue
Number/intestine
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N,D. = not determined.
.
.
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.
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.
N,D.
<102
<10 z
<102
<50
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Common carp
Cyprinus carpio
Natural population
0 3 5 x 10 ~ 0 ' 8 x 10 ~
.
Number/gill
Number/cm 2 skin
Body part of ~
.
N.D.
<102
<10 z
<102
<50
7"5 x 106
<10 z
I x 103
to
<102
<50 tO 1'1 ×104
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N,D,
N.D.
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2"2 x I0"
2"0x 106
<102
<10 z
<10 /
<50 tO I ' 2 X 10 ~
Hypothalrnichthys molitrix
Silt'er carp
WaMe treatment syslem
2"5 x 102
<102
<102
<102
<50 tO 50
~
N.D.
<102
<102
3X104
1 ' 5 x 104
~ .~
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N.D,
N.D.
N.D.
N.D.
N,D.
g .~
N.D,
<10 z
<102
3xlO 3
1"5 x 10 3
~ .~
Ctenopharyn$odon idella
TABLE 3 The Bacteriological Quality of Carp from the Waste Treatment System and from a Natural Population
N.D,
<10 a
<102
3 . 2 x 10 ~
1"6 x 10 2
12
T. E. Cloete, D. F. Toerien, A. J. H. Pieterse
The second is that these pathogenic bacteria were not detected in the tissues and blood of the waste-grown fish; in fact no bacteria were detected in the tissues and blood. However, the sensitivity of the analysis only allowed the detection of bacterial concentrations above 102/sample and, although it is likely, we cannot state unequivocally that the tissues and blood were sterile. The third is that very high bacterial numbers were associated with the intestines and lower (but still high) numbers with the skin and gills of the fish. This was equally true for the naturally-grown fish and the waste-grown fish. Identification of 20 isolates of presumptive salmonellae isolated from the waste-grown fish, indicated that only three isolates in fact belonged to the genus Salmonella. This suggests that the use of Bismuth Sulphite Agar overestimated the presence of Salmonella spp., but nevertheless, that salmonellae were present in or on the fish. The bacterial flora offish with different feeding habits may show some differences. For instance, the total aerobic bacterial numbers in the whole intestines of the bottom-feeding common carp (Cyprinus carpio) from both waste-grown and natural fish populations were substantially higher than those of the filter-feeding silver carp (Hypothalmichthys molitrix).
DISCUSSION The growing realisation that natural resources are finite has in the past decade led to an increasing interest in western countries in recycling of natural resources. In eastern countries, particularly in Asia, the recycling of organic wastes into agriculture and fish production has been practised for centuries (Edwards, 1980). Acceptance and use of these practices in many countries will depend, amongst other things, on evaluations of the associated health risks, because of the presence of pathogens in domestic and animal wastes. Waste treatment or recycling systems will therefore have to achieve substantial improvements in the pathogen quality of waste waters and yield products with a low health risk. The Soetvelde waste treatment system which incorporates hydraulic manure removal, a high-rate algal pond and a fish pond, is fairly sophisticated from an agricultural point of view, but from a waste treatment point of view it represents a fairly low-level technology. In semiarid countries, such as South Africa, there is an urgent need to balance environmental protection with the provision of a balanced diet for a
Bacteria in a feedlot eJfluent fish pond
13
rapidly increasing population. Feedlots may play an ever increasing role in providing animals for slaughter and it is possible that the Soetvelde waste treatment system is an early example of waste treatment systems needed in the future for cattle feedlot operations. Pathogenic bacteria, in the form of Salmonella spp. (potential human pathogens) and Clostridium spp. (potential cattle pathogens) occurred in the feedlot waste waters (Table 1). Seasonal trends of increases and decreases were evident for some, e.g. total aerobic and coliform bacteria in the HRAP, but not all bacterial groups, e.g. salmonellae in the fish pond. The average reduction in numbers of all bacterial groups in the waste treatment system exceeded 99.6 % (Table 1) and was as good as that (99.6 to 99.8 %) reported for a series of sewage-maturation-pond systems (Drews, 1966). The health risks associated with the feedlot waste water were therefore greatly reduced by the waste treatment system. Fish raised in the waste treatment system will however be exposed to the presence of pathogens, and pathogens were indeed recorded in various body parts of the waste-grown fish (Table 3). An unexpected finding was the presence of pathogens in or on c o m m o n carp from a natural population (Table 3). The presence of such bacteria in or on fish from natural populations (also reported by Liston (1979)) which must serve as the norm for assessing the disease risk involved with waste-grown fish, indicates that assessments will have to be done carefully and thoroughly, and should include fish from natural populations as controls, The microbiology of fish, especially marine fish of commercial value, has been studied for a long time (Liston, 1979). The early researchers showed that muscle tissues in healthy fish are sterile, and our results (Table 3) and Allen et al. (1979) suggest the same for the waste-grown fish. We also found no evidence that the blood of the fish was contaminated with bacteria (Table 3). Large populations of bacteria were associated with the external surfaces, gills and intestines of marine fish by Liston (1979), a condition which was also observed in this study. The skin of marine fish can house bacterial populations of 102 to 10 6 c m - 2 , the gills similar or higher numbers, and the intestines from a few to 108 g - 1 depending on whether the fish are feeding (Liston, 1979). The skins of the waste-grown fish contained from 450 to 0.7 x 105 aerobes and 2.3 x 105 anaerobes cm -2, the gills from < 102 to 1.4 x 105 per gill and the intestines from 7.5 x 10 6 t o 3"0 x 10 8 bacteria/intestine. Naturally-grown c o m m o n carp contained bacterial numbers of 0-3 × 105cm -2 of skin, 13 x 102 per gill and 8-5 x 10 s per intestine
