Pathogen removal in experimental deep effluent storage reservoirs

e

Pergamon

Wal. Sci. Tech. Vol. 33, No.7, pp. 251-260, 1996. Copyright ~ 1996 IA WQ. Published by ElseviCl SCIence Ltd Printed '" Great Britam. All rights reserved, 0273-1223/96 SIS'OO + 0'00

PH: S0273-1223(96)OO360-5

PATHOGEN REMOVAL IN EXPERIMENTAL DEEP EFFLUENT STORAGE RESERVOIRS H. W. Pearson*, D. D. Mara**, L. R. Cawley*, J. I. Oragui** and S. A. Silva * Department ofGenetics and Microbiology, University of Liverpool, P.O. Box 147. Liverpool 1.69 3BX UK .. Department ofCivil Engineering, University ofLeeds, Leeds LS2 9J7. UK ... EXTRABES. Universidade Federal de Paraiba, Caixa Postal 306, Campina Grande CEP58./00. Paraiba. Brazil

ABSTRACT

Studies on balCh loaded pilot-scale deep eft1ucnt storage reservoirs (ESR) in NE Brazil using the operating regime of Fill, Rest and Use, rapidly produced a high microbiological quality effiuent suitable for unrestricted irrigation ie. within 28 days at water temperatures of 2SoC. There was little vertical variation in water quality in the ESR as the water column turned over at night Physico chemical quality rather than microbiological quality of the eft1ucnt was more likely to control effiuent re-usc strategies and purification times as there were persistantly high ammonia and sulphide levels. ESR's could be organically loaded at rates similar to those used for facultative lagoons either using anaerobic pond effiuent or raw sewage but the latter involved slightly longer purification times. The ESR's produced no odour problems. Fe proved a good indicator of microbiological effiucnt quality and helminths could not be detected in the water column by the time the reservoir was full.

Copynght ~ 1996 IA WQ. Published by ElseVIer Science Ltd

KEYWORDS

Wastewater storage reservoirs; effiucnt rc-use; pathogen removal; faeca1 coliforms.

INTRODUCTION

The usc of wastewater storage reservoirs to both treat and store wastewater for irrigation during the arid season was pioneered in Israel (AbeliO\'ich 1982; Dor and Raber 1990; Juanico and Shelef 1991, 1994). Most of these S}'stems allow treated water which has resided in the reservoir during the storage period to mix with influent wastewater refilling the reservoir as it is emptied during the irrigation period. This results in a steady reduction in the irrigation water quality during the crop growing season such that the crops receive the poorest quality eft1ucnt closest to their harvest time. Mara and Pearson (1991) therefore proposed treating the reservoirs as balCh loaded reactors by using parallel reservoirs operating on a regime of Fill, Rest and Usc so that rested and thus purified eft1ucnt was not mixed with untreated influent wastewater. Only once empty was the reservoir allowed to be refJIlcd. The purpose of these microbiological studies was to determine the speed of biological disinfection of

the wastewater during the resting (or purification) phase in the ESR.

2S1

252

H. W. PEARSON ~tal. MATERIALS AND METHODS

Details of the construction and the operational regimes for the experimental deep effiuent storage reservoirs (ESR) are described elsewhere in this volume (Mara et al 1996) but for complemess and ease of reference the reservoir filling times and organic loading strategies for the various experiments with and without anaerobic pond pretreatment are presented in Table I. Table I. Effiucnt Storage Reservoir Filling Times and Organic Loadings for Experiments 1.0. Experiment No.

ESR No.·

Filling time (d)

Organic loading (kg BODlha d)

Anaerobic pondHRT(h)

