Protozoa in wastewater treatment processes: A minireview

Abstract

Biological wastewater treatment is a process of increasing importance in a world with an ever-increasing human population. Wastewater treatment facilities are designed to maintain the high density and activity levels of those microorganisms that carry out the various purification processes. Protozoa are one of the most common components in these man-made ecosystems and play an important role in wastewater purification processes. Protozoa are responsible for improving the quality of the effluent, maintaining the density of dispersed bacterial populations by predation. Studies of the relationships between protozoa and physicochemical and operational parameters have revealed that the species structure of these communities is an indicator of plant efficiency. The Sludge Biotic Index (SBI), an index based on the structure and abundance of the microfauna inhabiting the activated sludge and mixed liquor, has been devised to monitor activated-sludge plant performance. Heavy metals and other pollutants are toxic to most microorganisms at certain concentrations. These toxicants are common pollutants of sewage, particularly where there is industrial waste input. The protozoa community is a complex assemblage of interacting organisms, often including species that are sensitive, resistant or intermediate in their tolerance to pollutants. Many studies conducted on contaminated activated sludge and mixed liquor have revealed changes in the dynamics of the protozoa community. Tests on the acute toxicity of pollutants on ciliates have revealed that these microorganisms are useful bioindicators for evaluating the toxicity of waters polluted by different concentrations of metals.

Keywords:

• Wastewater treatment
• monitoring
• community structure
• protozoa
• toxicants

Introduction

Biological wastewater treatment processes rely on the natural self-purification ability of microbial communities. Nevertheless, they differ from aquatic environments due to certain characteristics, such as a strong flow of organic load into the system, accelerated decomposition processes, short biomass turnover time, and the prevalence of heterotrophic organisms. Due to these characteristics, biological wastewater treatment processes can be regarded as man-made ecosystems subjected to extreme conditions (Antonietti et al. 1981), so that of all the heterotrophic organisms only protozoa and small metazoa with life cycles shorter than the sludge retention time are able to compete in these processes.

The presence of protozoa in biological wastewater treatment processes was observed almost as soon as each process was introduced, but it has only been in recent years that the significance of these microorganisms has been underlined. The organisms most directly involved in wastewater treatment are the bacteria. They dominate all other groups, in number and biomass, and affect the process of mineralization and elimination of organic and inorganic nutrients. In modern systems, where there is a low load and high sludge retention time, the presence of protozoa such as ciliates, flagellates, and amoebae, or even small metazoa, is very common. These eukaryotic organisms are able to feed on particulates, such as suspended bacteria. It is generally assumed that their primary role in wastewater treatment is the clarification of the effluent.

Occurrence and diversity of protozoa

Percolating filter process

Lists of the protozoa found in percolating filters have been given by many authors (Agersborg & Hatfield 1929; Barker 1943; Curds & Cockburn 1970a). More than 200 species have been identified by these authors, and a complete list has been reported by Curds (1975). Ciliophora is the phylum of protozoa that contributes the greatest number of individuals to the microfauna of a percolating filter. Ciliates range from 500 to 10,000 individuals per ml of liquor (Curds & Cockburn 1970b). Certain species have been found in very large numbers (Chilodonella uncinata, Vorticella convallaria, Opercularia microdiscum and Carchesium polypinum). However, their frequency has not necessarily been associated with a dominant numerical position in the population. One such species is Cinetochilum margaritaceum, which, although present in 54% of cases, has never been found in large numbers (Curds & Cockburn 1970b). Other species such as Acineria uncinata, Amphileptus pleurosigma and Vorticella microstoma, although frequently found, were usually present in small numbers. In a study of the structure of a protozoan community in a percolating filter (Mistri et al. 1994), large variations in the densities of some ciliate species were observed. The structure of the protozoa community was characterized by the high-density values of the crawling hypotrich Aspidisca cicada associated with a rich assemblage of attached peritrichs. Nevertheless, species blooms were frequent and stalked ciliates often exhibited large density oscillations. Sudden demographic blooms of some ciliate species (mainly Dexiotricha colpidiopsis, Uronema nigricans, Acineria uncinata, Chilodonella uncinata, Paramecium caudatum and Colpidium colpoda) were indicative of a less mature condition.