14
T. E. Cloete, D. F. Toerien, A. J. H. Pieterse
respectively (Table 3). The results suggest the numbers of the microbial flora of the waste-grown fish did not differ substantially from either naturally-grown freshwater fish or marine fish. Despite the potential of fish grown on human wastes to transmit disease, Bryan (1977) found only two references in a worldwide review of medical and engineering literature in which illness was caused by fish contaminated with human wastes. Edwards (1980) reported that no disease problems have been reported with fish grown on animal wastes, but that prior to 1949 the use of human wastes in fish ponds in China led to the widespread occurrence of diseases of insanitation. In most cases where fish are grown on animal wastes, the latter material is added directly to the fish ponds. Saigal (1972) suggested that fish grown in ponds receiving human waste do not feed directly on the waste but on the natural food, e.g. plankton, that develops as a result of the fertilising effect of the waste, but Vaas & Sachlan (1956) reported the presence of human faeces in the intestines of fish cultured in cages in ponds receiving human wastes. In the Soetvelde waste treatment system, the H R A P ensures that fish are not in contact with untreated animal wastes and that a substantial attenuation in pathogens has occurred before the fish ponds. This may be the reason why the bacteriological quality of the waste-grown fish did not differ substantially from that of naturally-grown fish. No excessive health risks might be associated with the consumption of the Soetvelde fish, but additional scientific evaluations are still needed to fully assess the health risks involved with consumption of waste-grown fish.
ACKNOWLEDGEMENTS The financial assistance of the Anglo-American Research Laboratories and Soetvelde Farms (Pty) Ltd, and the help of Mr M. Downes, officers of the Division of Nature Conservation, Transvaal Provincial Administration and Dr I.G. Gaigher in obtaining fish samples are gratefully acknowledged.
REFERENCES Allen, G. H., Busch, R. A. & Motton, A. W. (1979). Preliminary bacteriological studies on wastewater-fertilized marine fish ponds, Humboldt Bay,
Bacterh~ in a feedlot effluent fish pond
15
California. In Advances in aquaculture (T. V. R. Pillay & W. A. Dill (Eds)). Fishing News Books, Farnham, UK, pp. 492-8. Allen, G. H. & Gearheart, R. A. (1980). Public health aspects of a wastewater based California salmon ranching project. In Proceedings of a Symposium on Aquaculture in Wastewater. ISBN 0 79882012, Council for Scientific and Industrial Research, Pretoria, South Africa. Bryan, F. L. (1977). Diseases transmitted by foods contaminated by wastewater. J. Food Prot., 40, 45-56. Cloete, T. E., Le Roux, J. D., Toerien, D. F., Pieterse, A. J. H. & Downes, M. (1983). Oxygen dynamics and heterotropic aspects of a pond treatment system for cattle feedlot effluent. Agricultural Wastes, 7, 147-74. Drews, R. J. L. C. (1966). Field studies of large-scale maturation ponds with respect to their purification efficiency. J. Proc. Inst. Sew. Purif., (3), 2-16. Edwards, P. (1980). A review of recycling organic wastes into fish, with emphasis on the tropics. Aquaculture, 21,261-79. Guelin, A. (1962). Polluted waters and the contamination offish. In Fish as food, Vol. 2 (G. Borgstrom (Ed.)). Academic Press, New York, pp. 481-502. Hojovec, J. (1977). Health effects from waste utilisation. In Animal wastes (E. P. Taiganides (Ed.)). Applied Science Publishers Ltd, London, pp. 105 9. Janssen, W. A. (1970). Fish as potential vectors of human bacterial diseases. Spec. Publ. Amer. Fish. Soc., 5, 284 90. Liston, J. (1979). Microbiology in fishery science. In Advances infish science and technology (J. J. Connell et al. (Eds)). Fishing News Books, Farnham, UK, pp. 138 57. Muttamara, S. & Wattayakom, G. (1978). Optimization of sewage treatment and fish propagation in ponds. International Conference on Water Pollution Control in Developing Countries. Bangkok, Thailand, 21-25 February 1978, pp. 241-51. Orland, H. P. (Ed.) (1965). Standard methods for the examination of water and wastewater. American Public Health Association, New York. Saigal, B. N. (1972). Domestic sewage in aid of fish culture. In Souvenir Silver Jubilee of CIFRI. Barackpore, pp. 79-84. Schoonbee, H. J., Nakani, V. S. & Prinsloo, J. (1979). The use of cattle manure and supplementary feeding in growth studies of the Chinese silver carp in Transkei. S. Aft. J. Sci., 75, 489-95. Smith, P. B., Tomfohrde, K. M., Rhoden, L. D. & Balows, A. (1972). API system: A multitube micro-method for identification of Enterobacteriaceae. Appl. Microbiol., 24, 449 52. Taiganides, P. (1978). Wastes are resources out of place. Agricultural Wastes, 1, 1 19. Vaas, K. F. & Sachlan, M. (1956). Cultivation of common carp in running water in West Jarva. Proceedings of the Indo-Pacific Fisheries Council, 6, 187-96. Wolfarth, G. (1978). Utilization of manure in fish farming. In Proceedings of Conference on Fish Farming and Wastes. London, pp. 78-95.
