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Influence of raw water storage on Giardia, Cryptosporidium and nematodes
~
Pergamon PII:S0273-1223(98~32-8
Wal. Sci. r,ch. Vol. 37. No 2. pp. 261-267. 1998. C 1998 IAWQ. Pubhshed by ElseVIer Science Ltd Printed in Greal Britain. 0273-1223198 $19'00 + ()OOO
INFLUENCE OF RAW WATER STORAGE ON GIARDIA, CRYPTOSPORIDIUM AND NEMATODES G. C. Bertolucci*. G. Gilli**. E. Carraro**. D. Giacosa* and M. Puppo*** • Az;enda Acquedotto Municipale, corso Xlfebbraio. 14-10/52 Turin, Jraly .. Unillersita'degli Studio Diparlimento di San;ta Pubblica e Microbiologia. Ilia Samma, 5lbis. 10100 Turin, Italy
ABSTRACT A watershed derived from a disused gravel-quarry has been studied for the relocation of the catchment area of Turin surface water treatment plants. The improvement of river water quality as a consequence of short. tenn storage has been investigated, focusing the allention on three problem organisms, namely the parasitic protozoans Giardia spp. and Cryplosporidium spp. and the free-living Nematodes, which could be considered indicators of healthy compliance and product agreeability respectively. lC> 1998 fAWQ. Published by Elsevier Science Ltd
KEYWORDS
Cryptosporidium; Giardia; Nematodes; reservoir; surface water.
INTRODUCTION The Turin Water Authority produces about one fourth of the drinking water supplied to the city from surface water sources. The catchment area is located immediately upstream from the city; the Po river flows from an area of about 4,900 sqkm and includes human settlements to a total of nearly 700 thousand inhabitants; the water is moderately polluted by industrial effluents, while microbiological pollution from civil effluents and agricultural runoffs is more consistent. In order to improve raw water quality, a pretreatment has been experimented, consisting in relocating the catchment area 10 kilometres upstream of the present intake and in adopting a natural lagoon, derived from the abandonment of a gravel-quarry as short-term storage basin. At present raw water is pumped inside the reservoir at a flow-rate of 1,000 L s·I, then sent back to the river with the same flow-rate, resulting in a theoretical detection time which can be evaluated around eighteen days. A two years period of observation has been planned, in order to achieve information about the advantages and the disadvantages to water treatment and to drinking water quality, which can be expected from the adoption of this raw water storage.
261
262
G. C. BERTOLUCCI et al.
Many investigations of chemical, physical and microbiological parameters and simulation treatment tests have been performed. Particular attention has been paid to the removal of some organisms, such as pathogenic protozoans (Giardia and Cryptosporidium) and metazoans (Nematodes).
Cryptosporidium and Giardia are enteric pathogenic protozoa, obligate parasites, characterized by low host specificity. They may produce gastrointestinal disease, with a wide variety of symptoms and medical care needs. The infective form is the dormant one, referred to as cyst (Giardia spp.) and oocyst (Cryptosporidium spp.), which follows transmission through the water and food contamination (AWWA Manual. M7, I995b). Cysts and OOCYSts may survive for long periods in the environment and represent a great concern in the water industry, because of the health risk they present, their high resistance to disinfectants and their shapes and dimensions (ranging respectively from 5 to 18 11m for the oval Giardia cysts and from 4 to 811m for the round Cryptosporidium oocysts) which permit them to pass through the filter-bed. It is still unknown how much infectivity, and. as consequence, health risk. is influenced by cyst and oocyst age and permanence in the environment, as well as by previous host and strain (Haas et al.• 1996; Roefer et al., 1996). On the other hand, information on dose-effect is fairly poor, while better knowledge is available on the data concerning the more sensitive immuno-compromised people, who notice effects on their health (Roefer et al., 1996; Solo-Gabriele and Neumeister, 1996). Pathogenic protozoan cyst occurrence seems to concern a wide number of surface water sources (Le Chevallier et al., 1991a;b; 1993; Lisle and Rose, 1995, Ketelaars et ai, 1995, Roefer et ai, 1996, Solo• Gabriele and Neumeister. 1996). surely related to human and animal activities, but not easily foreseable as regards entity. in terms of weather and seasonal influence, and, from the epidemiological point of view, on the bases of hygienic conditions. activities, and water treatment efficiency. Scarce literature data concerning the occurrence of cysts and OOCYSts in watersheds are available, (Ketelaars et al. 1995, Ongerth et al., 1995) with dimensions and detection periods quite different with respect to the reservoir here considered. Nematodes are organisms spread worldwide. Their implication in pathogenesis. at least for the species more frequently found in Italy, which are not parasitic, should be considered only from the viewpoint of their role as carriers of ingested pathogens. Anyway. Nematodes have to be considered problem organisms from the drinking water production standpoint (AWWA Manual, M7, I995a). The free-living species found in freshwater are generally microscopic in size, ranging from 5-50 11m in width and from less than 100 to 1,000 11m in length. These smooth, cylindrical shaped worms move whiplike, rather ineffectively in water, very effectively in denser materials such as sediments, filter beds, soil, in which they survive and can reproduce. In addition. Nematodes show high resistance to adverse environmental conditions and to disinfectants. Because of these reasons, they are well adapted to survive in water works, to resist treatments, in some cases to harbour in distribution systems and also to emerge alive from domestic taps. A settling process seems to be the most effective method able to reduce the Nematode numbers in water works. Surface water content ranges from a few nematodes L-I up to several hundreds organisms L-l. expecially in relation to rainfalls. In the present paper, an investigation on Giardia cysts, Cryptosporidium oocysts and Nematodes has been carried out on the Po river water at the inlet and outlet of the aforementioned reservoir. Data previously obtained from monitoring the Po river at the present intake, showed that almost the totality of the samples of raw water was affected by the presence of the two protozoan parasites; treatment plant
Influence of raw water storage
263
efficiency in their removal was, for the period studied, excellent. About Nematodes, raw water contamination was always found, with occurrence peaks detected especially in rainy seasons; in those days treatment abatments have not been completely successful. METHODS Giardia cysts and Cryptosporidium oocysts were investigated according to the U.S.E.P.A. (ICR Laboratory Manual, 1996) and APHA-AWWA-WEF Standard Methods (19° ed., 1995) procedures.
