Our results suggest that total phenolic compound levels within seagrass tissue could be an indicator of site quality. Differences in production of phenolics in tissues from both species were noted between impacted and control sites. For foliage leaves and sheaths of *P. oceanica* collected in January, and above-ground tissue of *Z. marina* collected in July, total phenolic content was significantly lower in plants collected from industrial sites (Fig. 1 & 2). This is supported by Vergeer et al. (1995) who concluded that a decrease of total phenolic compounds in the tissue of *Z. marina* indicated plants may be growing in unsuitable environmental conditions. Noteworthy is that correlation analysis indicated a significant (p< 0.05) inverse relationship between heavy metal content and the health of plants as measured by phenolic content for *P. oceanica* (r2 = 69.8 %, linear model of regression: mercury = 0.22 – 0.0055 \* phenol

Additionally, gas chromatography analysis of volatile compounds from *Z. marina* indicated that above-ground tissue from plants growing in the impacted site was significantly lower in volatiles from the April collection, when growth begins in Northern Puget Sound (Phillips, 1984) compared to tissue from the control site (Fig. 3). However, no significant differences occurred in volatile compound production between impacted and control sites in

For *P. oceanica*, the concentration of phenolic compounds differed between foliage leaves and sheaths being higher in leaf tissue regardless of site. Similarly, Agostini et al. (1998) found higher concentrations (6 mg.g-1) in the apical parts and youngest leaves and lower concentrations in sheaths (0.1 mg.g-1). Also, in our study significant variation was observed between seasons; for example, phenolic levels were found to be higher in the January 2001

Differences occur in the natural products analyzed depending on month of collection for both *P. oceanica* and *Z. marina (Fig. 1-3)*. For example, *P. oceanica* foliage leaves and sheaths in January 2001 were higher in phenolic content than those collected in June 2000 (Fig. 1). While phenolic content in above-ground *Z. marina* tissue was similar in concentration between April and July (Fig. 2), but volatile compounds in above-ground tissue collected in April were significantly higher than those collected in July (Fig. 3). April and July were selected as sampling times for *Z. marina* because they represent early and mature tissue

**4.3 Phenolic compound production with regard to tissue and time collection**

water than in the water column at our Rosignano sampling location.

pattern described by Lynby & Brix (1984) (Table 2).

**and control sites**

for sheaths).

the July collection.

samples compared to the June 2000 samples.

Significant differences were found in the accumulation of mercury in leaf and sheath tissues of *P. oceanica* when plants were growing on impacted sites as compared to sites not impacted heavily by mercury (Table 1). *Z. marina* plants growing in a site impacted by heavy metals associated with industrial pollution accumulated significantly higher amounts of iron, aluminium, nickel, and copper in above-ground tissues as compared to a nonimpacted site, and higher amounts of barium, iron, aluminium, zinc, nickel, manganese, copper, cadmium, arsenic, chromium, and lead in below-ground tissues at the impacted site (Table 2). For *P. oceanica,* total phenolics were significantly higher in leaves at the control site when compared to the mercury impacted site for the January sampling period (Fig. 1). For sheath tissue total phenolics from the control site were significantly higher when compared to the mercury impacted site for both sampling periods (Fig. 1). For *Z. marina*, total phenolic content was higher in both sampling periods at the non-impacted site compared to the control site, but only significantly so for the July 2000 sampling period (Fig. 2). Total volatile content also was higher at the control site for both sampling periods, but only significantly higher for the April sampling period (Fig. 3). These results support the hypotheses that *P. oceanica* and *Z. marina* accumulate significant amounts of heavy metals from impacted sites, and that these accumulations are associated with reduced total phenolic and volatile compound content. Based on these supportive data, we conclude that *P. oceanica* and *Z. marina* are potential candidates as bio-surveillance organisms especially with regard to heavy metal pollution of coastal and estuarine ecosystems.

Since we observed variation in the production of phenolics and volatiles with regard to sampling time and season, a priority is the identification of individual phenolic and volatile compounds in the tissue of these two species. In our labs we have identified in one or both species using gas chromatography/mass spectroscopy and high pressure liquid chromatography several cinnamic acid and benzoic acid derivatives; these results are comparable to those found by Quackenbush et al. (1986). Additionally, these analyses indicate not only a quantitative decrease in total phenolic and volatile compounds, but also qualitative differences between plants growing on impacted and non-impacted sites (Ferrat et al., unpublished data for *P. oceanica*; Zou et al., unpublished data for *Z. marina*). Finally, since various environmental perturbations may adversely affect seagrass health (impact of human activity reviewed in Short & Wyllie-Echeverria, 1996), and thereby phenolic and volatile compound production, collaboration among scientists working at a diversity of sites would greatly facilitate progress toward this bio-surveillance effort.

#### **6. Acknowledgments**

We thank Tina, Victoria, Rebecca, and Tessa Wyllie-Echeverria and Carl Young for assistance with sample collection in Washington State. RGC and JZ thank the Department of Botany for the professional development funds supporting this research, and RGC is grateful for additional funding from the Karl G. Maeser Research Award. SWE gratefully acknowledges support from the Russell Family Foundation during the writing portion of this investigation.

#### **7. References**

control site, but only significantly so for the July 2000 sampling period (Fig. 2). Total volatile content also was higher at the control site for both sampling periods, but only significantly higher for the April sampling period (Fig. 3). These results support the hypotheses that *P. oceanica* and *Z. marina* accumulate significant amounts of heavy metals from impacted sites, and that these accumulations are associated with reduced total phenolic and volatile compound content. Based on these supportive data, we conclude that *P. oceanica* and *Z. marina* are potential candidates as bio-surveillance organisms especially with regard to

Since we observed variation in the production of phenolics and volatiles with regard to sampling time and season, a priority is the identification of individual phenolic and volatile compounds in the tissue of these two species. In our labs we have identified in one or both species using gas chromatography/mass spectroscopy and high pressure liquid chromatography several cinnamic acid and benzoic acid derivatives; these results are comparable to those found by Quackenbush et al. (1986). Additionally, these analyses indicate not only a quantitative decrease in total phenolic and volatile compounds, but also qualitative differences between plants growing on impacted and non-impacted sites (Ferrat et al., unpublished data for *P. oceanica*; Zou et al., unpublished data for *Z. marina*). Finally, since various environmental perturbations may adversely affect seagrass health (impact of human activity reviewed in Short & Wyllie-Echeverria, 1996), and thereby phenolic and volatile compound production, collaboration among scientists working at a diversity of sites

We thank Tina, Victoria, Rebecca, and Tessa Wyllie-Echeverria and Carl Young for assistance with sample collection in Washington State. RGC and JZ thank the Department of Botany for the professional development funds supporting this research, and RGC is grateful for additional funding from the Karl G. Maeser Research Award. SWE gratefully acknowledges support from the Russell Family Foundation during the writing portion of

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### **Biofilms Impact on Drinking Water Quality**

Anca Farkas1, Dorin Ciatarâş1 and Brânduşa Bocoş2

*1Someş Water Company, 2National Public Health Institute, Regional Public Health Center of Cluj, Cluj-Napoca, Romania*

#### **1. Introduction**

140 Ecological Water Quality – Water Treatment and Reuse

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*Zostera marina* (eelgrass) in the San Juan Archipelago, Washington, USA. *Estuaries*

A paradigm shift currently occurs in microbiology, with significant impacts in a variety of environmental, medical and industrial applications. The old misconception of free floating microbes is invalidated by a different knowledge pattern: the great majority of terrestrial microorganisms live in communities associated to surfaces, called biofilms (Costerton et al, 1987; Flemming, 2008; Muntean, 2009). This organisation mode is associated to all surfaces in contact with water in drinking water processing, storage and distribution. Such biofilms are represented by structured consortia of sessile microorganisms characterized by surface attachment, self-produced exopolymeric matrix, structural, functional and metabolic heterogeneity, capable of intercellular communication by quorum-sensing and plurispecific composition.

Biofouling in drinking and industrial water systems has detrimental effects such as microbiological and chemical deterioration in water quality, corrosion inducing, drinking water treatment yield loss, efficiency reducing in cooling and heating exchange and transport, as well as in membrane processes (White et al, 1999; Flemming et al, 2002; LeChevallier and Au, 2004; Coetser and Cloete, 2005).

Biofilms are playing a major role in drinking and waste water treatment processes due to their enhanced properties of mineralization, bioaccumulation and bioadsorbtion. Despite the beneficial effects of the biological filter known as *schmutzdecke* in slow sand filtration or of the bio-sand filters, biofilms occurrence in other treatment stages, in drinking water networks and reservoirs represents a continuous challenge to water professionals. Drinking water associated biofilms induce residual disinfectants depletion and may cause aesthetic problems consisting in colour, odour and taste degradation due to chemical compounds released and more important, they pose a threat to human and animal health by hosting pathogenic or toxins producing bacteria, viruses, protozoa, algae, fungi and invertebrates. The great majority of water related health problems are the result of microbial contamination (Riley et al, 2011). Considering these aspects, naturally occurring biofilms in contact with drinking water were identified and described as microbial reservoirs for further contamination (Szewzyk et al, 2000; Wingender and Flemming, 2011).

The complex structure of drinking water associated biofilms is influenced by the microbial composition of source water and sediments (LeChevallier et al, 1987; Szewzyk et al, 2000; Emtiazi et al, 2004). They may enter the distribution network, escaping the treatment and disinfection processes (known as breakthrough) and multiply in bulk water or biofilms. The two modes of multiplication are defined as regrowth (recovery of disinfectant injured cells) and aftergrowth (microbial growth in a distribution system) processes (Characklis, 1988; van der Kooij, 2003). Pathogenic microorganisms of concern may also emerge in drinking water systems by intrusion, due to external contamination events in different steps of water treatment, storage and transportation: cross connections, backflow events, pipe breaks, negative pressure and because of improper flushing and disinfection procedures.

#### **2. Drinking water biofilms, emerging pathogens and opportunistic pathogens**

The most alarming consequences as a result of biofouling in drinking water distribution systems consist in the presence, multiplication and dispersion into water of bacterial pathogens, opportunistic pathogens, parasitic protozoa, viruses and toxins releasing fungi and algae. They may appear as primary colonizers promoting the adhesion at the interface and subsequent biofilm formation (Costerton, 1994), but more often as secondary colonizers in ecological microniches offered by the existent attached community.

Emerging pathogens are those that have appeared in a human population for the first time, or have occurred previously but are increasing in incidence or expanding to areas where they have not previously been reported, usually over the last 20 years. They include: bacteria (pathogenic *E. coli*, *Helicobacter pylori*, *Campylobacter jejuni*, *Mycobacterium* avium complex), parasitic protozoa (*Cryptosporidium* spp., *Cyclospora cayetanensis*, *Toxoplasma gonidii*), viruses (noroviruses, hepatitis E) and toxic cyanobacteria (Hunter et al, 2003). Opportunistic pathogens are commonly members of water microbiota that would be normally harmless to a healthy individual but can infect a compromised host (US EPA, 2002).

Such microorganisms were detected worldwide in drinking water and associated biofilms and in raw water and sediments. In this context, establishing health-based targets, drinking water quality assurance implies effective preventive measures, such as sources water protection in order to reduce contamination risk in these strategic environments, as well as corrective actions stipulated in water safety plans prepared by water suppliers.

When cases of illness are registered, epidemiological studies are conducted in order to demonstrate similarities in genetic profiles of strains isolated from clinical and the environmental specimens, to track the source of infection. Drinking water and associated biofilms are often among the prime candidates tested when gastrointestinal diseases and different types of infections are recorded.

