Anclostoma duodenale and Necator americanus. As the number of helminth eggs in effluents of waste treatment plants is normally very low ( 4,. 5, 6) any method ...
Environmental Technology, VoL 12. pp 617-623. @Publications Div:i!Don Selper Ltd..1991
COMPARISON OF TECHNIQUES FOR THE ENUMERATION OF HUMAN PARASITIC HELMINTH EGGS IN TREATED WASTEWATER. R.M. AYRES*, R. STOTI', D.L. LEE, D.D. MARA, S.A. SILVA.
Depts. of Civil Engineering and Pure and Applied Biology, University of Leeds, LS2 9JT, U.K. (Received 17 June 1991; Accepted 1July1991)
ABSTRACT Four methods for the enumeration of human parasitic nematodes in treated wastewater were compared in field trials in the northeast of Brazil. Effluents from a series of waste stabilisation ponds and a physico-chemical lime treatment plant were used. Comparisons were made on a daily and 24 hour basis. The recovery of eggs was found to be consistently higher using the method currently recommended by the World Health organisation (1) (commonly known as the Bailenger method) but only when 10 l samples, rather than 11 samples, were processed.
INTRODUCTION
An extensive range of tests has been developed and standardized for the monitoring of physico-chemical and bacteriological parameters in sewage treatment systems. However, one important area for which there is still no single standardized method is the enumeration of parasitic helminth eggs. The current World Health Organisation guidelines for the microbiological quality of treated wastewaters used for crop irrigation, require an arithmetic mean of ::::1 intestinal nematode egg per litre (1, 2, 3), the species referred to being Ascaris lumbricoides, Trichuris trichiura, Anclostoma duodenale and Necator americanus. As the number of helminth eggs in effluents of waste treatment plants is normally very low (4, 5, 6) any method used must have a high recovery rate and/or use a large sample volume to allow detection to the guideline level; additionally it must also be suitable for routine use in only basically equipped laboratories. In this study four methods for egg recovery were compared on effluents of various qualities, and their speed, ease and cost were assessed with specific
reference to their use in a busy multidisciplinary laboratory where helminthology is likely to be only one part of a technician's daily routine. The work was carried out at EXTRABES, the Federal University of Paraiba's experimental research station in Campina Grande, northeast Brazil, using effluents from a series of waste stabilisation ponds (7) and from a physicochemical lime treatment plant (8). The methods tested were as follows: (A) the method currently recommended by the World Health organisation, more commonly known as the Bailenger technique (1); in its published form 1 litre samples are processed, but during this work samples of 1 and 10 litres were taken; (B) the method currently used at EXTRABES (4), in which 500 ml samples are processed; (C) a method developed specifically to look for very low numbers of helminth eggs in pond effluents, the Leeds II technique (9), where 4 x 1 litre samples are examined on each occasion; and (D) an adaptation of the modified Janeckso-Urbanyi method in which the final flotation solution was saturated sodium thiosulphate with a specific gravity of 1.3 (10); samples of 25 l were taken on each occasion.
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MATERIALS AND METHODS
Samples were taken at 0800 h on a daily basis from the effluents of a primary anaerobic pond (coded A8) its secondary facultative pond (Fl6) and a single primary facultative pond (Fl8); and at 0200 h on a daily basis from the effluent of the lime treatment system (Cl) (helminth eggs are most commonly found in the effluent of this system at night). Samples for each method of analysis were taken in immediate succession and processed as soon as possible after collection, except that samples from Cl were left to sediment after collection and were processed the following morning. A 24-hour study was also carried out on the effluents of Fl8 and Cl; samples were taken every three hours (0800-0800 h) and analysed by each technique. Throughout the work the more subjective aspects of each method were noted; these covered ease of processing, time taken for final microscopic analysis, likelihood of operator error and if the technique varied with the quality of the effluent. Suspended solids were measured by filtration through
Whatman GF/C glass fibre filter papers (11) and chlorophyll a was determined using a methanol extraction method (12). RESULTS AND DISCUSSION
Tables 1-4 show the results of the daily monitoring for helminth eggs in each reactor. The means were compared using the T-method (13) using Gabriel's 95% intervals where means whose intervals do not overlap are significantly different at p = 0.05, Ii and ui being the lower and upper limits respectively. In the effluent of Cl, method D detected 71 % A. lumbricoides and 29% T.trichiura. Methods A, B and C detected only A lumbricoides. The differences between the methods can be seen in Tables 1-4. There were no significant differences in the mean helminth egg counts, primarily because of the large standard deviations in methods A (using 1 L samples) and B. Moreover, these two methods detected eggs on very few occasions compared to the other methods. Method B detected eggs only in the effluent of pond A8 (Table 2).
Table 1. Total helminth eggs per litre in the effluent of Cl with Gabriel's 95% intervals for comparison of means.
