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Capability of ciliated protozoa as indicators of effluent quality in activated sludge plants
~ ) Pergamon
0043-1354(94)00258-4
Wat. Res. Vol. 29, No. 4, pp. 1041-1050, 1995 Copyright © 1995 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0043-1354/95 $9.50 + 0.00
CAPABILITY OF CILIATED PROTOZOA AS INDICATORS OF EFFLUENT QUALITY IN ACTIVATED SLUDGE PLANTS H. S A L V A D O * , M. P. G R A C I A and J. M. A M I G O Departament de Biologia Animal, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain (First received April 1994; accepted in revised form September 1994) A~tract--The aim of this study is to determine the relationship between the ciliate populations density and effluent quality in activated sludge plants. A total of 231 samples taken at three activated sludge plants were analyzed over a three year period. Seven physico-chemical parameters were examined and protozoa (in particular ciliate protozoa) and small metazoa were counted by means of optical microscopy. Effluent quality was determined from BOD 5 and suspended solids concentration. For data analysis the species were classified in ranges in terms of abundance. Mean and standard deviation of effluents BOD s and SS were calculated for each range. It was found that as the abundance of each species population increased, both BOD s and suspended solids from effluents tended to a particular range of values (the optimal range) whereas the standard deviation diminished. Thus the higher the ciliate species population density, the better the capability of the species as an indicator, which is something reflected in the standard deviation. This capability of ciliated protozoa to act as indicators of effluent quality will also be limited by the 6ther factors influencing the presence of species. It was observed that the correlation coefficients between ciliates and effluent quality depend on the range of physico-chemical values studied, i.e. whether they are superior or inferior to the "optimal range". The values of each species' BOD 5 optimal range varied from 4 mg/1 to 18 ppm, suggesting that ciliates are good indicators between 4 and 18 ppm in activated sludge plants. In all cases observed, less common species such as Acineta tuberosa, Euplotes sp. and Zoothamnium sp. were indicators of high effluent quality. By contrast, the species that reach the highest densities and are the most common, such as Uronema nigricans, Vorticella microstoma and Opercularia coarctata, indicated lower effluent quality. Key words--ciliate protozoa, activated sludge, indicator species, effluent quality
NOMENCLATURE BOD 5E = SS = SSE = STD = AVG = MAX = MIN =
BOD 5 effluent suspended solids suspended solids effluent standard deviation average maximum minimum
INTRODUCTION The importance of ciliated protozoa in wastewater treatment involving activated sludge has often been described. Their effectiveness in the purifying process is due to the fact that they feed off dispersed bacteria, thus eliminating them (see Curds et al., 1968). The presence of ciliate protozoa reflects an increase in effluent quality (Curds, 1982a, 1993) and can also be taken as indicators of effluent quality (BOD) (Curds and Cockburn, 1970; Sartory, 1976; Al-shahwani and Horan, 1991). They are also useful as indicators of
*Author to whom all correspondence should be addressed.
parameters other than effluent quality in the purifying process. The type of structure of the ciliate protozoa community can characterize various types of biological treatment (Madoni and Ghetti, 1981), sludge loading (Curds and Cockburn, 1970), organic loading rate (Salvad6 and Gracia, 1993), mean cellular retention time ( M C R T ) (Salvad6, 1994) and biological quality of the sludge (Madoni, 1994). It has often been observed that the relation between various physico-chemical parameters and a particular species does not follow a linear model. For example, in the relation between the abundance of a certain organism and pH (a parameter not included in this study) an optimal range is observed; above and below this range either the species' density diminishes or the species disappears altogether. However, statistical analyses used to relate physico-chemical parameters and organisms are generally based on linear models, by means of either bivariant or multivariant analysis. Consequently, when the relationship between a parameter and a particlar species is studied, if the range of study of the parameter is above or below the values of the optimal range, it is easy to see
1041
1042
H. Salvad6 et al.
that for the same species good correlation coefficients will be found with positive or negative values. The aim of this study was to examine the relation between the densities of the different species of ciliate protozoa that colonize activated sludge and effluent quality, by analyzing a wide range of BOD 5 and suspended solids values. MATERIALS AND M E T H O D S
Sampling Three activated sludge plants in the vicinity of Barcelona were sampled over a three-year period. Each sampling (165 at Plant A, 30 at Plant B and 36 at Plant C) included one sample from the primary settling tank, one from the secondary settling tank and one from the aeration tank. Primary and secondary settling tank samples were integrated for 24 h and collected (approx. 0.51) every hour. Sludge samples were taken from the aeration tank. The characteristics of each plant are outlined in Table 1. Physical and chemical parameters from effluent were measured on three separate occasions for each microorganism sample: two days before, one day before and on the same day as the microorganism sample. Physical and chemical monitoring BODs, suspended solids (SS), volatile suspended solids and dissolved oxygen concentration in the aeration tank were measured in accordance with APHA (1989). Hydraulic retention time was calculated as the ratio of aeration tank volume to flow rate. Identification and quantification of microorganisms Microorganism counting was carried out using 500 ml portions of mixed liquor from the aeration tank. Ciliates were counted immediately in the plant laboratory. The number of organisms for each taxon was counted under an optical microscope at x 100 magnification in four 50pl subsamples taken with an automatic micropipette. Small flagellates and gymnoamoebae were counted using × 400 magnification. Ciliate species were identified in accordance with Kahl (1930 35), Curds (1982b), Curds et al. (1983), Guhl (1979), Foissner et al. (1991, 1992), Foissner and Schiffman (1974), Augustin et al. (1987), Perez-Uz (1993), using special techniques: Protargol (Tuffrau, 1967), pyridinated ammonic silver carbonate (Fern~indez-Galiano, 1976) and silver nitrate (Klein, 1928). Non-ciliated protozoa and small metazoa were identified following Baldwin and Chandler (1966), Kudo (1966) and Lee et al. (1985). Filamentous microorganisms were quantified according to Salvad6 (1990). Data analysis Two parameters for effluent quality control were selected for data analysis: BOD 5 and suspended solids. A table, in which physical-chemical data were grouped according to different ranges of density corresponding to each species of ciliates found, was elaborated. Against each