Rotating biological contactor process

Protozoa are commonly present in rotating biological contactors (RBC), where they colonize the biofilm. Lists of identified species appearing in RBC systems have been given by certain authors (Madoni 1981; Madoni & Ghetti 1981). Comparative studies of the occurrence (Madoni & Ghetti 1981; Kinner & Curds 1987; Chung & Strom 1991; Hul 1992; Pérez-Uz et al. 1998) and distribution (Madoni et al. 1979; Luna-Pabello et al. 1992; Martin-Cereceda et al. 2002) of ciliate populations in RBC systems have also been made. A relationship between the organic load and the distribution of microorganisms in the reactor has been found; heterotrophic flagellates and free-swimming ciliates such as Cyclidium glaucoma and Colpidium colpoda are dominant in the early stages, where the organic load is higher, whilst testate amoebae and attached ciliates such as Epistylis plicatilis and Zoothamnium procerius are dominant in the final stages, characterized by low BOD₅ values. The quantitative importance of protozoa in RBC systems has been determined in terms of biomass (Madoni 1994a): a ciliate biomass value of 314 μg cm^–2 (dry weight) has been measured, corresponding to over 12% of the volatile solids on the RBC biofilm.

Activated-sludge process

Since the installation of the first activated-sludge treatment facilities in 1922, many authors have noted the presence of free-living protozoa. These microorganisms are commonly found in the mixed liquor of activated-sludge plants and numbers of the order of 3–20 × 10⁶ cells l^–1 are often reported. It has been estimated that the protozoa biomass can reach values of 250 mg l^–1 (dry weight), constituting over 9% of the volatile solids (Madoni 1994a). Lists of the protozoa species found in activated-sludge plants have been reported by a number of authors (Agersborg & Hatfield 1929; Ardern & Lockett 1936; Clay 1964; Brown 1965; Curds & Cockburn 1970a; Morisita 1970). A complete list of 228 species of protozoa has been reported by Curds (1975). Since wastewater treatment is a process of increasing importance in a world with an ever-growing human population, the presence and action of microbial communities has received particular attention in recent years. Consequently, revised lists of protozoa have been made (Madoni & Ghetti 1981; Augustin & Foissner 1992; Madoni et al. 1993; Foissner & Berger 1996; Martin-Cereceda et al. 1996; Amann et al. 1998; Ettl 2001; Madoni 2002; Chen et al. 2004). Of the 228 protozoa species listed for the activated-sludge plants, about 160 belong to the phylum Ciliophora, but only a limited number of these have been observed frequently (Table I). Despite the fact that the ciliate community of the activated sludge is slightly different from that of the percolating filters, some species, such as Acineria uncinata, Vorticella convallaria, Vorticella microstoma, Opercularia coarctata and Aspidisca cicada, are commonly observed in both processes. In a well-functioning activated-sludge plant, the protozoa community is dominated by peritrichs (Vorticella spp., Carchesium spp., Zoothamnium spp., Epistylis spp.) and hypotrichs (Aspidisca spp., Euplotes spp.). In some cases, the dominance of cyrtophorids (Chilodonella spp., Trochilia minuta) or testate amoebae (Arcella spp., Euglypha spp.) can be observed. In a study devoted to reconstructing the species structure of the ciliate community in activated-sludge processes (Madoni & Ghetti 1981), it was found that of the 45 species present in 39 different plants, five were recurrent and representative of the principal community (Aspidisca cicada, Vorticella convallaria, V. striata octava, Epistylis plicatilis and Trochilia minuta). Other species, such as Trithigmostoma cucullulus, Euplotes sp. and Zoothamnium pygmaeum, have no affinity with each other, but show affinity with some species of the recurrent group, and can therefore be regarded as associated species.

