Water Research Pergamon Press 1973. Vol. 7, pp. 963-973. Printed in Great Britain

THE CONSTRUCTION OF A SAND PROFILE SAMPLER: ITS USE IN THE S T U D Y OF THE VORTICELLA

POPULATIONS A N D THE GENERAL INTERSTITIAL M I C R O F A U N A OF SLOW SAND FILTERS

B. LLOYD

Department of Biological Sciences, University of Surrey, Guildford, Surrey, England

(Received 24 September 1972)

Abstraet--A simple and inexpensive method is described by which the component groups of the interstitial fauna can be examined undisturbed by direct microscopy. The method has been developed specifically to locate and enumerate the functional interstitial microfauna of slow sand filters used in water purification and it is designed to demonstrate the spatial relations of the constituent populations as they develop in time in a flowing system. The sampler has been successfully applied to monitoring the development of Protozoa and Rotifera in pilot scale and full scale slow sand filters at the London Metropolitan Water Board's Walton and Ashford Common Treatment Works. Results are presented for the incidence of the general microfauna and for the development, vertical distribution and effect of flow rate on the Vorticella populations.

INTRODUCTION

THE efficacy of slow sand filtration in removing bacteria has been documented by BURMAN (1962) and in the METROPOLITAN WATER BOARD (M.W.B.) Reports on the results of the bacteriological, chemical and biological examination of the London waters (1967-1968, 1969-1970). IVES and GREGORY (1967) have summarized the physical mechanisms involved in removing suspended particles by sand filtration. However, there is almost no published work on possible biological mechanisms respon- sible for removing bacteria and other particles in suspension in the interstitial sand habitat. I t has long been known that a complex microbiological community, the "smutzdecke", accumulates at the sand-water interface.

With the exception of the algae (RIDLEY, 1967), the components of this community have not been described. The penetration of microorganisms into slow sand filters has not been examined, neither has any attempt been made to assess their functional role.

In 1970, the World Health Organisation took a fresh look at this the oldest method of water purification and is now encouraging its use in some developing countries and rural areas: but the increasing demand for drinking water has called into question the capability of slow sand filtration in urban areas in coping with this requirement. Work is in progress at the M.W.B. (London) to establish whether it is feasible to filter at significantly higher rates than the traditional and arbitrary 7-15 cm h - 1

The experimental work described here involved the development of a simple sampling procedure to investigate some of the questions regarding the general micro- bial ecology of slow sand filters, and more specifically those constituents of the micro- fauna which were likely to be strong candidates as bacterial predators.

A variety of methods for sampling aquatic sediments was reviewed by EDMONSON and WINBERG (1971) but none of the methods listed by these authors was ideal for

963

964 B. LLOYD

sampling sand filters. The column sampler described here has the following features: (I) Simple construction and composed of cheap materials. (2) No mechanical closure device, therefore no mechanism to jam. (3) The depth distribution of living organisms can be examined directly and im-

mediately without disturbing the column or subsampling.

M A T E R I A L S

Top quality "picture" glass, 2 mm thick. Chance microscope slides, 1 mm thick. Araldite [Ciba-Geigy (U.K.) Limited]. Nichrome wire. Nylon fishing line. Corks. Tools: glass cutter, template, fine pliers, bulldog clips.

M E T H O D S

Two 2-5-cm wide strips of picture glass were cut in lengths ranging from I0 to 50 cm depending on the depth required to be sampled. All grease and dirt was removed

Cork marker

Nylon fishing line

Nichrome loop

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- - Picture glass 2mm thick

FIG. l . Sand profile sampler.

The Construction of a Sand Profile Sampler 965

from the glass by applying a volatile solvent such as acetone. A sutticient number of microscope slides were cut into 0.5 cm wide strips for spacers. A template facilitated cutting slides of equal size.

A 4-cm length of nichrome wire was cut and bent into an omega-shaped loop. This provided the attachment point for the fishing line. A small quantity of Araldite was smeared sparingly along both 0-5 cm margins of each"picture" glass strip. The micro- scope slide spacers were mounted on each side of one glass strip. The nichrome loop was mounted so that it was firmly anchored between two adjacent slides. No gaps remained between adjacent slides, unless this was specially required. The second "picture" glass strip was next placed on top. The column was secured with bulldog clips and allowed to set overnight or for 2 h at 37°C.