The Bacteriological Quality of Water and Fish of a Pond System for the Treatment of Cattle Feedlot Effluent T. E. Cloete, D. F. Toerien & A. J. H. Pieterse* Institute for Environmental Sciences, University of the Orange Free State, PO Box 339, Bloemfontein, South Africa 9300
ABSTRACT A cattle feedlot waste system consisting of a high-rate algal pond and a fish pond was regularly sampled for bacteriological analyses. High numbers of aerobic and anaerobic bacteria and potential pathogens (e.g. Salmonella spp.) were present in the waste water. The waste treatment system reduced all bacterial groups by more than 99"6 %. The skin, gills and intestines of the waste-grown fish housed large numbers of bacteria, including potential pathogens. However, similar bacterial numbers, including potential pathogens, were associated with the skins, gills and intestines of naturally-grown fish, which suggests that the health risk involved in the comsumption of waste-grown fish might not be substantially different to that of natural fish populations. In both cases the tissues and blood appeared to be sterile, which would contribute to a much reduced health risk.
INTRODUCTION Taiganides (1978) considered animal wastes to be resources out of place. Edwards (1980) pointed out the large potential o f such wastes for fish production. However, the microbial flora of fish directly reflect the microbiological condition of the water from which they are taken * Present address: Department of Botany, Universityof the Orange Free State, PO Box 339, Bloemfontein, South Africa 9300. 1
Agricultural Wastes 0141-4607/84/$03-00 © Elsevier Applied Science Publishers Ltd, England, 1984. Printed in Great Britain
2
T.E. Cloete, D. F. Toerien, A. J. H. Pieterse
(Guelin, 1962). Therefore the utilisation of animal or other wastes for fish production could result in a health risk because of the presence of pathogens in waste water (e.g. Janssen, 1970; Bryan, 1977; Hojovec, 1977; Wolfarth, 1978; Allen et al., 1979). In addition the high organic load, microbial activity and biological oxygen demand of waste water also create the risk of transmittance of fish diseases and parasites (Muttamara & Wattayakom, 1978; Schoonbee et al., 1979; Allen & Gearheart, 1980). Knowledge about the bacteriological quality of the water and fish of waste utilisation systems is needed to assess the associated health risks. The norm for assessing this health risk must be the bacteriological quality of naturally-grown or pond-grown (without waste addition) fish. An integrated cattle feedlot waste treatment system, which includes the production of fish, is operated by Soetvelde Farms Ltd of Vereeniging, South Africa. It consists of hydraulic removal of manure from concretefloored pens, the separation of the solids and slurry of the effluent, and the treatment of the slurry in a high-rate algal pond (HRAP) followed by passage through a fish pond. Water from the fish pond is used to move the manure (Cloete et al., 1982). To provide information on the bacteriological quality of the water in the different ponds of the waste treatment system, water samples were analysed at monthly intervals over the period March 1979 to June 1980. In addition, the bacteriological quality of different body parts of silver carp (Hypothalrnichthys molitrix), grass carp (Ctenopharyngodon idella) and common carp (Cyprinus carpio) harvested from the waste treatment system, was compared with that of common carp from a natural fish population.
MATERIALS A N D METHODS
Sampling procedures Water samples were collected at seven different sampling points in the system (Fig. 1). The samples were taken during monthly intervals during the period March 1979 to June 1980. The samples were collected in sterile 25 ml McCartney bottles and kept at 0 °C during transportation (360 km). Analyses were started within 24 h after sampling. In September 1979, fish (sized 400 to 500g each) were netted and transported live to the laboratory in a container holding 200 litres of fish
Bacteria in a feedlot effluent fish pond
DEPTH 1 . 2 m
"ff . . . . . . .
i !i
!4
DEPTH 0 , 4 5 m
~
300m
~' I1= ,--:.,~
--
I~
8~,
1J rcQ:
J
,g,,, .
szcr~or~ : B - - B M
f.