Developing countries are facing a major lack of safe drinking water, the transmission via faecal-oral route causing enormous numbers of severe water related illness and cases of deaths, especially in infants (Riley et al, 2011). Microbial source tracking approach is particularly important for drinking water sources, in order to identify the origin of contamination. Increasing population's access to clean drinking water and sanitation facilities is one of the first priorities of local and global authorities.

Even if the access to high quality drinking water is provided, neither humans in rich countries have definitely won the fight against microbes, yet. Tap water quality assurance is facing new challenges, consisting mainly in biofouling issues, emergent waterborne

Emtiazi et al, 2004). They may enter the distribution network, escaping the treatment and disinfection processes (known as breakthrough) and multiply in bulk water or biofilms. The two modes of multiplication are defined as regrowth (recovery of disinfectant injured cells) and aftergrowth (microbial growth in a distribution system) processes (Characklis, 1988; van der Kooij, 2003). Pathogenic microorganisms of concern may also emerge in drinking water systems by intrusion, due to external contamination events in different steps of water treatment, storage and transportation: cross connections, backflow events, pipe breaks,

**2. Drinking water biofilms, emerging pathogens and opportunistic pathogens**  The most alarming consequences as a result of biofouling in drinking water distribution systems consist in the presence, multiplication and dispersion into water of bacterial pathogens, opportunistic pathogens, parasitic protozoa, viruses and toxins releasing fungi and algae. They may appear as primary colonizers promoting the adhesion at the interface and subsequent biofilm formation (Costerton, 1994), but more often as secondary colonizers

Emerging pathogens are those that have appeared in a human population for the first time, or have occurred previously but are increasing in incidence or expanding to areas where they have not previously been reported, usually over the last 20 years. They include: bacteria (pathogenic *E. coli*, *Helicobacter pylori*, *Campylobacter jejuni*, *Mycobacterium* avium complex), parasitic protozoa (*Cryptosporidium* spp., *Cyclospora cayetanensis*, *Toxoplasma gonidii*), viruses (noroviruses, hepatitis E) and toxic cyanobacteria (Hunter et al, 2003). Opportunistic pathogens are commonly members of water microbiota that would be normally harmless to a

Such microorganisms were detected worldwide in drinking water and associated biofilms and in raw water and sediments. In this context, establishing health-based targets, drinking water quality assurance implies effective preventive measures, such as sources water protection in order to reduce contamination risk in these strategic environments, as well as

When cases of illness are registered, epidemiological studies are conducted in order to demonstrate similarities in genetic profiles of strains isolated from clinical and the environmental specimens, to track the source of infection. Drinking water and associated biofilms are often among the prime candidates tested when gastrointestinal diseases and

Developing countries are facing a major lack of safe drinking water, the transmission via faecal-oral route causing enormous numbers of severe water related illness and cases of deaths, especially in infants (Riley et al, 2011). Microbial source tracking approach is particularly important for drinking water sources, in order to identify the origin of contamination. Increasing population's access to clean drinking water and sanitation

Even if the access to high quality drinking water is provided, neither humans in rich countries have definitely won the fight against microbes, yet. Tap water quality assurance is facing new challenges, consisting mainly in biofouling issues, emergent waterborne

negative pressure and because of improper flushing and disinfection procedures.

in ecological microniches offered by the existent attached community.

healthy individual but can infect a compromised host (US EPA, 2002).

facilities is one of the first priorities of local and global authorities.

different types of infections are recorded.

corrective actions stipulated in water safety plans prepared by water suppliers.

pathogens, toxins releasing and opportunistic pathogens occurrence. Besides households, different branches of industry, especially food and pharmaceutical, health care facilities, schools, nursing homes and other critical areas are carefully included in monitoring assays. Recently is seriously considered the ability of some species, referred as opportunistic pathogens, to induce disease under certain circumstances in immunocompromised individuals: immunosuppressed, malnourished, diabetic, burn, cancer, AIDS, on haemodialysis, respiratory, with organ transplants patients (Rusin et al, 1997; Payment and Robertson, 2004). Sensitive subpopulations such as young children, elderly persons or pregnant women are also vulnerable to infections caused by opportunistic pathogens (Reynolds et al, 2008). Other special categories of exposed subjects consist in patients with indwelling cannulae and catheters, implant devices and contact lenses wearers. Opportunistic pathogens are becoming a major issue, causing from allergy or superficial infections to life-threatening systemic infections, since the ascending trend in congenital and acquired immunodeficiency affecting global population. Such species presence is often investigated, in addition to routine monitoring, within drinking water and associated biofilms, as wide-occurring bacteria of concern in the continuous increasing category of hospitalised and ambulatory immunocompromised persons (Glasmacher et al, 2003).

Even in drinking water carefully treated and distributed at high standards, pathogenic contamination and disease outbreaks may occur (Szewzyk et al, 2000; Wingender & Flemming, 2011) demonstrating the imperative requirement for comprehensive water safety plans implementation.

#### **2.1 Drinking water quality assessment – Microbiological aspects and biofilms**

Tap water supposes not to be and is not sterile, microbial load in bulk water consisting mainly in inoffensive heterotrophs, presumably coming from associated biofilms by detachment during dispersion. Routine monitoring of raw water sources, finishing water at the exit from treatment plant, drinking water in pipe networks, service reservoirs and finally at the consumers implies periodically investigations of a number of water samples collected with a frequency depending on the population deserved. According to European regulations, microbial indicators assessed by standardised conventional culturing techniques are: colony count at 37⁰C, colony count at 22⁰C, total coliforms, *Escherichia coli*, intestinal enterococci and *Clostridium perfringens*. The greatest microbial risk being associated with ingestion of water contaminated with human or animal faeces, thus the potential presence of pathogenic bacteria, viruses and cysts of protozoan parasites; faecal indices (*E. coli*, intestinal enterococci and *C. perfringens*) presence is routinely investigated.

The shortcomings of water quality monitoring based on faecal indicators and heterotrophic plate count, resulting in underestimation of drinking water microbial populations in numbers and composition are discussed worldwide considering the following:

are indicating bacterial numbers characterizing the biomass in pipe biofilms being 25 times more abundant than the suspended cells (Servais et al, 2004). Although, the common notion that biofilms dominate the distribution systems has been proven to be not true under all conditions by Srinivasan (2008), whose findings suggest that bulk bacteria may dominate in network sections containing chlorine residuals lower than 0.1mg/L and having residence time longer than 12 hours.

Many studies targeting attached microbial communities have been performed for quantification of the total number of germs, by different methods. They offer an unspecific overview upon microbial load, bringing certain information about drinking water treatment process efficiency and distribution system integrity. Still, the real composition and dynamics of microbial populations within drinking water associated biofilms represents a continuous challenge. Experimental biofilm succession monitored for a long term development indicated a stable population state after 500 days in a model drinking water distribution system. A homogenous composition of the population in the mature biofilm could mask a dynamic situation at a smaller scale (Martiny et al., 2003). Quantitative and prescriptive evaluation is the next target of scientific community. Prediction of microorganisms' behaviour in the distribution system water and biofilms requires greater understanding of the effects in microbial attachment, detachment, survival, multiplication and viability of three groups of abiotic and biotic factors: substratum physicochemical properties (type of materials), biofilm composition (microbial intra- and interspecific interactions) and bulk water characteristics (disinfectants residuals, oxygen and nutrients concentrations, system hydraulics, temperature).

• A significant percent of water and biofilm bioburden may be in a viable but non cultivable state, unable to grow on artificial growth media but alive and capable of renewed activity and so hygienically relevant (Moritz et al, 2010). A small fraction of waterborne microorganisms (0.01%) are estimated to be culturable heterotrophic

• Limitations of detection methods (Lehtola et al, 2006; 2007; September et al, 2007). The investigation of drinking water associated biofilms from four European countries (France, Great Britain, Portugal and Latvia) confirmed *E. coli* presence by culturing techniques in one out of five pipes whereas all networks except one were positive for *E. coli* using the PNA FISH methods; their viability was also demonstrated. *E.coli* contributed with percents from 0.001% to 0.1% in the total bacterial numbers (Juhna et

• Faecal indicators are the best predictors of potential risks, but their concentrations rarely correlate perfectly with those of pathogens (Payment and Locas, 2011). Although in freshwater significant correlations have been established between faecal indices and pathogenic species, their presence in drinking water showed limited or no correlation with different species of pathogenic or opportunistic pathogenic bacteria, viruses, protozoa and fungi. Water quality assessment based only on the investigation of faecal indicators' presence proved to be insufficient when many waterborne outbreaks emerged. Still, until more reliable indices and methods of detection will be wider implemented, the well- known standardised procedures are applied in routine

Many studies targeting attached microbial communities have been performed for quantification of the total number of germs, by different methods. They offer an unspecific overview upon microbial load, bringing certain information about drinking water treatment process efficiency and distribution system integrity. Still, the real composition and dynamics of microbial populations within drinking water associated biofilms represents a continuous challenge. Experimental biofilm succession monitored for a long term development indicated a stable population state after 500 days in a model drinking water distribution system. A homogenous composition of the population in the mature biofilm could mask a dynamic situation at a smaller scale (Martiny et al., 2003). Quantitative and prescriptive evaluation is the next target of scientific community. Prediction of microorganisms' behaviour in the distribution system water and biofilms requires greater understanding of the effects in microbial attachment, detachment, survival, multiplication and viability of three groups of abiotic and biotic factors: substratum physicochemical properties (type of materials), biofilm composition (microbial intra- and interspecific interactions) and bulk water characteristics (disinfectants residuals, oxygen and nutrients concentrations, system

0.1mg/L and having residence time longer than 12 hours.

bacteria (Watkins and Jian, 1997; Exner et al, 2003);

al., 2007);

monitoring across the globe.

hydraulics, temperature).

are indicating bacterial numbers characterizing the biomass in pipe biofilms being 25 times more abundant than the suspended cells (Servais et al, 2004). Although, the common notion that biofilms dominate the distribution systems has been proven to be not true under all conditions by Srinivasan (2008), whose findings suggest that bulk bacteria may dominate in network sections containing chlorine residuals lower than

#### **2.1.1 Occurrence of bacteria in drinking water and associated biofilms**

Among the nuisance bacteria regularly found in drinking water and biofilms, species that are not characteristic to the water environment may appear due to contamination events, with major impacts upon human health. Enteric bacteria such as *Escherichia coli*, *Klebsiella pneumoniae*, *K. oxytoca*, *Enterobacter cloacae*, *E. aglomerans*, *Helicobacter pylori, Campylobacter* spp., *Shigella* spp., *Salmonella* spp., *Clostridium perfringens*, *Enterococcus faecalis*, *E. faecium*, as well as environmental bacteria becoming opportunistic pathogens *Legionella pneumophila*, *Pseudomonas aeruginosa*, *P. fluorescens*, *Aeromonas hydrophila*, *A. caviae*, *Mycobacterium avium*, *M. xenopi*, together with other waterborne agents have been indicated to live in ecological microniches offered by drinking water associated biofilms (table 1).