When set hard the sampler column was packed with washed and dried sand by temporarily sealing one end with tape and pouring in dry sand obtained previously from the site to be sampled. As described the sampler was limited to medium to fine sand grades. The recommended size range of sand grains in relation to percentage of total weight in slow sand filters was as follows:

Up to 0.3 mm dia. = I0 per cent by weight; In the range 0.3-0.5 mm dia. = 50 per cent by weight; In the range 0.5-1.0 mm dia. = 40 per cent by weight. The size grading of sub samples was checked using British Standard test sieves to

ensure that the sand used in the sampler conformed to this. The volume ratio in the sampler was 46 per cent water: 54 per cent sand, which also conforms to the conditions in the filter bed.

At least 50 samplers were prepared as described above. All of this first batch were 25 cm long. They were used in two adjacent pilot scale slow sand filters, East and West, at Walton treatment works where the sand depth was approximately 0.7 m.

POSITIONING THE COLUMNS AND SAMPLING

The, samplers were introduced into the slow sand filters while the beds were drained for cleaning. Since these filters function by gravity under a head of 1.5 m of water, an equivalent length of fishing line was attached by one end to the nichrome loop of the sampler and by the other to a cork marker float. The entire sampler column was then driven into the filter until flush with the top of the bed using a mallet. The vertical positioning of the column was checked using a spirit level.

Slow sand filter beds are normally bottom-charged by a gentle upward flow of water through the sand bed until the entire bed is covered by several cm of water and then top charging commences until the desired head of about 1"5 m water is reached. After filtration has commenced, the filtrate is allowed to run to waste for a variable period, depending on the bacteriological quality of the filtrate. The average duration of filtration is about 50 days before cleaning is necessary.

SAMPLE COLLECTION

Samplers were removed at regular intervals during the entire period of filtration, thus allowing the development of the constituent populations to be traced from day 0 to the last day of filtration. The samplers were collected by taking hold of the cork

966 B. LLoyD

marker and pulling firmly. As soon as the sampler emerged from the water it was briskly dried externally with blotting paper and both the open ends were immediately sealed with polythene adhesive tape (Lassotape). Due to surface tension and capillarity no sand or interstitial water was lost from the samples during their removal, neither was sand displaced. The spatial relations of the non-living constituents and sedentary microorganisms were thus preserved and microscopic examination was commenced immediately in case animal migration occurred rapidly.

DIRECT MICROSCOPIC EXAMINATION

The most convenient system for scanning whole samplers was found to be a Wild stereo binocular microscope with a × 2 supplementary objective and × 15 eye piece. The routine scanning magnification was × 75 and the field width 3.3 mm. For species identification the magnification was changed to x 150, and, where this was inadequate the sample was transferred to a Leitz Orthoplan using ×400 magnification. The Wild microscope was used in conjunction with a 30-cm long perspex sampler carrier, designed specifically for this purpose (Plate 1). Illumination using transmitted light was preferred to incident light.

The whole area of one face of each sampler was scanned starting at the top taking transverse fields and then moving on one field. Simultaneously the microscope was racked up and down and, having the advantage of a good depth of focus, it was estimated that one-third of the 1 mm thickness of sand-water could be examined.

Organisms were scored for every I cm step. All organisms above about 20/~m (overall length) were counted, except flagellates, the majority of which were less than 20/~m. The dimensions of the sampler permit the examination of a mere 0.05 ml combined sand and water per cm step. This weakness was manifested by the low numbers recorded in TABLE 4. In the light of this experience triplicate sampling was introduced.

It was feared that animal migration during the course of examination would produce the error of counting the same animal more than once. In practice this did not occur since, with the exception of the Oligochaeta, the sand habitat imposes a relatively sedentary mode of life upon the microfauna.

RESULTS AND DISCUSSION

The normal quality parameters of the filtrate from the two filters were monitored weekly throughout the year. (M.W.B. Report 1971-1972, in press). These parameters were coliform, Escherichia eoli Migula, and pour plate colony counts as well as turbidity, colour, ammonia and albumenoid nitrogen. To effect the normal quality standards it is clear that the functional constituents in the sand bed must be consistently active and present in adequate numbers. TABLES 1 and 2 show clearly that only two main groups of the interstitial microfauna were present in a high percentage of samples throughout the 1-yr period. These groups are the ciliated Protozoa and the Rotifera.