Table I. Occurrence of protozoa in activated sludge plants in Italy (V, very common; C, common; F, frequent; R, rare)

Group	Species	Frequency
Large flagellates	Euglena sp.	F
	Peranema sp.	F
Amoebae
Testate amoebae	Arcella sp.	F
	Euglypha sp.	F
	Centropyxis sp.	R
Bacterivorous ciliates
Free-swimming	Cinetochilum margaritaceum	F
	Colpidium colpoda	R
	Cyclidium glaucoma	R
	Dexiostoma campylum	R
	Glaucoma scintillans	R
	Paramecium aurelia complex	R
	Paramecium caudatum	R
	Pseudocohnilembus pusillus	R
	Spirostomum teres	F
	Uronema nigricans	R
Crawling	Acineria uncinata	V
	Aspidisca cicada	V
	Aspidisca lynceus	C
	Chilodonella uncinata	F
	Drepanomonas revoluta	F
	Euplotes aediculatus	F
	Euplotes affinis	F
	Euplotes patella	R
	Trithigmostoma cucullulus	F
	Trochilia minuta	C
	Stylonychia sp.	R
Attached	Carchesium polypinum	C
	Epistylis chrysemidis	C
	Epistylis entzii	F
	Epistylis plicatilis	V
	Opercularia articulata	F
	Opercularia coarctata	F
	Opercularia nutans	R
	Stentor coeruleus	R
	Vaginicola ingenita	F
	Vorticella aquadulcis complex	V
	Vorticella convallaria complex	V
	Vorticella infusionum complex	R
	Vorticella microstoma complex	R
	Vorticella octava complex	C
	Zoothamnium arbuscula	F
Carnivorous ciliates
	Acineta tuberosa	C
	Amphileptus claparedei	F
	Coleps hirtus	F
	Litonotus crystallinus	C
	Litonotus cygnus	R
	Litonotus lamella	F
	Plagiocampa rouxi	V
	Podophrya sp.	R
	Prorodon sp.	C
	Spathidium sp.	R
	Tokophrya lemnarum	F
	Tokophrya quadripartita	C

The role of protozoa in wastewater treatment

Protozoa were originally thought to be harmful to the activated-sludge process (Fairbrother & Renshaw 1922). However, some authors (Curds et al. 1968) were able to assess the role of these organisms and to quantify the magnitude of their effect upon the effluent quality. These authors found that with no protozoa in the mixed liquor the BOD₅ of the effluent and other parameters (e.g. the level of organic carbon and mixed liquor suspended solids (MLSS)) were higher. The positive effects of protozoa on carbon mineralization by bacteria in activated sludge are well known (Ratsak et al. 1996). The excretion of mineral nutrients by protozoa results in an accelerated usage of the carbon source by the bacteria (Coleman et al. 1978; Bloem et al. 1988; Tezuka 1990). Furthermore, protozoa excrete growth-stimulating compounds that can enhance bacterial activity (Nisbet 1984; Horan 1990). Nevertheless, these indirect effects of protozoa on bacterial growth cannot increase carbon mineralization under carbon-limitation conditions (Curds 1982). Thus, in wastewater systems with a low substrate concentration, the process is of little importance. Among the direct effects of protozoa on bacteria, grazing plays an important role. Protozoa, in fact, are considered to be the most important bacterivorous grazers. The clearance rates of several protozoa inhabiting activated sludge range from 4 × 10^–7 to 1 × 10^–6 ml medium protozoa^–1 h^–1 (Bloem et al. 1988, 1989). Even though some protozoa (crawling ciliates and other forms) can eat flocculated bacteria, most protozoa (prevalently attached ciliates) can only graze on suspended bacteria and particles; in this way they have a significant effect on the effluent quality. It is generally assumed that the primary role of protozoa in wastewater treatment is the clarification of the effluent (Curds et al. 1968; Madoni 2003). In the presence of ciliates, a reduction of the density of viable Escherichia coli was also observed (Curds & Fey 1969; Mallory et al. 1983). Several studies have focused specifically on the role of protozoa predation in nitrogen cycling in activated sludge (Verhagen & Laanbroek 1991; Petropoulos & Gilbride 2005; Pogue & Gilbride 2007). It has been observed that the presence of protozoa increases the per-cell nitrification rate, probably because of the ability of protozoa to influence bacterial growth. Protozoa release inorganic and organic products into their surroundings. These products are mainly recycled nutrients, such as nitrogen, phosphorous, and organic carbon, but might also include stimulatory compounds that contribute to the dissolved organic carbon pool and affect the physiological state and growth of bacteria (Jurgens & Matz 2002).