range of density the mean and standard deviation of BOD 5E and SSE were calculated. In analyzing these data we should bear in mind that effluent quality is found for a particular ciliate species' density and not the other way round. The correlation coefficients for each group classified according to abundance is given for each species, BODsE and SSE, using the logarithmic transformation (x = Log(x + 1)). Data table, described above, was used in order to select the best sampling day for further statistical analysis. The selection criteria of this best sampling day was made separately for BODsE and SSE and corresponded to the day with lowest STD. RESULTS
Table 1 shows the values of the parameters of the process in order to characterize the various plants studied. The best effluent quality was found at Plant A, which had highest hydraulic retention time and M C R T , and lowest BOD 5 of settled sewage. Measurements of B O D 5E and SSE taken on the same day as the microorganism sample were found to be the best since STD between those and the density ranges of ciliates were the least. Samples of BOD5 E and SSE taken one and two days before microscopic analyses were 5 - 3 0 % and 5 - 2 6 % higher respectively than the sampling analysis taken on the same day. Contrary to this apparent pattern, the STD between carnivorous ciliates ( Acineria uncinata and Suctoria ) and B O D s E and SSE samples taken two days previously was found to be 20% lower than those samples taken on the same day. But over all the measurements of B O D 5E and SSE taken on the same day as the microorganism (ciliates) sample were considered as giving the most significant with the lowest STD value. In general it can be seen that the higher the density range, the greater the relationship between that particular density of ciliates and B O D s E and SSE (see Tables 2, 3 and 4). In these tables it can be observed that as the density of organisms increased there was a tendency towards certain values of BOD5 E and SSE while the standard deviation diminished. In the study of the relationship between some effluent quality parameters (BOD 5 and SS) and ciliates, BOD5 emerged as having a closer relationship with ciliates, as a higher fall in its standard deviation was observed as ciliate density increased. Correlations without mathematical transformation based on the results obtained presented very low correlation coefficients. After logarithmic transformation of each ciliate species' density, the correlation coefficients
Table 1. Values of the physico-chemical parameters measured at the three plants Plant A
BOD~ Settled sewage (rag/l) Retention time in aeration tank (h) Organic Loading Rate [kg BODs/kg MLVSS)d] MLVSS (mg/l) M C R T (days) Oxygen concentration in aeration tank (final track) (mg/l) BOD5 in ettiuent (mg/l)
Plant B
Plant C
AVG
STD
AGV
STD
AVG
STD
194 9.6 0.44 1513 7.4 3.15 12.6
102 2.4 0.22 605 3,5 0.88 7.6
210 5.4 0.76 1367 4.4 1.2 23
77 1.8 0.7 616 1.5 0.35 13
313 6.4 0,9 1240 2.47 1.1 29
89 1.8 0.4 496 0.95 0.6 18
12.3 12.3 64
12.8 10.4 116
13.8 10.8 115
13.1 10 142
13.5 10.3 132
12.8 9.9 146 Paramecium aurelia (complex) BOD s A V G 13.3 BOD 5 STD 10.5 n 119
BOD 5 AVG BOD 5 S T D n
13.2
16 19.4 23
10.1 130
Cinetochilum margaritaceum
BOD 5 A V G BOD 5 STD n
Uronema nigricans
BOD s A V G BOD 5 S T D n
Podophrya fixa
BOD 5 AVG BOD 5 S T D n
Tokophrya ciclopum
BOD 5 A V G BOD 5 S T D n
Acineta tuberosa
BOD 5 A V G BOD 5 S T D n
Trochilia minuta
B O D 5 STD n
Chilodonella uncinata BOD 5 AVG
BOD~ A V G BOD 5 STD n
Acineria uncinata
< 10
9.7 4.7 4
--
1
11.5 7.9 23
--
16 13 13
9.6 4.5 8
8.1 3.6 7
7.5 2.6 4
----
8 4 5
12 5.6 15
100-200
3
10.9 6.3 29
13.3 8.2 27
9.4 5.1 23
5 3.4 5
11.3 3.4 7
9.1 6.7 12
14.1 9.3 27
1(~100
6.8 1.7 5
I
4
13 8.9 14
8 1.4 3
7.3 3.7 4
----
12.5 5.7 24
200-400
2
3.9
--
--
12.7 2.2 6
2
1
13.4 3.3 7
14
3.3 0.25 2
I
5.5
12.1 6.7 14
800-1600
16
7 3.9 4
9.3 5.3 3
11 6 21
400-800
1
4
13.4 5.8 9
3.4 0.43 2
10.7 3.5 15
1600-3200
Range o f ciliate density (ind/ml)
14 2 4
12.3 5.29 10
3200-6400
1280~30000
Table 2--continued overleaf
18 3.2 5
10.5 2.5 2
6400-12800
Table 2. Values of mean BOD 5 and standard deviation of the various ranges of ciliate densities in aeration tank at Plant A. The number of findings o f each species are indicated for each range of ciliate density (n)
¢3
BOD 5 AVG BOD5 S T D n Ciliates BOD 5 AVG BOD 5 STD n
Aspidisca cicada
BOD 5 AVG BOD 5 STD n Oxytricha sp. BOD 5 AVG BOD 5 STD n Euplotes sp. BOD 5 AVG BOD 5 STD n
Carchesium polypinum
BOD5 A V G BOD 5 STD n
Vaginicola cristallina
BOD 5 AVG BOD 5 STD n
Epistylis plicatilis
BOD 5 AVG BOD5 S T D n
Opercularia coarctata
BOD 5 AVG BOD 5 STD n
Zoothamnium sp.
BOD s AVG BOD 5 STD n
Vorticella microstoma
BOD 5 AVG BOD~ S T D n
Vorticella convallaria
Table 2--continued
3
70 --
--
--
--
---
8.6 5.4 5
1
12.6 8.6 27
1
6
6.5 3.9 6
13 10 143 19.2 17.5 23
5
11 5.8 7
9.3 6 17
13.7 10.7 114
--
--
--
12.3 5.2 12
8.8 4.8 6
2
1
2
11
3.3 0.5 3
9.3 4.9 9
10
1
4
7.7 2.5 3
23 11.2 5
2
1
19.8 11.3 5
5
8.6 6.4 13
9.9 5.7 19
200~,00
10
11.2 4.7 6
11.4 4.6 15
100-200
3
3.83 1.9 3
10.4 4.4 18
9.9 5.8 11
17.8 17.4 3
14.3 9.3 24
10-100
13.4 10.2 135
13.3 9.9 139
14.7 1I 103
12.1 I 1.7 78
12.6 9.9 148
11.3 15.8 31
15.4 13.9 53
< 10
4
4
12
21
29 6
9.6 5.2 26
I
8
2
10.5
1
3
6.6 2 5
11.4 3.2 16
11.9 3.5 17
9.8 4.4 13
12.5 9.7 26
12.2 8.4 6
2
7.5
5.1 0.7 4
5.25 1.5 4
14.4 7.4 9
11.5 7.3 13
10.3 3.4 12
density
800-1600
of ciliate
400-800
Range
12
5
8.7
12.8 3.8 14
6.3 2.3 4
9.6 1.3 3
13.3 6.4 12
12.6 5.9 21
9.8 3.9 12
1600-3200
(ind/ml)
51
6.2
11.3
12.7 5.2 10
4.3 1.3 3
6 3.6 6
13 3.8 12
14.6 6.4 29
9.7 3.4 3
3200~400
54
8
11.5
9.5 3 7
10.4 3.8 3
17.3 9.7 13
6400-12800
23
4.3
13.6
1
11
16 3.3 5
12800-30000
t:x o2
ct)
BOD 5 AVG BOD 5 STD n Ciliates BOD s AVG BOD s STD n
Aspidisca cicada
BOD~ A V G BOD 5 STD n Euplotes sp. BOD 5 AVG BOD 5 STD n
Epistylis plicatilis
BOD 5 AVG BOD 5 STD n
Opercularia coarctata
BOD~ A V G BOD 5 STD n
Vorticella microstoma
BOD 5 AVG BOD 5 STD n
Vorticella convallaria
BOD S AVG BOD 5 STD n
Uronema nigricans
BOD 5 AVG BOD s STD n
Podophrya fixa
BOD 5 AVG BOD s STD n
Chilodonella uncinata
BOD 5 AVG BOD 5 STD n
Acineria uncinata
62.7 34. l 10
12.9 8 33
10.6 5.7 7
13 7.6 4
11.5 8.4 9
17.9 17.6 191
30.2 25 61
7.7 2.5 3
24.4 14.2 7
23 23 7
11.4 4.6 15
18.1 13.2 17
9.9 4.4 9
11.5 8.1 4
13.3 5.9 19
100-200
10.4 4.4 18
14.6 14.9 14
27.3 16.2 6
14.3 9.3 24
13.4 10.1 33
13.9 8 31
10 5.7 13
17.5 14.8 39
10-100
20.3 18.7 153
17.9 22.2 93
23 22 40
23 21.4 107
20 24.7 81
18.5 18.8 159
18.3 17.7 184
28.4 26.4 54
< I0
13.5 9.9 28 45.5 25.8 5
59.5 7 2
6.4 2.7 5
15.6 7.43 14
14,7 10.9 17
10.3 3.4 12
16 5 12
20 8.2 5
9.3 5.3 3
11.2 5.8 24
400-800
12.2 4.8 14
I
5
9.7 4.8 10
27 12.4 17
12.7 9.6 17
9.9 5.7 19
15.2 9.7 18
1
12
----
13 5.9 26
200~,00
25.25 I 1.4 9
9.6 5.2 26
8.5 3.8 7
14.2 5.35 24
14 5 27
9,8 4.4 13
18.7 10.2 10
1
5.5
12 6.5 15