Field water samples of 500 L were collected, eleven at the reservoir inlet and nine at the reservoir outlet by using a pressurized tap, and filtered at a flow-rate of 4-10 L min-I through (25.4 cm) wound polypropylene cartridge filters having a nominal porosity of I ~m (Fluxa, Micro-Wind II). Samples were processed within 24-48 h. Filters have been cut down to the core by using a bistoury, teased and washed with three Htres of PBS eluting solution with O. I % 5DS (Sigma Chimica) and 0.1 % Tween-SO (F1uka Chemika-BioChemika), Solution has been concentrated into a single pellet by centrifugation (1050 x G, IS min, 4°C) and purified by flotation on Percoll-sucrose gradient (specific gravity 1.10; Sigma Chimica) (1050 x G, 15 min, 4°C). Samples washed with PBS Tween-20 have been analyzed using an indirect immunofluorescence antibody assay (Hydrofluor- Combo, Ensysis Inc.). Microscopic examination has been performed using a microscope equipped with phase contrast and epifluorescence optics (exciter wavelength: 450-490 nm). The screening examination has been carried out at 400x, confirming the proper shaped apple-green labelled structures at IOOOx, with the aid of a linear micrometer. Presumptive vitality has been evaluated by the examination of marked cysts and oocysts in contrast-phase microscopy. Nematode analyses (AWWA Manual, M7. 199530 APHA-AWWA-WEF Standard Methods _19° ed., 1995) were performed on 46 10 L water samples (23 collected at the prereservoir inlet and 23 at the outlet), which were filtered through a 5 ~m membrane filter. The membrane has been then rinsed with several milli1itres of distilled water, and eluting solution has been displaced and settled for at least one hour in a plankton cell before examination with reversed microscopy (Ioox). Particle counts have been performed on 120 mL samples by means of a laser particle counter working in a particle range between 2 and 400 11m Microbiological analyses followed the APHA-AWWA-WEF Standard Methods (19° ed., 1995) procedures. RESULTS AND DISCUSSION In Table I data about microbiological, chemical and physical analyses on raw water entering the reservoir are reported, and in Table 2 data on water effluent are shown. As a preliminary overview, the presence of the protozoans investigated was detected in all the water samples collected at the reservoir inlet, with an average concentration of 137 Giardia cysts per 100 L (range 10-561) and 70 Cryptosporidium oocysts per 100 L (range 3-536). Data on the percentage of presumptively viable cysts and oocysts vary from 25.0 to 100.0 per cent for Giardia, and from 13.5 to 60.0 per cent for Cryptosporidium, being the mean values 51.5 and 33.2 per cent, respectively. In the water effluent from the reservoir, protozoan cysts and oocysts were still present in most samples, but their occurrence decreased to a mean value of 46 Giardia cyst per 100 L and 7 Cryptosporidium oocysts per 100 L. The mean removal percentage, calculated date by date (Figure I), is quantified in 55.8% for Giardia
264
G. C. BERTOLUCCI et al.
cysts, and in 78.1 % for Cryptosporidium oocysts; similarly, removal calculated on mean value of all samples at the inlet (Table I) and at the outlet (Table 2) is 66.2% for Giardia and 90.0% for Cryptosporidium. On the other hand, viability percentages do not seem to differ from those evaluated in raw water. This fact seems to indicate that only a typical sedimentation removal occurs in the reservoir, while there is no evidence about the inactivation of the two pathogens, also considering the short reservoir retention time. Correlations, expressed as
Px,y
=Cov (x,y)· (ax· ay)-I,
have been evaluated for cyst or oocyst occurrence and the other analytical parameters reported in the tables, indicating a poor correlation with microbiological as well as with physico-chemical parameters; it has to be noticed, anyway, that in the first samples, and particularly at the reservoir inlet, correlations were found with the microbial and particle counts, suggesting an increase of investigations on seasonal correlations.