Protozoa as indicators of activated-sludge performance

Ciliates are present in wastewater treatment processes in terms of large numbers of individuals and species. Even though some ciliates are carnivorous or omnivorous, most of these microorganisms feed upon dispersed bacteria populations. Bacterivorous ciliates colonizing activated sludge can be subdivided into three functional groups on the basis of their behaviour: free-swimming ciliates, which swim in the liquor phase; crawling ciliates, which move on the surface of the sludge floc; and sessile ciliates, which are firmly attached to the sludge floc. Studies of the dynamics and succession of protozoa in activated sludge have suggested that flagellates predominate in the system in the early stages only because of their lower energy requirements (Curds 1966). As the flagellates decrease, they are replaced by free-swimming ciliates and then by crawling and attached ciliates. Three phases in the time span can be identified from the beginning to the stabilization of the system (Madoni 1982; Madoni & Antonietti 1984). The plant start-up phase is characterized by the presence of species typical of raw sewage: free-swimming bacterivorous ciliates and small heterotrophic flagellates. These microorganisms cannot be considered typical of these environments because they are not linked to the presence of sludge flocs. With the growth of sludge, they are replaced by other functional groups. The second phase is characterized by the growth of ciliates typical of the activated-sludge habitat: crawling and attached ciliates. In this phase, a species-rich community can be observed, but its species structure changes with the progressive formation of activated sludge. The third phase (the stabilization phase) is characterized by a ciliate community whose structure reflects the stable condition of the aeration tank environment, with a balance between the organic loading and the sludge that is produced, removed and recycled. A fully functioning plant need not host species characteristic of one of the colonization phases, unless dysfunctions, due to the amount of sludge, the degree of aeration, the sewage retention time, and the organic load at the input, cause regression in the environmental conditions (Madoni 1982). For example, crawling ciliates (hypotrichs) decrease with increasing organic loading (no hypotrichs are observed in sludge loaded above 0.6 kgBOD₅ kg MLSS^–1 day^–1), while attached ciliates (peritrichs) are able to grow over a large range of sludge loadings (Curds & Cockburn 1970b). Of the attached ciliates, Vorticella convallaria and V. microstoma characterize the first phase of colonization, but the latter is replaced by V. convallaria, which may reach high numbers during the second and third sludge phases. In the case of a drastic reduction in the dissolved oxygen (DO) content of the mixed liquor, an alternation of these two species can be observed due to their different degrees of tolerance to the lack of oxygen. Large numbers of V. microstoma thus indicate a poorly aerated sludge (Madoni & Antonietti 1984). Low numbers of Opercularia often occur in activated sludge; these attached ciliates increase in number when the activated sludge performance is poor due to their association with high final effluent BOD₅ concentrations (Curds & Cockburn 1970b; Klimowicz 1970; Esteban et al. 1991; Salvadò et al. 1995). Testate amoebae, mostly Arcella and Euglypha, are normally found in the aeration tanks of activated-sludge plants that operate the biological removal of nitrogen. These plants are characterized by low loading, a long sludge retention time and high oxygen content (Poole 1984; Madoni et al. 1993). Under these conditions, the quality of the final effluent is excellent and the plant is considered to have a high biological performance. The number of ciliates living in a normally functioning activated-sludge plant is over 10³ ml^–1, but the number and diversity of the ciliate communities change according to the quality of the settled sewage and the operating conditions of the plant (Drakides 1980; Esteban et al. 1991; Esteban & Tellez 1992). The most common limiting conditions are generally the presence of a shock load of toxic material, a lack of aeration, and excess sludge wastage. Since the species and functional groups of the protozoa depend on the environmental conditions in the aeration tank, the structure of the protozoa community can be considered a valid indicator of purification plant performance. Any major variations in the plant performance are thus indicated by the dominant group of protozoa. For this reason, the routine analysis of these eukaryotic microorganisms is becoming increasingly common to determine activated-sludge plant performance. Curds and Cockburn (1970b) were probably the first to use protozoa as indicators of the effluent quality of activated-sludge plants. In recent years, the biotechnology of activated-sludge processes has improved and important innovations have been made (i.e. tertiary treatment or advanced processes, such as biological phosphorous and nitrogen removal). These new processes are often incorporated in the plant between the aeration tank and the final clarifier. The management of these processes and their associated problems in the final clarifier (i.e. rising, unfit sludge recycle rate) can affect the effluent quality. Since these symptoms originate downstream from the aeration tank (in which the microorganisms develop), the ciliate community is unable to indicate them. At present, protozoa are effectively used to indicate changes in the performance of specific activated-sludge plants (Al-Shahwani & Horan 1991; Esteban et al. 1991), but these methods cannot always be applied directly to other, similar plants. An objective index, based on the protozoa community and applicable to all types of activated-sludge plants, has been proposed for the evaluation of the biological performance of the sludge (Madoni 1994b). The method, called the Sludge Biotic Index (SBI), is based on two principles. First, the dominance of the microfauna key groups changes in relation to the environmental and operational conditions of the plant. Second, the number of morphological species is reduced as the plant performance deteriorates. The SBI method was set up to investigate the relationship between the microfauna groups and the principal physicochemical and operational parameters. The high correlations obtained (Table II) made it possible to select and group the microfauna organisms into positive and negative key groups. Positive key groups are crawling and attached bacterivorous ciliates, and testate amoebae; negative key groups are small heterotrophic flagellates, free-swimming bacterivorous ciliates, and the peritrich ciliates Vorticella microstoma, V. infusionum and Opercularia spp. The density and diversity of the microfauna, moreover, were shown to be highly correlated to the plant performance.