Day
Egg count per litre by method: AlL
AlOL
B
c
D
1 2 3 4 5 6
10 5 0 0 0 0
1.25 0 0 0 0 0
0 0 0 0 0 0
3.5 4 0.25 0 0.5 0.5
2.2 0.39 0 0 0 0.26
Mean ui Ii SD
2.5 4.38 0.62 4.18
0.25 2.13 0 0.56
0
1.46 3.34 0 1.79
0.48 2.36 0 0.86
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Table 2. Total helminth eggs per litre in the effiuent of A8 with Gabriel's 95% intervals for comparison of means.
Day
Egg count per litre by method: AlL
AlOL
B
c
D
1 2 3 4 5 6 7
5 0 8 0 0 0 5
9.55 0 2 3 1.5 0.5 0.5
10 0 0 0 0 0 10
11.25 4 2.75 4.25 4.25 5.0 4.25
0.79 0.13 0.61 0.19 0.39 0.26 0.39
Mean ui li SD
2.57 5.13 0.02 3.36
2.44 4.99 0 3.30
2.86 5.41 0.31 4.89
5.11 7.66 2.56 2.79
0.39 2.94 0 0.23
Table 2a. The relative percentage of each species of nematode in the effiuent of A8.
Method
Percentage of each species. lumbricoides T. trichiura hookworms A.
A( 1 L) A( 10 L) B
72 84 100 99 44
c
D
0 8 0 0 22
28 8 0 1 34
In Table 4, method D recovered 71 % A. lumbricoides and 29% T.trichiura; all other methods detected only A. lumbricoides. Eliminating exaggerated results and increasing the rate of egg detection can be achieved in two ways: the original volume sampled or the final proportion of the sample examined under the microscope can be increased, or both. The number of positive samples is increased greatly in method A, by increasing the sample size to 10 L, as shown in Tables 2, 3 and 4. The proportion of the final sample examined remained unchanged.
The assumption is made in methods A and B that eggs are distributed uniformly within the final step of the sample processing, thus a multiplication step is used to convert the number of eggs found to eggs per litre. If the only egg in the sample is detected, the final egg count will be greatly exaggerated, increasing the standard deviation in a series of samples. The small sample size plus this subsampling stage makes detection of very low numbers improbable. In Table 3, method A (10 L) recovered 60% A. lumbricoides and 40% T.trichiura. 100% of eggs recovered by method C were A. lumbricoides.
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Table 3. Total helminth eggs per litre in the effiuent of Fl6 with Gabriel's 95% intervals for comparison of means.
Day
Egg count per litre by method: AlL
AlOL
B
c
D
1 2 3 4 5 6 7
0 0 0 0 0 0 0
0 0 0 0.5 0.5 1.5 0
0 0 0 0 0 0 0
0 0.5 0 0 0.5 0.5 0
0 0 0 0 0 0 0
Mean ui Ii
0
0.36 0.62 0.11 0.56
0
0.21 0.47 0 0.27
0
SD
Table 4. Total helminth eggs per litre in the effiuent of Fl8 with Gabriel's 95% intervals for comparison of means.
Day
Egg count per litre by method: AlL
AlOL
B
c
D
1 2 3 4 5 6 7
5 0 0 0 0 0 0
1.0 1. 7 1.25 0 0.5 1.0 0
0 0 0 0 0 0 0
0 0.25 0.25 0.25 1.0 0 0
0.26 0 0.13 0 0.13 0.13 0
Mean ui li
0.7 1.45 0 1.89
0.78 1.53 0.03 0.64
0
0.25 1.00 0 0.35
0.09 0.83 0 0.10
SD
The elimination of a final subsampling step and the larger sample size increased the rate of egg detection in method C as compared to methods A (1 L) and method B (Tables 1, 2 and 3). The improved rate of detection in method D was due to a combination of larger sample size and a
decrease in the proportion of the final sample examined. The last step of this method assumes that all the eggs have been concentrated in the meniscus of the flotation solution, 0.3 ml of which is removed and examined. The majority of suspended solids found in
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there was no positive correlation between suspended solids and egg numbers from any of the methods. The sedimentation tank had a mean hydraulic retention time of 12 hours and was shown to produce an effluent well within the guidelines for wastewater reuse, both in the 24 h and daily monitoring (Tables 1 and 6). The same time is required to look at the McMaster slides from either the 1 litre or 10 litre samples of method A (Table 7); normally two slides per sample are examined and a mean calculated. For method D only one McMaster slide can be examined as the sample is taken from the meniscus which, once disturbed cannot be sampled again. In method C it is recommended that 4 x 1 L samples are examined on each occasion and this greatly increases the amount of time required at the microscope. Preparation and processing for each method are straightforward: they require the same basic laboratory equipment, a bench top centrifuge and microscope, and take roughly the same time. Method B requires no chemicals at all and just simple microscope slides for the final analysis. Method C needs technical grade sodium chloride and the use of a Doncaster counting dish (which needs to be ordered from the United Kingdom).
