Sanitary Microbiological

Full text

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INTRODUCTION
Recent research emphasizes the importance
of studying the microbiota of aquatic ecosystems
to assess surface water quality. Coliform bacteria,
particularly E. coli, have proven reliable indica-
tors of fecal pollution and potential risks to pub-
lic health (Gauthier et al., 2001; Pekarova et al.,
2009). Additionally, these bacteria serve as indi-
cators of fecal contamination in pulp and paper
production euents, highlighting their signi-
cance for risk assessment (Gauthier et al., 2001).
Water quality assessment and the implementation
of innovative treatment technologies remain sig-
nicant public interests in developed countries
(Pekarova et al., 2009; Kostenko et al., 2017;
Malovanyy et al., 2019; Malovanyy et al., 2020).
Pathogenic bacteria identication, according to
numerous researchers (Fey et al., 2004; Tymchuk
et al., 2020), remains a major concern for assess-
ing environmental safety and its impact on human
health and the ecosystem. In tropical and temper-
ate regions, microbiological E. coli contamina-
tion serves as a specic indicator of fecal pollu-
tion. Monitoring bacterial density aids in evaluat-
ing data reliability (Besemer et al., 2005). Faecal
Sanitary, Microbiological Condition, and Ecological State of Surface
Water Quality in the Upper Siret River Basin (Ukraine)
Ivan Burdenyuk1, Аndrii Masykevich1, Konstantin Dombrovskiy2, Olexandr Rylskyi2,
Yurij Masykevich1, Svyatoslav Deyneka1, Myroslav Malovanyy3, Ivan Tymchuk3*
1 Department of Hygiene and Ecology, Bucovinian State Medical University, 2 Teatralna sq., Chernivtsi, 58000,
Ukraine
2 Faculty of Biology, Department of General and Applied Ecology and Zoology, Zaporizhzhia National
University , 66, Zhukovsky Str., Zaporizhzhia, 69600, Ukraine
3 Viacheslav Chornovil Institute of Sustainable Development, Department of Ecology and Sustainable
Environmental Management, Lviv Polytechnic National University, S. Bandera St, 12, Lviv, 79013, Ukraine
* Corresponding author’s e-mail: i.s.tymchuk@gmail.com
ABSTRACT
This study aimed to spatially assess the ecological state of the upper Siret River basin in Ukraine, along with its
main tributaries, using physicochemical and microbiological indicators. Additionally, we evaluated the impact of
anthropogenic activities on surface water quality in the region. The research spanned one year, with nine sampling
points ranging from the Siret River sources in the Pokutsko-Bukovynian Carpathians to the Romania border cross-
ing area. Notably, a signicant portion of the upper basin lies within the Vyzhnytskyi National Nature Park. Physi-
cochemical analysis involved measuring pH, water turbidity, and concentrations of NH4+, NO3-, and CI- ions. Mi-
crobiological analysis focused on total and fecal bacteria, specically E. coli species. Results revealed a signicant
correlation between population density, the absence of centralized sewage treatment facilities in large settlements,
and the level of surface water pollution in the Ukrainian segment of the Siret River basin. The upper part of the
river network, where the Vyzhnytskyi National Nature Park is located, displayed the cleanest waters. Downstream,
surface water pollution increased, particularly near certain points outside large settlements and tourist complexes
(Myhove, Berehomet, and Storozhynets). Our ndings highlight the importance of using microbiological indica-
tors to monitor the ecological state of Danube sources in the Eastern Carpathians.
Keywords: Siret River basin, water quality, coliforms E. coli, anthropogenic inuence.
Received: 2023.08.07
Accepted: 2023.09.26
Published: 2023.11.15
Ecological Engineering & Environmental Technology 2023, 24(9), 55–63
hps://doi.org/10.12912/27197050/172930
ISSN 2719-7050, License CC-BY 4.0
ECOLOGICAL ENGINEERING
& ENVIRONMENTAL TECHNOLOGY