Table II. Pearson correlation test between protozoa and plant operational conditions, obtained from 44 activated-sludge plants (from Madoni 1994b; – = negative correlation, * P < 0.01, ** P < 0.001)

	D.O.	Nitrifying ability	BOD5 removed	Effluent colour	Sludge age	MLSS
Small flagellates	–0.652**	–0.596**	–0.798**	0.836**	–0.583**	–0.736**
Free-swimming ciliates	–0.651**	–0.549**	–0.829**	0.725**	–0.668**	–0.753**
Crawling ciliates	0.616**	0.620**	0.784**	–0.611**	0.534**	0.534**
Attached ciliates	0.340	−0.029	0.432*	–0.516**	0.171	0.266
Vorticella microstoma
and/or V. infusionum	–0.676**	–0.504**	–0.679**	0.648**	–0.638**	–0.722**
Opercularia spp.	–0.745**	–0.597**	–0.763**	0.676**	–0.008	–0.181
Testate amoebae	0.727**	0.912**	0.760**	–0.583**	0.464**	0.464**
Microfauna abundance	0.626**	0.429*	0.762**	–0.768**	0.335	0.495**
Number of species	0.778**	0.645**	0.923**	–0.841**	0.487**	0.591**

The advantage of this method over others is that it provides numerical values, which enable the operator to monitor the prevalent plant operating conditions on a daily basis.

Effect of toxicants on protozoa in sewage treatment processes

In recent years, the presence of toxicants in both surface waters and sewage has become a common occurrence. Originating mainly in industrial sewage, they affect the performance of biological wastewater treatment processes, reducing or inhibiting the activity of microorganisms. Numerous papers have been published which deal with the occurrence of heavy metals in the various sections of sewage treatment systems (Yetis & Gokcay 1989; Cimino & Caristi 1990; Dilek & Yetis 1992; Melcer et al. 1992; Mazierski 1995). The effect of metal pollutants on activated-sludge microorganisms varies with the concentration and exposure time. The toxic effects of copper on ciliate communities in activated-sludge plants have been studied using concentrations from 1 to 10 mg l^–1 (Gracia et al. 1994). The mean total number of ciliates and the specific diversity were reduced by the toxic action of Cu. However, detailed observations of copper-induced reduction of organism concentration have shown that not all species are reduced in the same way, causing changes in the dynamics of the ciliate communities that inhibit the precise calculation of the median tolerance-limit concentration. In experiments using the free-swimming ciliate Tetrahymena pyriformis, copper above 200 mg l^–1 inhibit growth significantly (Nicolau et al. 1999). In a study in which ciliate species were monitored in relation to metal concentration over a two-year period (Abraham et al. 1997), it was found that the major ciliate species present were able to tolerate high concentrations of contaminant metals, including Fe (>2 mg l^–1), Zn (>0.5 mg l^–1), Cu (>0.06 mg l^–1) and Cr (0.1 mg l^–1). This suggests that acclimatization can reduce the toxicity of heavy metals upon ciliates. Moreover, increasing the sludge age favours the retention of heavy metals in the biofloc, thus reducing their toxicity (Neujeed & Herman 1975). The cytotoxic effect of the heavy metals Cd, Zn and Cu on three different species of ciliated protozoa (Drepanomonas revoluta, Uronema nigricans and Euplotes sp.) isolated from an urban wastewater treatment plant has been studied (Martin-Gonzales et al. 2006). The order of toxicity was Cd > Cu_Zn or Cu > Cd_Zn, depending on the microbial species. In bimetallic (Cd + Zn) treatments, the results indicated that, in general, the presence of Zn in the same medium decreased Cd cytotoxicity. Both cellular assays and microscopic observations have shown that bioaccumulation is an important mechanism of resistance to these toxic environmental pollutants in such eukaryotic microorganisms. However, bioaccumulation might not be the main mechanism involved in Cu resistance.