.
M a x DEPTH0 ~ 5 m
,,o~1
r,..
------i"~.-~
!1
.
f"
AI
Fig. 1. Schematicrepresentation of the Soetveldecattle feedlot system. A, confinement unit; B, concrete platform where particulate wastes are separated from slurried waste; C, storage pond; D, high-rate algal pond (HRAP); E, fish pond. Cage aerator is between sampling points 3 and 4. Bacteriologicalsamples were taken at points 1 (screen water), 2 (storage pond), 4 (below stream of cage aerator), 3 (upstream of cage aerator), 5 (outflow of HRAP), 6 (fish pond inflow) and 8 (fish pond outflow). pond water. On arrival the fish were killed, immediately dissected and bacterial enumerations performed. The same procedure was followed for fish collected from Wuras Dam, a freshwater impoundment. In September 1980, fish were again harvested from the waste treatment system and immediately dissected. The samples for bacteriological analyses were prepared on site and were then transported in an ice-box at 0 °C to the laboratory, where the analyses were completed.
Sample pretreatment Appropriate dilutions of the water samples were made using 9ml quantities of sterile water in test tubes. Analyses of the fish were done on different body parts, i.e. gill surfaces, skin, blood, tissue and intestinal contents. Sterile swabs were used to sample the gill surfaces and skin (2 cmZ). Sterile swabs were prepared in 10 ml sterile distilled water. Blood samples were obtained by separating the tail of the fish with a sterile scalpel, inserting the needle of a sterile 5 ml syringe into the caudal vein and withdrawing I ml of blood. Fish tissue samples were collected by either dissection with a sterile scalpel (September 1979) or by using a
4
T.E. Cloete, D. F. Toerien, A. J. H. Pieterse
sterile corkscrew after removing the scales with the aid of a sterile scalpel. The samples were transferred to sterile homogeniser tubes, weighed and homogenised. Intestinal samples were obtained by removing the intestines with a sterile scalpel and transferring them to a jar containing 50 ml sterile water. After shaking vigorously for 5 rain, a dilution series was prepared. Bacteriological analyses of intestinal contents were done only in September 1979 and analyses of blood in September 1980.
Bacteriological analyses Total viable aerobic and facultative anaerobic bacteria (hereafter referred to as total aerobic bacteria) were enumerated on Bacto Nutrient Agar by incubating at 37 °C for 48 h. Presumptive salmonellae were enumerated on Difco Bismuth Sulphite Agar by incubating at 37 °C for 48 h. Black colonies with a metallic sheen were counted. Coliform bacteria were enumerated with the MPN technique using MacConkey Broth (Oxoid) and incubating at 37°C for 24h. Presumptive clostridia (hereafter referred to as total anaerobic bacteria) were enumerated with the MPN technique using Reinforced Clostridial Medium (Oxoid) and incubating at 37 °C for 24 h. The basic procedures followed were as described by Orland (1965). Twenty randomly isolated presumptive Salmonella colonies on Bismuth Sulphite Agar were purified by repeated streaking on Nutrient Agar. These isolates were identified by the Gram stain and the use of the API-20E microtube system (e.g. Smith et al., 1972). RESULTS
Water quality The variations of the total aerobic bacteria, total anaerobic bacteria, coliforms and presumptive salmonellae for different stages of the system and with time are presented in Figs 2-5. Total aerobic bacteria (Fig. 2) The total aerobic bacterial numbers in the screen water did not show any distinct seasonal pattern and ranged from 1 x 10 6 t o 38 × 106m1-1 (Fig. 2(A)). In the storage pond these numbers were apparently higher (about 30 × 106 ml- 1) in the period August to January (spring-summer) than in autumn-winter (March to July) (Fig. 2(A)).
Bacteria in a feedlot effluent fish pond
5
1 •r
E
B
8
U
u
x4
3
t I MAM
I
I I I J J A 1979
I I SONDJJ
I
I J
I i I FMAMJ
I
1980
Fig. 2. Aerobic bacterial numbers in the waste treatment system during the study period. (A) H screen water, H storage pond; (B) HRAP, H below stream of cage aerator, O O upstream of cage aerator, [] [ ] outflow; (C) fish pond, ~ inflow, O O outflow.
The total aerobic bacteria in the H R A P tended to be higher in the outflow than in the vicinity of the cage aerator (Fig. 2(B)). Higher numbers also occurred in the s p r i n g - s u m m e r - a u t u m n period than in winter. Maximum numbers were about 8 x 106m1-1 and minimum numbers were about 0.2 x 106 ml-1. The fish pond did not show any distinct seasonal pattern for total aerobic bacteria, but the numbers in the inflow were about ten thousand times higher than at the outflow (Fig. 2(C)).