It was expected that those groups found characteristically in fresh water and marine sands would also be present in slow sand filters. PENNAK (1950) surveyed the general ecology of the interstitial fauna of the first two habitats, and all three are summarized in TABLE 3. It can be seen that all groups listed as occurring in natural sand habitats were also found in the slow sand filters.

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968 B. LLOYD

TABLE 2. COMPARISON OF THE INCIDENCE OF MICROFAUNAL GROUPS OBSERVED IN SAMPLER COLUMNS

FROM TWO PILOT SCALE SLOW SAND FILTERS

Protozoa Rotifera (Ciliophora)

Sample date Bdelloid Non-Bdelloid Peritrichia Spirotrichia Holotrichia Suctoria

1971 W E W E W E W E W E W E Aug. 20 + - - - - + - - + + + + +

23 + + + + + + + + - - + 24 + + + + + - - + + + + + - - 26 + + + + + + + + + + - - + 29 + + + + + + + + + + + - - 31 + + + - - + - - + + + + + - -

Sept. 3 + + + + + - - + + + + + - - 6 + + + + + + + + + + 9 + + + + + + + + + + - - +

13 + + + + + + + + + + - - + 16 + + + + + + + + + + - - + 20 + + + + + + 27 + + + + + + + + + + + + 29 - - + + + + + + + + +

Oct. 4 + + + + - - + + + + + 6 + + + + + + + + + +

11 + + + + + + 18 - - + + + + - - 25 + + + + + - -

Nov. 1 + + + + + + + + + + + - - 8 + + + + + - -

1972 Filter bed sand completely replaced with washed sand

Jan. 7 - - + + - - + + + + + + 17 + + + + + + + + 24 + + + + + + + + + + - - +

Feb. 7 + - - - - + + + + + + +

Mar. 27 - - + + + +

Apr. 13 - - + + + + + + + + + 19 + + - - + + + + + + + + - - 24 + + + + + + - - + + + __ +

May 8 + + + + + + - - + + + - - + 15 + + + + + + + - - + + 20 + + + + + _ _

July 6 4- + + + + + + + + + + _ 10 + + + + + + + + + +

Yo incidence 85 92 85 85 94 89 94 96 100 100 29 33

W = west filter (15 cm h -1 flow rate). E = east filter (45 cm h - t flow rate). + = present. - - = absent.

The Construction of a Sand Profile Sampler 969

TABLE 3. COMPARISON OF THE INCIDENCE OF THE MICROFAUNAL GROUPS IN DIFFERENT SAND HABITATS

Slow sand filters Lake beaches* Intertidal* marine beaches

Very abundant Common, sometimes Fewer present, very abundant seldom abundant

Protozoa

Rotifera

Turbellaria

Nematoda

Very abundant. About 10 species are regular inhabitants

Occasionally abundant

Common in mature filters when detritus is present

Gastrotricha Small numbers regularly present

Tardigrada Small numbers occasionally

Oligochaeta Common in mature filters when detritus is present

Copepoda Few present, seldom abundant

Extremely abundant. About 40 species are regular inhabitants

Rare. Several species

Few individuals and few species

Variable numbers; many species present

Not usually abundant

Sometimes abundant Few individuals

Small numbers Small to medium numbers

Common to abundant; Common; several only three species hundred species

* Data of PENNAK (1950).

The largest organisms observed in the sand filter interstitial habitat were members of the Oligochaeta. Only three genera of this group were recorded Nais Miiller, Stylaria Lamarck and Aelosoma Ehrenberg. The last two were exclusively Stylaria fossularis Leidy and Aelosoma hemprichii Ehrenberg. The Oligochaeta were absent f rom newly cleaned sand filters in which the bed had been completely replaced with washed sand (TABLE 1). Since the Oligochaeta are reported to be detritus feeders it was reasonable to expect their return to coincide with the accumulation of interstitial silt. Likewise the Nematoda, Gastrotrichia and Turbellaria were only abundant in mature beds into which detritus had penetrated 2-10 cm. The Turbellaria, however, consumed a variety of algae when these penetrated the filters.