------------------------Giardia cysts
5OOlk-----l
... 4Xl 8
ii :m IA------~
jCIN..ET
!- 3Xl1A------1
I
.0U1lET
100 Ik;. .- - - - l
2
3
8
4
9
Cryptosporkllum oocysts
f - . - - - - ...
500
-
-------- - - - - - -
g4Xl
i
r
:m1k-------
I
ICIN..ET .00000T
3Xl 1. . - t - - - - l
100
Ll-1I.1!!!I'I• •..I.~-~IIIII~~.-~-Ili!-lIIqAIL.~~~ ... 2 3 458 789
o
.mpllng
Figure I. Pathogenic protozoa occurrence at the prereservoir inlet and outlet.
Table I. PhysilXKhemical and microbiological analyses at the prereservoir inlet
pH temperature conductivity tmbidity particles count (4-7 lUll) particles count (8-15 lUll) Giardia vital cysts cyst viability Cryptosporidium vital oocysts
oocyst viability total coliforms feca.l coIiforms fecal StreplocOcClls microbial count (J7"C) microbial count (22"Cl heterolrophic bacteria
unit
·C
uScm-1 N1U
particles mL-1 particles mL'l cysts looL·1 cysts 100L" % oocysts 100L" oocysts looL- 1
".
CFUmL" CFUmL" CFUmL-1 CFUmL" CFUmL'l CFUmL-t
1 7.8S 7.2 350 4.0
2 7.82 7.0 360
3 7.72 8.0 340 2.4
93 37 39.8 37 14 37.8 56 4 29
75 20 26.7 14 3 21.4 24 218 5
561 144 25.7 536 187 34.9 132 180
4 7.70 8.S 330 4.8 5103 4308 10 10 100.0 20 10 SO.O 130 2S
160000
12ססoo
1.8
90000
258000 421000
IS
12
90000
10ססoo
260000
S 7.43 12.6 310 1.1 9984 8581 20 8 40.0 20 4 20.0 424 S4
130 6000 16400 392000
6 7.6S 7.6 285 6.8 SI96 1686 36 12 33.3 3 1 20.0 79400 44800 87000 86000 31000
7 7.SS 7.2 280 7.8 469S 3815 26 IS 57.7 5 I 13.S 0 0 0 11000 282000
23ססoo
31ססoo
8 7.S7 S.7 285 3.4 S142 1862 159 82 51.6 44 17 38.6 3100
sao
5200 8000 46000
39000
9 7.56 7.1 275 2.9 5340 3723 403 ISO 37.2 25 8 32.0 0 0 200 3000 51000 40ססoo
10 7.71 15.2 360 15.4 107075 2444 7 7 100.0 35 21 60.0 4800 720 34200 2400 2840 12100
11 7.73 6.7 320 7.5 4501 3979 112 62 5SJ 32 12 37.5 100 0 0 49000 345000 374000
!i' => c:
il"
..."iii 0
~
Table 2. PhysllXKhemica\ and microbiological analyses at prereservoir outlet.
~
~
unit pH temperature
conductivity turbidity particles count (4-7 jUIl) particles count (8-lS jUIl) Giardia vital cysts cyst viability Cryptosporidium
vita1 OOC)'Sfs
oocyst viability tota1 coliforms
feca1 coliforms
fecal Strcptococcus microbial count (J7'C) microbial count (22"C) heterolrophic bacteria
·C uScm·t N1U
particles mL" particles mL-' cysts 100L-' cysts lOOL" % oocysts 1001.;' oocysts looL- 1 % CFUmL-1 CFUmL-1 CFUmL-1 CFUmL'\ CFUmL'\ CFUmL-1
I 7.79 6.7 390
1.1
9.2 4.6 SO.O 27.6 4.6 16.7 2 I I
2 7.86 6.9 310 1.8 4.8 4.8 100.0 0 20 5 1ססoo
196000
15900 402000
3 7.88 10.3 360 3.3 5213 3360 1.4 1.4 100.0 0 25 20 7 9500 11ססoo
83000
4 7.53 13.9 310 3.8 6225 3020 8.4 4.2 50.0 2.8 1.4 SO.O 26 4 0
800 1380 16700
5 7.87 13.2 360 2.2 3895 1513 0 S.5 S.S 100.0 4.7 8 54 860 820 2900
6 7.73 9.5 580 2.0 4187 1051 9.0 2.8 30.2 0 100 0 4200 11000 8000
24000
7 7.56 6.3 360
1.8
5108 1548 136 72 52.2 0 0 0 100 0 19000 344000
8 747 6.1 360 1.3 4389 1273 80 37 46.3 16 9 56.3 0 0 2500 1000 8000 2ססoo
9
7.SS 5.7 340 1.4 4787 1525 168 120 71.4 12.2 5.6 50.0 200 0 200 0 3000 61000
II
•~ "
GO
.,
e:
266
G. C. BERTOLUCCI et al.
Nematodes (Figure 2 and Table 3) were always present in raw water (average concentration 19.0 ind L-t, range 1.0-132.0 ind VI), but in very few cases they were not found in the effluent (average concentration 4.3 ind L-I, range 0-37.0 ind L-I). Nematodes 14)
100
-
o
1.1.l1..