Total anaerobic bacteria (Fig. 3) The total anaerobic bacterial numbers in the screen water and storage pond were lower in winter (June to August) than the rest of the year (Fig. 3(A)). The numbers varied from 10 x 106 to 150 x 106 ml-1. In the H R A P the total anaerobic numbers were appreciably lower (0.5 to 1 "8 x
6
T.E. Cloete, D. F. Toerien, A. J. H. Pieterse 15
~1o 5
"7 E
~
A
16
Z
14
0 u
12
~, "o I° u~8
~
6
10 8 ~6 4
MAMJ
JASONDIJ 1979
F M A M J 1980
Fig. 3. Anaerobic bacterial numbers in the waste treatment system during the study period. (A) n screen water, H storage pond; (B)HRAP, H below stream of cage aerator, O O upstream of cage aerator, ~ outflow; (C)fish pond, H inflow, O O outflow. 10 6 ml - 1) than those of the screen water and storage pond and no distinct
seasonal pattern was observed, although the mid-winter ( J u n e - J u l y ) values were very low (0.5 to 1.5 x 10Sm1-1) (Fig. 3(B)). The total anaerobic bacterial numbers of the fish pond ranged from 1.5 x 105 to 10 x 105 ml-1 (Fig. 3(C)). The inflow values were somewhat higher than those of the outflow, but not as markedly so as for the total aerobic bacteria. Coliform bacteria (Fig. 4) N o seasonal pattern was evident for the coliforms in the screen water and the storage pond and the numbers varied from 0.3 × 10 6 to 6"5 × 10 6 ml - 1 (Fig. 4(A)). In the H R A P the numbers were appreciably lower than in the screen water and storage pond (0-2 x 104 to 8.3 × 104m1-1), and lower numbers occurred in winter (June to August) and higher numbers in
Bacteria in a feedlot effluent fish pond
7
6 S •o
4
2 1
E 0
8 6 5
3 2 1
12 6 :3
I I I I I I I I I I I I I I I I M A M J J A S O N DJJ 1979
F M A M 1980
Fig. 4.
Numbers of coliforms in the waste treatment system during the study period. (A) screen water, I I - - - - U storage pond; (B) H R A P , H below stream of cage aerator, O O upstream of cage aerator, [ ] [] outflow; (C) fish pond, 4 - - - - - - 4 inflow, ~ outflow,
summer (November to December) (Fig. 4(B)). The coliform numbers in the fish pond were still lower (2.5 × 102 to 17 × 102 ml-1) and the inflow usually had somewhat higher numbers than the outflow (Fig. 4(C)).
Presumptive salmonellae (Fig. 5) In the storage pond and screen water the numbers were highest during the late winter (July-September 1979), but were lower in M a r c h - M a y 1979 and variable in M a r c h - M a y 1980 and ranged from 0.2 × 10 to 23 x 104 (Fig. 5(A)). In the H R A P the numbers ranged from 20 to 800 m l - ~ and showed no distinct seasonal pattern (Fig. 5(B)). There was also no distinct pattern in the fish pond where the numbers were from about 80 to 520 ml - 1 (Fig. 5(C)).
8
T . E . Cloete, D. F. Toerien, A. J. H. Pieterse
4 2 1 E
8
°°
!
~,~5 u ~ 4
o
3
a 1
4 2 1
M A M J J A S O N DIJ 1979
F M A M J 1980
Fig. 5, Numbers of salmonellae in the waste treatment system during the study period. (A) H screen water, H storage pond; (B) HRAP, H below stream of cage aerator, O O upstream of cage aerator, I-q[] outflow; (c) fish pond,
H
inflow, O
O outflow.