When compared with planktonic counterparts, biofilm bacteria and other inhabitants display superior characteristics due to specialization within this emergent structure and to complex relationships established (Costerton, 1994). Community belonging, from a microbial perspective represents a benefit materialized in increasing the chances of survival in this oligotrophic environment by offering ecological microniches, establishing intra- and interspecific cooperation relationships by communication via quorum sensing and perpetuating individuals' resistance to disinfection agents. Even species not able to survive and most of them incapable of growth and multiplication in water were identified in associated biofilms; recent studies have demonstrated their ability to grow in those microniches. For example, *Legionella pneumophila* survives but does not multiply in sterile drinking water, its proliferation being dependent on parasitic relationship with other microorganisms: 14 species of amoebae, two species of ciliated protozoa, and one slime mould - *L. pneumophila* being described as protozoonotic bacteria (Murga et al, 2001; Fields et al, 2002; Declerck, 2010). In many outbreaks, the presence of pathogenic bacteria was not detected by routine monitoring, the correlation with faecal indicators found in tap water samples being defective. *E. coli* bacillus, the most popular faecal indicator, was chosen inter alia based on its incapacity of growth in water. Recent studies have shown its ability to multiplicate in drinking water associated biofilms under strictly anaerobic conditions (Latimer et al., 2010), so the indicative value of the faecal index of choice becomes questionable.

One of the advantages offered by drinking water biofilm organization to its members is represented by the enhanced resistance to disinfection residuals. The four hypothetical mechanisms of biofilm resistance involve slow antimicrobial penetration, deployment of adaptative stress responses, physiological heterogeneity in biofilm population and the presence of phenotypic variants or persister cells (Chambless et al, 2005). Another benefit from the microbial perspective consists in the emergence of genetically encoded resistance to biocides and antibiotics, and the spread of antimicrobial resistance genes in bacterial populations via mobile genetic elements, by lateral gene transfer. Integrons are genetic elements possessing a site-specific recombination system for assembling of resistance genes in gene cassettes. They play a major role in the rapid spread of antibiotic resistance in clinical environments. Gene cassettes encoding resistance to quaternary ammonium compounds (*qac*) and integron-integrase (*intlI*) genes characteristics for class 1 integron were recently recovered from environmental samples, including biofilms from a groundwater treatment plant (Gillings et al, 2009). The proximity of individuals in biofilm consortia and the extremely short generation times in bacteria multiplication are prerequisites for intensive rates of lateral gene transfer and thus resistance spreading and perpetuation in diverse natural or artificial ecosystems.

Table 1. Pathogenic and opportunistic pathogenic bacteria detected in association with drinking water; WDS – water distribution systems, DWTP - drinking water treatment plant.

USA Germany France, England, Portugal, Latvia South Africa Romania

> Korea South Africa Portugal Romania

> > Portugal Greece Romania

USA Korea South Africa

Germany Germany Korea South Africa Portugal Romania

Scotland USA Australia Korea South Africa Brasil Romania

Korea South Africa Bangladesh Sudan

Germany South Africa Greece USA

> Germany Korea

Finland France

England Portugal

Germany The Netherlands Portugal USA

Table 1. Pathogenic and opportunistic pathogenic bacteria detected in association with drinking water; WDS – water distribution systems, DWTP - drinking water treatment plant.

LeChevallier et al, 1987 Schmeisser et al, 2003 Juhna et al, 2007

September et al, 2007 Farkas et al, 2011

Lee et al, 2006 September et al, 2007 Menaia et al, 2008 Farkas et al, 2011

Menaia et al, 2008 Kormas et al, 2010 Farkas et al, 2011

LeChevallier et al, 1987 Lee et al, 2006 September et al, 2007

Schmeisser et al, 2003 Emtiazi et al, 2004 Lee et al, 2006 September et al, 2007 Menaia et al, 2008 Farkas et al, 2011

Gavriel et al, 1998 Chauret et al, 2001 Bomo et al, 2004 Lee et al, 2006 September et al, 2007 Razzolini et al, 2008 Farkas et al, 2011

Lee et al, 2006 September et al, 2004; 2007 Alam et al, 2007 Shanan et al, 2011

Schmeisser et al, 2003 September et al, 2004 Kormas et al, 2010 Marciano-Cabral et al, 2010

Schmeisser et al, 2003 Lee et al, 2006

Hänninen et al, 2002 Gallay et al, 2006

Watson et al, 2004 Bragança et al, 2005

Emtiazi et al, 2004 Diederen et al, 2007 Menaia et al, 2008 Marciano-Cabral et al, 2010

**Bacteria Samples type/Origin Country References**

Biofilm - WDS Biofilm - WDS Biofilm – WDS

Biofilm, Water - WDS Biofilm - DWTP

> Water - WDS Water - WDS Biofilm - WDS Biofilm - DWTP

Biofilm - WDS Water - WDS Biofilm - DTP

Biofilm - WDS Water - WDS Biofilm, Water - WDS

Biofilm - WDS Biofilm - WDS Water - WDS Biofilm, Water - WDS Biofilm - WDS Biofilm - DWTP

Water - WDS Biofilm, Water - WDS Biofilm - WDS Water - WDS Biofilm, Water - WDS Water - WDS Biofilm - DWTP

Water - WDS Biofilm, Water - WDS Biofilm - pond Water - reservoirs

> Biofilm - WDS Biofilm - WDS Water - WDS Water - WDS

> Biofilm - WDS Water - WDS

Water - WDS Raw water

Biofilm - WDS

Biofilm - WDS Water - WDS Biofilm - WDS Water - WDS

*Escherichia coli*

Faecal enterococci

*Clostridium* spp.

*Klebsiella* spp.

*Pseudomonas* spp.

*Aeromonas* spp.

*Vibrio* spp. *V. cholerae*

*Mycobacterium* spp.

*Shigella* spp., *Salmonella* spp.

*Campylobacter* spp.

*Legionella pneumophila*

*Helicobacter pylori* Biofilm - WDS

Experimental studies emphasized on bacteria ability of colonization, survival and multiplication in water associated biofilms, followed by dispersion in water phase in a planktonic state. The findings of Banning et al. (2003) suggested that the ability of *P. aeruginosa* to survive longer than *E. coli* in water associated biofilm could not be attributed to the association with the biofilm, rather than to the ability to utilize a wider range of organic molecules as carbon and energy sources compared to other *Enterobacteriaceae*. An increment in available nutrients may reduce *E. coli* survival in enhanced competition for nutrients and increased antagonism by the indigenous microbial population.

Lehtola et al. (2007) investigated the survival of faecal indices versus pathogenic bacteria and viruses, in drinking water biofilms experimentally infested with *E. coli*, *L. pneumophila*, *Mycobacterium avium* and canine calcivirus (as a surrogate for human norovirus). The results proved that pathogenic bacteria and virus particles entering water distribution systems can survive in biofilms for weeks, even in conditions of high-shear turbulent flow and may pose a risk to the consumers. Meanwhile, *E. coli* registered a limited survival to a few days in water and in biofilms, being a poor indicator of certain pathogens in biofilms. The study also showed that standard culture methods may seriously underestimate the real numbers of bacteria in water and biofilms.

Comparative evaluation of classical techniques involving bacterial growth on specific selective media and molecular methods based on 16s rDNA sequence identity reveals a high discrepancy between what was expected to grow and the species isolated from specific selective growth media. Bacterial analyses of water based on selective isolation and culturing approach is recommended to be interpreted with caution (September et al, 2007).

Experimental studies revealed also low detectable numbers by culture-based technique in case of potable water biofilms infected with *Campylobacter jejuni* (Lehtola et al, 2006). *C. jejuni* and *C. coli* waterborne epidemics registered in Finland (Hänninen et al, 2000) and France (Gallay et al, 2000) were associated to consumption of contaminated tap water with origin in polluted sources.

Severe outbreaks such as cholera caused by the ingestion of water contaminated with *Vibrio cholerae*, typhoid and paratyphoid enteric fevers caused by *Salmonella enterica* subsp. *enterica* serovar *Typhi*, respective serovar *Paratyphi*, shigellosis due to infections with *Shigella* species still occur in countries with insufficient access to safe water. But even in developed countries, outbreak events involving emerging pathogenic bacteria like *Legionella pneumophila*, waterborne E.coli O157:H7 and foodborne *E.coli* O104:H4 demonstrate the microbes' versatility and the fragility of humanity's victory over the nature.

#### **2.1.2 Occurrence of protozoa in drinking water and associated biofilms**

The food web in drinking water microbial consortia is based on heterotrophic bacteria, the next trophic level being represented by protozoa. Species of parasitic protozoa, including free living amoebae associated with infections in humans have been isolated from source waters and drinking water (table 2). Their presence represents a double threat to human health, being also related to amoeba-resisting bacteria, such as *Legionella* spp. and *Mycobacterium* spp., which proliferate in protozoa thus increasing the probability of causing diseases in humans (Marciano-Cabral et al, 2010).

Some protozoa, for example *Giardia* spp. and *Cryptosporidium* spp. may persist to hostile environment in drinking water, resist to different disinfection procedures and accumulate in biofilms under the form of cysts, respective oocysts. Experimental introducing *Cryptosporidium* oocysts for the prediction of behaviour in drinking water distribution system showed surface attachment and subsequent intermittent detachment, with exposure to high doses of chlorine (20mg/L) needed for the removal of substantial numbers of oocysts attached to pipe walls (Warneke et al, 2006). The study of Helmi et al. (2008) investigating the interaction of *Giardia lamblia* and *Cryptosporidium parvum* (oo)cysts in drinking water biofilms, revealed that protozoa are able to attach in biofilm matrix from the first day and survive extended periods of time, longer for *Cryptosporidium*. Viable (oo)cysts were recovered from biofilm and water phase for the whole period of investigation, of 34 days, turbulent shear stress influencing the detachment.

Table 2. Protozoa detected in raw water sources, water treatment plants (DWTP) and drinking water networks (WDS) and associated biofilms.

#### **2.1.3 Occurrence of viruses in drinking water and associated biofilms**

Sources of drinking water were investigated for the presence of enteric viruses, especially when gastrointestinal outbreaks occurred, and the results revealed episodes of faecal contamination in raw water. Epidemiological studies conducted supported the association between drinking water consumption and illness (table 3).

Table 3. Viruses identified in raw water sources and water distribution networks (WDS).

Some protozoa, for example *Giardia* spp. and *Cryptosporidium* spp. may persist to hostile environment in drinking water, resist to different disinfection procedures and accumulate in biofilms under the form of cysts, respective oocysts. Experimental introducing *Cryptosporidium* oocysts for the prediction of behaviour in drinking water distribution system showed surface attachment and subsequent intermittent detachment, with exposure to high doses of chlorine (20mg/L) needed for the removal of substantial numbers of oocysts attached to pipe walls (Warneke et al, 2006). The study of Helmi et al. (2008) investigating the interaction of *Giardia lamblia* and *Cryptosporidium parvum* (oo)cysts in drinking water biofilms, revealed that protozoa are able to attach in biofilm matrix from the first day and survive extended periods of time, longer for *Cryptosporidium*. Viable (oo)cysts were recovered from biofilm and water phase for the whole period of investigation, of 34

**Protozoa Samples type/Origin Country References**

Table 2. Protozoa detected in raw water sources, water treatment plants (DWTP) and

Sources of drinking water were investigated for the presence of enteric viruses, especially when gastrointestinal outbreaks occurred, and the results revealed episodes of faecal contamination in raw water. Epidemiological studies conducted supported the association

Coxsakie A viruses Raw water Taiwan Hsu et al, 2009

Table 3. Viruses identified in raw water sources and water distribution networks (WDS).