I 2 3 4 5 6

ESRI ESRI ESRl ESR3 ESRI ESR3

30 18 42 77 35 30

126 324 162 127 103 326

24 16 16 16

·ESRJ was filled with raw sewage, and ESRs I and 2 with anaerobic pond effiuenl Previous studies with pond systems confirmed that both faecal coliforms (FC) and faecal streptococci (FS) behaved similarly as indicators of faecal bacterial di~ff or removal, and thus only FC were studied in these experiments. Clostridium perfringens was monitored as an indicator ofanaerobic spore-fonning bacterial survival. and the di~ff of pathogenic salmonellae, Vibrio cholerae 01 and campylobactcrs were also evaluated. Since the use of treated wastewalcr effiuents for restricted irrigation requires no FC standard, only the helminth guideline of < I egg per litre, the numbers of helminths in the water column of the ESR were also enumerated. Sampling was routinely carried out at 08.00h by drawing off the required volume of sample with a variable speed peristaltic pump whose tube was attached to a rigid pair of discs (200 nun diameter) fixed O.OS m apart. The depths sampled were O.OS m, 0.2S m, O.SO m, 0.7S m, 1.00 m, I.SO m, 2.00 m, 3.00 m, 4.00 m, S.OO m, 600 m. During stratification studies samples were also taken at 14.00 h During the filVemptying phases samples were taken after the depth had risenlfallen by I m; during the rest phase the samples were taken at weekly intervals. During the filling and emptying cycles the Faecal coliforms (FC) present were analysed to indicate the number of pathogens present. When the reservoir was in the rest phase Clostridium perfringens (CP), Salmonella spp., Campylobacter spp. numbers were also determined. FC enumeration followed the membrane filtration technique using 0.4SIW Gelman celulose ester filters and oxoid membrane Iauryl sulphate broth with incubation at 44.S·C. CP were analysed for by first treating the sample to kill all the vegetative cells, leaving only sporefonning bacteria. This treatment involved leaving the sample in a water bath at 70 • 7SoC for twenty minutes. Perfringens Agar Base (Oxoid) was made up according to the manufacturer's instructions and used in SS nun Petri dishes. The sample was filtered through a 0.4S jIlII membrane in a manner similar to FC analysis. Each membrane was then placed in a Petri dish containing the agar medium and the plates put into a BBL Gas Jar with an anaerobic gas generating kit (Oxoid), the jar sealed and put in an incubator at 37.0°C for 48 hours. After incubation the visible black colonies were counted asCP.

Pathogen removal in effluent storage reservoirs Sa/monella spp. were analysed by the DlCIhod described in Oragui et a/. (1993a). A series of VOIURlCS from each depth sample (50 mI, 10 mI and I ml) were pipeued into sterile Rappaport Vassilidis broth (Oxoid), then incutxlled at J7.0·C for 24 hours. A loopful of this culture was then spread OrllO a 80 mm plate of Xylose Lysine Dc
serology was done on any large black shiny colonies using Polyvalent.Q Groups A-S (Murex) and Polyvalent • H phases I & 2 (Murex) agglutinating sera. Plates with colonies giving a negative reaction or which did not have colonies appropriate for serology were incubated for a further 24 hours at 37.0·C. Appropriate colonies wc:re again screened using slide serology, but this time any showing a negative n:action or black colonies too small for serology were subcultured onlo both &0 nun plate of MacConkey Agar (Oxoid) and a Lysine Iron At;.r (LlA) (Oxoid) tube slope; both these subcultures were incubated for 24 hours at 37.0·C. Any plates which showed no black colonies were recorded as negative. If the LlA tube showed black colouration after incubation, the corresponding MacConkey subculture was examined and any almost clear colonies underwerll slide serology. Any colonies gi\ing a negative reaction now or plates not showing growth appropriate for serology were recorded as negative. The results were analysed with the aid of a Most Probable Number (MPN) table to dctennine the number of Salmonella spp. present. Campy/abaete,. spp. identification was done by first inoculating media with a sample - single strength for sample volumes I mI or less and double strength for volumes of 10 mI and above. The medium was Nutrient Broth No.2 (Oxoid), which after sterilization had Campylobacte,. selective supplement

and So/. 1ysed defibrinolated horse blood added. A series of volumes for each sample depth was pippctted tnto a series of bottles containing the prepared medium, then the bottle was filled to the neck with media (i.e. to creale micracrobic conditions) and incubated at 37.0·C for 24 hours. Caml'}'1otlacter Blood Free Agar (Oxoid) was prepared and the incubated samples streaked onto this. The plates ""-ere incubated at 37.0·C for 24 hours in a BBL Gas Jar with a gas generating leit suitable for Campy/abaete,.. The plates were examined under a strong light for the typical Campylabacler appearance of a clear slime. Again a MPN table was used to determine the numbers for each sample depth.

Hbrlo cho/e,.oe 01 was determined by the MPN method previously deseribed (Oragui et a/., 1993b).