Reduction in bacterial numbers An examination of the average bacterial numbers at the various stages of the treatment system (Table l) showed that an overall reduction exceeding 99.6 % was achieved in the waste treatment system. Whereas the total aerobic bacteria and the coliforms increased in numbers between the screen water and the storage pond, the total anaerobic bacteria and presumptive salmonellae decreased throughout the waste treatment system. A comparison of the ratios between the different bacterial groups in the system (Table 2) reflects the rates at which the groups were either stimulated or eliminated by conditions in the different stages of the waste treatment system. High ratios of aerobic bacteria to the other bacterial groups in the HRAP indicate a more rapid elimination of anaerobes,
7x 70 x 1× 1-8 x
106 106 106 10 s
Screen water
12 × 106 55 x 106 4 x 106 1.8 x 104
Storage pond
2.1 3.3 2 2
x x x x
106 105 104 102
at cage aerator x 106 x l0 s x 104 × 103
outflow 4 6 3-5 2
HRA P
3.5 3,4 2 2.2
x 106 x l0 s × 103 x 102
inflow 2-5 2.8 1.8 2
x 104 x l0 s x 103 × 102
outflow
Fish pond
Coliforms
Total anaerobic
0.10 0-22 6,36 6.67 1.03 0.09
Screen water Storage pond HRAP inflow HRAP outflow Fish pond inflow Fish pond outflow
7.0 3-0 105.0 114.3 175,0 13.9
Total aerobic
Total aerobic
Stage of treatment system 38.9 666.7 10 500.0 20 000.0 1 590.9 125-0
Salmonellae
Total aerobic
70-0 13.8 16.5 17.1 170.0 155.6
Coliforms
Total anaerobic
388.8 3 055.6 165.0 300.0 154.5 1400.0
Salmonellae
Total anaerobic
99.64 99.60 99.98 99.89
% Overall reduction
5.6 222,2 100.0 17,5 9-1 9.0
Salmonellae
Coliforms
TABLE 2 Ratios of the Different Bacterial Groups in the Treatment System (Based on Average Bacterial Numbers)
Total aerobic bacteria Total anaerobic bacteria Coliforms Salmonellae
Bac~r~
TABLEI ComparisonoftheAverageBacterialNum~rsattheVariousSta~sofTreatment
10
T. E. Cloete, D. F. Toerien, A. J. H. Pieterse
coliforms and salmonellae by the conditions in the HRAP than aerobes (since there was a decrease in aerobes between storage pond and the HRAP, the high ratios of aerobes to the other groups are not a result of increased numbers of aerobic bacteria). The reduction in the ratios of aerobes to the other groups in the fish pond (except for the inflow of the fish pond) suggests a more rapid rate of elimination of aerobes other than coliforms and salmonellae in the fish pond. This could indicate a higher grazing pressure by the organisms living in the fish pond on these aerobes. The anaerobic bacterial numbers were drastically reduced between the storage pond and the HRAP, suggesting that the oxygenated conditions which occurred in the H R A P (Cloete et al., 1982) could have been the cause of an elimination of obligate anaerobes. On the other hand fairly high numbers of apparently anaerobic bacteria were recorded in the H R A P and fish pond (Table 1). If the reduction in numbers between the storage pond and the HRAP was due to the elimination of obligate anaerobes, then the anaerobes detected in the HRAP must have been facultative anaerobes or bacterial spores able to survive oxygenated conditions. The ratios of anaerobes to coliforms were relatively constant in the screen water, the storage pond and the HRAP, but increased in the fish pond. A more rapid rate of coliform elimination in the fish pond was the reason for this. The anaerobe/salmonellae ratio varied widely and did not show a distinct pattern. Salmonellae were eliminated more rapidly than coliforms in the storage pond and the HRAP (higher coliform/salmonellae ratios), but less rapidly in the fish pond (lower coliform/salmonellae ratios). Selective grazing by bacterivorous organisms and differential behaviour towards environmental conditions in the different ponds probably contributed to this phenomenon. The above illustrates that the dynamics of the bacterial population and groups in the system are complex and probably influenced by factors such as state of oxygenation, selective grazing, etc. Quality of the fish Some significant facts emerged from a comparison of the bacterial numbers of different body parts of waste-grown and natural fish (Table 3). The first is that potential pathogens in the form of salmonellae were present in all fish, including those derived from a natural population.
N.D.
ND.
8"5 x 108
Number/ml blood
Number/g tissue
Number/intestine
13X103
N,D. = not determined.
.
.
1'5 x 102
.
4"5 x 102 to 0"7× 10 s
.
l ' 2 x 107
ND.
N.D.
2"9 X 10"
1'5 x 106
N.D.
ND,
|XI0*
4×10 ~
6x10 ~
ND.
N.D,
1"8 x 105
3 ' 0 x 10"
<102
<102
1"4 x 10 s
to
<10 z
<5x103
N.D.
N,D.
1×103
2"3 x 10 ~
.
N,D.
<102
<10 z
<102
<50
Common carp Cyprinus carpio
Common carp
Cyprinus carpio
Natural population
0 3 5 x 10 ~ 0 ' 8 x 10 ~
.
Number/gill
Number/cm 2 skin
Body part of ~
.
N.D.
<102
<10 z
<102
<50
7"5 x 106
<10 z
I x 103
to
<102
<50 tO 1'1 ×104
1 ' 5 x 10°
N,D,
N.D.
1"1X 103
2"2 x I0"
2"0x 106
<102
<10 z
<10 /
<50 tO I ' 2 X 10 ~
Hypothalrnichthys molitrix
Silt'er carp
WaMe treatment syslem
2"5 x 102
<102
<102
<102
<50 tO 50
~
N.D.
<102
<102
3X104
1 ' 5 x 104
~ .~
Grass carp
N.D,
N.D.
N.D.
N.D.