**2.1.3 Occurrence of viruses in drinking water and associated biofilms**

Well water Water - WDS

Well water Spring water Groundwater Groundwater

Drinking water sources

USA Canada Australia Spain

USA Canada Spain

USA USA USA Sudan

**type/Origin Country References**

USA India

USA Finland New Zeeland Korea

LeChevallier et al, 1991 Chung et al, 1998 Hellard et al, 2001 Carmena et al, 2007

LeChevallier et al, 1991 Chung et al, 1998 Carmena et al, 2007

Blair et al, 2008 Marciano-Cabral et al, 2010 Shoff et al, 2010 Shanan et al, 2011

> Bloch et al, 1990 Hazam et al, 2010

Parshionikar et al, 2003 Maunula et al, 2005 Hewitt et all, 2007 Koh et al, 2011

West Africa Verheyen et al, 2009

days, turbulent shear stress influencing the detachment.

Filtered water - DWTP Water - WDS Water - WDS Water - WDS

Filtered water - DWTP Water - WDS Water - WDS

Well water Biofilm, Water - WDS Biofilm, Water - WDS Water reservoir

drinking water networks (WDS) and associated biofilms.

between drinking water consumption and illness (table 3).

**Viruses Samples**

Flagellates: *Giardia lamblia*

Apicomplexa (Sporozoans):

Amoebae: *Naegleria fowleri Acanthamoeba* spp. *Hartmannella* spp. *Vahlkampfia* spp.

*Cryptosporidium parvum*

Hepatitis A virus Hepatitis E virus

Noroviruses

Adenoviruses Rotaviruses

The presence of enteric viruses associated with inadequate water supplies, poor sanitation and hygiene is mostly affecting developing countries (Ashbolt et al, 2004). Episodes of gastroenteritis caused especially by noroviruses attributed to contaminated drinking water have been reported also in developed countries. Inefficient raw water treatment and secondary contamination of distribution systems with sewage are of high concern, enteric viruses being generally more resistant than enteric bacteria to widely used free chlorine, chlorine dioxide and monochloramine disinfectants (LeChevallier & Au, 2004). Although no complete investigations regarding faecal indicators presence were performed in the considered studies (especially for intestinal enterococci as index of viruses), coliforms and *E. coli* have been detected in water samples in many cases of gastroenteritis outbreaks investigated (Parshionikar et al, 2003; Hewitt et al, 2007; Koh et al, 2011).

There is no evidence of viruses ability of multiplication in environmental biofilms, but they may survive for extended periods of time trapped in the matrix, similarly to protozoan (oo)cysts, and be detached in water column, where remain inactive until they find a host. Experimental studies using pilot scale systems demonstrated the ability of viruses to attach and accumulate into drinking water biofilms within one hour after inoculation, while their detachment in water phase is influenced by flow velocity (Lehtola et al, 2007; Helmi et al, 2008). The viral genomes were detected in biofilms over the whole period of both experiments (for 21, respectively 34 days). Helmi and co-workers, investigating the poliovirus infectivity, recovered the infectious viruses only for 6 days, when flow velocity increment from laminar to turbulent regimen was applied, concluding that detection of viral genome in biofilms is not sufficient to assess a risk associated with the presence of infectious particles.

#### **2.1.4 Occurrence of fungi in drinking water and associated biofilms**

Initially considered to be airborne, fungal infections in immunocompromised patients hospitalized in controlled atmospheric conditions raised the hypothesis of waterborne origin of aspergillosis (Anaissie & Costa, 2001). Opportunistic pathogens, potentially causing superficial or systemic infections, allergenic or toxigenic species of fungi (yeasts and moulds) have been isolated from drinking water worldwide, their presence being primary attributed to the ability of surfaces colonization as biofilms (table 4).

Table 4. Fungi identified in drinking water distribution systems (WDS) and associated biofilms.

In some studies, the correlation with standard hygiene indicators was not found (Göttlich, 2002), other authors described negative correlations between bacteria and filamentous fungi, which may be explained either by competition for nutrients either by inhibiting toxins produced (Gonçalves et al, 2006) while in other investigations positive significant correlations were found between the presence of filamentous fungi, yeasts and bacteria in drinking water (Sammon et al, 2010). Regarding filamentous fungi behaviour in water distribution systems, deposition is attributed to highly resistant spores, while mycotoxins, taste and odour changing compounds producing implies germination and hyphal growth in biofilms. The occurrence of fungi in drinking water systems may have significant impact due to health effects of mycotoxins (such as aflatoxins): mutagenic, teratogenic, oestrogenic, carcinogenic and allergenic, although no reports of disease attributed to mycotoxins produced in the water distribution systems have been reported (Sonigo et al, 2011).

#### **2.1.5 Occurrence of algae in drinking water and associated biofilms**

Algae are assumed not to be characteristic to water distribution system biofilms due to the absence of light (Wingender and Flemming, 2011), but algal biomass is a major component of biofilms in surface source waters, water treatment and storage, in areas exposed to air and light. Experimental research designed by Chrisostomou et al (2009) emphasized on airdispersed phytoplankton diversity and colonization potential of algal taxa in drinking water reservoir systems. Algal communities are associated to biofilms and may support bacterial growth, for example *Legionella* species (Declerck, 2010). Few recent studies investigating the presence of algae in drinking water are available (table 5).

Table 5. Algae identified in drinking water distribution systems (WDS).

Algal toxins, of which the most dangerous for humans is cyanobacterial microcystin, are considered chemical hazards in drinking water, especially when open-air reservoirs are used in water storage (Lymperopoulou et al, 2011). Algal growth and eutrophication in surface waters are widely investigated, with respect to ecological effects. In drinking water sources and throughout the water treatment process, distribution and storage, algal blooms raise issues about toxins releasing and aesthetic problems inducing, such as colour and smell. Algal removal in drinking water treatment is recommended to be carefully performed, in order not to disrupt the cells and release toxins in drinking water (LeChevallier and Au, 2004). Epidemiological studies are conducted worldwide in order to demonstrate the evidence of algal toxins in the environment and to evaluate their relatedness to illness in humans. Possible linkages between algae toxins in drinking water and health effects, including liver problems and diarrhoea in children were indicated by a survey in Namibia, although microcystin never exceeded the tolerable daily intake (Gunnarsson and Sanseovic, 2001).

In some studies, the correlation with standard hygiene indicators was not found (Göttlich, 2002), other authors described negative correlations between bacteria and filamentous fungi, which may be explained either by competition for nutrients either by inhibiting toxins produced (Gonçalves et al, 2006) while in other investigations positive significant correlations were found between the presence of filamentous fungi, yeasts and bacteria in drinking water (Sammon et al, 2010). Regarding filamentous fungi behaviour in water distribution systems, deposition is attributed to highly resistant spores, while mycotoxins, taste and odour changing compounds producing implies germination and hyphal growth in biofilms. The occurrence of fungi in drinking water systems may have significant impact due to health effects of mycotoxins (such as aflatoxins): mutagenic, teratogenic, oestrogenic, carcinogenic and allergenic, although no reports of disease attributed to mycotoxins

produced in the water distribution systems have been reported (Sonigo et al, 2011).

Algae are assumed not to be characteristic to water distribution system biofilms due to the absence of light (Wingender and Flemming, 2011), but algal biomass is a major component of biofilms in surface source waters, water treatment and storage, in areas exposed to air and light. Experimental research designed by Chrisostomou et al (2009) emphasized on airdispersed phytoplankton diversity and colonization potential of algal taxa in drinking water reservoir systems. Algal communities are associated to biofilms and may support bacterial growth, for example *Legionella* species (Declerck, 2010). Few recent studies investigating the

**Algae Samples type/Origin Country References**

Algal toxins, of which the most dangerous for humans is cyanobacterial microcystin, are considered chemical hazards in drinking water, especially when open-air reservoirs are used in water storage (Lymperopoulou et al, 2011). Algal growth and eutrophication in surface waters are widely investigated, with respect to ecological effects. In drinking water sources and throughout the water treatment process, distribution and storage, algal blooms raise issues about toxins releasing and aesthetic problems inducing, such as colour and smell. Algal removal in drinking water treatment is recommended to be carefully performed, in order not to disrupt the cells and release toxins in drinking water (LeChevallier and Au, 2004). Epidemiological studies are conducted worldwide in order to demonstrate the evidence of algal toxins in the environment and to evaluate their relatedness to illness in humans. Possible linkages between algae toxins in drinking water and health effects, including liver problems and diarrhoea in children were indicated by a survey in Namibia, although microcystin never exceeded the tolerable daily intake

Water - WDS Spain Codony et al, 2003

Water - WDS Argentina Ricardo et al, 2006

Water reservoirs Greece Lymperopoulou et al, 2011

**2.1.5 Occurrence of algae in drinking water and associated biofilms**

Table 5. Algae identified in drinking water distribution systems (WDS).

presence of algae in drinking water are available (table 5).

*Oocystis* spp. *Xenococcus* spp.

*Anabaena* spp. *Microcystis* spp.

*Microcystis aeruginosa Chroococcus dispersus*

*Oscillatoria* spp. and many more

(Gunnarsson and Sanseovic, 2001).

#### **3. Drinking water and associated biofilms – Chemical aspects**

Detrimental effects of biofouling in drinking water distribution systems include chemical aspects, involving organic and inorganic compounds produced by the microorganisms inhabiting water phase, biofilms and sediments. Different volatile compounds, organic and inorganic acids, metal oxides and enzymes resulted in microbial metabolism or decay may cause aesthetic problems in water: colour, taste and odours and may also have an impact on the substratum, leading to microbially influenced corrosion.

#### **3.1 Drinking water aesthetic problems**

Aesthetic and organoleptic characteristics of water may be affected by a series of chemical substances, resulting in colour, odour and taste degradation. Such substances originate in microbial activity and decomposition in source waters and in distribution systems, disinfectants used in water treatment, materials used in pipes and joints in water networks. A list of these substances, related to microbial activity and decay that may be produced in the journey of drinking water from drinking water sources to the tap, that may influence consumers perception, is presented in table 6 (after the UK Environment Agency, 2004). These chemical compounds are usually attributed to microbial biofilms associated to drinking water processing and distribution.

Investigating the sources of taste and odour in drinking water in order to find their sources and mitigation strategies, Peter (2008) concluded that low concentrations in chlorine residuals, stagnant water, plastic pipes and particles accumulation in distribution systems may increase the generation of taste and odour compounds by favouring biofilm formation and microbial activity. Other sources of aesthetic problems in water may reside in the activity of bacteria involved in sulphur cycle, producing sulphur odours and yellow discoloration (US EPA, 2002). Oxidation and reduction of soluble metals may produce metal oxides, leading to consumer complaints about the metallic taste and yellow, black or brown staining water (Cerrato et al, 2006).

#### **3.2 Microorganisms – Surface interactions and microbially influenced corrosion**

Biofouling proved to be interdependent on surface characteristics. Investigations of microbial reversible and irreversible attachment in primary or secondary colonization and in drinking water biofilms composition concluded as following:

• The hydrophobic/hydrophilic properties of the substrate are influencing biofilm formation. Exopolysacharides produced by some bacteria facilitate cell adhesion to hydrophilic surfaces, while exopolymers of other bacteria may show a preference for hydrophobic substrata (Beech et al, 2005).

Regarding the influence of the substratum on biofilm composition, copper materials appear to be colonized just by *L. pneumophila* in low numbers, inhibiting *P. aeruginosa* integration, while drinking water biofilms on elastomeric and polyethylene materials proved to be a better support for pseudomonads (Moritz et al, 2010).

• Pipe materials may be corroded, influencing disinfection effectiveness: corrosion products in iron pipes react with free chlorine and lead to residual disinfectants depletion. LeChevallier et al. (1987) detected high concentrations of coliforms only in tubercles formed on iron pipes and suggested few possible explanations: coliform growth stimulated by iron oxides; nutrient syntropy; sourface roughness; protection from disinfection. Iron pipes may be a better support for fungi also, when compared to PVC pipes (Doggett, 2000).