The protozoan classes Flagellata and Rhizopoda were regretfully excluded f rom this study although it is now clear that both are regular inhabitants of the filters. The stalked choanoflagellates are particularly numerous on sand grains.

Of the ciliated Protozoa the least frequent subclass was the predatory Suctoria, but the remaining three subclasses are known to be primarily bacteria-feeding ciliates. TAaLE 4 gives a fair representation of the relative numbers and vertical distribution of Protozoa and Rotifera in one sample. Despite the 100 per cent incidence of Holo- trichia recorded in TABLE 2, TABLE 4 shows that in terms of population the Holo- trichia are infrequent by comparison with the remaining groups. From a functional aspect there is a significant difference in their feeding mechanisms. In the sand habitat the commonly occurring species of Rotifera and Spirotrichia are strongly thigmotactic,

970 B. LLOYD

TABLE 4. ABBREVIATED SPECIMEN RESULTS SHEET FOR ONE SAMPLER COLUMN ALL COUNTS PER 0"05 ml COMBINED SAND AND WATER

Protozoa Rotifera (Ciliophora)

Sand depth Bdelloid Non Peritrichia Spirotrichia Holotrichia Suctoria (cm) Bdelloid Vorticella spp

0 4 - - 37 1 1 - - 1 1 7 1 3 - - - -

2 4 8 2 1 - - - - 3 2 6 1 5 - - - - 4 2 3 - - 5 - - - - 5 2 10 2 6 - - - - 6 4 4 - - 4 - - - - 7 - - 1 - - 1 - - - - 8 3 2 - - 1 - - - -

9 2 - - 2 1 - - - - 10 2 - - - - 1 - - - - 11 2 2 - - 2 - - - - 12 2 - - - - 5 1 - - 13 1 1 1 2 - - - - 14 - - - - - - 2 - - - - 15 - - - - - - 1 - - - -

16 1 1 - - 2 - - - - 17 11 . . . . . 18 - - - - - - 5 3 - - 19 1 1 - - 3 - - 1 20 - - 1 - - 5 - - - -

- - = None observed. Location = Walton West Pilot. Date = 1 November 1971. Filtration period = 20 days. Water temperature = 12.3°C.

t h a t is t h e y m o v e c lose t o t h e s a n d g r a i n su r face a n d a p p e a r t o f eed la rge ly o n t h a t

sur face . By c o n t r a s t , t h e Pe r i t r i ch ia , a l t h o u g h a t t a c h e d to t h e s a n d g r a i n s feed

exc lus ive ly o n p a r t i c l e s s u s p e n d e d in t h e w a t e r cu r r en t .

DRAOESCO (1962) e x a m i n e d t h e s a n d - d w e l l i n g c i l ia ted P r o t o z o a o c c u r r i n g in f r e sh

a n d sa l t wa te r . H e s t a t e d ca t ego r i ca l ly t h a t sessile a n d s e d e n t a r y a n i m a l s w e r e a b s e n t

f r o m t h e s a n d - w a t e r h a b i t a t . In s low s a n d filters, h o w e v e r , t h e s i t u a t i o n is c o m p l e t e l y

d i f fe ren t . T h e vo r t i ce l l a p o p u l a t i o n , f o r e x a m p l e , f o r m s one o f t h e n u m e r i c a l l y d o m i -

n a n t g r o u p s . In a d d i t i o n t h e S u c t o r i a are n o t u n c o m m o n in s l o w s a n d fi l ters. But

D r a g e s c o s t a t e s " t h e r e a re n o s and - l i v ing r ep re s en t a t i v e s o f t h e o r d e r s o f per i t r ic ! s

o r s u c t o r i a n s " . T h e m a i n d i f f e rence b e t w e e n l i t to ra l s a n d s a n d s a n d f i l ters is t h a t in

t h e l a t t e r case t h e r e is a c o n s t a n t u n i d i r e c t i o n a l f low b r i n g i n g a c o n s t a n t s u p p l y o f

s u s p e n d e d n u t r i e n t s w h i c h is l ack ing , o r a t bes t i n t e r m i t t e n t , in t he f o r m e r h a b i t a t .