N,D.
g .~
N.D,
<10 z
<102
3xlO 3
1"5 x 10 3
~ .~
Ctenopharyn$odon idella
TABLE 3 The Bacteriological Quality of Carp from the Waste Treatment System and from a Natural Population
N.D,
<10 a
<102
3 . 2 x 10 ~
1"6 x 10 2
12
T. E. Cloete, D. F. Toerien, A. J. H. Pieterse
The second is that these pathogenic bacteria were not detected in the tissues and blood of the waste-grown fish; in fact no bacteria were detected in the tissues and blood. However, the sensitivity of the analysis only allowed the detection of bacterial concentrations above 102/sample and, although it is likely, we cannot state unequivocally that the tissues and blood were sterile. The third is that very high bacterial numbers were associated with the intestines and lower (but still high) numbers with the skin and gills of the fish. This was equally true for the naturally-grown fish and the waste-grown fish. Identification of 20 isolates of presumptive salmonellae isolated from the waste-grown fish, indicated that only three isolates in fact belonged to the genus Salmonella. This suggests that the use of Bismuth Sulphite Agar overestimated the presence of Salmonella spp., but nevertheless, that salmonellae were present in or on the fish. The bacterial flora offish with different feeding habits may show some differences. For instance, the total aerobic bacterial numbers in the whole intestines of the bottom-feeding common carp (Cyprinus carpio) from both waste-grown and natural fish populations were substantially higher than those of the filter-feeding silver carp (Hypothalmichthys molitrix).
DISCUSSION The growing realisation that natural resources are finite has in the past decade led to an increasing interest in western countries in recycling of natural resources. In eastern countries, particularly in Asia, the recycling of organic wastes into agriculture and fish production has been practised for centuries (Edwards, 1980). Acceptance and use of these practices in many countries will depend, amongst other things, on evaluations of the associated health risks, because of the presence of pathogens in domestic and animal wastes. Waste treatment or recycling systems will therefore have to achieve substantial improvements in the pathogen quality of waste waters and yield products with a low health risk. The Soetvelde waste treatment system which incorporates hydraulic manure removal, a high-rate algal pond and a fish pond, is fairly sophisticated from an agricultural point of view, but from a waste treatment point of view it represents a fairly low-level technology. In semiarid countries, such as South Africa, there is an urgent need to balance environmental protection with the provision of a balanced diet for a
Bacteria in a feedlot eJfluent fish pond
13
rapidly increasing population. Feedlots may play an ever increasing role in providing animals for slaughter and it is possible that the Soetvelde waste treatment system is an early example of waste treatment systems needed in the future for cattle feedlot operations. Pathogenic bacteria, in the form of Salmonella spp. (potential human pathogens) and Clostridium spp. (potential cattle pathogens) occurred in the feedlot waste waters (Table 1). Seasonal trends of increases and decreases were evident for some, e.g. total aerobic and coliform bacteria in the HRAP, but not all bacterial groups, e.g. salmonellae in the fish pond. The average reduction in numbers of all bacterial groups in the waste treatment system exceeded 99.6 % (Table 1) and was as good as that (99.6 to 99.8 %) reported for a series of sewage-maturation-pond systems (Drews, 1966). The health risks associated with the feedlot waste water were therefore greatly reduced by the waste treatment system. Fish raised in the waste treatment system will however be exposed to the presence of pathogens, and pathogens were indeed recorded in various body parts of the waste-grown fish (Table 3). An unexpected finding was the presence of pathogens in or on c o m m o n carp from a natural population (Table 3). The presence of such bacteria in or on fish from natural populations (also reported by Liston (1979)) which must serve as the norm for assessing the disease risk involved with waste-grown fish, indicates that assessments will have to be done carefully and thoroughly, and should include fish from natural populations as controls, The microbiology of fish, especially marine fish of commercial value, has been studied for a long time (Liston, 1979). The early researchers showed that muscle tissues in healthy fish are sterile, and our results (Table 3) and Allen et al. (1979) suggest the same for the waste-grown fish. We also found no evidence that the blood of the fish was contaminated with bacteria (Table 3). Large populations of bacteria were associated with the external surfaces, gills and intestines of marine fish by Liston (1979), a condition which was also observed in this study. The skin of marine fish can house bacterial populations of 102 to 10 6 c m - 2 , the gills similar or higher numbers, and the intestines from a few to 108 g - 1 depending on whether the fish are feeding (Liston, 1979). The skins of the waste-grown fish contained from 450 to 0.7 x 105 aerobes and 2.3 x 105 anaerobes cm -2, the gills from < 102 to 1.4 x 105 per gill and the intestines from 7.5 x 10 6 t o 3"0 x 10 8 bacteria/intestine. Naturally-grown c o m m o n carp contained bacterial numbers of 0-3 × 105cm -2 of skin, 13 x 102 per gill and 8-5 x 10 s per intestine
14
T. E. Cloete, D. F. Toerien, A. J. H. Pieterse
respectively (Table 3). The results suggest the numbers of the microbial flora of the waste-grown fish did not differ substantially from either naturally-grown freshwater fish or marine fish. Despite the potential of fish grown on human wastes to transmit disease, Bryan (1977) found only two references in a worldwide review of medical and engineering literature in which illness was caused by fish contaminated with human wastes. Edwards (1980) reported that no disease problems have been reported with fish grown on animal wastes, but that prior to 1949 the use of human wastes in fish ponds in China led to the widespread occurrence of diseases of insanitation. In most cases where fish are grown on animal wastes, the latter material is added directly to the fish ponds. Saigal (1972) suggested that fish grown in ponds receiving human waste do not feed directly on the waste but on the natural food, e.g. plankton, that develops as a result of the fertilising effect of the waste, but Vaas & Sachlan (1956) reported the presence of human faeces in the intestines of fish cultured in cages in ponds receiving human wastes. In the Soetvelde waste treatment system, the H R A P ensures that fish are not in contact with untreated animal wastes and that a substantial attenuation in pathogens has occurred before the fish ponds. This may be the reason why the bacteriological quality of the waste-grown fish did not differ substantially from that of naturally-grown fish. No excessive health risks might be associated with the consumption of the Soetvelde fish, but additional scientific evaluations are still needed to fully assess the health risks involved with consumption of waste-grown fish.