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Ecological Engineering & Environmental Technology 2023, 24(9), 55–63
indicator bacteria, including total coliforms, fae-
cal coliforms, Escherichia coli, and intestinal
enterococci, are excreted by humans and warm-
blooded animals into wastewater, maintaining
viability and pathogenicity for extended periods
(Besemer et al., 2005).
Anthropogenic activity has led to an increase
in fecal bacteria in the river network of the Car-
pathians, particularly in the Danube basin. Despite
eorts to preserve its ecosystem, the Danube faces
severe physical, chemical, and biological pollu-
tion, especially in its upper part, the Pokutsko-Bu-
kovynian Carpathians (Pall et al., 2013; Kolarevich
et al., 2011). Researchers have classied the mi-
crobiological quality of water in the Danube basin
into dierent classes based on standard indicators
of fecal and organic pollution (Eiler et al., 2022;
Kavka et al., 2006; Winter et al., 2007; Kirschner
et al., 2009; Kirschner et al., 2017). The Siret and
Prut rivers, originating in the Ukrainian Carpathi-
ans, were categorized as critical and strongly fecal
pollution classes, respectively. In Romania, stud-
ies have focused on priority pollutants in the Siret
River Basin, originating from municipal, indus-
trial, and agricultural wastewater discharges (Zait
et al., 2022). The assessment included monitoring
inorganic (e.g., As, Cd, Hg, Ni, Pb) and organic
(e.g., naphthalene, anthracene, phenanthrene,
uoranthene, benzo(a)anthracene, benzo(b)uor-
anthene, benzo(k)uoranthene, benzo(a)pyrene,
benzo(hy)perylene, indeno(1,2,3-cd)pyrene, α, β,
and γ-hexachlorocyclohexane and di-2-ethylhex-
yl phthalate) priority pollutants in the Siret River
Basin from 2015 to 2020. Findings revealed that
the water quality generally corresponds to the 2nd
or 3rd class and is mostly unsuitable for drink-
ing water supply, inuenced by main tributaries’
quality and treatment facility euents. The sani-
tary and microbiological state of the Siret River in
Romania has been comprehensively studied.
As for the upper part of the Siret River
(Ukraine), it should be noted that the issue of
the ecological state of this part of the basin has
not been suciently studied. Siret is a river that
ows in Ukraine (Chernivtsi region) and Roma-
nia. Siret is a left tributary of the Danube. The
length of 513 km, including the upper part of 110
km ows through the territory of Ukraine, then
through Romania to the conuence with the Dan-
ube near the city of Galati. In the upper part of
the stream (Ukraine), the channel of the Siret is
moderately winding, with a width of 7–10 m and
a depth of 0.2–0.7 m, the current speed varies
from 1.5 to 2–3 m/s. It crosses the Ukrainian-
Romanian border in the area of Novy Vovchynets
village. More than twenty tributaries ow into the
Siret on the territory of Ukraine, among them:
right tributaries Lustun, Myhyvka, Maly Siret,
Siretul, etc., left tributaries Lapushna, Lekechi,
Stebnyk, Sukhyy, Hlybochok, Molnytsia, etc.
Due to the lack of centralized sewage treatment
facilities, there are large settlements in the up-
per part of the basin: the village of Berehomet,
the city of Storozhynets, the villages of Ropcha,
Panka, Yordaneshti, Karapchiv, Kamianka, Pet-
richanka, etc. are potential polluters of the Siret
Basin (Baseinove upravlinnia …, 2022).
Control over the quality of water resources of
the Prut and Siret in Ukraine at the state level is
carried out by the basin management of this river
network (BUVR), which publishes the results of
research on the corresponding website (Baseinove
upravlinnia…, 2022; Derzhavne ahenstvo…,
2022). However, there are no sanitary-microbio-
logical indicators among the indicators by which
the certied laboratories of BUVR assess the con-
dition of surface, underground, return (wastewa-
ter) and drinking water. Therefore, scientically
based bioindication of the upper part of the Siret
River (Ukraine) has not been carried out until now.
Separate studies on the study of phytoperiph-
yton to establish anthropogenic regression of wa-
ter ecosystems of the Siret River basin are known
(Karavan et al., 2013; Karavan et al., 2019). The
authors show that algae are a convenient display
object for bioindication of the water environment
of mountain and foothill rivers. Our earlier re-
search made it possible to nd out the sanitary
and microbiological state of the sources of the
Siret River (Masikevych et al., 2018; Masikevych
et al., 2022). It was shown that there is a progres-
sive pollution of watercourses by discharges of
an organic nature downstream, as evidenced by
BSK5 indicators, an increase in indicators of the
total microbial number and Сoli index, etc. These
studies are aimed at assessing the sanitary and
microbiological state of the Siret River from its
source in the Pokutsko-Bukovynian Carpathians
to the border with Romania.
MATERIALS AND METHODS
The material was prepared on the basis of
joint research carried out in the laboratories of Bu-
kovynian State Medical University, Zaporizhzhia