Acute toxicity tests of five heavy metals on the protozoa community inhabiting activated sludge have also been performed (Madoni et al. 1996). The experimental results demonstrated the relative toxicity of the tested metals, and indicated that the order of toxicity of the five metals on the studied microbial community was generally: Cd, Cu > Pb > Zn > Cr. Lethal concentrations (24-h LC₅₀) of heavy metals on ciliates from activated-sludge plants have been determined during different acute toxicity tests (Madoni et al. 1992, 1994, 1996; Madoni 2000; Madoni & Romeo 2006), and the values obtained are reported in Table III. Large differences appeared in the sensitivity of the species to the metals. Crawling ciliates such as Chilodonella uncinata and Trochilia minuta showed a high sensitivity to all the studied metals, while the attached ciliates Opercularia coarctata and O. minima were the most tolerant species. Nevertheless, the results obtained in these studies suggest large differences in the tolerance levels of the tested ciliated protozoan to heavy metals. Euplotes aediculatus and Spirostomum teres showed the highest sensitivity to nickel, while Blepharisma americanum showed a high sensitivity to copper. The derived toxicity revealed in these studies is of potential relevance for the microbial food web in wastewater treatment plants since sewage water often contains considerable amounts of toxic metals.

Table III. 24-h LC₅₀ mean values (mg l^–1) of some protozoa from activated sludges tested with heavy metals

Species	Metal	LC50	Ref.	Species	Metal	LC50	Ref.
Aspidisca cicada	Cd	0.31	a	E. patella	Pb	2.18	b
A. cicada	Cu	0.021	a	E. aediculatus	Cd	0.59	d
A. cicada	Hg	0.07	a	E. aediculatus	Cu	0.01	d
A. cicada	Zn	2.4	a	E. aediculatus	Cr	0.10	d
A. cicada	Pb	1.26	b	E. aediculatus	Ni	0.03	d
A. cicada	Cr	2.35	b	E. aediculatus	Pb	0.50	d
A. lynceus	Cd	2.21	c	Opercularia coarctata	Cd	3.75	c
A. lynceus	Cu	1.82	c	O. coarctata	Cr	211	c
A. lynceus	Cr	14.5	c	O. coarctata	Pb	3.28	c
A. lynceus	Pb	0.004	c	O. coarctata	Zn	10.3	c
A. lynceus	Zn	0.05	c	O. minima	Cd	5.55	c
Blepharisma americanum	Cd	1.4	a	O. minima	Cr	164	c
B. americanum	Cu	0.001	a	O. minima	Pb	5.0	c
B. americanum	Hg	0.017	a	O. minima	Zn	84.7	c
B. americanum	Zn	1.05	a	Paramecium caudatum	Cd	0.18	a
Chilodonella uncinata	Cd	<0.0001	c	P. caudatum	Cu	0.011	a
C. uncinata	Cu	<0.00004	c	P. caudatum	Cr	2.57	b
C. uncinata	Cr	0.011	c	P. caudatum	Hg	0.02	a
C. uncinata	Pb	<0.0015	c	P. caudatum	Ni	0.49	e
C. uncinata	Zn	<0.0002	c	P. caudatum	Pb	2.26	b
Colpidium colpoda	Cd	0.89	d	P. caudatum	Zn	2.50	a
C. colpoda	Cu	0.05	d	Spirostomum teres	Cr	3.23	b
C. colpoda	Cr	108	d	S. teres	Ni	0.17	e
C. colpoda	Ni	1.19	d	S. teres	Pb	1.08	e
C. colpoda	Pb	0.23	d	Trochilia minuta	Cd	0.27	c
Dexiostoma campylum	Cd	0.2	a	T. minuta	Cu	0.31	c
D. campylum	Cu	0.01	a	T. minuta	Cr	9.25	c
D. campylum	Cr	3.29	b	T. minuta	Zn	0.20	c
D. campylum	Hg	0.017	a	Uronema nigricans	Cd	0.62	a
D. campylum	Ni	1.05	e	U. nigricans	Cu	0.014	a
D. campylum	Pb	1.1	b	U. nigricans	Cr	2.18	b
Drepanomonas revoluta	Cr	45.6	b	U. nigricans	Hg	0.004	a
D. revoluta	Pb	0.88	b	U. nigricans	Pb	1.62	b
Euplotes affinis	Cd	0.4	a	U. nigricans	Zn	0.003	a
E. affinis	Cu	0.064	a	Vorticella convallaria	Cd	3.81	c
E. affinis	Cr	2.73	b	V. convallaria	Cr	101	c
E. affinis	Hg	0.19	a	V. convallaria	Pb	2.29	c
E. affinis	Pb	2.32	b	V. convallaria	Zn	3.79	c
E. affinis	Zn	3.10	a	V. octava	Cd	3.24	c
E. patella	Cd	2.65	a	V. octava	Cr	80	c
E. patella	Cu	0.011	a	V. octava	Cu	2.05	c
E. patella	Cr	9.47	b	V. octava	Pb	2.81	c
E. patella	Hg	0.13	a	V. octava	Zn	0.57	c
E. patella	Ni	7.70	e
The letters in the bibliography columns refer to published references, as follows: a, Madoni et al. (1992); b, Madoni et al. (1994); c, Madoni et al. (1996); d, Madoni & Romeo (2006); e, Madoni (2000).