Helminth eggs: the number of human inlestinal nematode eggs was determined by the method of Mara and Silva (1986). Physicochemical parameters were analysed according to the methods described APHA (1989). RESULTS AND DISCUSSION Samples laken at 08.00 h at dilfererll levels throughout the depth of the ESR during the rest phase failed to detect significant differences in bacterial numbers at different levels in the water column (fable 2). Similar results were obtained with the physiCCKhemical parameters deseribed in Mara et a/ (1996). This indicates that the water column in the ESRs destratifies and turns over within each 24 hour period, principally at night. Thus sampling at 08.00 h provides a conservative estimate of wastewater quality, as it undergoes purification. Although routine sampling involved continuing the collection of samples from all depths, only bacterial values of the O. S m level are included in the tables showing the results of the punfication process during the resting phase of ESR operation.

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H. W. PEARSON er al.

254

Table 2. Faecal Bacterial Numbers at Different Depths in the ESR at 08.00 h on the First Day of the Rest Phase in Experiment 5.

Bacterial numbers per 100 00 Depth from surface (m)

FC

C. perfringens

Salmonellae

Campylobacters

0.05

7.02 x 105

0.25

8.32 x 105

8.00 x 10 1 8.36 x 10 1

2.20 x 10 1 1.30 x 10 1

3.00 x 100 2.00 x 100

2.40 x 101 1.00 x 10 1

0.50

7.94 x 105

7.84 x 101

0.75

7.24 x 105

1.00

8.54 x 105

7.28 x 10 1 7.54 x 10 1

7.90 x 105

7.80 x 10 1

2.00

7.48 x 105

3.00

7.94 x 105

8.06 x 10 1 7.78 x 10 1

4.00

8.68

5.00

8.50 x 105 8.32 x 105

1.50

6.00

x 105

7.64x 10 1 7.60 x 10 1 9.38 x 10 1

1.40 x 10 1 2.10 x 10 1

3.00 x 100 5.00 x 100 1.00 x 100

2.00 x 10 1

2.00 x 100 7.00 x 100

3.80 x 10 1 1.20 x 10 1

4.00 x 100 5.40 x 10 1

2.40 x 10 1 2.70 x \01

5.00 x 100

5.00 x 100

The di~ff of FC under the different loading regimes using anaerobic pond emuent are presented in Table 3. These results show that even at the most rapid filling rate of 18 days, equivalent to a surface BODS loading of 324 kglha d (Experiment 2) the entire wastewater contents of the ESR were suitable for unrestricted irrigation (I.e. it had an FC concentration of< 1000 per 100 00), after 21-28 days into the rest phase.

The Clostridium perjnngens data (Table 4) also show that no funher decrease in CP numbers occurred after 28 days of resting at which point a 2-3 log reduction had occurred. The continued presence of CP in the water column suggests that the daily turnover in the ESR brings the settled spores back into the water column from the sediments. but this is unlikely to represent a health hazard as few pathogenic anaerobic spore-formers occur in wastewaters.

Pathogen removal in effluent storage reservoirs

25S

Table 3 Faecal coJifonn numbers in the ESR (filled with anaerobic pond eft1uent) from the start of the rest phase (experiments 1·3 and S)·

Fe numbers per 100 ml Days from stan of rest phase Raw sewage Anaerobic pond emuent 0 7 14 21 28 35 42

Experiment 1

Experiment 2

Experimenl 3

Experimenl S

2.29 x 107 5.45 x 106

3.01 x 107 7.84 x 106

4.10x 107 1.97 x 107

5.39 x 107 9.98 x 106

7.94 x lOS 1.62 x 104 S.10 x 102 2.24 x 102 4.20 x 10 1 2.60 x 10 1 3.60 x 10 1

8.92 x lOS 1.34 x lOS 2.50 x 104 1.80 x 103 S.OO x 10 1 2.70 x 102 6.06 x 10 1

1.33 x 106 1.87 x 105 7.17xl02 3.00 x 102 8.67 x 10 1 6.67 x 100 1.27 x 10 1

3.7 x 105 3.3 x 103 1.6 x 103 6.0x 102 1.4 x 102 8.h 10 1 7.7x 10 1

• The samples were taken from a depb of O.S m in the ESR water column.