57
Ecological Engineering & Environmental Technology 2023, 24(9), 55–63
National University. As a result of the conducted
research, the sanitary and ecological condition was
analyzed in terms of a number of microbiological,
as well as the content of surface-active substances
and Ukrainian oil products in 9 sampling points
in the upper part (territory of Ukraine) of the Siret
River basin. Below, Figure 1 presents water sam-
pling points in the Siret river basin.
Based on the data in Table 1, taking into ac-
count the indicator of the area of the basin, it is
possible to calculate the average population den-
sity of the river basin. It is 37.5 people per km2,
while the average rate in Chernivtsi region is
109.9 people per km2 and 72 people per km2 in
Ukraine. Therefore, the basin of the Siret River
within the borders of Ukraine is characterized by
rather low average indicators of population den-
sity. This territory is inhabited mainly by the rural
population, which is approximately 60% and is
mainly engaged in cattle breeding, logging and
Fig. 1. Hydrological network of the Siret River basin within the territory
of Ukraine with the indication of water sampling points
Table 1 Characteristics of population indicators and characteristics of the landscape of the Siret River basin within
Ukraine
Range between
sampling sites
The number
of settlements
within the range
Population,
in thousands of
people
General characteristics of the landscape
1–2 3 2385 mountainous area, mostly covered with spruce-r forest
2–3 1 7647 foothills, mostly covered with r-beech forest
3–4 3 6933 at terrain, sparse forests, deciduous species prevail, meadows
4–5 5 6799 at area, sparse forests, deciduous species prevail, arable land
5–6 1 14428 urban landscape
6–7 4 8471 at and rough terrain, deciduous species prevail, arable land
7–8 2 2302 at and rough terrain, deciduous species prevail, arable land
8–9 1 6284 at terrain, deciduous species prevail, arable land
In all 20 55 249
Note: the length of the Siret river basin within Ukraine is 110 km, the area of the basin is 2.07 thousand km2
(Baseinove upravlinnia …, 2022).

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Ecological Engineering & Environmental Technology 2023, 24(9), 55–63
agriculture. It should also be noted that central-
ized sewage treatment facilities are completely
absent in this region, and therefore waste from
economic activities and communal discharges to
a large extent enter the surface waters of the Siret
River network. In the land cover of the Siret basin,
three vegetation zones can be distinguished: the
upper mountain zone (spruce-r forest), the foot-
hill zone (Carpathian spruce-beech forest) and the
cross-plain zone (broad-leaved species prevail).
RESULTS AND DISCUSSION
In order to determine the microbiological in-
dicator of minor components of surface waters,
a brous carrier of the „Viya” type was used,
made of textured plait thread (Hvozdiak, 2003).
It is shown (Fig. 2) that bacteria are able to ac-
cumulate on brous material, which facilitates
their identication. Figure 2 shows the coecient
of bacterial accumulation on the brous carrier,
ranging from 6.5 to 8.8 units. The enumeration
of total coliforms and thermotolerant (fecal) coli-
forms, represented by purple-red colonies with a
metallic sheen on Endo agar, was conducted us-
ing the membrane ltration method. For total and
thermotolerant E. coli identication, blue-green
colonies were observed on TBX - Tryptone Bile
X agar, incubated at 37°C and 44°C for 48 hours,
respectively (Sanitarno-virusolohichnyy con-
trol…, 2007). The morphology and other prop-
erties of the microbial cultures were conrmed
through microscopy, followed by identication
based on Bergey’s determinant (Khoult et al.,
1997). Physico-chemical indicators of water were
determined in accordance with (Voda pytna…,
2014; Pro vidpovidnist…, 2005). The results of
the study were processed statistically at the signi-
cance level of p<0.05 (Herych et al., 2021). The
data presented in Table 2 reect the average value
of the physico-chemical and microbiological indi-
cators of the ood waters of the Siret River basin
(within the territory of Ukraine) at 9 investigated
sampling points. The average value of the pH in-
dicator was 6.5–6.8 units, which indicates slight
acidication of ood waters of the river network.
In the upper part of the basin (sampling points
1–4), the water of the river network was character-
ized by a high degree of transparency, which sub-
sequently undergoes changes (acquires turbidity).
As for microbiological indicators, the water in-
take points we studied are characterized by quite
diverse values. Thus, the average concentrations
of fecal E. coli range from 150 CFU/100 ml (in
the main part of the Siret River basin, in the head-
waters) to 10,800 CFU/100 ml (at intake point 6,
after discharges in the city of Storozhynets). The
range of dierences is even greater when compar-
ing the total number of BGCP and ranges from
360 CFU/100 ml in the upper reaches of the Siret
River to 25,650 CFU/100 ml after the river passes
through large settlements (Berehomet and Storo-
zhynets). It should be noted, that the maximum
values of physico-chemical indicators (NH4, NO3,
CI) were also recorded for sampling point No. 6
(Storozhynets). The obtained result can be ex-
plained by the lack of centralized sewage treat-
ment facilities and a signicant number of people
in this settlement, as evidenced by the data of
Table 1. On the other hand, the minimum value of
the studied physico-chemical and microbiological
Fig. 2. Bacterial accumulation coecient on the brous carrier “VIYA”