It
11If1 ....
..-~ ..... 1'1. .. rI U.......... 1~ ~ ~ ~ ~ ~ _mpIlng
~ ~
~ ~ ~ ~ ~
Figure 2. Nematodes occurrence at the prereservoir inlet and ouUeL
Good removal was always observed (average value calculated date by date is 60.3%, and on mean values 77.2%, which can be considered very interesting in comparison with results obtained in conventional drinking water treatments). Table 3. Nematodes (ind L-I) at the prereservoir inlet and outlet sampling I
2
3 4 5 6 7 8 9 10 11 12 13 14
IS
16 17
18 19 20 21
22 23
inlet outlet 14.0 2.8 16.2 3.2 10.0 3.2 13.4 1.4 6.6 1.0 12.5 0.8 11.4 1.6 7.5 0.3 16.5 2.0 45.0 1.9 48.0 2.3 31.2 10.0 11.0 2.0 8.0 0.0 7.0 0.0 5.0 4.0 1.0 3.0 2.5 1.0 5.0 5.5 132.0 37.0 16.0 8.6 6.0 4.0 10.0 40
Influence ofraw water storage
267
This effect has to be considered a very relevant advantage in drinking water production. As aforementioned nematode harbouring in filter beds could take place in case of treatment troubleshootings, involving als~ colonization of treatment plants and distribution systems. The removal obtained by means of such pretreatment, of the nematode load entering the water work, together with the equalization of raw water quality, involves a higher degree of plant automation and avoidance of critical interventions, as supply interruption or heavy disinfection procedures.
CONCLUSIONS Even if investigations are still in progress, raw water storage seems to pennit a good removal of the nematodes and of the pathogenic protozoans considered. Despite the improvement of hygienic conditions as well as treatments efficacy, and considering also the difficulty to identify the etiology in waterborne outbreaks and diseases, Giardia and Cryptosporidium have been involved in an increasing number of such occurrences, expecially in industrialized countries. Reported outbreaks occurred in case of drinking water treatment troubleshootings, as well as in regular efficient operating conditions. Nematode occurrence, although it seems to have no relation with health problems, does not obviously portray drinking water agreeability. Because of the difficulty in removing these organisms in drinking water production, the reduction of their presence in raw water seems to guarantee definite quality and safety improvements. The use of reservoir pretreatments therefore represents an interesting procedure which advantages the raw water quality.
REFERENCES APHA-AWWA-WEF. (1995). Standard methods for the examination of water and wastewater. M.A.H. Franson (Ed.); 19th edn, APHA. Washington, DC 20005. AWWA. (l995a). Nematodes. In: Problem organisms in water: identification and treatment. Manual of water supply practices M7. American Water Works Association (Ed.), 2nd edn, Denver, CO, pp. 33-38. AWWA. (l995b). Protozoa. In: Problem organisms in water: identification and treatment. Manual of water supply practices M7. American Water Works Association (Ed.), 2nd edn, Denver. CO. pp. 81-89. Frost, F. J., Craun, G. F. and Calderon, R. L. (1996). Waterborne disease surveillance. Jour. AWWA, 88, 66-75. Haas. C. N., Crockett, C. S.• Rose, J. B., Gerba, C. P. and Fazi!, A. M. (1996). Assessing the risk posed by oocysts in drinking water. Jour. AWWA, 88(9) 131-136. Ketelaars. H. A. M., Medema, G., van Breemen. L. C. W. A., van der Kooij, D., Nobel, P. J. and Nuhn. P. (1995). Occurrence of Cryptosporidium oocysts and Giardia cysts in river Meuse and removal in the Biesbosch reservoirs, J. Water SRT-Aqua, 44, (supp!. I) J08-11 I. LeChevallier, M. W., Norton. W. D. and Lee, R. G. (1991). Occurrence of Giardia and Cryptosporidium spp.. in surface water supplies, Appl. Environ. Microbiol.• 57, 2610-2616. LeChevallier. M. W., Norton. W. D. and Lee, R. G. (1991). Giardia and Cryptosporidium spp.. in filtered drinking water supplies, Appl. Environ. Microbiol., 57, 2617·2621. LeChevallier. M. W. and Norton. W. D. (1993). Giardia and Cryptosporidium in raw and finished water, Jour. AWWA, 87(9),54• 68. Lisle, J. T. and Rose, J. B. (1995). Cryptosporidium contamination of water in the USA and UK: a mini·review. J. Water SRTAqua, 44(3), 108-111. Ongerth, J. E., Hunter, G. D. and DeWalle, F. B. (1995). Watershed use and Giardia cyst presence, Wat. Res., 29(5), 1291-1299. Roofer. P. A., Monscvitz. J. T. and Rexing, D. J. (1996). The Las Vegas cryptosporidiosis outbreak. Jour. AWWA, 88(9).95·106. Solo-Gabriele, H. and Neumeister. S. (1996). US outbreaks of Cryptosporidium. Jour. AWWA, 88(9),76-86. U.S. Environmental Protection Agency. Information Collection Rule (1996). Laboratory Manual, Section VII, 1·44.