800-1600
15.9 14.7 18
12.8 3.8 14
9.6 1,3 3
14.7 9.03 17
16.8 11.6 31
9.8 3.9 12
16.2 8.6 16
10.7 3,5 15
1600-3200
R a n g e o f ciliate density (ind/ml)
10.4 3.6 8 12.9 8.5 66
12.9 8.4 61
10.6 3.01 5
18.1 10.7 18
16.8 3.4 6
10.5 2.5 2
6400-12800
12.4 4.6 13
6 3.6 6
15.1 6.2 14
15.2 6.4 35
9.7 3.4 3
17.6 7.3 5
12.3 5 I1
3200-6400
14.3 4.6 34
1
11
15.3 3.5 7
18.2 4.7 7
12800-30000
Table 3. Values o f m e a n B O D 5 and standard deviation o f the various ranges o f abundance o f ciliates (in aeration tank) c o m m o n to at Plants A, B and C, The number of findings o f each species are indicated for each range o f ciliate density (n)
e~ r~
SS AVG SS STD n Ciliates SS AVG SS STD n
Aspidisca cicada
SS AVG SS STD n Euplotes sp. SS AVG SS STD n
Epistylis plicatilis
SS AVG SS STD n
Opercularia coarctata
SS AVG SS STD n
Vorticella microstoma
SS AVG SS STD n
Uronema nigricans
SS AVG SS STD n
Podophrya fixa
SS AVG SS STD n
Chilodonella uncinata
SS AVG SS STD n
Acineria uncinata
13.4 103 33
m
85.1 45.1 11
13.7 8.6 6
16.2 6.2 4
12.6 9 10
22.9 28,8 186
41.2 41.9 62
9.7 5.25 3
21.4 13.3 7
40 42 6
21.7 18.9 17
16.8 11.9 9
10 5.5 5
13.7 7.8 19
100-200
9 4.9 18
18 25.7 15
36.3 15.7 5
15.8 15 33
14.2 8,6 30
8.3 4.7 13
23,7 25 38
10-100
26 30.1 148
22.7 37 88
28.5 41.5 41
26.6 40.4 75
23.8 30 154
23.7 28 177
37.3 37.2 53
< 10
15.9 14.6 27 78 86.8 4
92.5 19.5 2
9.6 5.8 5
17.8 9.3 14
16.8 13.4 16
22.3 11.5 II
24.8 9 5
7.8 4.6 3
13,7 8 23
40~800
14.9 7.9 14
1
7
15.6 12 11
30.3 18.2 17
18.6 20.9 16
23 17.8 17
I
26
----
12.1 6.2 26
200-400
26.9 15 8
11.7 7.3 25
10.3 6,3 7
19.6 15 24
19.2 12.5 27
22.7 13.3 10
I
9
14 7.6 15
800-1600
Range of ciliate density
20.6 18,6 16
13.5 6 14
7.2 1.3 3
20.2 11.8 17
19.6 13 30
20.35 10.7 16
13,3 5.7 14
1600-3200
(ind/ml)
14.3 11.2 59
16.8 9.5 13
7.7 1.67 6
16.4 10.4 14
14.7 7 35
23.8 12.3 5
21.2 17.3 11
3200-6400
16.1 14 66
8.9 4.9 7
16.8 6.2 5
21.2 17.5 17
19.8 14.5 6
8 3.5 2
6400-12800
15,9 6.8 35
19.8 7.4 7
19.7 6.5 7
12800-30000
Table 4, Values of effluent suspended solids: mean and standard deviation of the various ranges of abundance (in aeration tank) of ciliates common to Plants A, B and C. The number of findings of each species are indicated for each range of ciliate density (n)
O-
o~
1047
C i l i a t e s in a c t i v a t e d s l u d g e e f f l u e n t s Table 5. Correlation coefficients between ciliates and BOD~E or SSE at Plants A, B and C. These values were found using the data obtained in the Tables 2, 3 and 4 BOD5 Plant A
BOD 5 Plants A, B and C
Suspended solids Plants A, B and C
Acineria uncinata Chilodonella uncinata Trochilia minuta Acineta tuberosa Tokophrya ciclopum Podophrya .[ixa Uronema nigricans Cinetochilum rnargaritaceum Paramecium aurelia (complex) Vorticella cont~allaria Vorticella rnicrostoma Zoothamnium sp. Opercularia coarctata Epistylis plicatilis Vaginicola cristallina Carchesium polypinurn Oxytricha sp. Euplotes sp. Aspidisca cicada
-0.894 -0,900 - 0,937
-0.925 -0.950 - 0.940
-0.814 -0.902 - 0.930
0,007
-0.435 - 0.281 -0.114 -0.864 -0.989 -0.938 -0.608
0.046 0.032 -0.361 -0.827 -0.922 -0.878 -0.572
-0.680
-0.373 - 0.883 - 0.906 - 0.683 -0.856 -0.853 -0.791
-0.384 - 0.847 - 0.884 - 0.780 -0.837 -0.824 -0.820
Ciliates
- 0.964
- 0.868
0.000
0.528 -0,849 -0.872 -0.920 0.377 0.067 - 0.831 - 0.906 - 0.510 -0.678
were slightly higher, but were still very low--all below 0.3, and most below 0.05. But correlations based on the data from Tables 2, 3 and 4 presented high correlation coefficients between control parameters and logarithms of organism density (Table 5). It can thus be seen, that for a small change in BODsE there is a large change in the ciliated populations structure. Most ciliate species studied at Plant A showed the existence of an inverse relation with BOD5 E; however certain species had very low correlation coefficients, such as Opercularia coarctata. There were still some species with a positive correlation, such as Vorticella microstorna and Uronema nigricans. After joint examination of data from all three plants presented negative correlations for the species mentioned the averages and standard deviations of the three species increased at relatively low population densities, but at high densities BOD 5E and SSE values remained practically even. The other ciliate species that clearly showed negative correlation coefficients, continued to present high, negative correlation coefficients: Chilodonella uncinata, Aspidisca cicada and Epistylis plicatilis. In the suctoria, with the exception of Acineta tuberosa, which only appeared in periods of high effluent quality, there was no close relationship with effluent quality. In addition, suctoria possessed extremely low regression coefficients; when their population increased they did not clearly tend towards a certain value of BODsE or SSE. A comparison of the results presented in Tables 2 and 3 can be found in Table 6, which shows the average density of each ciliate species or group of organisms found at the plants for seven BODsE ranges. In this table it can be observed that the highest ciliate densities are found between 10 and 26 mg/l of BODsE while ciliate densities are reduced above and under this range. It should also be stressed that the number of ciliate and other protozoan taxons WR
2914~E
are reduced as BODsE increases. Thus in ranges of low effluent quality (i.e. above 42 mg/1 of BOD 5E), densities of ciliate populations are progressively reduced being almost zero above 67 mg/l. The most abundant protozoa at higher BODsE levels were small protozoa such as flagellates < 20 #m and gymnoamoebae < 50 pm. The high values of standard deviations in Table 6 show that a particular BODsE range does not establish which protozoan community structure will be found in the activated sludge. The lowest standard deviations are just found when assessing the total number of ciliates between 0 and 6 mg/l BODsE, which indicates that good effluent qualities are linked to a minimum ciliate density without establishing the species composition of the community. DISCUSSION
The fact that the ciliates show a closer relationship to BOD 5E than to SSE may be due to various causes: suspended solids in the effluent may be more affected by the system's hydrodynamics than BODsE, since variations in flow can produce sudden sludge rises; only a percentage of the suspended solids in the effluent consists of dispersed bacteria, since flocculi, filamentous organisms and protozoa are also present; Curds (1993) observed that the presence of ciliate protozoa not only reduces the density of dispersed bacteria in the environment by feeding, but also produces a reduction in the concentration of soluble BODs, although the causes have not been determined. BODs, as an estimation itself of the biodegradable material in the medium, also reflects the medium's saprobity better than suspended solids. As can be seen in Tables 2, 3 and 4, certain species are abundant and frequent in all ranges of ciliate population density, and some are infrequent. For species that appear in a small number of samples it