O n e o f t he m o s t c o n s t a n t m e m b e r s o f t h e in te rs t i t i a l m i c r o f l o r a o f s l o w s a n d fi l ters

was t h e vor t i ce l l a p o p u l a t i o n . T h e spec ies m o s t c o m m o n l y iden t i f i ed u s i n g KAIaL

(1930-1935) w e r e II. campanula E h r e n b e r g , V. convallaria L i n n a e u s , a n d , less f re-

quen t ly , V. fromenteli K a h l , V. communis F r o m e n t e l a n d 11". picta E h r e n b e r g .

T h e ro le o f t h e P e r i t r i c h i a in r e m o v i n g b a c t e r i a has been e s t a b l i s h e d f o r t h e act i-

v a t e d s ludge p r o c e s s a n d f o r p e r c o l a t i n g fi l ters in s e w a g e t r e a t m e n t (CUROS a n d

VANOYKE, 1966). H o w e v e r , th i s h a s n o t p r e v i o u s l y b e e n e s t a b l i s h e d f o r s a n d f i l ters

The Construction of a Sand Profile Sampler 971

for domestic water supplies, and since the water quality is very different it was to be expected that vast qualitative and quantitative differences would be found in this habitat within the order Peritrichia. In biological aerobic sewage processes the colonial peritrichs are numerous; these have never so far been observed in slow sand filters. Those species of Vorticella listed above are oligosaprobic to mesosaprobic types (SL,~DE~EK, 1971). In activated sludge plants producing effluents in the BOD range 0-10 (CURDS and COCKBURN, 1970) V. campanula is given an association rating of 8/10, whereas V. convallaria is only 3/10.

P O P U L A T I O N D E V E L O P M E N T

As a first step towards assessing the functional role of Vorticella spp. as a group in sand filtration the rate of colonisation during the filtration period was examined for a number of filtration runs. This was executed by plotting the populations from regular samples and comparing these with the bacterial quality of the filtrate. Rapid colonisa- tion of clean sand within the first several days of filtration is a necessary prerequisite for a functional constituent. It can be seen from FIC. 2 that this requirement is ful- filled. Furthermore, the incidence of Vorticella as a percentage of all samples examined

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FIo. 2. Population development of Vortlcella spp. in the Walton West Filter. O Number of Vorticella c m - 2 in a 20-cm column of sand. /x Number of bacterial colonies 1 ml -x filtrate water developing in a yeastrel peptone agar pour plate incubated at 37°C for

24 h.

was very high (94 per cent ÷ 89 per cent), TABLE 2 thus fulfilling the requirements of constancy and persistence at high levels throughout the filtration period.

The effect of different rates of filtration on the Vorticella population

The effect of flow rate on the distribution of Vorticella spp. can be most readily assessed by summing replicate sample results for the low rate of filtration and com- paring them with a parallel series of results for the high rate of filtration. Since both

972 B. LLOYD

pilot filters studied were simultaneously receiving the same source of primary rapid sand filtered water and used the same source of sand this comparison is valid. The combined results of 20 samples from each filter are illustrated graphically in Fit;. 3.

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FIG. 3. Relationship between flow rate and the dispersal of the Vorticella spp. populations in two pilot scale filters. (Counts plotted represent 1 ml combined sand and water, obtained by

combining the counts for 20 samples from each filter.)

The low rate west filter was run at 15 cm h - i and the graph shows the vorticella populations clearly limited to the top of the sand bed and mainly the top 1.0 cm. On the other hand the high rate East filter, running at 45 cm h - 1 forces Vorticella deep into the filter. Since data had not been collected below 20 cm sand depth, a standard Olivetti programme was used to plot the linear regression and extinction of the popula- tion in the East filter. The extinction was found to be at 35 cm and is plotted accord- ingly in FIG. 3.

Very similar graphs, not figured here, were obtained for the dispersion of two com- monly occurring species of the Spirotrichia. One was an Oxytricha sp., the other Tachysoma pellionella M OLLER--STFIN. Both were identified from KAHL (1930-1935).