Drinking water flowing through PVC pipes contains three times the aqueous concentration of soluble manganese and 35 times the concentration of total manganese than present in the drinking water transported by iron pipes (Cerrato et al, 2006).

Table 6. Chemical compounds produced by microbial decomposition and metabolism, affecting taste and odour of drinking water.

Microbially influenced corrosion represents another undesirable impact of biofilms associated to drinking water treatment and distribution, involving metallic or non-metallic materials deterioration as a result of pipes inner surface biofouling.

Physiological groups of bacteria classified on account of the ability to use different substrates in their nutrition or in respiration are summarized in table 7 (Drăgan-Bularda & Kiss, 1986; Drăgan-Bularda & Samuel, 2006; Muntean, 2009). Their representative species may belong to microbial communities of source waters and sediments, enter drinking water

Drinking water flowing through PVC pipes contains three times the aqueous concentration of soluble manganese and 35 times the concentration of total manganese than present in the

**Microorganisms Chemical substances produced Aesthetic effects**

Indole, skatole, putrescine, cadaverine, β-phenylethylamine, butyric, propionic and stearic acids

polysulphides

n-hexanal, n-heptanal, isomers of decadienal sulphur compounds, terpenes, aromatic compounds, esters

Geosmin Cadin-4-ene-1-ol 2-isopropyl-3-methoxypyrazine

> Geosmin 2-methylsorboneol

Sulphuric acid, sulphates, sulphur, methyl mercaptan, hydrogen sulphide Metal sulphides (ferrous sulphide)

Microbially influenced corrosion represents another undesirable impact of biofilms associated to drinking water treatment and distribution, involving metallic or non-metallic

Physiological groups of bacteria classified on account of the ability to use different substrates in their nutrition or in respiration are summarized in table 7 (Drăgan-Bularda & Kiss, 1986; Drăgan-Bularda & Samuel, 2006; Muntean, 2009). Their representative species may belong to microbial communities of source waters and sediments, enter drinking water

Table 6. Chemical compounds produced by microbial decomposition and metabolism,

materials deterioration as a result of pipes inner surface biofouling.

Algae decomposition Mercaptan, dimethyl sulphide,

Microbial decomposition

Algae decomposition/activity

Sulphur oxidizing/reducing

Metals oxidizing/ reducing

affecting taste and odour of drinking water.

Sulphate reducing bacteria

*Pseudomonas* spp*. Flavobacterium* spp*. Aeromonas* spp*. Paenicillium caeseicolum*

*Chaetomium globosum Basidiobolus ranarumi Actinomycetes*

Actinomycetes*: Streptomyces* spp*. Nocardia* spp*. Microbiospora* spp Cyanobacteria*: Anabaena* spp*. Microcystis* spp*. Oscillatoria* spp*. Aphanizomenon* spp*.*

Fungi

Algae*:*  Chlorophyceae Bacillariophyceae

bacteria

bacteria

formed on iron pipes and suggested few possible explanations: coliform growth stimulated by iron oxides; nutrient syntropy; sourface roughness; protection from disinfection. Iron pipes may be a better support for fungi also, when compared to PVC pipes (Doggett, 2000).

treatment plants and distribution systems in a planktonic state and adhere to surfaces or become members of established biofilms (Costerton, 1994). Their metabolites have significant impacts on drinking water quality, either being released in bulk water where may react with other compounds, for example with disinfectants, leading to toxic disinfection-by products (as trihalomethanes), or by remaining in biofilm matrix where acting upon pipes surfaces and inducing corrosion.

Table 7. Physiological groups of bacteria, their representatives and metabolites.

Chemical and enzymatic microbial products resulted in biofilms activity may induce corrosion and related effects by different mechanisms:

Some of the recommended strategies in drinking water associated biofilm control are: source waters protection, appropriate treatment, infrastructure contamination prevention, pipes and reservoirs maintenance, corrosion control, appropriate disinfection practices, nutrient levels reducing, water quality monitoring, personnel training, water safety plans implementation.

We are still living in an age of surfaces, even the remark was first said by Oscar Wild's character in 1895. Having in mind the virtual idea of self-cleaning surfaces, researchers in nanotechnology field are targeting innovative repellent materials with a wide range of applications, for the biofouling control. The superhydrophobicity models such as "the lotus effect" characterizing the lotus (*Nelumbo nucifera*) leaf, offered by natural patterns are investigated at nanoscale. The interdependence between surface roughness, reduced particle adhesion and water repellence proved to be the keystone in the self-cleaning mechanism of many biological surfaces (Barthlott & Neinhuis, 1997).

#### **4. Conclusions**

The present review emphasize on the following recent and relevant findings:

#### **5. References**

154 Ecological Water Quality – Water Treatment and Reuse

Chemical and enzymatic microbial products resulted in biofilms activity may induce

• oxygen concentration cells and anaerobic sites generation (promoting growth of

• formation of iron concentration cells by the activity of iron and manganese oxidizing

• metabolites such as acids produced by bacteria have corrosive action upon the surface; • production of depolarizing enzymes within the biofilm matrix, which may persist

• exopolymers produced by slime forming bacteria stimulate biofilm formation and

• the binding capacity of biofilm matrix which may lead to deposits accumulation with

Some of the recommended strategies in drinking water associated biofilm control are: source waters protection, appropriate treatment, infrastructure contamination prevention, pipes and reservoirs maintenance, corrosion control, appropriate disinfection practices, nutrient levels reducing, water quality monitoring, personnel training, water safety plans implementation. We are still living in an age of surfaces, even the remark was first said by Oscar Wild's character in 1895. Having in mind the virtual idea of self-cleaning surfaces, researchers in nanotechnology field are targeting innovative repellent materials with a wide range of applications, for the biofouling control. The superhydrophobicity models such as "the lotus effect" characterizing the lotus (*Nelumbo nucifera*) leaf, offered by natural patterns are investigated at nanoscale. The interdependence between surface roughness, reduced particle adhesion and water repellence proved to be the keystone in the self-cleaning mechanism of

clogging effects (Beech et al, 2005; Coetser and Cloete, 2005).

The present review emphasize on the following recent and relevant findings:

quality and may threat human health when contamination events occur;

• Biofilms associated with drinking water are ubiquitous, harbouring bacterial pathogens, opportunistic pathogens, parasitic protozoa, viruses, toxins releasing fungi and algae; • Microbial consortia in contact with drinking water have significant impacts upon water

• Access to safe water continues to be a target for developing countries, unfulfilled at the

• Even in developed countries, where substantial efforts are submitted in order to ensure population's access to a high quality drinking water, microbial versatility represents an

• Further research is needed in order to understand attached microbial consortia for biofouling prevention and control in drinking water industry, as a matter of public

endless source of problems, with respect to opportunistic pathogens emergence; • Microbial communities in water networks and biofilms represent complex ecosystems; their ecology is influenced by a series of abiotic and biotic factors: raw water sources quality, temperature, flow rate and system hydraulics, nutrient concentration, pipe material, particles accumulation, ingress and intrusion, water treatment, water

corrosion and related effects by different mechanisms:

many biological surfaces (Barthlott & Neinhuis, 1997).

disinfection and microbial interactions;

anaerobic bacteria);

longer than viable cells;

biomass accumulation;

bacteria;

**4. Conclusions**

moment;

security.

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### **Water Quality After Application of Pig Slurry**

#### Radovan Kopp

*Mendel University in Brno, Department of Fisheries and Hydrobiology Czech Republic*

#### **1. Introduction**

160 Ecological Water Quality – Water Treatment and Reuse

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Pig slurry is a complex organic-mineral fertilizer with high fertilising efficiency and it is comparable with farmyard manure. The pig house with excremental ending and subsequent use of slurry is very popular in agriculturally advanced countries because of economic efficiency, work culture and hygiene. Pig house is operationally by 30 - 40 % cheaper than using bedding. Properly made and treated slurry means a significant source of organic matter, nutrients, bacteria and stimulators which if correctly applied, increase fertility and provide a significant cost saving. These slurry nutrients are easily usable for phytoplankton. Nitrogen is found mainly in inorganic form (50 – 60%), just 10% is nitrate and rest forms organic nitrogen. Phosphorus is bound to organic matter and potassium is included in the urine. Chemically the high fertilising efficiency of slurry depends on C:N ratio, which is usually 4-8:1. This ratio affects the mineralization of organic matter, release of N from organic fixation and energy utilization for multiplication of microorganisms. Composition of the slurry is in the Table No.1.

Table 1. Values of physical and chemical parameters in pig slurry applied into ponds. (minmax interval, amended by more authors), ANC - acid neutralization capacity, COD chemical oxygen demand, TP - total phosphorus, TN – total nitrogen

An additional fertilizer in intensively managed fish-ponds usually does not mean that significant increase of fish production as observed in nutrient-poor waters. Moreover, excessive application of fertilizer may lead to environmental pollution and sanitary problems. Fairly high fish production can be achieved by organic fertilizers. The application of pig slurry to ponds as a fertilizer is widely used in many countries in order to increase plankton production and fish growth. That's why manuring is considered to be a cheap way to increase carp production in the pond. Some fish species also feed directly upon these waste, which is enriched by microorganisms with high protein value. Further, different types of manure can be also added into the low-level protein feeds. The type of pig slurry, quality, quantity and the season affects water quality and production of the pond and finally on well-being and growth of fish. Additionally, recycling of pig slurry could be interesting for fish farming in order to reduce the impact of intensive pig farming on environment.

Pig slurry applied to ponds as organic fertilizer necessary needs a decomposition before their nutritional contents are released, assimilated and utilized by plankton. The nutrient content of pig slurry may vary with time, and nutrient availability to phytoplankton growth remains unclear. The rate of nutrients released from animal manure is an important factor to regulate the frequency and amount of manure required to fertilize fish ponds. Pig slurry is destined mainly for refilling of carbon into water and modification of proportion basic biogenic elements (C, N, P). During the water treatment by slurry the amount of carbon dioxide in pond was relatively higher (Fig. 1). After the last application of pig slurry in early may in 2001 alkalinity increased and value of pH decreased which caused a significant increased in values of TIC due to the weather conditions and lower intensity of photosynthesis. On the other hand another TIC value decrease occurred in 2002 (at the end of March) due to higher phytoplankton development. This expansion decreased carbonate content, alkalinity and increased pH.

Fig. 1. Variation of total inorganic carbon (TIC) during application of pig slurry into the pond. Level of total doses 16 kg . m-2 in the year 2001 and 2002. (Arrows indicate term of pig slurry application)

directly upon these waste, which is enriched by microorganisms with high protein value. Further, different types of manure can be also added into the low-level protein feeds. The type of pig slurry, quality, quantity and the season affects water quality and production of the pond and finally on well-being and growth of fish. Additionally, recycling of pig slurry could be interesting for fish farming in order to reduce the impact of intensive pig

Pig slurry applied to ponds as organic fertilizer necessary needs a decomposition before their nutritional contents are released, assimilated and utilized by plankton. The nutrient content of pig slurry may vary with time, and nutrient availability to phytoplankton growth remains unclear. The rate of nutrients released from animal manure is an important factor to regulate the frequency and amount of manure required to fertilize fish ponds. Pig slurry is destined mainly for refilling of carbon into water and modification of proportion basic biogenic elements (C, N, P). During the water treatment by slurry the amount of carbon dioxide in pond was relatively higher (Fig. 1). After the last application of pig slurry in early may in 2001 alkalinity increased and value of pH decreased which caused a significant increased in values of TIC due to the weather conditions and lower intensity of photosynthesis. On the other hand another TIC value decrease occurred in 2002 (at the end of March) due to higher phytoplankton development. This expansion decreased carbonate

Fig. 1. Variation of total inorganic carbon (TIC) during application of pig slurry into the pond. Level of total doses 16 kg . m-2 in the year 2001 and 2002. (Arrows indicate term of pig

farming on environment.

content, alkalinity and increased pH.

slurry application)

Negative balance of carbon dioxide in ponds, evoked by plants assimilation at high content of nutrients in water, leads to high values of pH that could be the cause of fish gill necrosis. When using the pig slurry for ponds treatment, some bacteria and other microorganisms are also required to commence decomposition of the slurry and they have positive influence on zooplankton development, especially cladocerans. Bacteria that come into the water environment together with pig slurry are also a direct food source of zooplankton.