H. S a l v a d 6 et al.
1048
Table 6. Aeration tank microorganism variation according to BOD 5 effluent at Plants A, B and C. Abundances, averages and standard deviations of organisms are represented in individuals/ml
Abundance (ind/ml)
Acineria uncinata Amphileptus pleurosigma Chilodonella uncinata Trochilia minuta Acineta tuberosa Tokophrya ciclopum Podophya fixa Uronema nigricans Cinetochilum margaritaceum Paramecium aurelia Vortieella convallaria Vorticella microstoma Zoothamnium sp. Opercularia coarctata Epistylis plieatilus Vaginicola cristallina Carchesium polypinum Oxytricha sp. Euplotes sp. Aspidisca c&ada Total eiliates Flagellates < 2 0 # m Flagellates > 2 0 p r o Gymnamoebae < 50/~m Gymnamoebae > 50/~m
Testate amoebae Rotifers
Nematodes Oligochaeta Filamentous organisms (m/ml)
Range of BOD s (mg/I)
0 4
4 6
No. of samples
31
AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD
717 885 4 10 74 263 252 546 13 40 12 25 20 46 66 221 94 369 30 72 476 990 1125 1928 18 103 836 1699 404 892 140 280 611 1367 65 136 16 49 884 1653 6094 3092 334474 826422 36 128 10307 22140 107 129 65 98 I 10 200 5 16 12 36 99 137
is harder to calculate their value as indicators and often their standard deviation cannot be determined. Although it is difficult to calculate their relationship to the environment, they are considered valuable as indicators. According to Sartory (1976) if these species are stenosaprobic, that is to say, if they "occur at a single level of saprobity", they will be good indicators of effluent quality (BOD). Table 6 shows that the least common species such as Acineta
10 16
16 26
26 42
42 67
67 107
41
54
40
15
12
12
717 1031 0 0 14 66 13 75 7 31 67 158 29 86 1274 5808 0 0 32 73 463 711 2295 3577 8 49 672 1406 686 1747 1 5 40 142 87 208 3 15 934 1949 8048 6786 249417 348815 0 0 13308 26617 284 677 40 145 16 31 6 12 0 0 92 110
1048 1852 0 0 8 54 22 91 1 6 17 66 32 96 2310 8161 0 0 9 30 423 849 3528 5459 0 0 1015 1731 149 517 0 0 70 374 24 108 2 I1 1809 2765 11328 11313 276711 539811 0 0 31392 73952 189 489 0 0 8 26 3 8 0.2 1 210 202
410 898 0 0 9 34 I 6 0 0 2 12 18 79 5705 16061 0 0 4 15 289 892 4427 4608 0 0 1079 1477 32 186 0 0 0 0 10 40 7 26 861 1615 13241 16350 701904 813976 0 0 72542 76986 29 58 0 0 0 0 11 17 0 0 359 421
143 263 7 26 2 7 0 0 0 0 0 0 84 218 1322 2555 0 0 0 0 5 I1 2764 2661 0 0 680 1085 0 0 0 0 0 0 42 155 5 20 179 299 5269 5235 733258 747372 0 0 76285 93827 21 44 0 0 0 0 0 0 0 0 283 303
2 7 0 0 0 0 0 0 0 0 0 0 1 4 115 240 0 0 1 2 6 18 210 541 0 0 304 652 0 0 0 0 0 0 0 0 0 0 4 14 641 985 13826 38210 0 0 39875
2 12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 36 123 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 38 199 19720 40277 0 0 43960 125402 0 0 0 0 0 0 0 0 0 0 0 0
42022 2 7 0 0 0 0 9 15 0 0 55 120
tuberosa, Euplotes sp. and Zoothamnium sp. are, in all observed cases, indicators of high effluent quality; in contrast, the species that reach the highest densities and are the most common, such as Uronema nigricans, Vorticella microstoma and Opercularia coarctata are indicators o f lower effluent quality. The relationship observed between the population density of ciliate species and effluent quality mainly tends to a narrow range of values. Abundance of
Ciliates in activated sludge effluents individuals, though in a relative form, is already habitually used to calculate saprobity indices of an environment on the basis of indicator species (Zelinka and Marvan, 1961; Sladecek, 1973). However, to calculate saprobity indices only a fixed significance value is used for any of the population density found. When the population density increases, the quality indicator parameter tends to a narrower range of values, which we call the optimal range, and the lower the standard deviation obviously the higher will be the capability as an indicator. Consequently when the population density of a certain species rises, its capability as an indicator also increases; as is reflected by its standard deviation values, The results of the correlation coefficients found reflect clearly the capability of ciliates as indicators. The coefficients may not necessarily be very high, since the tendency towards a narrow range of values makes it difficult to perform the correlation analyses, because BOD 5E values remain similar as ciliate density increases. Thus, correlation results show that negative or positive correlation coefficients vary according to the studied BOD 5E range. As an example we have observed positive and negative coefficients in Vorticella microstoma. Such variations are also found in literature, while Madoni (1994) found positive correlation coefficients for V. microstoma and BODsE, Esteban et al. (1991) found negative correlations with the same parameter and species. Thus the results with low correlation coefficients do not necessarily imply a particular ciliate species is not related with the indicator parameters of effluent quality. We observe how Uronema nigricans, with low regression coefficients, has a strong relation to effluent quality which becomes clear as the standard deviation gradually falls. On the other hand suctors such as Podophyafixa and Tokophrya ciclopum have low regression coefficients as they do not have a close relation with the indicator parameters of effluent quality. We do not believe that the suctors' ciliophagous feeding contributes directly to the clearing of the effluent, as the bacteriophagous ciliates do. The capability of ciliates as indicators of effluent quality is limited by a number of factors. The presence of particular ciliate species in the activated sludge obviously depends on various factors, among which we should mention those which are not to do with the plant, such as the organic matter in the effluent (or volumetric loading rate), and those caused by the operational parameters, such as dissolved oxygen and MCRT (Curds and Cockburn, 1970; Curds, 1982a; Al-shahwani and Horan, 1991; Madoni and Ghetti, 1981; Madoni, 1994; Esteban et al., 1991; Salvad6 and Gracia, 1993; Salvad6, 1994). Furthermore, communities of protozoa in activated sludge in plug flow systems tolerate a wide variety of environmental conditions; at different stages in the treatment the availability of food can decrease markedly in a short time. For this reason it is difficult
1049
to find good indicators of effluent quality (Curds, 1993). Best results have been obtained using the effluent quality value of the same day as the microscopic analysis, suggesting that although each species' capability as an indicator of effluent quality is influenced and also limited by the sum of the factors at work in the process, the optimal range of each species will be more the reflection of the action of ciliates as purifying agents or indicators of this activity than of the influence of the values of the effluent itself on the species' abundance. The values of the optimal ranges of the BODsE of the species found vary from 4 to 18mg/l which suggests that the ciliate species that colonize the activated sludges can be good indicators between 4 and 18 mg/l. Outside these values accurate diagnoses are not possible, although the low densities of ciliates ( < 1000 ind/ml) or their absence easily cause BOD 5 values of over 30 mg/l in the conventional activated sludge purifying plants studied (see Table 6). Curds and Cockburn (1970) stated in their observations that when ciliates were absent the effluent was over 30 mg/l BOD 5E. CONCLUDING REMARKS
The most useful finding in this study was that after grouping data of physical and chemical parameters according to ranges of ciliate abundance, a decrease in standard deviation of BOD~E or SSE as the ciliate population density increased could be observed. Thus, the increase in abundance of a particular species indicates more accurately its optimal range; this optimal range of each species can be used as an indicator of effluent quality. The capability of assessing effluent quality by the study of ciliate densities has limitations. Thus even though a density of ciliates can indicate a range of values of BOD 5E or SSE, the presence of a particular species of ciliate will also qualitatively depend on the nature of the effluent, other operational parameters and moreover the relations with the other species of the community. In addition this study indicates that the gradient of the correlation between effluent quality (BODs) and the abundance of ciliates populations is positive or negative depending on the range of data values established in the samplings. This gradient may be positive or negative if the range of effluent quality parameter values is above or below the optimal indicator value of the species. Such observations may be also considered in further correlation studies of other physico-chemical parameters and ciliate populations. Acknowledgements--The authors wish to express their gratitude to Dr Miquel Salicrfi, Departament d'Estadistica, Universitat de Barcelona, for technical advice on statistical analysis. We also wish to mention our gratitude to Empresa Metropolitana de Sancjament S.A. (EMSSA), and the management of C.I.C.Y.T. (Comisi6n Interministerial de Ciencia y Tecnologia) Project PB 88-0212.