Pergamon PII:S0273-1223(98~32-8
Wal. Sci. r,ch. Vol. 37. No 2. pp. 261-267. 1998. C 1998 IAWQ. Pubhshed by ElseVIer Science Ltd Printed in Greal Britain. 0273-1223198 $19'00 + ()OOO
INFLUENCE OF RAW WATER STORAGE ON GIARDIA, CRYPTOSPORIDIUM AND NEMATODES G. C. Bertolucci*. G. Gilli**. E. Carraro**. D. Giacosa* and M. Puppo*** • Az;enda Acquedotto Municipale, corso Xlfebbraio. 14-10/52 Turin, Jraly .. Unillersita'degli Studio Diparlimento di San;ta Pubblica e Microbiologia. Ilia Samma, 5lbis. 10100 Turin, Italy
ABSTRACT A watershed derived from a disused gravel-quarry has been studied for the relocation of the catchment area of Turin surface water treatment plants. The improvement of river water quality as a consequence of short. tenn storage has been investigated, focusing the allention on three problem organisms, namely the parasitic protozoans Giardia spp. and Cryplosporidium spp. and the free-living Nematodes, which could be considered indicators of healthy compliance and product agreeability respectively. lC> 1998 fAWQ. Published by Elsevier Science Ltd
KEYWORDS
Cryptosporidium; Giardia; Nematodes; reservoir; surface water.
INTRODUCTION The Turin Water Authority produces about one fourth of the drinking water supplied to the city from surface water sources. The catchment area is located immediately upstream from the city; the Po river flows from an area of about 4,900 sqkm and includes human settlements to a total of nearly 700 thousand inhabitants; the water is moderately polluted by industrial effluents, while microbiological pollution from civil effluents and agricultural runoffs is more consistent. In order to improve raw water quality, a pretreatment has been experimented, consisting in relocating the catchment area 10 kilometres upstream of the present intake and in adopting a natural lagoon, derived from the abandonment of a gravel-quarry as short-term storage basin. At present raw water is pumped inside the reservoir at a flow-rate of 1,000 L s·I, then sent back to the river with the same flow-rate, resulting in a theoretical detection time which can be evaluated around eighteen days. A two years period of observation has been planned, in order to achieve information about the advantages and the disadvantages to water treatment and to drinking water quality, which can be expected from the adoption of this raw water storage.
261
262
G. C. BERTOLUCCI et al.
Many investigations of chemical, physical and microbiological parameters and simulation treatment tests have been performed. Particular attention has been paid to the removal of some organisms, such as pathogenic protozoans (Giardia and Cryptosporidium) and metazoans (Nematodes).
Cryptosporidium and Giardia are enteric pathogenic protozoa, obligate parasites, characterized by low host specificity. They may produce gastrointestinal disease, with a wide variety of symptoms and medical care needs. The infective form is the dormant one, referred to as cyst (Giardia spp.) and oocyst (Cryptosporidium spp.), which follows transmission through the water and food contamination (AWWA Manual. M7, I995b). Cysts and OOCYSts may survive for long periods in the environment and represent a great concern in the water industry, because of the health risk they present, their high resistance to disinfectants and their shapes and dimensions (ranging respectively from 5 to 18 11m for the oval Giardia cysts and from 4 to 811m for the round Cryptosporidium oocysts) which permit them to pass through the filter-bed. It is still unknown how much infectivity, and. as consequence, health risk. is influenced by cyst and oocyst age and permanence in the environment, as well as by previous host and strain (Haas et al.• 1996; Roefer et al., 1996). On the other hand, information on dose-effect is fairly poor, while better knowledge is available on the data concerning the more sensitive immuno-compromised people, who notice effects on their health (Roefer et al., 1996; Solo-Gabriele and Neumeister, 1996). Pathogenic protozoan cyst occurrence seems to concern a wide number of surface water sources (Le Chevallier et al., 1991a;b; 1993; Lisle and Rose, 1995, Ketelaars et ai, 1995, Roefer et ai, 1996, Solo• Gabriele and Neumeister. 1996). surely related to human and animal activities, but not easily foreseable as regards entity. in terms of weather and seasonal influence, and, from the epidemiological point of view, on the bases of hygienic conditions. activities, and water treatment efficiency. Scarce literature data concerning the occurrence of cysts and OOCYSts in watersheds are available, (Ketelaars et al. 1995, Ongerth et al., 1995) with dimensions and detection periods quite different with respect to the reservoir here considered. Nematodes are organisms spread worldwide. Their implication in pathogenesis. at least for the species more frequently found in Italy, which are not parasitic, should be considered only from the viewpoint of their role as carriers of ingested pathogens. Anyway. Nematodes have to be considered problem organisms from the drinking water production standpoint (AWWA Manual, M7, I995a). The free-living species found in freshwater are generally microscopic in size, ranging from 5-50 11m in width and from less than 100 to 1,000 11m in length. These smooth, cylindrical shaped worms move whiplike, rather ineffectively in water, very effectively in denser materials such as sediments, filter beds, soil, in which they survive and can reproduce. In addition. Nematodes show high resistance to adverse environmental conditions and to disinfectants. Because of these reasons, they are well adapted to survive in water works, to resist treatments, in some cases to harbour in distribution systems and also to emerge alive from domestic taps. A settling process seems to be the most effective method able to reduce the Nematode numbers in water works. Surface water content ranges from a few nematodes L-I up to several hundreds organisms L-l. expecially in relation to rainfalls. In the present paper, an investigation on Giardia cysts, Cryptosporidium oocysts and Nematodes has been carried out on the Po river water at the inlet and outlet of the aforementioned reservoir. Data previously obtained from monitoring the Po river at the present intake, showed that almost the totality of the samples of raw water was affected by the presence of the two protozoan parasites; treatment plant
Influence of raw water storage
263
efficiency in their removal was, for the period studied, excellent. About Nematodes, raw water contamination was always found, with occurrence peaks detected especially in rainy seasons; in those days treatment abatments have not been completely successful. METHODS Giardia cysts and Cryptosporidium oocysts were investigated according to the U.S.E.P.A. (ICR Laboratory Manual, 1996) and APHA-AWWA-WEF Standard Methods (19° ed., 1995) procedures.