#### **2. Fertilizer management**

The amount of applied pig slurry depends on natural production of the pond, altitude, depth, control of the water inflow, fish stock etc. Due to the fish stock safety it is better to apply a slurry before vegetation season when a lower water temperature reduces the risk of oxygen deficit (Olah et al. 1986). Abnormal phytoplankton development is reduced in cold weather us well, which is associated with low pH and limited effect of toxic ammonia nitrogen on aquatic organisms. To maintain the stability of the pond ecosystem is not suitable to use a slurry in case of abnormal growth of submerged vegetation or water bloom, when water transparency is less than 40 cm (measured by Secchi disc), in case of abundance of basic nutrients or zooplankton overgrowth. For stability of the physical – chemical parameters it is necessary to properly organize an areal distribution, dose and interval of slurry application (Wohlfarth and Schroeder, 1979).

Knud-Hansen and Batterson (1994) tested four fertilization frequencies (daily, twice per week, weekly, and once every two weeks). The study showed that fertilization frequency had a strong linear relationship to net primary production. Zhu et al. (1990) showed that applying pig slurry daily produced higher yields of fish than five or seven day application. Garg and Bhatnagar (2000) also stated that fertilization frequency influenced fish yield. Variation of manuring frequency often depended on convenience, labour saving, the need to dispose the waste and availability of fertilizer, rather than increasing of the productivity of pond ecosystem. Under the experimental conditions it appears that the frequency of fertilizers application to maintain optimum primary production, should be every eight to ten days (Kestemont, 1995**;** Kumar et al. 2004). Planning of the fertilization frequencies should predict the time frame between each treatment which has got the potential of production enhancing and also reducing costs. There is a factor to be considered when fertilizing with manure: possibility of de-oxygenation of the water, which is quite likely when large quantities of manure are added at a time.

Fertilizers batching at doses 0.5-2.0 kg.m-2 of ponds water increased live weight gain of fish about 30-450 kg.ha-1 without supplementary feeding of fish (Hartman et al., 1973; Dhawan and Kaur, 2002). The effect of organic fertilizers from animal breeding farm is higher in ponds with polycultural fish stock where is the highest weight-gain per fish (Buck et al., 1978; Dhawan and Kaur, 2002; Zoccarato et al., 1995). Woynarovich (1976) reported pig slurry conversion of 3–5 % into fish body mass. In the case of polycultural fish stock is ratio higher than in the case of carp monoculture (Prinsloo and Schoonbee, 1984). Zhu et al. (1990) mentioned demand of 8.3 kg dry weight of pig slurry to weightgain of 1 kg fish flesh. The maximal increases in fish production from fertilizers is especially in the tropics, on average, 4 kg fresh pig slurry can produce 1 kg of fish (Biro, 1995). Variously high doses of pig slurry were tried in ponds experiments. Pig slurry doses around 15-16 kg.m-2 were acceptable but they should be applied mainly before vegetation season, when a water temperature is lower (Kopp et al. 2008). Ponds have had concentrate polycultural fish stock during the experiment and fish were intensively fed by cereals. The increased production of the pond supported by pig slurry was 270–630 kg.ha-1. In these experiments the fish production in the high fertilized ponds appears the same values that obtained other researchers.

#### **3. Autotrophs and heterotrophs**

A successful fertilization programme in ponds should develop adequate amounts of food organisms for fish. Organic fertilizer (e.g. pig slurry) has been used to stimulate the development of heterotrophs (bacteria), autotrophs (algae) and other food organisms (zooplankton) to increase fish production in ponds (Schroeder, 1978). Bacteria and algae are important food organism for the herbivorous zooplankton that is consumed by many fish species (Wylie and Currie, 1991). In additional to the bulk of photosynthetic production passing directly to higher trophic levels, in some cases more than 50% of primary production is derived from a microbial loop (Hepher, 1992; Qin et al. 1995; Biro, 1995). Bacterivorous flagellates are a key link in the microbial loop to transfer organic carbon from bacteria to zooplankton and fish (Sanders and Porter, 1990). An increasing amount of evidence shows that bacteria together with algae provide an efficient energy pathway from low trophic levels to zooplankton. In natural waters, bacterial production can be enhanced by increasing organic matter loading, but the excessive application of organic matter into fish ponds can reduce dissolved oxygen and can cause fish kills (Qin and Culver, 1992). It is crucial to keep the amounts of organic fertilizer within limits such that adequate amount of heterotrophic organisms are produced yet without oxygen depletion occurring.

The reduction of algal biomass through grazing of zooplankton may indirectly regulate the amount of substrate available for bacterial growth. Bacterial and algal abundances were negatively correlated in fertilized ponds. This may have resulted from the die-off of algae that released dissolved organic compounds into the water, which in turn stimulated bacterial growth (Qin et al. 1995). In natural lakes, zooplankton populations crash after the decline of algae. In ponds, the zooplankton population might be sustained after algal decline by adding organic fertilizer to promote bacterial growth. Thus a direct input of pig slurry could increase bacterial productivity, which in turn could serve as a valuable food source for zooplankton.

When organic fertilizers are applied to ponds the first link of the food chain is heavily influenced, principally phytoplankton (Dhawan and Toor, 1989). High doses of pig slurry supported typically groups of phytoplankton favour higher amount of organic matter. Dominant algal genera often are *Cryptomonas*, *Chroomonas*, *Monodus* and *Euglena*. Generally also are occurred small genera of centric diatoms (*Stephanodiscus*, *Cyclotella*) and chlorococcal green algae (*Desmodesmus*, *Scenedesmus*, *Chlorella*, *Monoraphidium*). Cyanobacteria usually are not dominant groups in fertilizing ponds. High nitrogen to phosphorus ratios non favour dominance by cyanobacteria in pond phytoplankton (Smith, 1983). Typically genus *Microcystis*, *Aphanizomenon* and *Dolichospermum* in many fish-ponds and eutrophic lakes, cyanobacteria constitute the greater part of the summer phytoplankton biomass, causing regular water blooms and massive fish mortalities by depletion of oxygen after the bloom

vegetation season, when a water temperature is lower (Kopp et al. 2008). Ponds have had concentrate polycultural fish stock during the experiment and fish were intensively fed by cereals. The increased production of the pond supported by pig slurry was 270–630 kg.ha-1. In these experiments the fish production in the high fertilized ponds appears the same

A successful fertilization programme in ponds should develop adequate amounts of food organisms for fish. Organic fertilizer (e.g. pig slurry) has been used to stimulate the development of heterotrophs (bacteria), autotrophs (algae) and other food organisms (zooplankton) to increase fish production in ponds (Schroeder, 1978). Bacteria and algae are important food organism for the herbivorous zooplankton that is consumed by many fish species (Wylie and Currie, 1991). In additional to the bulk of photosynthetic production passing directly to higher trophic levels, in some cases more than 50% of primary production is derived from a microbial loop (Hepher, 1992; Qin et al. 1995; Biro, 1995). Bacterivorous flagellates are a key link in the microbial loop to transfer organic carbon from bacteria to zooplankton and fish (Sanders and Porter, 1990). An increasing amount of evidence shows that bacteria together with algae provide an efficient energy pathway from low trophic levels to zooplankton. In natural waters, bacterial production can be enhanced by increasing organic matter loading, but the excessive application of organic matter into fish ponds can reduce dissolved oxygen and can cause fish kills (Qin and Culver, 1992). It is crucial to keep the amounts of organic fertilizer within limits such that adequate amount of

heterotrophic organisms are produced yet without oxygen depletion occurring.

The reduction of algal biomass through grazing of zooplankton may indirectly regulate the amount of substrate available for bacterial growth. Bacterial and algal abundances were negatively correlated in fertilized ponds. This may have resulted from the die-off of algae that released dissolved organic compounds into the water, which in turn stimulated bacterial growth (Qin et al. 1995). In natural lakes, zooplankton populations crash after the decline of algae. In ponds, the zooplankton population might be sustained after algal decline by adding organic fertilizer to promote bacterial growth. Thus a direct input of pig slurry could increase bacterial productivity, which in turn could serve as a valuable food source for

When organic fertilizers are applied to ponds the first link of the food chain is heavily influenced, principally phytoplankton (Dhawan and Toor, 1989). High doses of pig slurry supported typically groups of phytoplankton favour higher amount of organic matter. Dominant algal genera often are *Cryptomonas*, *Chroomonas*, *Monodus* and *Euglena*. Generally also are occurred small genera of centric diatoms (*Stephanodiscus*, *Cyclotella*) and chlorococcal green algae (*Desmodesmus*, *Scenedesmus*, *Chlorella*, *Monoraphidium*). Cyanobacteria usually are not dominant groups in fertilizing ponds. High nitrogen to phosphorus ratios non favour dominance by cyanobacteria in pond phytoplankton (Smith, 1983). Typically genus *Microcystis*, *Aphanizomenon* and *Dolichospermum* in many fish-ponds and eutrophic lakes, cyanobacteria constitute the greater part of the summer phytoplankton biomass, causing regular water blooms and massive fish mortalities by depletion of oxygen after the bloom

values that obtained other researchers.

**3. Autotrophs and heterotrophs**

zooplankton.

collapsed are not common in fertilizing ponds. Most often are occurred genus of cyanobacteria *Pseudanabaena*, *Planktothrix* and *Aphanocapsa* (Kopp and Sukop, 2003; Qui et al. 1995; Terziyski et al. 2007). Higher fish stock, sufficiency of carbon dioxide, suitable ratio of nutrients (C, N, P) in ponds after applications of pig slurry handicapped cyanobacteria forming water blooms. Cyanobacteria are of poor food value to zooplankton, their large size making them inaccessible to the filter-feeding entomostraca. Even the substances produced by many species of cyanobacteria are toxic to aquatic plants and animals (Sevrin-Reyssac and Pletikosic, 1990).

In ponds treated with slurry they have positive influence on zooplankton development. Though, the results of the experiments indicate that the fish stock with its predaceous pressure exerts a more significant influence on zooplankton than application of the slurry. Nevertheless, the initial development of large species of cladocerans was in ponds fertilized by slurry the more intensive to compare with control ponds (Sukop, 1980). When comparing different treatments, zooplankton was significantly higher in ponds manured with pig slurry. Higher zooplankton density as a result of manuring has also been reported in ponds receiving pig dung (Govind et al. 1978; Dhawan and Kaur, 2002). Dominant of zooplankton genera often are Copepoda (*Cyclops*, *Thermocyclops* and *Acanthocyclops*), nauplius and copepodits stages, small and middle genus of cladocerans (*Bosmina, Chydorus, Moina* and *Daphnia*) and Rotatoria (*Brachionus, Keratella* and *Asplanchna*) (Kopp and Sukop, 2003; Terziyski et al. 2007). Development biomass of planktonic communities in pond after pig slurry application is demonstrated in Figures 2-4.