With this type of data it should be possible to predict the maximum acceptable flow rate for a given depth of filter bed, that is to prevent the wash out of the functional components of the interstitial microfauna. This temptation has been resisted as being premature. However, it is probable that the normal quality parameters would begin to deteriorate simultaneously, and clearly filtrate quality is more simply monitored.

A more pertinent consideration in respect of the functional interstitial microfauna is the question of filter bed cleaning. A knowledge of the stratification of the micro- fauna is of use in deciding how deep to allow bed skimming equipment to operate, particularly in prolonged cold weather when the functional microfauna may be very slow to re-establish itself in the filter bed. Such information is likewise relevant to the use of in situ jet washing procedures investigated on a pilot scale by BUXMAN and LEWIN (1961) and currently undergoing modifications at Ashford Common treatment works on full scale slow sand filters.

The Construction of a Sand Profile Sampler 973

C O N C L U S I O N S

(1) The c o m p o n e n t s o f the interst i t ia l mic ro fauna most l ikely to p lay the ma jo r role in removing bac te r i a f rom the fi l trate are the P r o t o z o a and Rot i fera . These groups have the a p p r o p r i a t e ci l iary feeding appara tus .

(2) The only g r o u p which obvious ly feeds upon bac te r ia in suspension are the Per i t r ichia . W i t h i n this o rde r the genus Vorticella is dominan t .

(3) Since the in ters t i t ia l mic ro fauna may become dispersed in the dep th o f the sand bed in -depth pur i f ica t ion is possible , par t i cu la r ly under high flow rates, whereas it had long been bel ieved tha t pur i f ica t ion occurred exclusively at the s a n d - w a t e r

interface. (4) W h e n sand g rad ing is ma in ta ined cons tan t re tent ion or wash-out o f the inter-

st i t ial m ic ro fauna is p r inc ipa l ly dependen t on two factors : flow rate and filter dep th .

Acknowledgements The author is grateful to the Metropolitan Water Board for facilities provided, and in particular to Dr. N. P. BURMAN and Mr. A. STEELE for their advice. Special thanks go to the following members of the University of Surrey Biological Sciences Department: Dr. M. O. Moss for commenting on the script, Mr. B. NAISH for constructing the microscope stage and SHARON BAXTER for technical assistance.

R E F E R E N C E S BURMAN N. P. (1962) Bacteriological control of slow sand filtration. Eft. Wat. Treat. J. 2, 674-677. BURMAN N. P. and LEWIN J. (1961) Microbiological and operational investigation of relative effects

of skimming and in situ sand washing of two experimental slow sand filters. J. Inst. Water Engng 15, 355-367.

CURDS C. R. and VANDYKE J. M. (1966) The feeding habits and growth rates of some fresh-water ciliates found in activated-sludge plants. J. appl. Ecol. 3, 127-137.

CURDS C. R. and COCKBURN A. (1970) Protozoa in biological sewage--treatment processes. Water Research 4, 237-249.

EDMONSON W. T. and WINBERG G. G. (1971) I.B.P. Handbook No. 17. Secondary Productivity in Fresh Waters.

DRAGESCO J. (1962) On the biology of sand-dwelling ciliates. Sci. Prog. 199, 353-363. IrES K. J. and GREGORY J. (1967) Basic concepts of filtration. Proe. Soc. Wat. Treat. Exam. 16,

147-169. KAHL A. (1930-1935) Wimpertiere oder Ciliata. Die Tierwelt Deut. 18, 21, 25, 30. PENNAK R. W. (1950) Comparative ecology of the interstitial fauna of fresh-water and marine beaches.

Ann. Biol. 27, 217-248. RIDLEY J. E. (1967) Experience in the use of slow sand filtration, double sand filtration and micro-

straining. Proc. Soc. Wat. Treat. Exam. 16, 170-191. SLADE~EK V. (1971) Saprobic sequence within the genus Vorticella. Water Research 5, 1135-1140.

Reports

METROPOLITAN WATER BOARD. (1967--1968, 1969-1970, 1971-1972) Report on the results of the bacteriological, chemical and biological examination of the London waters. No. 43, 29-32 and 105-106. No. 44, 16-23 and 138-139. No. 45 (in press).

WORLD HEALTH ORGANISATION (1970) Community Water Supply Research and Development Programme. Background Paper: "Biological" or "Slow Sand" Filters.