Field water samples of 500 L were collected, eleven at the reservoir inlet and nine at the reservoir outlet by using a pressurized tap, and filtered at a flow-rate of 4-10 L min-I through (25.4 cm) wound polypropylene cartridge filters having a nominal porosity of I ~m (Fluxa, Micro-Wind II). Samples were processed within 24-48 h. Filters have been cut down to the core by using a bistoury, teased and washed with three Htres of PBS eluting solution with O. I % 5DS (Sigma Chimica) and 0.1 % Tween-SO (F1uka Chemika-BioChemika), Solution has been concentrated into a single pellet by centrifugation (1050 x G, IS min, 4°C) and purified by flotation on Percoll-sucrose gradient (specific gravity 1.10; Sigma Chimica) (1050 x G, 15 min, 4°C). Samples washed with PBS Tween-20 have been analyzed using an indirect immunofluorescence antibody assay (Hydrofluor- Combo, Ensysis Inc.). Microscopic examination has been performed using a microscope equipped with phase contrast and epifluorescence optics (exciter wavelength: 450-490 nm). The screening examination has been carried out at 400x, confirming the proper shaped apple-green labelled structures at IOOOx, with the aid of a linear micrometer. Presumptive vitality has been evaluated by the examination of marked cysts and oocysts in contrast-phase microscopy. Nematode analyses (AWWA Manual, M7. 199530 APHA-AWWA-WEF Standard Methods _19° ed., 1995) were performed on 46 10 L water samples (23 collected at the prereservoir inlet and 23 at the outlet), which were filtered through a 5 ~m membrane filter. The membrane has been then rinsed with several milli1itres of distilled water, and eluting solution has been displaced and settled for at least one hour in a plankton cell before examination with reversed microscopy (Ioox). Particle counts have been performed on 120 mL samples by means of a laser particle counter working in a particle range between 2 and 400 11m Microbiological analyses followed the APHA-AWWA-WEF Standard Methods (19° ed., 1995) procedures. RESULTS AND DISCUSSION In Table I data about microbiological, chemical and physical analyses on raw water entering the reservoir are reported, and in Table 2 data on water effluent are shown. As a preliminary overview, the presence of the protozoans investigated was detected in all the water samples collected at the reservoir inlet, with an average concentration of 137 Giardia cysts per 100 L (range 10-561) and 70 Cryptosporidium oocysts per 100 L (range 3-536). Data on the percentage of presumptively viable cysts and oocysts vary from 25.0 to 100.0 per cent for Giardia, and from 13.5 to 60.0 per cent for Cryptosporidium, being the mean values 51.5 and 33.2 per cent, respectively. In the water effluent from the reservoir, protozoan cysts and oocysts were still present in most samples, but their occurrence decreased to a mean value of 46 Giardia cyst per 100 L and 7 Cryptosporidium oocysts per 100 L. The mean removal percentage, calculated date by date (Figure I), is quantified in 55.8% for Giardia
264
G. C. BERTOLUCCI et al.
cysts, and in 78.1 % for Cryptosporidium oocysts; similarly, removal calculated on mean value of all samples at the inlet (Table I) and at the outlet (Table 2) is 66.2% for Giardia and 90.0% for Cryptosporidium. On the other hand, viability percentages do not seem to differ from those evaluated in raw water. This fact seems to indicate that only a typical sedimentation removal occurs in the reservoir, while there is no evidence about the inactivation of the two pathogens, also considering the short reservoir retention time. Correlations, expressed as
Px,y
=Cov (x,y)· (ax· ay)-I,
have been evaluated for cyst or oocyst occurrence and the other analytical parameters reported in the tables, indicating a poor correlation with microbiological as well as with physico-chemical parameters; it has to be noticed, anyway, that in the first samples, and particularly at the reservoir inlet, correlations were found with the microbial and particle counts, suggesting an increase of investigations on seasonal correlations.