the effluents of waste stabilisation ponds are algae, many of which are motile species (14). These motile algae tend to move up and down the pond profile in response to sunlight and temperature, and therefore effiuent suspended solids concentration tends to follow a diurnal pattern (14). Table 5 shows clear correlations between suspended solids and chlorophyll a, ( r = 0.989, p = 0. 001) and temperature and suspended solids (r = 0.792, p = 0.01). AB in earlier studies (9) no correlation could be found between the numbers of eggs in the effiuent and its suspended solids concentration with any of the methods used, suggesting that helminth eggs appear in the effluent independently of the suspended solids, probably by hydraulic short circuiting or resuspension by gases moving up from the anaerobic sludge layer. Tables 4 and 5 show that pond F18, which had a mean hydraulic retention time of 15 days, was producing an effluent within the WHO guidelines. The suspended solids in the effluent of Cl also varied over the 24 h period (Table 6) with a slightly higher concentration at night, presumably due to thermal mixing within the pond as the air temperature dropped. Very few helminth eggs were found in the effluent and
Table 5. Comparison of techniques for the enumeration of helminth eggs in the effiuent of pond F18 over 24 h.
Time
0800 1100 1400 1700 2000 2300 0200 0500 0800
Tss Chl
Temp Eff°C
27.5 31.0 32.0 30.0 27.5 26.0 26.0 26.0 26.0
TSS
(mg l·l)
102 182 122 111 63 73 77 64 88
Total Helminth Eggs per litre
Chl (µg l·l)
888 2379 1221 896 234 477 467 397 759
AlL
AlOL
B
c
D
0 0 0 0 0 0 0 5 0
0 0 0 0 0 0.5 0 0.5 0.5
0 0 0 0 0 0 0 0 0
0.25 0 0 0 0.25 0 0 0.5 1.0
0 0.131 0 0.653 0 0.131 0 0 0.131
Total suspended solids. Chlorophyll a.
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Table 6. Comparison of techniques for the enumeration of helminth eggs in the effluent of the lime treatment plant settlement tank (Cl) over 24 h.
Time
0800 1100 1400 1700 2000 2300 0200 0500
0800
Temp Efl'°C
25.0 28.8 30.4 28.6 24.2 23.0 22.3 22.3 24.4
TSS (mg 1·1)
31 36 28 34 39 38 40 34 32
Total Helminth Eggs per litre AlL
AlOL
B
c
D
0 0 0 0 0 5 0 0 0
0 0 0 0 0 0.5 0 0 0
0 0 0 0 0 0 0 0 0
0 0 0 0 0.5 0.25 0 0 0.5
0 0.131 0 0 0 0.131 0 0 0
Table 7. Mean time (min) for final microscopic examination.
Reactor
Cl Fl6 Fl8 A8
Method A
B
c
D
1.0 2.0 2.0 2.0
5.0 5.0 5.0 6.0
4.5 6.5 9.0 10.0
3.0 3.0 3.5 3.0
Methods A and D call for ether, glacial acetic acid, sodium acetate and zinc sulphate, or sodium thiosulphate respectively for the final flotation. The McMaster slides needed for enumeration are normally available through local suppliers. Method D and the 10 L version of method A produced considerable quantities of solids in the ether layer, particularly algae from the pond effluents. The band of algae in the 25 1 samples was so dense that the persistently lower egg count may have been due to eggs being retained in this layer during centrifugation. CONCLUSIONS
Method A using 1 litre samples is inefficient with low egg counts. However with 10 litre samples its detection rate is much higher and compares with that of method C. Sample
preparation is straight forward and in terms of microscope work it is the least time consuming method. A few special reagents are required, but they are all usually available locally and are inexpensive. McMaster slides are routinely used in parasitology laboratories and should be easily available. The method can also be used for raw sewage, and it has been shown (15) that all species of helminth eggs routinely found in wastewater can be detected. Method B is the cheapest and easiest and samples such as raw sewage, where egg concentration is much higher, can be analysed using this method. However, it is inadequate for the detection of very low numbers of helminth eggs due to both the small sample size and the subsampling step involved. Method C is cheap and easy to perform. When the total suspended solids concentration is low,
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each dish can be examined in about 5 - 10 min. However effluent quality is very variable and in certain effluents algae and heavier debris will not float in the salt solution; this leaves a dirty final sample which is both time consuming and tiring to examine. The main advantage of the technique is its high recovery rate, which is due to the fact that there is no subsampling stage and all the eggs from any individual sample are directly counted. Method D detected eggs with the same frequency as methods A (10 l) and C. The method was considered slightly less easy than the others due to handling the large samples, and in the last step it was found that, in certain samples, floating debris made sampling the meniscus difficult. For routine work in a busy laboratory
method A using a large sample size seemed to be the most useful way to enumerate helminth eggs in treated wastewater.
ACKNOWLEDGEMENTS
The technical help of Emilia Savana Maia Honorio and the financial support of the Overseas Development Administration (Research Scheme No.R4497) are both greatly appreciated. The suggestion from Prof. J. Schwartzbrod and Monsieur J.L. Stien for the use of sodium thiosulphate as a low cost alternative flotation solution for method D is gratefully acknowledged.
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