.. All counts are the means of allcast lriplicate determinations for all bacterial types. Table 4. Clostridillm perfnngens numbers in the ESR (filled with anaerobic pond effluent) from the start of the rest phase of operation (experiments 1·3 and 5)

ClostridJ 11m perfringens numbers per 100 ml

Days from stan of rest phase Raw sewage Anaerobic pond emuent 0 7 14 21 28 3S 42

Experimenl 1

Experimen12

Experiment 3

Experiment 5

7.95 x loS

3.40 x lOS

3.2S x lOS

4.70 x lOS

1.07 x 103

l.lRx lOS

1.10 xlOS

9.60 x 104

7.84 x 10 1 2.42 x 10 3 2.02 x 103 4.22 x 102 7.52 x 102 2.41 x 102 6.12 x 102

8.33 x 100 1.S7 x 102 lOO x 10 1 1.37 x 102 5.08 x 10 1 5.92 II 10 1 NO

5.42 x 104 1.67 x 104 1.40 x 103 US x 103 4.63 x 10 3 2.60 x 103 1.73 x 103

S.33 x 104 2.58 x 104 3.33 x 102 8.4Rx 10 3 NO

1.17 x 104 4.38 x 10 3

H. W. PEARSON et at.

256

Table S. Salmonellae numbers in the ESR (filled with anaerobic pond emuent) from the start of the rest phase of operation (experiments 1-3 and 5) Salmonellae numbers

Days from start of rest phase

Experiment I

Experiment 2

Experiment 3

Experiment S

Raw sewage

1.27 x 102

3.80x 102

2.4 x 103

2.20 x 103

Anaerobic pond emuent

6.22 x 10 1

S.19x 102

4.9 x 102

1.7 x 103

2.40 x 10 1 7.00 x 100 3.00 x 10 1

7.90 x 10 1 1.00 x 100 <1.00 x 100

3.30x 10 1 5.00 x 100 <1.00 x 100

<1.OOxOO <1.00 x 100

2.30 x 10 1 <1.00 x 100 1.00 x 100

<1.00 x 100 <1.00 x 100

<1.00 x 100

NO NO

2.00 x 100 <1.00 x 100

0 7 14 21 35

3.00 x 100 <.00 x 100 <1.00 x 100

42

<1.00 x 100

28

NO

<1.00 x 100 <1.00 x 100

Table 6 Campylobacter numbers in the ESR (filled with anaerobic pond eft1uent) from the start of the rest phase of operation (experiments 1-3 and 5) Campylobacter numbers per 100 ml

Days from start of rest phase

Experiment I

Experiment 2

Experiment 3

Experiment S

Raw sewage

31

11

NO

54

Anaerobic pond emuent

13

5

NO

18

3


NO NO NO NO NO NO NO

3 3 6

°

7 14 21

28

3S 42

9
NO NO

<1
NO

2

<1
Table 7 Bacterial numbers in the ESR (filled with raw sewage) from the start of the rest phase of operation. (experiments 4 and 6). Bacterial numbers per 100 mI

251

Pathogen removal in effluent storage reservoirs

Days from start of

Faecal colifonns

C. perfringens

Salmonellae sp

Carnpylobaeter

sp

rest phase

Raw sewage

0 7 14

21 28

3S 42

Expt4

Expt6

Expt4

Expt6

Expt4

Expt6

Expt4

Expt6

1.18 x 107

1.59 x 107

6.55 x 105

4.1x lOS

2.13 x 103

2.13 x 103

7

7

3.2 x lOS 4.0 x 104 7.5 x 10 3

2.3 x lOS

2.48 x 104 1.60 x 104

4.9 x 104 8.9 x 104

7

4.9 x 10 1 l.l x 10 1

1.4 x 10 3

2.95 x 104

7.8 x 103• 1.8 x 104 • 2.6 x 106 •

1.3 x 103

4.07 x 103

1.17 x 104

1.3 x 103

7.73 x 10 3

1.7 x 10 3 4.2 x 102

4.3 x 10 1

2.9 x lOS 1.6 x 104

5

3


5



9

l.S3 x 104


NO

3

2.08 x 103

2.50 x 104

NO


NO

2

1.34 x 10 3

1.30 x 104

NO


NO


2


5

6

• Apparent increase in Fe numbers (yellow colonies on membrane with lauryl sulphate broth) was found by biochemical tests (indole production and gas from lactose) to be due to non-Fe organisms.