------------------------Giardia cysts
5OOlk-----l
... 4Xl 8
ii :m IA------~
jCIN..ET
!- 3Xl1A------1
I
.0U1lET
100 Ik;. .- - - - l
2
3
8
4
9
Cryptosporkllum oocysts
f - . - - - - ...
500
-
-------- - - - - - -
g4Xl
i
r
:m1k-------
I
ICIN..ET .00000T
3Xl 1. . - t - - - - l
100
Ll-1I.1!!!I'I• •..I.~-~IIIII~~.-~-Ili!-lIIqAIL.~~~ ... 2 3 458 789
o
.mpllng
Figure I. Pathogenic protozoa occurrence at the prereservoir inlet and outlet.
Table I. PhysilXKhemical and microbiological analyses at the prereservoir inlet
pH temperature conductivity tmbidity particles count (4-7 lUll) particles count (8-15 lUll) Giardia vital cysts cyst viability Cryptosporidium vital oocysts
oocyst viability total coliforms feca.l coIiforms fecal StreplocOcClls microbial count (J7"C) microbial count (22"Cl heterolrophic bacteria
unit
·C
uScm-1 N1U
particles mL-1 particles mL'l cysts looL·1 cysts 100L" % oocysts 100L" oocysts looL- 1
".
CFUmL" CFUmL" CFUmL-1 CFUmL" CFUmL'l CFUmL-t
1 7.8S 7.2 350 4.0
2 7.82 7.0 360
3 7.72 8.0 340 2.4
93 37 39.8 37 14 37.8 56 4 29
75 20 26.7 14 3 21.4 24 218 5
561 144 25.7 536 187 34.9 132 180
4 7.70 8.S 330 4.8 5103 4308 10 10 100.0 20 10 SO.O 130 2S
160000
12ססoo
1.8
90000
258000 421000
IS
12
90000
10ססoo
260000
S 7.43 12.6 310 1.1 9984 8581 20 8 40.0 20 4 20.0 424 S4
130 6000 16400 392000
6 7.6S 7.6 285 6.8 SI96 1686 36 12 33.3 3 1 20.0 79400 44800 87000 86000 31000
7 7.SS 7.2 280 7.8 469S 3815 26 IS 57.7 5 I 13.S 0 0 0 11000 282000
23ססoo
31ססoo
8 7.S7 S.7 285 3.4 S142 1862 159 82 51.6 44 17 38.6 3100
sao
5200 8000 46000
39000
9 7.56 7.1 275 2.9 5340 3723 403 ISO 37.2 25 8 32.0 0 0 200 3000 51000 40ססoo
10 7.71 15.2 360 15.4 107075 2444 7 7 100.0 35 21 60.0 4800 720 34200 2400 2840 12100
11 7.73 6.7 320 7.5 4501 3979 112 62 5SJ 32 12 37.5 100 0 0 49000 345000 374000
!i' => c:
il"
..."iii 0
~
Table 2. PhysllXKhemica\ and microbiological analyses at prereservoir outlet.
~
~
unit pH temperature
conductivity turbidity particles count (4-7 jUIl) particles count (8-lS jUIl) Giardia vital cysts cyst viability Cryptosporidium
vita1 OOC)'Sfs
oocyst viability tota1 coliforms
feca1 coliforms
fecal Strcptococcus microbial count (J7'C) microbial count (22"C) heterolrophic bacteria
·C uScm·t N1U
particles mL" particles mL-' cysts 100L-' cysts lOOL" % oocysts 1001.;' oocysts looL- 1 % CFUmL-1 CFUmL-1 CFUmL-1 CFUmL'\ CFUmL'\ CFUmL-1
I 7.79 6.7 390
1.1
9.2 4.6 SO.O 27.6 4.6 16.7 2 I I
2 7.86 6.9 310 1.8 4.8 4.8 100.0 0 20 5 1ססoo
196000
15900 402000
3 7.88 10.3 360 3.3 5213 3360 1.4 1.4 100.0 0 25 20 7 9500 11ססoo
83000
4 7.53 13.9 310 3.8 6225 3020 8.4 4.2 50.0 2.8 1.4 SO.O 26 4 0
800 1380 16700
5 7.87 13.2 360 2.2 3895 1513 0 S.5 S.S 100.0 4.7 8 54 860 820 2900
6 7.73 9.5 580 2.0 4187 1051 9.0 2.8 30.2 0 100 0 4200 11000 8000
24000
7 7.56 6.3 360
1.8
5108 1548 136 72 52.2 0 0 0 100 0 19000 344000
8 747 6.1 360 1.3 4389 1273 80 37 46.3 16 9 56.3 0 0 2500 1000 8000 2ססoo
9
7.SS 5.7 340 1.4 4787 1525 168 120 71.4 12.2 5.6 50.0 200 0 200 0 3000 61000
II
•~ "
GO
.,
e:
266
G. C. BERTOLUCCI et al.
Nematodes (Figure 2 and Table 3) were always present in raw water (average concentration 19.0 ind L-t, range 1.0-132.0 ind VI), but in very few cases they were not found in the effluent (average concentration 4.3 ind L-I, range 0-37.0 ind L-I). Nematodes 14)
100
-
o
1.1.l1..