1050
H. Salvad6 et al. REFERENCES
AI-Shahwani S. M. and Horan N. J. (1991) The use of protozoa to indicated changes in the performance of activated sludge plants. Wat. Res. 25, 633-638. APHA (1989) Standard Methods for the Examination of Water and Waste Water, 17th Edition. American Public Health Association, Washington, DC. Augustin H., Foissner W. and Adam H. (1987) Revision of the genera Acinera, Trimyema and Trochiliopsis (Protozoa, Ciliophora). Bull. Br. Mus. nat. Hist. (Zool) 52, 197-224. Baldwin H. and Chandler G. (1966) Freshwater Biology (Edited by Edmonson W. T.), Washington, p. 1248. Curds C. R. (1982a) The ecology and role of protozoa in aerobic sewage treatment processes. A. Rev. Microbiol. 36, 27-46. Curds C. R. (1982b) British and Other Freshwater Ciliated Protozoa. Part I. Cambridge University Press, Cambridge. Curds C. R. (1993) Le r61e des protozoaires dans la purification de l'eau. Ann. biol. 5-6, 193-219. Curds C. R., Cockburn A. (1970) Protozoa in biological sewage-treatment processes. II. Protozoa as indicators in the activated-sludge processes. Wat. Res. 4, 237-249. Curds C. R., Cockburn A. and Vandyke J. M. (1968) An experimental study of the role of ciliated protozoa in the activated-sludge process. Wat. Pollut. Contr. 67, 312-329. Curds C. R., Gates M. A., Roberts D. McL. (1983) British and Other Freshwater Ciliated Protozoa. Part II. Cambridge University Press, Cambridge. Eikelboom D. H. (1977) Identification of filamentous organism in bulking activated sludge. Prog. Wat. Tech. 8, 153-161. Esteban G., Tellez C and Bautista L. M. (1991) Dynamics of ciliated protozoa communities in activated-sludge process. Wat. Res. 25, 967-972. Fernandez-Galiano D. (1976) Silver impregnation ofciliated protozoa: procedure yielding good results with the pyridinated silver carbonate method. Trans. Am. Micr. Soc. 95, 557-560. Foissner W., Blattener H., Berger H. and Kohmann F. (1991) Taxonomische und 6kologische Revision der Ciliaten des Saprobiensystem. Band 2. pp. 1 471. Bayerisches Landesamt f/Jr Wasserwirtschaft, Miinchen. Foissner W., Berger H. and Kohmann F. (1992) Taxonomische und iJkologische Revision der Ciliaten des Saprobien-
system. Band II, pp. 1-502. Bayerisches Landesamt fiir Wasserwirtschaft, M/inchen. Foissner W. and Schiffmann H. (1974) Vergleichende Studien an argerophilen Structuren von vierzehn peritrichen Ciliaten. Protistologica 10, 489-508. Guhl W. (1979) Opercularia coarctata, ein variables Peritrich. Arch. Protistenk. 121, 308 346. Kalh A. (1930-35) Urtiere oder Protozoa. I. Wimpertiere oder Ciliata (Infusoria). In Die Tierwelt Deutschlands (Edited by Dahl F.), pp. 1-886. G. Fischer, Jena. Klein B. M. (1928) Die Silberlinensysteme der Ciliaten. Weitere Resultate. Arch. Protistenk. 62, 177-260. Kudo R. R. (1966) Protozoology. pp. 1174, 5th Edition, Charles C. Thomas, Springfield. Illinois. Lee J. J., Hunter S. H. and Bovee E. C. (1985) An Illustrated Guide to the Protozoa. pp. 629 Edited by Lawrence, Kansas. Madoni P. (1994) A sludge biotic index (SB1) for the evaluation of the biological performance of activated sludge plants based on the microfauna analysis. War. Res. 28, 67-75. Madoni P. and Ghetti P. F. (1981) The structure of ciliated protozoa communities in biological sewage-treatment plants. Hydrobiologia 83, 207 215. Perez-Uz B. (1993) Tesis Doctoral, Facultad de Biologia, Universidad Complutense de Madrid. Salvad6 H. (1990) Mrtodo rfipido para el control del bulking, trcnica simple y rfipida de contaje de microorganismos filamentosos. Tecn. Agua 67, 60 63. Salvad6 H. and Gracia M. P. (1993) Determination of organic loading rate of activated sludge plants based on protozoa analysis. Wat. Res. 27, 891-895. Salvad6 H. (1994) Effect of mean cellular retention time on ciliated protozoan populations in urban wastement plants based on a proposed model. Wat. Res. 28, 1315-1321. Sartory D. P. (1976) The peritrich ciliates as biological indicators for activated sludge. Microscopy 33, 85-89. Slfidecek V. (1973) System of water quality from the biological point of view. Arch. Hydrobiol. Engeb. Limnol. 7, 1-217. Tuffrau M. (1967) Perfectionements et practique de la technique d'impr~gnation au protargol des infusoires cilirs. Protistologica 3, 91 98. Zelinka M. and Marvan P. (1961) Zur Prfizisierug der bilogischen Klassification der Reinheit fliessender Gerwasser. Arch. Hydrobiol. 57, 389 407.
0043-1354(94)00258-4
Wat. Res. Vol. 29, No. 4, pp. 1041-1050, 1995 Copyright © 1995 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0043-1354/95 $9.50 + 0.00
CAPABILITY OF CILIATED PROTOZOA AS INDICATORS OF EFFLUENT QUALITY IN ACTIVATED SLUDGE PLANTS H. S A L V A D O * , M. P. G R A C I A and J. M. A M I G O Departament de Biologia Animal, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain (First received April 1994; accepted in revised form September 1994) A~tract--The aim of this study is to determine the relationship between the ciliate populations density and effluent quality in activated sludge plants. A total of 231 samples taken at three activated sludge plants were analyzed over a three year period. Seven physico-chemical parameters were examined and protozoa (in particular ciliate protozoa) and small metazoa were counted by means of optical microscopy. Effluent quality was determined from BOD 5 and suspended solids concentration. For data analysis the species were classified in ranges in terms of abundance. Mean and standard deviation of effluents BOD s and SS were calculated for each range. It was found that as the abundance of each species population increased, both BOD s and suspended solids from effluents tended to a particular range of values (the optimal range) whereas the standard deviation diminished. Thus the higher the ciliate species population density, the better the capability of the species as an indicator, which is something reflected in the standard deviation. This capability of ciliated protozoa to act as indicators of effluent quality will also be limited by the 6ther factors influencing the presence of species. It was observed that the correlation coefficients between ciliates and effluent quality depend on the range of physico-chemical values studied, i.e. whether they are superior or inferior to the "optimal range". The values of each species' BOD 5 optimal range varied from 4 mg/1 to 18 ppm, suggesting that ciliates are good indicators between 4 and 18 ppm in activated sludge plants. In all cases observed, less common species such as Acineta tuberosa, Euplotes sp. and Zoothamnium sp. were indicators of high effluent quality. By contrast, the species that reach the highest densities and are the most common, such as Uronema nigricans, Vorticella microstoma and Opercularia coarctata, indicated lower effluent quality. Key words--ciliate protozoa, activated sludge, indicator species, effluent quality
NOMENCLATURE BOD 5E = SS = SSE = STD = AVG = MAX = MIN =
BOD 5 effluent suspended solids suspended solids effluent standard deviation average maximum minimum
INTRODUCTION The importance of ciliated protozoa in wastewater treatment involving activated sludge has often been described. Their effectiveness in the purifying process is due to the fact that they feed off dispersed bacteria, thus eliminating them (see Curds et al., 1968). The presence of ciliate protozoa reflects an increase in effluent quality (Curds, 1982a, 1993) and can also be taken as indicators of effluent quality (BOD) (Curds and Cockburn, 1970; Sartory, 1976; Al-shahwani and Horan, 1991). They are also useful as indicators of
*Author to whom all correspondence should be addressed.
parameters other than effluent quality in the purifying process. The type of structure of the ciliate protozoa community can characterize various types of biological treatment (Madoni and Ghetti, 1981), sludge loading (Curds and Cockburn, 1970), organic loading rate (Salvad6 and Gracia, 1993), mean cellular retention time ( M C R T ) (Salvad6, 1994) and biological quality of the sludge (Madoni, 1994). It has often been observed that the relation between various physico-chemical parameters and a particular species does not follow a linear model. For example, in the relation between the abundance of a certain organism and pH (a parameter not included in this study) an optimal range is observed; above and below this range either the species' density diminishes or the species disappears altogether. However, statistical analyses used to relate physico-chemical parameters and organisms are generally based on linear models, by means of either bivariant or multivariant analysis. Consequently, when the relationship between a parameter and a particlar species is studied, if the range of study of the parameter is above or below the values of the optimal range, it is easy to see