Fig. 2. Quantity of phytoplankton (individuals 103 . mL-1) and zooplankton (individuals . L-1) in pond during applications of pig slurry in total doses 16 kg . m-2. (Application began at 5.3.)

Fig. 3. Quantity of main groups of phytoplankton in pond during applications of pig slurry in total doses 16 kg . m-2. (Application began at 5.3.)

Fig. 4. Quantity of main groups of zooplankton in pond during applications of pig slurry in total doses 16 kg . m-2. (Application began at 5.3.)

#### **4. Organic matter**

166 Ecological Water Quality – Water Treatment and Reuse

Fig. 3. Quantity of main groups of phytoplankton in pond during applications of pig slurry

Fig. 4. Quantity of main groups of zooplankton in pond during applications of pig slurry in

in total doses 16 kg . m-2. (Application began at 5.3.)

total doses 16 kg . m-2. (Application began at 5.3.)

Application of pig slurry to the water environment is bringing a huge amount of organic matter. Compare to other organic fertiliser the pig slurry has got lower content of organic substances, usually not exceed 10% in total (Hennig and Poppe, 1975). Majority of organic substances in pig slurry is biodegradable and relatively quickly mineralized by microorganisms. During the higher doses of manure to the fish ponds is important to control oxygen content which is intensively consumed due the decomposition of organic matter (Kopp et al. 2008). Application of organic fertiliser into the pond is immediately reflected in the increase of biological oxygen demand (BOD) and chemical oxygen demand (COD). As shows Figures 5 and 6, values change in wide range depending on dosage and elapsed time since last application. Important role for degradation of organic matter plays mainly water temperature and amount of microorganisms that decompose present organic matter. Significant reduction of COD and BOD observed during pig slurry application (Fig. 5 and 6) were mainly caused by changes of water temperature and the rate of degradation of organic substances. In water unloaded organic substances is value of BOD under 8 mg . L**-1** and COD under 35 mg . L**-1**, due to manuring could be these values doubled (Zalud, 2008). Just one month after last application of pig slurry into the pond are decreasing values of organic load to a level comparable with ponds without fertilization (Kopp et al. 2008).

Fig. 5. Variation values of chemical oxygen demand (CODCr) during application of pig slurry into the ponds. Level of total doses 16 kg . m-2 (2001, 2002) and 0.10 kg . m-2 (2000). (Arrows indicate term of application of pig slurry)

Fig. 6. Variation values of biological oxygen demand (BOD5) during application of pig slurry into the ponds. Level of total doses 16 kg . m-2 (2001, 2002) and 0.10 kg . m-2 (2000). (Arrows indicate term of application of pig slurry)

#### **5. Chlorophyll-a**

Chlorophyll content in surface water is an important indicator of trophic level and one of the most often used indicators of biomass of primary producers. Concentration of chlorophyll in clear water usually does not exceed 10 µg . L-1. In fish ponds and water reservoirs are values during the vegetation season in the tens to hundreds µg . L-1. In case of hypertrophic water area with massive cyanobacteria water bloom could be chlorophyll content over thousands µg . L-1 (Zalud, 2008).

The average amount of phytoplankton in ponds, expressed as chlorophyll-a concentration, has increased since 1960s from 30-35 µg . L-1 to 140-150 µg . L-1 till then 1990s. The impact of organic fertilization on increasing values of chlorophyll-a is well documented (Potužák et al., 2007). The application of organic fertilizer at regular intervals caused regular fluctuations of chlorophyll-a in the ponds.

After the application of pig slurry is increasing phytoplankton biomass as well as content of chlorophyll-a. The subsequent increase of zooplankton is lowering phytoplankton biomass by predation and also cause decreasing of chlorophyll–a. Fluctuations in value of chlorophyll–a during the pig slurry application is shows in Figure 7. Despite bigger doses of pig slurry are values of chlorophyll–a not so high. High stock of carp fish is increasing inorganic water turbidity by the feed pressure on the ponds bottom, decreasing

Fig. 6. Variation values of biological oxygen demand (BOD5) during application of pig slurry into the ponds. Level of total doses 16 kg . m-2 (2001, 2002) and 0.10 kg . m-2 (2000). (Arrows

Chlorophyll content in surface water is an important indicator of trophic level and one of the most often used indicators of biomass of primary producers. Concentration of chlorophyll in clear water usually does not exceed 10 µg . L-1. In fish ponds and water reservoirs are values during the vegetation season in the tens to hundreds µg . L-1. In case of hypertrophic water area with massive cyanobacteria water bloom could be chlorophyll

The average amount of phytoplankton in ponds, expressed as chlorophyll-a concentration, has increased since 1960s from 30-35 µg . L-1 to 140-150 µg . L-1 till then 1990s. The impact of organic fertilization on increasing values of chlorophyll-a is well documented (Potužák et al., 2007). The application of organic fertilizer at regular intervals caused regular fluctuations

After the application of pig slurry is increasing phytoplankton biomass as well as content of chlorophyll-a. The subsequent increase of zooplankton is lowering phytoplankton biomass by predation and also cause decreasing of chlorophyll–a. Fluctuations in value of chlorophyll–a during the pig slurry application is shows in Figure 7. Despite bigger doses of pig slurry are values of chlorophyll–a not so high. High stock of carp fish is increasing inorganic water turbidity by the feed pressure on the ponds bottom, decreasing

indicate term of application of pig slurry)

content over thousands µg . L-1 (Zalud, 2008).

of chlorophyll-a in the ponds.

**5. Chlorophyll-a**

transparency and limiting abnormal phytoplankton development due to inadequate lighting conditions. Lower water temperature and predation of zooplankton is limiting phytoplankton as well. (Kopp et al. 2008).

Fig. 7. Variation values of chlorophyll-a during the application of pig slurry into the pond. Level of total doses 16 kg . m-2 (2001, 2002) and without application of pig slurry (2003). (Application began at 28.2. in the year 2001 and 5.3. in the year 2002 respectively)

#### **6. Dissolved oxygen**

Dissolved oxygen concentrations in the ponds are most affected by phytoplankton biomass. Greater oxygen production and consumption occurs in ponds with higher phytoplankton biomass. The use of pig slurry increased the biological oxygen demand in ponds and could result in periods of low dissolved oxygen levels. The aerobic decomposition of organic matter by bacteria is an important drain of oxygen supplies in ponds. When added to ponds organic fertilizers exert an oxygen demand and an excessive application may result in depletion of dissolved oxygen (Schroeder, 1974, 1975). Qin et al., (1995) observed that dissolved oxygen in fish ponds with organic fertilizer was lower than in those without organic fertilizer, and dissolved oxygen in enclosures decreased with the increase of organic fertilizer loading.

Oxygen consumption in pond is regulated by biological and sediment oxygen demand and fish. In intensive fish aquaculture, BOD reached 0.29 mg O2 . L-1 . h-1 in Israel (Schroeder, 1975), and 0.14 mg O2 . L-1 . h-1 in fertilized ponds in USA (Boyd 1973). Qin et al., (1995) described that the BOD value varied with organic matter loadings and temperature. The BOD of fertilized ponds water varied from 0.19 to 0.34 mg O2 . L-1 . h-1. Similar results described Kopp et al. (2008) from Czech Republic, when the BOD value varied from 0.12 to 0.34 mg O2 . L-1 . h-1 in pond during application high doses of pig slurry (Figure 8). It is certain that excessive organic fertilizer inputs can be a major factor depleting oxygen in ponds, especially during the clear-water phase when algae are less abundant (Qin and Culver, 1992). Analogous situation can be turn up during application of organic fertilizers, when higher development of zooplankton reduced the quantity of phytoplankton and dissolved oxygen concentrations declined. At high temperatures a heavy organic fertilizer application should also be avoided, otherwise a fish kill is likely to occur due to oxygen depletion.

On the second hand Dhawan and Kaur, (2002) reported that pig slurry even at higher dose (36 t . ha-1 . yr-1) had no adverse effect on the dissolved oxygen content, which is an important parameter for the survival and growth of fish.

Fig. 8. Regression of temperature with biological oxygen demand (BOD). Pond water without pig slurry was used from pond a year before application and pond water with pig slurry was used during application of 16 kg . m-2 of pig slurry (February-May)

Sharma and Das, (1988) also reported that in a fish-pig pond, even higher organic loading by pig slurry did not deteriorate the oxygen content of water. This may be attributed to the higher plankton abundance in manure-loaded ponds, leading to higher dissolved oxygen level by photosynthetic activity especially during day. The values of dissolved oxygen varied during day and night in large interval and after application of pig slurry in during higher development of phytoplankton biomass can be exceed to 250 % saturation within day

described that the BOD value varied with organic matter loadings and temperature. The BOD of fertilized ponds water varied from 0.19 to 0.34 mg O2 . L-1 . h-1. Similar results described Kopp et al. (2008) from Czech Republic, when the BOD value varied from 0.12 to 0.34 mg O2 . L-1 . h-1 in pond during application high doses of pig slurry (Figure 8). It is certain that excessive organic fertilizer inputs can be a major factor depleting oxygen in ponds, especially during the clear-water phase when algae are less abundant (Qin and Culver, 1992). Analogous situation can be turn up during application of organic fertilizers, when higher development of zooplankton reduced the quantity of phytoplankton and dissolved oxygen concentrations declined. At high temperatures a heavy organic fertilizer application should also be avoided, otherwise a fish kill is likely to occur due to oxygen

On the second hand Dhawan and Kaur, (2002) reported that pig slurry even at higher dose (36 t . ha-1 . yr-1) had no adverse effect on the dissolved oxygen content, which is an

Fig. 8. Regression of temperature with biological oxygen demand (BOD). Pond water without pig slurry was used from pond a year before application and pond water with pig

Sharma and Das, (1988) also reported that in a fish-pig pond, even higher organic loading by pig slurry did not deteriorate the oxygen content of water. This may be attributed to the higher plankton abundance in manure-loaded ponds, leading to higher dissolved oxygen level by photosynthetic activity especially during day. The values of dissolved oxygen varied during day and night in large interval and after application of pig slurry in during higher development of phytoplankton biomass can be exceed to 250 % saturation within day

slurry was used during application of 16 kg . m-2 of pig slurry (February-May)

important parameter for the survival and growth of fish.

depletion.

(Behrends et al. 1980; Kopp et al., 2008). Figure 9 shows fluctuations in oxygen content during the application of pig slurry at low and high values. In both cases oxygen varied in wide range but has not fall below 50% of saturation, which is limit value ensure survival of carp fish. A wide range of values of oxygen during the application of pig slurry is determined by the intensity of photosynthesis, which is mainly influenced by the amount of available nutrients, water temperature and feed pressure of the zooplankton.

Fig. 9. Variation values of dissolved oxygen during application of pig slurry into the ponds. Level of total doses 16 kg . m-2 ( 2002) and 0.10 kg . m-2 (2000) (application began at 9.5. in the year 2000 and 5.3. in the year 2002 respectively)

#### **7. pH**

The pH value of water depends on the alkalinity and hardness of the water. Algal photosynthesis causes an increase in pH due to removal of H+ ions. Algal assimilation of nitrate and its subsequent reduction within the algal cell to ammonium also increases the pH. On the second hand pH decreases with organic fertilization because bacterially generated carbon dioxide from manure decomposition. It was clear that the fertilization regimes were able to maintain adequate buffering capacity so that the pH fluctuations were within the desired limits for fish culture.