It
11If1 ....
..-~ ..... 1'1. .. rI U.......... 1~ ~ ~ ~ ~ ~ _mpIlng
~ ~
~ ~ ~ ~ ~
Figure 2. Nematodes occurrence at the prereservoir inlet and ouUeL
Good removal was always observed (average value calculated date by date is 60.3%, and on mean values 77.2%, which can be considered very interesting in comparison with results obtained in conventional drinking water treatments). Table 3. Nematodes (ind L-I) at the prereservoir inlet and outlet sampling I
2
3 4 5 6 7 8 9 10 11 12 13 14
IS
16 17
18 19 20 21
22 23
inlet outlet 14.0 2.8 16.2 3.2 10.0 3.2 13.4 1.4 6.6 1.0 12.5 0.8 11.4 1.6 7.5 0.3 16.5 2.0 45.0 1.9 48.0 2.3 31.2 10.0 11.0 2.0 8.0 0.0 7.0 0.0 5.0 4.0 1.0 3.0 2.5 1.0 5.0 5.5 132.0 37.0 16.0 8.6 6.0 4.0 10.0 40
Influence ofraw water storage
267
This effect has to be considered a very relevant advantage in drinking water production. As aforementioned nematode harbouring in filter beds could take place in case of treatment troubleshootings, involving als~ colonization of treatment plants and distribution systems. The removal obtained by means of such pretreatment, of the nematode load entering the water work, together with the equalization of raw water quality, involves a higher degree of plant automation and avoidance of critical interventions, as supply interruption or heavy disinfection procedures.
CONCLUSIONS Even if investigations are still in progress, raw water storage seems to pennit a good removal of the nematodes and of the pathogenic protozoans considered. Despite the improvement of hygienic conditions as well as treatments efficacy, and considering also the difficulty to identify the etiology in waterborne outbreaks and diseases, Giardia and Cryptosporidium have been involved in an increasing number of such occurrences, expecially in industrialized countries. Reported outbreaks occurred in case of drinking water treatment troubleshootings, as well as in regular efficient operating conditions. Nematode occurrence, although it seems to have no relation with health problems, does not obviously portray drinking water agreeability. Because of the difficulty in removing these organisms in drinking water production, the reduction of their presence in raw water seems to guarantee definite quality and safety improvements. The use of reservoir pretreatments therefore represents an interesting procedure which advantages the raw water quality.
REFERENCES APHA-AWWA-WEF. (1995). Standard methods for the examination of water and wastewater. M.A.H. Franson (Ed.); 19th edn, APHA. Washington, DC 20005. AWWA. (l995a). Nematodes. In: Problem organisms in water: identification and treatment. Manual of water supply practices M7. American Water Works Association (Ed.), 2nd edn, Denver, CO, pp. 33-38. AWWA. (l995b). Protozoa. In: Problem organisms in water: identification and treatment. Manual of water supply practices M7. American Water Works Association (Ed.), 2nd edn, Denver. CO. pp. 81-89. Frost, F. J., Craun, G. F. and Calderon, R. L. (1996). Waterborne disease surveillance. Jour. AWWA, 88, 66-75. Haas. C. N., Crockett, C. S.• Rose, J. B., Gerba, C. P. and Fazi!, A. M. (1996). Assessing the risk posed by oocysts in drinking water. Jour. AWWA, 88(9) 131-136. Ketelaars. H. A. M., Medema, G., van Breemen. L. C. W. A., van der Kooij, D., Nobel, P. J. and Nuhn. P. (1995). Occurrence of Cryptosporidium oocysts and Giardia cysts in river Meuse and removal in the Biesbosch reservoirs, J. Water SRT-Aqua, 44, (supp!. I) J08-11 I. LeChevallier, M. W., Norton. W. D. and Lee, R. G. (1991). Occurrence of Giardia and Cryptosporidium spp.. in surface water supplies, Appl. Environ. Microbiol.• 57, 2610-2616. LeChevallier. M. W., Norton. W. D. and Lee, R. G. (1991). Giardia and Cryptosporidium spp.. in filtered drinking water supplies, Appl. Environ. Microbiol., 57, 2617·2621. LeChevallier. M. W. and Norton. W. D. (1993). Giardia and Cryptosporidium in raw and finished water, Jour. AWWA, 87(9),54• 68. Lisle, J. T. and Rose, J. B. (1995). Cryptosporidium contamination of water in the USA and UK: a mini·review. J. Water SRTAqua, 44(3), 108-111. Ongerth, J. E., Hunter, G. D. and DeWalle, F. B. (1995). Watershed use and Giardia cyst presence, Wat. Res., 29(5), 1291-1299. Roofer. P. A., Monscvitz. J. T. and Rexing, D. J. (1996). The Las Vegas cryptosporidiosis outbreak. Jour. AWWA, 88(9).95·106. Solo-Gabriele, H. and Neumeister. S. (1996). US outbreaks of Cryptosporidium. Jour. AWWA, 88(9),76-86. U.S. Environmental Protection Agency. Information Collection Rule (1996). Laboratory Manual, Section VII, 1·44.