1041
1042
H. Salvad6 et al.
that for the same species good correlation coefficients will be found with positive or negative values. The aim of this study was to examine the relation between the densities of the different species of ciliate protozoa that colonize activated sludge and effluent quality, by analyzing a wide range of BOD 5 and suspended solids values. MATERIALS AND M E T H O D S
Sampling Three activated sludge plants in the vicinity of Barcelona were sampled over a three-year period. Each sampling (165 at Plant A, 30 at Plant B and 36 at Plant C) included one sample from the primary settling tank, one from the secondary settling tank and one from the aeration tank. Primary and secondary settling tank samples were integrated for 24 h and collected (approx. 0.51) every hour. Sludge samples were taken from the aeration tank. The characteristics of each plant are outlined in Table 1. Physical and chemical parameters from effluent were measured on three separate occasions for each microorganism sample: two days before, one day before and on the same day as the microorganism sample. Physical and chemical monitoring BODs, suspended solids (SS), volatile suspended solids and dissolved oxygen concentration in the aeration tank were measured in accordance with APHA (1989). Hydraulic retention time was calculated as the ratio of aeration tank volume to flow rate. Identification and quantification of microorganisms Microorganism counting was carried out using 500 ml portions of mixed liquor from the aeration tank. Ciliates were counted immediately in the plant laboratory. The number of organisms for each taxon was counted under an optical microscope at x 100 magnification in four 50pl subsamples taken with an automatic micropipette. Small flagellates and gymnoamoebae were counted using × 400 magnification. Ciliate species were identified in accordance with Kahl (1930 35), Curds (1982b), Curds et al. (1983), Guhl (1979), Foissner et al. (1991, 1992), Foissner and Schiffman (1974), Augustin et al. (1987), Perez-Uz (1993), using special techniques: Protargol (Tuffrau, 1967), pyridinated ammonic silver carbonate (Fern~indez-Galiano, 1976) and silver nitrate (Klein, 1928). Non-ciliated protozoa and small metazoa were identified following Baldwin and Chandler (1966), Kudo (1966) and Lee et al. (1985). Filamentous microorganisms were quantified according to Salvad6 (1990). Data analysis Two parameters for effluent quality control were selected for data analysis: BOD 5 and suspended solids. A table, in which physical-chemical data were grouped according to different ranges of density corresponding to each species of ciliates found, was elaborated. Against each
range of density the mean and standard deviation of BOD 5E and SSE were calculated. In analyzing these data we should bear in mind that effluent quality is found for a particular ciliate species' density and not the other way round. The correlation coefficients for each group classified according to abundance is given for each species, BODsE and SSE, using the logarithmic transformation (x = Log(x + 1)). Data table, described above, was used in order to select the best sampling day for further statistical analysis. The selection criteria of this best sampling day was made separately for BODsE and SSE and corresponded to the day with lowest STD. RESULTS
Table 1 shows the values of the parameters of the process in order to characterize the various plants studied. The best effluent quality was found at Plant A, which had highest hydraulic retention time and M C R T , and lowest BOD 5 of settled sewage. Measurements of B O D 5E and SSE taken on the same day as the microorganism sample were found to be the best since STD between those and the density ranges of ciliates were the least. Samples of BOD5 E and SSE taken one and two days before microscopic analyses were 5 - 3 0 % and 5 - 2 6 % higher respectively than the sampling analysis taken on the same day. Contrary to this apparent pattern, the STD between carnivorous ciliates ( Acineria uncinata and Suctoria ) and B O D s E and SSE samples taken two days previously was found to be 20% lower than those samples taken on the same day. But over all the measurements of B O D 5E and SSE taken on the same day as the microorganism (ciliates) sample were considered as giving the most significant with the lowest STD value. In general it can be seen that the higher the density range, the greater the relationship between that particular density of ciliates and B O D s E and SSE (see Tables 2, 3 and 4). In these tables it can be observed that as the density of organisms increased there was a tendency towards certain values of BOD5 E and SSE while the standard deviation diminished. In the study of the relationship between some effluent quality parameters (BOD 5 and SS) and ciliates, BOD5 emerged as having a closer relationship with ciliates, as a higher fall in its standard deviation was observed as ciliate density increased. Correlations without mathematical transformation based on the results obtained presented very low correlation coefficients. After logarithmic transformation of each ciliate species' density, the correlation coefficients
Table 1. Values of the physico-chemical parameters measured at the three plants Plant A
BOD~ Settled sewage (rag/l) Retention time in aeration tank (h) Organic Loading Rate [kg BODs/kg MLVSS)d] MLVSS (mg/l) M C R T (days) Oxygen concentration in aeration tank (final track) (mg/l) BOD5 in ettiuent (mg/l)
Plant B
Plant C
AVG
STD
AGV
STD
AVG
STD
194 9.6 0.44 1513 7.4 3.15 12.6
102 2.4 0.22 605 3,5 0.88 7.6
210 5.4 0.76 1367 4.4 1.2 23
77 1.8 0.7 616 1.5 0.35 13
313 6.4 0,9 1240 2.47 1.1 29
89 1.8 0.4 496 0.95 0.6 18
12.3 12.3 64
12.8 10.4 116
13.8 10.8 115
13.1 10 142
13.5 10.3 132
12.8 9.9 146 Paramecium aurelia (complex) BOD s A V G 13.3 BOD 5 STD 10.5 n 119
BOD 5 AVG BOD 5 S T D n
13.2
16 19.4 23
10.1 130
Cinetochilum margaritaceum
BOD 5 A V G BOD 5 STD n
Uronema nigricans
BOD s A V G BOD 5 S T D n
Podophrya fixa
BOD 5 AVG BOD 5 S T D n
Tokophrya ciclopum
BOD 5 A V G BOD 5 S T D n
Acineta tuberosa
BOD 5 A V G BOD 5 S T D n
Trochilia minuta
B O D 5 STD n
Chilodonella uncinata BOD 5 AVG
BOD~ A V G BOD 5 STD n
Acineria uncinata
< 10
9.7 4.7 4
--
1
11.5 7.9 23
--
16 13 13
9.6 4.5 8
8.1 3.6 7
7.5 2.6 4
----
8 4 5
12 5.6 15
100-200
3
10.9 6.3 29
13.3 8.2 27
9.4 5.1 23
5 3.4 5
11.3 3.4 7
9.1 6.7 12
14.1 9.3 27
1(~100
6.8 1.7 5
I
4
13 8.9 14
8 1.4 3
7.3 3.7 4
----
12.5 5.7 24
200-400
2
3.9
--
--
12.7 2.2 6
2
1
13.4 3.3 7
14
3.3 0.25 2
I
5.5
12.1 6.7 14
800-1600
16
7 3.9 4
9.3 5.3 3
11 6 21
400-800
1
4
13.4 5.8 9
3.4 0.43 2
10.7 3.5 15
1600-3200
Range o f ciliate density (ind/ml)
14 2 4
12.3 5.29 10
3200-6400
1280~30000
Table 2--continued overleaf
18 3.2 5
10.5 2.5 2
6400-12800
Table 2. Values of mean BOD 5 and standard deviation of the various ranges of ciliate densities in aeration tank at Plant A. The number of findings o f each species are indicated for each range of ciliate density (n)
¢3
BOD 5 AVG BOD5 S T D n Ciliates BOD 5 AVG BOD 5 STD n
Aspidisca cicada
BOD 5 AVG BOD 5 STD n Oxytricha sp. BOD 5 AVG BOD 5 STD n Euplotes sp. BOD 5 AVG BOD 5 STD n
Carchesium polypinum
BOD5 A V G BOD 5 STD n
Vaginicola cristallina
BOD 5 AVG BOD 5 STD n
Epistylis plicatilis
BOD 5 AVG BOD5 S T D n
Opercularia coarctata
BOD 5 AVG BOD 5 STD n
Zoothamnium sp.
BOD s AVG BOD 5 STD n
Vorticella microstoma
BOD 5 AVG BOD~ S T D n
Vorticella convallaria
Table 2--continued
3
70 --
--
--
--
---
8.6 5.4 5
1
12.6 8.6 27
1
6
6.5 3.9 6
13 10 143 19.2 17.5 23
5
11 5.8 7
9.3 6 17
13.7 10.7 114
--
--
--
12.3 5.2 12
8.8 4.8 6
2
1
2
11
3.3 0.5 3
9.3 4.9 9
10
1
4
7.7 2.5 3
23 11.2 5
2
1
19.8 11.3 5
5
8.6 6.4 13
9.9 5.7 19
200~,00
10
11.2 4.7 6
11.4 4.6 15
100-200
3
3.83 1.9 3
10.4 4.4 18
9.9 5.8 11
17.8 17.4 3
14.3 9.3 24
10-100
13.4 10.2 135
13.3 9.9 139
14.7 1I 103
12.1 I 1.7 78
12.6 9.9 148
11.3 15.8 31
15.4 13.9 53
< 10
4
4
12
21
29 6
9.6 5.2 26
I
8
2
10.5
1
3
6.6 2 5
11.4 3.2 16
11.9 3.5 17
9.8 4.4 13
12.5 9.7 26
12.2 8.4 6
2
7.5
5.1 0.7 4
5.25 1.5 4
14.4 7.4 9
11.5 7.3 13
10.3 3.4 12
density
800-1600
of ciliate
400-800
Range
12
5
8.7
12.8 3.8 14
6.3 2.3 4
9.6 1.3 3
13.3 6.4 12
12.6 5.9 21
9.8 3.9 12
1600-3200
(ind/ml)
51
6.2
11.3
12.7 5.2 10
4.3 1.3 3
6 3.6 6
13 3.8 12
14.6 6.4 29
9.7 3.4 3
3200~400