Properly chosen dosage and interval of slurry application should keep pH in optimum range for fish farming. To avoid pH increasing (over 8.5) due to intensive photosynthesis of phytoplankton what can happen especially in pond with a low alkalinity it is recommended to add calcium. Dose of calcium fertilizer is set up along of water alkalinity and depends on amount and timing of slurry application. In intensively fertilized ponds is pH usually under 9.0 (Kopp et al. 2008). Figure 10 shows pH fluctuations during the high dose application of pig slurry into intensively managed pond for two years. Significant changes in pH depending on phytoplankton development were apparent in 2001. First two years after slurry application is decreasing pH due to reduced nutrients supply and lower development of primary producers.

Fig. 10. Variation values of pH during application of pig slurry into the pond. Level of total doses 16 kg . m-2 (2001, 2002). (Arrows indicate term of pig slurry application)

#### **8. Transparency**

Application of fertilizer at regular intervals affects the transparency of the water. The transparency was significantly worsened within several weeks in the ponds after pig slurry application. Organic particles from manure had a significant influence in water turbidity. Minimum transparency was measured in ponds with abnormal phytoplankton biomass and higher fish stock of cyprinids, which reducing transparency by feeding by pressure on the bottom. High abundance of autotrophs may cause higher development of zooplankton and better transparency. During applications of pig slurry was transparency usually in interval 20-40 cm (Kumar et al. 2004; Kopp et al. 2008). Figure 11 shows different transparency during the high dose application in 2001 and 2002 compare to water transparency in 2003 without pig slurry application. Higher transparency in 2003 was due to lower level of nutrients and lower phytoplankton development in cooler period of the year compare to years with pig slurry application.

pig slurry into intensively managed pond for two years. Significant changes in pH depending on phytoplankton development were apparent in 2001. First two years after slurry application is decreasing pH due to reduced nutrients supply and lower development

Fig. 10. Variation values of pH during application of pig slurry into the pond. Level of total

Application of fertilizer at regular intervals affects the transparency of the water. The transparency was significantly worsened within several weeks in the ponds after pig slurry application. Organic particles from manure had a significant influence in water turbidity. Minimum transparency was measured in ponds with abnormal phytoplankton biomass and higher fish stock of cyprinids, which reducing transparency by feeding by pressure on the bottom. High abundance of autotrophs may cause higher development of zooplankton and better transparency. During applications of pig slurry was transparency usually in interval 20-40 cm (Kumar et al. 2004; Kopp et al. 2008). Figure 11 shows different transparency during the high dose application in 2001 and 2002 compare to water transparency in 2003 without pig slurry application. Higher transparency in 2003 was due to lower level of nutrients and lower phytoplankton development in cooler period of the year compare to

doses 16 kg . m-2 (2001, 2002). (Arrows indicate term of pig slurry application)

of primary producers.

**8.

Fig. 11. Variation values of transparency during application of pig slurry into the pond. Level of total doses 16 kg . m-2 (2001, 2002) and without application of pig slurry (2003). (Application began at 28.2. in the year 2001 and 5.3. in the year 2002 respectively)

#### **9. Alkalinity**

The alkalinity of the water is associated with the level of carbonates and bicarbonates content. The bicarbonate and carbon dioxide in the water has a buffering effect, preventing sudden changes in pH. Low alkalinity (under 1 mmol . L-1) leads to poor buffering capacity and generally, a high fluctuation of pH. A high buffering capacity prevents fluctuation in the acid-base equilibrium caused by strong photosynthetic activity. Alkalinity increases with pig slurry application because bacterially generated carbon dioxide from manure decomposition dissolves calcium carbonate present in the pond sediments and slurry (Kumar et al. 2005). The gradual reduction in alkalinity occurred during the prime period of primary production. According to Boston et al. (1989), a decrease in pond water alkalinity can occur when algae remove bicarbonates. This may occur during periods of high photosynthetic activity which is promoted by application of pig slurry. Figure 12 shows fluctuations in alkalinity during the application of pig slurry into the fishpond. Alkalinity rises at the beginning of application (in accordance with literature) and after that change due to phytoplankton biomass development. Significant decrease of alkalinity during the pig slurry application in 2002 was caused by abnormal plankton development, high intensity of photosynthesis and depletion of carbonates. The alkalinity was quite stable or fluctuates during the year in small interval only in the fishponds that was not fertilizer.

Fig. 12. Variation values of alkalinity during application of pig slurry into the ponds. Level of total doses 16 kg . m-2 (2001, 2002). (Arrows indicate term of application pig slurry)

#### **10. Nutrients**

The nutrient content of animal manure may vary with time an d the quantity of nutrient released from the manures over time is a key factor in determining the fertilization regime. According to Muck and Steenhuis (1982), manure nutrient concentrations, and the percentage of P, N and C which become available for algal uptake, depend primarily on the animal´s diet, whether the manure is liquid or solid, and the age and storage conditions of the manure. The primary productivity pattern in the pond indicated a gradual built-up phase, a peak phase followed by a decline phase, this can be related to the nutrient release pattern. In pig slurry application, the primary productivity attained its peak 5-7 days after the application of slurry (Kumar et al. 2004).

Culver (1991) and Culver et al. (1993) introduced a fertilization regimen which attempts to optimize the algal composition by maintaining nitrogen and phosphorus at a specific ratio in ponds. They suggested that green algae could be maintained whereas cyanobacterial blooms could be suppressed in ponds by manipulating the N:P ratio (20:1) with fertilizer. Oin et al. (1995) showed that it is difficult to reach the N:P ratio recommended by Culver et al. (1993) by using only organic fertilizer, because most organic fertilizers have a low N and P content and inappropriately low N:P ratios.

Sediment is usually considered the major sink of orthophosphates in fishponds. The sediments absorb soluble forms of P from pond water until fully saturated. The rate of

Fig. 12. Variation values of alkalinity during application of pig slurry into the ponds. Level of total doses 16 kg . m-2 (2001, 2002). (Arrows indicate term of application pig slurry)

The nutrient content of animal manure may vary with time an d the quantity of nutrient released from the manures over time is a key factor in determining the fertilization regime. According to Muck and Steenhuis (1982), manure nutrient concentrations, and the percentage of P, N and C which become available for algal uptake, depend primarily on the animal´s diet, whether the manure is liquid or solid, and the age and storage conditions of the manure. The primary productivity pattern in the pond indicated a gradual built-up phase, a peak phase followed by a decline phase, this can be related to the nutrient release pattern. In pig slurry application, the primary productivity attained its peak 5-7 days after

Culver (1991) and Culver et al. (1993) introduced a fertilization regimen which attempts to optimize the algal composition by maintaining nitrogen and phosphorus at a specific ratio in ponds. They suggested that green algae could be maintained whereas cyanobacterial blooms could be suppressed in ponds by manipulating the N:P ratio (20:1) with fertilizer. Oin et al. (1995) showed that it is difficult to reach the N:P ratio recommended by Culver et al. (1993) by using only organic fertilizer, because most organic fertilizers have a low N and

Sediment is usually considered the major sink of orthophosphates in fishponds. The sediments absorb soluble forms of P from pond water until fully saturated. The rate of

**10. Nutrients**

the application of slurry (Kumar et al. 2004).

P content and inappropriately low N:P ratios.

absorption decreases with an increase in partial saturation of sediments (Boyd and Musig, 1981). The P probably accumulates in the newly created sludge layer. Thus, the retained P does not appear to seep into the underground water, but remains in the pond. The P-containing sediment is the basis of the fertility of the pond. The higher P- input into the pond, the more P was retained (Knosche et al. 2000). Pig slurry has a low P content relatively, and application of pig slurry to pond has not negatively impact on higher value of P in outlet water (Kopp et al., 2008; Blažková et al., 1987). Balances of total P and N during the application of pig slurry into the ponds are demonstrated in the Figure 13 and 14.

Nitrogen is one of the most important agents of eutrophication, but it is not the most decisive factor in the eutrophication process of surface waters. Its balance is also influenced by the fixation of N from the air caused by cyanobacteria and by bacterial denitrification. The N balances in carp pond shows the similar picture as the P-retention, i.e. a clear increase in retention with N load (Olah et al. 1994). On the second hand Pechar (2000) reported that the rise in P and N in carp ponds during the 1990s is a result of the higher loading of organic fertilizers, particularly pig and cattle manure. Higher doses of pig slurry increase amount of nitrogen in outlet water from manure pond, especially ammonium for define time (Kopp et al. 2008).

Fig. 13. Balance of total P and N (output by outflow minus input by inflow) during the application of pig slurry (total doses 16 kg . m-2) into the pond (February-April), (Arrows indicate term of application pig slurry)

Fig. 14. Balance of total P and N (output by outflow minus input by inflow) during the application of pig slurry (total doses 0.10 kg . m-2) into the pond (May-July), (Arrows indicate term of application pig slurry)

#### **11. Ammonia nitrogen**

The critical water quality parameter during application of pig slurry is ammonia nitrogen, a principal excretory product of fish metabolism, which in the un-ionized form is highly toxic to fish. The oxidation of ammonia requires oxygen in the water, for this reason during the initial period of pig slurry application, low levels of nitrate were recorded.

The amount of toxic ammonia depends mainly on pH and water temperature. The maximum allowable (safe) concentration for carp is very low (0.05 mg . L-1 NH3), LC50 (lethal concentration) for carp is 1.0 -1.5 mg . L-1 NH3 (Svobodová et al. 1971; Russo and Thurson, 1991). During the application of higher dose of pig slurry has been level of ammonia nitrate increased for a short time which could endanger fish stock by not just acute toxic effects of ammonia but by autointoxication of fish body as well (Kopp et al. 2008). The dosage of pig slurry must follow hydrochemical conditions in pond and application is not possible in high pH or higher water temperature.

Figure 15 shows fluctuations of ammonia nitrogen during the high dose application of pig slurry. The graph clearly demonstrates the significant increase of ammonia in 2002, when were applied higher doses of slurry then a year ago. Despite high level of toxic ammonia in the water there was no fish kills or noticeable reduction of food intake but we can expect some negative impact of sublethal level of ammonia on fish organism. Arrilo et al. (1981)

Fig. 14. Balance of total P and N (output by outflow minus input by inflow) during the application of pig slurry (total doses 0.10 kg . m-2) into the pond (May-July), (Arrows

The critical water quality parameter during application of pig slurry is ammonia nitrogen, a principal excretory product of fish metabolism, which in the un-ionized form is highly toxic to fish. The oxidation of ammonia requires oxygen in the water, for this reason during the

The amount of toxic ammonia depends mainly on pH and water temperature. The maximum allowable (safe) concentration for carp is very low (0.05 mg . L-1 NH3), LC50 (lethal concentration) for carp is 1.0 -1.5 mg . L-1 NH3 (Svobodová et al. 1971; Russo and Thurson, 1991). During the application of higher dose of pig slurry has been level of ammonia nitrate increased for a short time which could endanger fish stock by not just acute toxic effects of ammonia but by autointoxication of fish body as well (Kopp et al. 2008). The dosage of pig slurry must follow hydrochemical conditions in pond and application is not possible in high

Figure 15 shows fluctuations of ammonia nitrogen during the high dose application of pig slurry. The graph clearly demonstrates the significant increase of ammonia in 2002, when were applied higher doses of slurry then a year ago. Despite high level of toxic ammonia in the water there was no fish kills or noticeable reduction of food intake but we can expect some negative impact of sublethal level of ammonia on fish organism.  Ammonia nitrogen**

pH or higher water temperature.