258

H. W. PEARSON el al. Table 8 Faecal coliform numben at different depths in the water column of ESRJ (filled with raw sewage) during the resting phase of Experiment 6. Samples were collected at 08.00 and 14.00 h for comparison. FC numbers per 100 mI at 14-28 days into the rest phase

Depth from surface (m)

0.05 0.25 0.50 0.75 1.00 1.50 2.00 3.00 4.00

14 days

21 days

28 days

08.00h

14.00 h

08.00h

14.00 h

08.00h

14.00 h

9.73" 105 1.14" 106

1.25" 105 2.15" 104

1.67" 103 2.00" 103 1.33" 10 3 5.50" 103

4.00" 103 5.33" 10 3 1.33" 10 3 1.67,,103

3.47" 102 1.05 x 103 1.65 x 103 3.60 x 102

8.86" 102 4.87 x 102 4.80 x 102 6.20 x 102

2.40 x 104 2.67 x 103 2.00 x 10 3

2.50" 10 3 2.00 x 103 1.33 x 103

2.67 x 10 3 4.00" 103

2.80 x 102 2.80 x 102 4.60 x 102

3.47 x 102 2.07 x 102

2.00 x 10 3 1.67 x 103 3.50 x 103

3.40" 102 3.90 x 102 4.90 x 10 2

4.33 x 103

3.30 x 102

2.93 x 105

7.55" 105 2.23" 10 5

6.90 x 105 3.87 x 10 5 8.03 x 10 5 2.67 x 10 5

1.55" 104 9.00" 104 5.85" 104 3.10 x 104 3.45 x 104 2.75 x 104

5.00

1.51 x 106

2.17xl04 2.17 x 104

6.00

1.40 x 10 5

1.93 x 104

2.00 x 103 5.00 x 103

7.67 x 102

3.13 x 102 5.47 x 102 2.60 x 102 3.13 x 102

Numbers of salmonellae and campylobacters in the influent wastewater were low compared to Fe numbers, and these organisms were absent from the water column within 21 days in all the experiments in which the ESRs were filled with anaerobic emuent (Tables 5 and 6). In experiments 4 and 6 in which the ESRs were filled with raw sewage in n days (127 kg BODIha d) and 30 days (326 kg BOOIha d) respectively, the purification process took longer (Table 7), but wastewater quality still met the WHO guideline (WHO 1989) for unrestricted irrigation within 42 days into the rest phase. After this period of resting Salmonella and Campylobaeter numbers were negligible. The lack of faecal bacterial stratification in the water column at 08.00 h was compared with the situation later in the day when physico-chemical stratification had re~lished itself. The results presented in Tables 8 and 9 for FC and CP respectively confirm that at 14.00 h (when physicochemical stratification was pronounced), bacterial stratification was not significant, although there was some evidence of reduced bacterial numbers in the water column at 14.00 h, panicularly in the case of Clostridium perfringens. Vibrio cholerae 01 was monitored throughout the experimental period. However, the cholera epidemie had passed in Campina Grande at the time of this study and the numbers of V. cholerae in the raw sewage rarely exceeded 1 per 100 mi. Consequently, V. cholerae was never detected in the emuent from the anaerobic ponds or in the ESRs.

Helminth numbers averaged < 10 per litre in the emuents of the anaerobic ponds, and none was detected in the water columns of the ESRs during the fill or rest phases, regardless of whether they were filled with anaerobic pond emuent or raw sewage.

Pathogen removal in effluent storage reservoirs Table 9 Clostric6um perfringens numbers at different depths in the water column of ESR I (filled with anaerobic pond effluent) during the late resting phase (day 56) in Experiment I. Samples were coIlceted at 08.00 and 14.00 for comparison. Clostric6um perfringens per 100 ml

Depth from

08.00h

14.00 h

1.19" 10 3 1.28" 103 1.19 x 10 3 1.05" 103 9.98 x 102

1.49" 102 4.70 x 10 1 6.20 x 10 1 6.40 x 10 1

surface (m) 0.05 0.25 0.50 0.75 1.00 I. SO

2.00 3.00 4.00 5.00 6.00

9.86 x 102 9.96 x 102 7.64 x 102 8.96" 102

8.96 x 102 9.10 x 102

3.00" 10 1 2.10 x 10 1 2.80 x 10 1 2.30 x 10 1 1.30 x 10 1 2.20" 10 1 1 1.90 " 10