54
8
11.5
9.5 3 7
10.4 3.8 3
17.3 9.7 13
6400-12800
23
4.3
13.6
1
11
16 3.3 5
12800-30000
t:x o2
ct)
BOD 5 AVG BOD 5 STD n Ciliates BOD s AVG BOD s STD n
Aspidisca cicada
BOD~ A V G BOD 5 STD n Euplotes sp. BOD 5 AVG BOD 5 STD n
Epistylis plicatilis
BOD 5 AVG BOD 5 STD n
Opercularia coarctata
BOD~ A V G BOD 5 STD n
Vorticella microstoma
BOD 5 AVG BOD 5 STD n
Vorticella convallaria
BOD S AVG BOD 5 STD n
Uronema nigricans
BOD 5 AVG BOD s STD n
Podophrya fixa
BOD 5 AVG BOD s STD n
Chilodonella uncinata
BOD 5 AVG BOD 5 STD n
Acineria uncinata
62.7 34. l 10
12.9 8 33
10.6 5.7 7
13 7.6 4
11.5 8.4 9
17.9 17.6 191
30.2 25 61
7.7 2.5 3
24.4 14.2 7
23 23 7
11.4 4.6 15
18.1 13.2 17
9.9 4.4 9
11.5 8.1 4
13.3 5.9 19
100-200
10.4 4.4 18
14.6 14.9 14
27.3 16.2 6
14.3 9.3 24
13.4 10.1 33
13.9 8 31
10 5.7 13
17.5 14.8 39
10-100
20.3 18.7 153
17.9 22.2 93
23 22 40
23 21.4 107
20 24.7 81
18.5 18.8 159
18.3 17.7 184
28.4 26.4 54
< I0
13.5 9.9 28 45.5 25.8 5
59.5 7 2
6.4 2.7 5
15.6 7.43 14
14,7 10.9 17
10.3 3.4 12
16 5 12
20 8.2 5
9.3 5.3 3
11.2 5.8 24
400-800
12.2 4.8 14
I
5
9.7 4.8 10
27 12.4 17
12.7 9.6 17
9.9 5.7 19
15.2 9.7 18
1
12
----
13 5.9 26
200~,00
25.25 I 1.4 9
9.6 5.2 26
8.5 3.8 7
14.2 5.35 24
14 5 27
9,8 4.4 13
18.7 10.2 10
1
5.5
12 6.5 15
800-1600
15.9 14.7 18
12.8 3.8 14
9.6 1,3 3
14.7 9.03 17
16.8 11.6 31
9.8 3.9 12
16.2 8.6 16
10.7 3,5 15
1600-3200
R a n g e o f ciliate density (ind/ml)
10.4 3.6 8 12.9 8.5 66
12.9 8.4 61
10.6 3.01 5
18.1 10.7 18
16.8 3.4 6
10.5 2.5 2
6400-12800
12.4 4.6 13
6 3.6 6
15.1 6.2 14
15.2 6.4 35
9.7 3.4 3
17.6 7.3 5
12.3 5 I1
3200-6400
14.3 4.6 34
1
11
15.3 3.5 7
18.2 4.7 7
12800-30000
Table 3. Values o f m e a n B O D 5 and standard deviation o f the various ranges o f abundance o f ciliates (in aeration tank) c o m m o n to at Plants A, B and C, The number of findings o f each species are indicated for each range o f ciliate density (n)
e~ r~
SS AVG SS STD n Ciliates SS AVG SS STD n
Aspidisca cicada
SS AVG SS STD n Euplotes sp. SS AVG SS STD n
Epistylis plicatilis
SS AVG SS STD n
Opercularia coarctata
SS AVG SS STD n
Vorticella microstoma
SS AVG SS STD n
Uronema nigricans
SS AVG SS STD n
Podophrya fixa
SS AVG SS STD n
Chilodonella uncinata
SS AVG SS STD n
Acineria uncinata
13.4 103 33
m
85.1 45.1 11
13.7 8.6 6
16.2 6.2 4
12.6 9 10
22.9 28,8 186
41.2 41.9 62
9.7 5.25 3
21.4 13.3 7
40 42 6
21.7 18.9 17
16.8 11.9 9
10 5.5 5
13.7 7.8 19
100-200
9 4.9 18
18 25.7 15
36.3 15.7 5
15.8 15 33
14.2 8,6 30
8.3 4.7 13
23,7 25 38
10-100
26 30.1 148
22.7 37 88
28.5 41.5 41
26.6 40.4 75
23.8 30 154
23.7 28 177
37.3 37.2 53
< 10
15.9 14.6 27 78 86.8 4
92.5 19.5 2
9.6 5.8 5
17.8 9.3 14
16.8 13.4 16
22.3 11.5 II
24.8 9 5
7.8 4.6 3
13,7 8 23
40~800
14.9 7.9 14
1
7
15.6 12 11
30.3 18.2 17
18.6 20.9 16
23 17.8 17
I
26
----
12.1 6.2 26
200-400
26.9 15 8
11.7 7.3 25
10.3 6,3 7
19.6 15 24
19.2 12.5 27
22.7 13.3 10
I
9
14 7.6 15
800-1600
Range of ciliate density
20.6 18,6 16
13.5 6 14
7.2 1.3 3
20.2 11.8 17
19.6 13 30
20.35 10.7 16
13,3 5.7 14
1600-3200
(ind/ml)
14.3 11.2 59
16.8 9.5 13
7.7 1.67 6
16.4 10.4 14
14.7 7 35
23.8 12.3 5
21.2 17.3 11
3200-6400
16.1 14 66
8.9 4.9 7
16.8 6.2 5
21.2 17.5 17
19.8 14.5 6
8 3.5 2
6400-12800
15,9 6.8 35
19.8 7.4 7
19.7 6.5 7
12800-30000
Table 4, Values of effluent suspended solids: mean and standard deviation of the various ranges of abundance (in aeration tank) of ciliates common to Plants A, B and C. The number of findings of each species are indicated for each range of ciliate density (n)
O-
o~
1047
C i l i a t e s in a c t i v a t e d s l u d g e e f f l u e n t s Table 5. Correlation coefficients between ciliates and BOD~E or SSE at Plants A, B and C. These values were found using the data obtained in the Tables 2, 3 and 4 BOD5 Plant A
BOD 5 Plants A, B and C
Suspended solids Plants A, B and C
Acineria uncinata Chilodonella uncinata Trochilia minuta Acineta tuberosa Tokophrya ciclopum Podophrya .[ixa Uronema nigricans Cinetochilum rnargaritaceum Paramecium aurelia (complex) Vorticella cont~allaria Vorticella rnicrostoma Zoothamnium sp. Opercularia coarctata Epistylis plicatilis Vaginicola cristallina Carchesium polypinurn Oxytricha sp. Euplotes sp. Aspidisca cicada
-0.894 -0,900 - 0,937
-0.925 -0.950 - 0.940
-0.814 -0.902 - 0.930
0,007
-0.435 - 0.281 -0.114 -0.864 -0.989 -0.938 -0.608
0.046 0.032 -0.361 -0.827 -0.922 -0.878 -0.572
-0.680
-0.373 - 0.883 - 0.906 - 0.683 -0.856 -0.853 -0.791
-0.384 - 0.847 - 0.884 - 0.780 -0.837 -0.824 -0.820
Ciliates
- 0.964
- 0.868
0.000
0.528 -0,849 -0.872 -0.920 0.377 0.067 - 0.831 - 0.906 - 0.510 -0.678
were slightly higher, but were still very low--all below 0.3, and most below 0.05. But correlations based on the data from Tables 2, 3 and 4 presented high correlation coefficients between control parameters and logarithms of organism density (Table 5). It can thus be seen, that for a small change in BODsE there is a large change in the ciliated populations structure. Most ciliate species studied at Plant A showed the existence of an inverse relation with BOD5 E; however certain species had very low correlation coefficients, such as Opercularia coarctata. There were still some species with a positive correlation, such as Vorticella microstorna and Uronema nigricans. After joint examination of data from all three plants presented negative correlations for the species mentioned the averages and standard deviations of the three species increased at relatively low population densities, but at high densities BOD 5E and SSE values remained practically even. The other ciliate species that clearly showed negative correlation coefficients, continued to present high, negative correlation coefficients: Chilodonella uncinata, Aspidisca cicada and Epistylis plicatilis. In the suctoria, with the exception of Acineta tuberosa, which only appeared in periods of high effluent quality, there was no close relationship with effluent quality. In addition, suctoria possessed extremely low regression coefficients; when their population increased they did not clearly tend towards a certain value of BODsE or SSE. A comparison of the results presented in Tables 2 and 3 can be found in Table 6, which shows the average density of each ciliate species or group of organisms found at the plants for seven BODsE ranges. In this table it can be observed that the highest ciliate densities are found between 10 and 26 mg/l of BODsE while ciliate densities are reduced above and under this range. It should also be stressed that the number of ciliate and other protozoan taxons WR
2914~E
are reduced as BODsE increases. Thus in ranges of low effluent quality (i.e. above 42 mg/1 of BOD 5E), densities of ciliate populations are progressively reduced being almost zero above 67 mg/l. The most abundant protozoa at higher BODsE levels were small protozoa such as flagellates < 20 #m and gymnoamoebae < 50 pm. The high values of standard deviations in Table 6 show that a particular BODsE range does not establish which protozoan community structure will be found in the activated sludge. The lowest standard deviations are just found when assessing the total number of ciliates between 0 and 6 mg/l BODsE, which indicates that good effluent qualities are linked to a minimum ciliate density without establishing the species composition of the community. DISCUSSION
The fact that the ciliates show a closer relationship to BOD 5E than to SSE may be due to various causes: suspended solids in the effluent may be more affected by the system's hydrodynamics than BODsE, since variations in flow can produce sudden sludge rises; only a percentage of the suspended solids in the effluent consists of dispersed bacteria, since flocculi, filamentous organisms and protozoa are also present; Curds (1993) observed that the presence of ciliate protozoa not only reduces the density of dispersed bacteria in the environment by feeding, but also produces a reduction in the concentration of soluble BODs, although the causes have not been determined. BODs, as an estimation itself of the biodegradable material in the medium, also reflects the medium's saprobity better than suspended solids. As can be seen in Tables 2, 3 and 4, certain species are abundant and frequent in all ranges of ciliate population density, and some are infrequent. For species that appear in a small number of samples it