Toxic substances other than the heavy metals can also enter the plant and damage the protozoa community. The effects of the shock load of salt (NaCl) on protozoa communities have also been evaluated (Salvadò et al. 2001), and the results have shown that salt concentrations from 3000 to 10,000 mg l^–1 gradually affected the microbial community and few protozoa survived to 96 h. The attached ciliates Vorticella spp. and Opercularia articulata resisted the highest dosages of NaCl better than the other ciliates. The toxic effect of chemical disinfection of sewage treatment plants has been tested on three ciliates inhabiting both the receiving water bodies and the activated sludge (Madoni et al. 1998). Three chemical disinfectants were tested: sodium hypochlorite (NaClO), chlorine dioxide (ClO₂) and peracetic acid (PAA). The effluent treated with ClO₂ was highly toxic for Spirostomum teres but was only slightly toxic for the other two ciliate species. The effluent treated with NaClO had a moderate toxic effect only on Euplotes patella. The free-swimming ciliate Dexiostoma campylum showed the lowest sensitivity to all the chemical disinfectants. Rehman et al. (2008) reported that the ciliates Stylonychia mytilus and Paramecium caudatum are resistant to the organophosphate endosulfan and have the capacity to utilize it as a carbon source. This allows these ciliates to be used for bioremediation of toxic xenobiotics. The introduction of phenol to the wastewater resulted in a change in the dominance of the sessile species, favouring the dominance of Opercularia sp. rather than Vorticella sp. (Papadimitriu et al. 2007). Phenol enhanced the abundance of the suctorian Podophrya sp. and the free-swimming Colpidium sp., the latter being typical of wastewater treatment plants receiving industrial inputs. The suctorian Podophrya sp. showed a significant negative correlation to the removal of phenol in the effluent, suggesting that its presence may be an indicator of phenol removal efficiency. The introduction of cyanide to the wastewater increased the abundance of Opercularia sp. and Colpidium sp. suggesting their tolerance under highly toxic conditions. Chilodonella sp. showed a negative correlation to the cyanide content of the effluent (Papadimitriu et al. 2007).

These studies of ciliate sensitivity to a wide number of toxic substances provide a yardstick for identifying the intensity and potential for ecological damage caused by anthropogenic pollutants discharged into surface waters.

Notes

^†This paper is dedicated to Maria Umberta Delmonte Corrado for her contribution to the development of Italian Protistology.