CONCLUSIONS This study has clcarly shown that adoption of the strategy of separating the fill, rest and use phases in ESR, as proposed by Mara and Pearson (1992), ensures rapid microbiological purification of the stored wastewalcr to a level suitable for unrestricted irrigation. At temperatures of 25°C ESRs filled with anaerobic pond effiuent at organic loading rates comparable to those used for facultative waste stabilization ponds at similar temperatures, produced purified effiuents (FC < 1000 per 100 mI) within 28 days. This is well within any likely minimum storage time employed in irrigation programmes involving ESR. The wastewater was found to purifY at the same rate throughout its depth, and therefore ESR effluent usage can be safely matched to any likely irrigation regime and docs not impose restrictions on the speed of ESR draw down. The results from the FC studies confirmed their applicability as indicators of pathogen removal in ESR S)'SlCrns.

The numbers of helminth eggs do not represent an impediment on the use of ESR effluent, as thejr presence could not be detected in the water columns of the ESR once they had filled. Pbysico-chemical water quality (sulphide levels), rather than microbial quality, is likely to control the length of the rest phase before the ESR contents are used for irrigation.

ACKNOWLEDGEMENTS We wishto express our gratitude to the Overseas Development Administration of the United Kingdom Government, for financing this two year research programme from April 1993 - May 1995. We are also greatly indebted to the Universidadc Federal da Parm'ba - UFPB, and the Companhia de Aguas e

259

H. W. PEARSON et al.

260

Esgotos da Paraiba - CAGEPA, for the provision of research facilities at EXTRABES and Catingueira. Almost all the academic, technical, clerical and maintenance staff at EXTRABES were involved in one way or another with this research project. Indeed without them and their unstinting devotion the project would never have drawn to a successful conclusion. We are very pleased to thank thcm all, especially Miss Salena Tatiana Silva, Mr Jose Soares and Mr Andre Araujo (doctoral students) and our own three field assistants who worked such long hours so cheerfully and so efficiently: Lenirnar de Andrade Oliveira, Jose Wanderley do Nascimento Silva, and AntOnio Minervino da Silva to whom we say a heartfelt thanks.

REFERENCES Abeliovich, A (1982). Biological equilibrium in a wastewater reservoir. Wat",. Research, 16(7), 1135-1138. APHA (1989). Standard Methods for the F.xamination of Water and Wastewater, 17th edition. Washington: American Public Health Association. Oor,l. And Raber, M. (I 99O).Deep wastewater reservoirs in Israel: Empirical data for monitoring and control. Water Research, 24(9), lOn-10M. Juanico, M. and Shelef, G. (1991). The performance of stabilization reservoirs as a function of design and operation parameters. Wat",. ScIence and Technology 23 (7/9), 1509-1516. Juanico, M. And Shelcf, G. (1994). Design, operation and performance of stabilisation reservoirs for wastewater irrigation in Israel. Wate,. Research, 28(1),175-186. Mara, D.O. and Pearson, H. W. (1992). Sequential batch-fed effiuent storage reservoirs: a new concept of wastewater treatment prior to unrestricted crop irrigation. Wate,. Science and Technology 26 (718), 1459-1464. Mara, D.O. and Silva, SA (1986). Removal ofintcstinal nematode eggs in tropical waste stabilization ponds. Journal ofT,.opical Medicine and Hygiene 89, 71-74. Mara, D.O., Pearson, H. W., Silva, SA and de Oliveira, R (1996). Process performance of experimental deep effluent storage reservoirs under different organic loadings and operating regimes. Water Science and Technology xxxxxxxxxxxx. Oragui, J.I., Arridge, H., Mara, D.O., Pearson, H.W. and Silva, SA (1993a). Vibrio Cholerae 01 (El Tor) removal in waste stabilisation ponds in northeast Brazil. Wale,. Research, 27(4), 727728. Oragui, J.I., Arridge, H., Mara, D.O., Pearson, H.W. and Silva, SA (1993b). Enumeration of saImonallae in wastewater by the MPN technique. Water ReseQr'ch, 27(11), 1697·1699. WHO (1989). Health Guidelines for the Use of Wastewater in Agricultun and Aquacullun. Technical Report Series No. 778. Geneva: World Health Organization.