H. S a l v a d 6 et al.
1048
Table 6. Aeration tank microorganism variation according to BOD 5 effluent at Plants A, B and C. Abundances, averages and standard deviations of organisms are represented in individuals/ml
Abundance (ind/ml)
Acineria uncinata Amphileptus pleurosigma Chilodonella uncinata Trochilia minuta Acineta tuberosa Tokophrya ciclopum Podophya fixa Uronema nigricans Cinetochilum margaritaceum Paramecium aurelia Vortieella convallaria Vorticella microstoma Zoothamnium sp. Opercularia coarctata Epistylis plieatilus Vaginicola cristallina Carchesium polypinum Oxytricha sp. Euplotes sp. Aspidisca c&ada Total eiliates Flagellates < 2 0 # m Flagellates > 2 0 p r o Gymnamoebae < 50/~m Gymnamoebae > 50/~m
Testate amoebae Rotifers
Nematodes Oligochaeta Filamentous organisms (m/ml)
Range of BOD s (mg/I)
0 4
4 6
No. of samples
31
AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD AVG STD
717 885 4 10 74 263 252 546 13 40 12 25 20 46 66 221 94 369 30 72 476 990 1125 1928 18 103 836 1699 404 892 140 280 611 1367 65 136 16 49 884 1653 6094 3092 334474 826422 36 128 10307 22140 107 129 65 98 I 10 200 5 16 12 36 99 137
is harder to calculate their value as indicators and often their standard deviation cannot be determined. Although it is difficult to calculate their relationship to the environment, they are considered valuable as indicators. According to Sartory (1976) if these species are stenosaprobic, that is to say, if they "occur at a single level of saprobity", they will be good indicators of effluent quality (BOD). Table 6 shows that the least common species such as Acineta
10 16
16 26
26 42
42 67
67 107
41
54
40
15
12
12
717 1031 0 0 14 66 13 75 7 31 67 158 29 86 1274 5808 0 0 32 73 463 711 2295 3577 8 49 672 1406 686 1747 1 5 40 142 87 208 3 15 934 1949 8048 6786 249417 348815 0 0 13308 26617 284 677 40 145 16 31 6 12 0 0 92 110
1048 1852 0 0 8 54 22 91 1 6 17 66 32 96 2310 8161 0 0 9 30 423 849 3528 5459 0 0 1015 1731 149 517 0 0 70 374 24 108 2 I1 1809 2765 11328 11313 276711 539811 0 0 31392 73952 189 489 0 0 8 26 3 8 0.2 1 210 202
410 898 0 0 9 34 I 6 0 0 2 12 18 79 5705 16061 0 0 4 15 289 892 4427 4608 0 0 1079 1477 32 186 0 0 0 0 10 40 7 26 861 1615 13241 16350 701904 813976 0 0 72542 76986 29 58 0 0 0 0 11 17 0 0 359 421
143 263 7 26 2 7 0 0 0 0 0 0 84 218 1322 2555 0 0 0 0 5 I1 2764 2661 0 0 680 1085 0 0 0 0 0 0 42 155 5 20 179 299 5269 5235 733258 747372 0 0 76285 93827 21 44 0 0 0 0 0 0 0 0 283 303
2 7 0 0 0 0 0 0 0 0 0 0 1 4 115 240 0 0 1 2 6 18 210 541 0 0 304 652 0 0 0 0 0 0 0 0 0 0 4 14 641 985 13826 38210 0 0 39875
2 12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 36 123 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 38 199 19720 40277 0 0 43960 125402 0 0 0 0 0 0 0 0 0 0 0 0
42022 2 7 0 0 0 0 9 15 0 0 55 120
tuberosa, Euplotes sp. and Zoothamnium sp. are, in all observed cases, indicators of high effluent quality; in contrast, the species that reach the highest densities and are the most common, such as Uronema nigricans, Vorticella microstoma and Opercularia coarctata are indicators o f lower effluent quality. The relationship observed between the population density of ciliate species and effluent quality mainly tends to a narrow range of values. Abundance of
Ciliates in activated sludge effluents individuals, though in a relative form, is already habitually used to calculate saprobity indices of an environment on the basis of indicator species (Zelinka and Marvan, 1961; Sladecek, 1973). However, to calculate saprobity indices only a fixed significance value is used for any of the population density found. When the population density increases, the quality indicator parameter tends to a narrower range of values, which we call the optimal range, and the lower the standard deviation obviously the higher will be the capability as an indicator. Consequently when the population density of a certain species rises, its capability as an indicator also increases; as is reflected by its standard deviation values, The results of the correlation coefficients found reflect clearly the capability of ciliates as indicators. The coefficients may not necessarily be very high, since the tendency towards a narrow range of values makes it difficult to perform the correlation analyses, because BOD 5E values remain similar as ciliate density increases. Thus, correlation results show that negative or positive correlation coefficients vary according to the studied BOD 5E range. As an example we have observed positive and negative coefficients in Vorticella microstoma. Such variations are also found in literature, while Madoni (1994) found positive correlation coefficients for V. microstoma and BODsE, Esteban et al. (1991) found negative correlations with the same parameter and species. Thus the results with low correlation coefficients do not necessarily imply a particular ciliate species is not related with the indicator parameters of effluent quality. We observe how Uronema nigricans, with low regression coefficients, has a strong relation to effluent quality which becomes clear as the standard deviation gradually falls. On the other hand suctors such as Podophyafixa and Tokophrya ciclopum have low regression coefficients as they do not have a close relation with the indicator parameters of effluent quality. We do not believe that the suctors' ciliophagous feeding contributes directly to the clearing of the effluent, as the bacteriophagous ciliates do. The capability of ciliates as indicators of effluent quality is limited by a number of factors. The presence of particular ciliate species in the activated sludge obviously depends on various factors, among which we should mention those which are not to do with the plant, such as the organic matter in the effluent (or volumetric loading rate), and those caused by the operational parameters, such as dissolved oxygen and MCRT (Curds and Cockburn, 1970; Curds, 1982a; Al-shahwani and Horan, 1991; Madoni and Ghetti, 1981; Madoni, 1994; Esteban et al., 1991; Salvad6 and Gracia, 1993; Salvad6, 1994). Furthermore, communities of protozoa in activated sludge in plug flow systems tolerate a wide variety of environmental conditions; at different stages in the treatment the availability of food can decrease markedly in a short time. For this reason it is difficult
1049
to find good indicators of effluent quality (Curds, 1993). Best results have been obtained using the effluent quality value of the same day as the microscopic analysis, suggesting that although each species' capability as an indicator of effluent quality is influenced and also limited by the sum of the factors at work in the process, the optimal range of each species will be more the reflection of the action of ciliates as purifying agents or indicators of this activity than of the influence of the values of the effluent itself on the species' abundance. The values of the optimal ranges of the BODsE of the species found vary from 4 to 18mg/l which suggests that the ciliate species that colonize the activated sludges can be good indicators between 4 and 18 mg/l. Outside these values accurate diagnoses are not possible, although the low densities of ciliates ( < 1000 ind/ml) or their absence easily cause BOD 5 values of over 30 mg/l in the conventional activated sludge purifying plants studied (see Table 6). Curds and Cockburn (1970) stated in their observations that when ciliates were absent the effluent was over 30 mg/l BOD 5E. CONCLUDING REMARKS
The most useful finding in this study was that after grouping data of physical and chemical parameters according to ranges of ciliate abundance, a decrease in standard deviation of BOD~E or SSE as the ciliate population density increased could be observed. Thus, the increase in abundance of a particular species indicates more accurately its optimal range; this optimal range of each species can be used as an indicator of effluent quality. The capability of assessing effluent quality by the study of ciliate densities has limitations. Thus even though a density of ciliates can indicate a range of values of BOD 5E or SSE, the presence of a particular species of ciliate will also qualitatively depend on the nature of the effluent, other operational parameters and moreover the relations with the other species of the community. In addition this study indicates that the gradient of the correlation between effluent quality (BODs) and the abundance of ciliates populations is positive or negative depending on the range of data values established in the samplings. This gradient may be positive or negative if the range of effluent quality parameter values is above or below the optimal indicator value of the species. Such observations may be also considered in further correlation studies of other physico-chemical parameters and ciliate populations. Acknowledgements--The authors wish to express their gratitude to Dr Miquel Salicrfi, Departament d'Estadistica, Universitat de Barcelona, for technical advice on statistical analysis. We also wish to mention our gratitude to Empresa Metropolitana de Sancjament S.A. (EMSSA), and the management of C.I.C.Y.T. (Comisi6n Interministerial de Ciencia y Tecnologia) Project PB 88-0212.
1050
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