ACKNOWLEDGEMENTS The financial assistance of the Anglo-American Research Laboratories and Soetvelde Farms (Pty) Ltd, and the help of Mr M. Downes, officers of the Division of Nature Conservation, Transvaal Provincial Administration and Dr I.G. Gaigher in obtaining fish samples are gratefully acknowledged.
REFERENCES Allen, G. H., Busch, R. A. & Motton, A. W. (1979). Preliminary bacteriological studies on wastewater-fertilized marine fish ponds, Humboldt Bay,
Bacterh~ in a feedlot effluent fish pond
15
California. In Advances in aquaculture (T. V. R. Pillay & W. A. Dill (Eds)). Fishing News Books, Farnham, UK, pp. 492-8. Allen, G. H. & Gearheart, R. A. (1980). Public health aspects of a wastewater based California salmon ranching project. In Proceedings of a Symposium on Aquaculture in Wastewater. ISBN 0 79882012, Council for Scientific and Industrial Research, Pretoria, South Africa. Bryan, F. L. (1977). Diseases transmitted by foods contaminated by wastewater. J. Food Prot., 40, 45-56. Cloete, T. E., Le Roux, J. D., Toerien, D. F., Pieterse, A. J. H. & Downes, M. (1983). Oxygen dynamics and heterotropic aspects of a pond treatment system for cattle feedlot effluent. Agricultural Wastes, 7, 147-74. Drews, R. J. L. C. (1966). Field studies of large-scale maturation ponds with respect to their purification efficiency. J. Proc. Inst. Sew. Purif., (3), 2-16. Edwards, P. (1980). A review of recycling organic wastes into fish, with emphasis on the tropics. Aquaculture, 21,261-79. Guelin, A. (1962). Polluted waters and the contamination offish. In Fish as food, Vol. 2 (G. Borgstrom (Ed.)). Academic Press, New York, pp. 481-502. Hojovec, J. (1977). Health effects from waste utilisation. In Animal wastes (E. P. Taiganides (Ed.)). Applied Science Publishers Ltd, London, pp. 105 9. Janssen, W. A. (1970). Fish as potential vectors of human bacterial diseases. Spec. Publ. Amer. Fish. Soc., 5, 284 90. Liston, J. (1979). Microbiology in fishery science. In Advances infish science and technology (J. J. Connell et al. (Eds)). Fishing News Books, Farnham, UK, pp. 138 57. Muttamara, S. & Wattayakom, G. (1978). Optimization of sewage treatment and fish propagation in ponds. International Conference on Water Pollution Control in Developing Countries. Bangkok, Thailand, 21-25 February 1978, pp. 241-51. Orland, H. P. (Ed.) (1965). Standard methods for the examination of water and wastewater. American Public Health Association, New York. Saigal, B. N. (1972). Domestic sewage in aid of fish culture. In Souvenir Silver Jubilee of CIFRI. Barackpore, pp. 79-84. Schoonbee, H. J., Nakani, V. S. & Prinsloo, J. (1979). The use of cattle manure and supplementary feeding in growth studies of the Chinese silver carp in Transkei. S. Aft. J. Sci., 75, 489-95. Smith, P. B., Tomfohrde, K. M., Rhoden, L. D. & Balows, A. (1972). API system: A multitube micro-method for identification of Enterobacteriaceae. Appl. Microbiol., 24, 449 52. Taiganides, P. (1978). Wastes are resources out of place. Agricultural Wastes, 1, 1 19. Vaas, K. F. & Sachlan, M. (1956). Cultivation of common carp in running water in West Jarva. Proceedings of the Indo-Pacific Fisheries Council, 6, 187-96. Wolfarth, G. (1978). Utilization of manure in fish farming. In Proceedings of Conference on Fish Farming and Wastes. London, pp. 78-95.