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Ecological Engineering & Environmental Technology 2023, 24(9), 55–63
Table 2. Mean values of physico-chemical and microbiological indicators of water quality in the examined
catchments River Siret
Sampling
site (N0)
Parameter
BOC5
mg О2 /
dm3
O2
mg/
dm3
PP
mg/
dm3
NH4
+NO3
-Cl-Fecal
E.coli
Fecal
сoli-forms
Total
E.coli
Total
сoli-forms
mg l-1 CFU/100ml
1 1.5 10.5 - 0.015 1.55 2.95 150 260 210 360
2 1.9 9.8 - 0.018 2.77 2.93 178 285 240 450
3 6.2 4.6 0.012 0.055 6.80 13.44 1020 1470 2300 3800
4 6.1 4.0 0.027 0.057 6.84 14.22 2600 3430 3100 6900
5 7.0 4.5 0.055 0.051 6.95 15.21 8200 9100 6350 11800
6 12.8 2.2 0.090 0.204 25.22 20.43 10800 10300 12460 25650
7 6.4 5.3 0.015 0.030 7.20 14.68 2450 6200 8300 5130
8 3.5 5.8 0.013 0.031 6.91 12.71 2120 3900 2200 3950
9 3.2 7.0 0.010 0.028 6.65 8.55 840 950 11 45 2210
Note: РР – petroleum products; BOC5 – biochemical oxygen consumption; CFU – Colony-forming units.
Fig. 3. Changes in the physico-chemical and microbiological indicators of water
quality along the course of the Siret River within the territory of Ukraine

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Ecological Engineering & Environmental Technology 2023, 24(9), 55–63
indicators was registered in points 1-2 of the wa-
ter intake of the Siret river basin. These points are
located in the upper part of the stream, where sur-
face water enters mainly from the protected terri-
tories of the national natural park „Vyzhnytskyi”,
and where there is a slight impact from economic
activity. The studied territories are characterized
by a sparse population of approximately 2,385
people. Previous research has demonstrated a
strong positive correlation (r = 0.95) between bio-
chemical oxygen demand (BOC), humic organic
carbon (HOC), and microbiological indicators.
Moving downstream, the river shows an increase
in suspended matter content, leading to reduced
dissolved oxygen levels and elevated BOC5 val-
ues. Additionally, there is an increase in chloride
and nitrite concentrations (salts of hydrochloric
and nitric acids). These ndings suggest a rise in
organic pollution in the river network, potentially
indicating the presence of fecal compounds, par-
ticularly in areas with economic landscapes lack-
ing functional treatment facilities. The observed
dierences in water quality indicators are primar-
ily attributed to variations in land use, manage-
ment practices of subsidiary farms, and the ab-
sence of centralized treatment facilities within the
Siret River basin. The data are presented in Fig.
3. testify to the existence of spatial diversity of
physico-chemical and microbiological indicators
along the Siret River bed.
Thus, in the upper reaches of the basin (sam-
pling points No. 1-2), where the population den-
sity is insignicant and the territories of the nature
reserve fund prevail, the lowest concentrations of
nitrogen compounds in the water occur and the
minimum number of coliforms bacteria is record-
ed. The surface waters of this region are character-
ized by high transparency. Along the course, the
value of these indicators increases. Deterioration
of water quality at sampling points No. 3-4 is most
likely caused by the placement of a developed net-
work of private „green” tourism in the basin of the
Sukhyy, Solonets, and Myhivka tributaries with-
out the presence of appropriate cleaning spores.
The maximum values of most of the investigated
indicators were recorded below the sources of
communal discharges in the city of Storozhynets,
where about 15,000 people live (Table 1). In this
way, the dierences in the values of water quality
indicators obtained by us are mainly the result of
land use, management of subsidiary farms and the
absence of centralized treatment facilities in the
Siret River basin. The classes of microbiological
quality of water in the investigated bodies were
determined on the basis of (Directive 2006/7/
EC…, 2006), which regulates the quality of water
for domestic purposes (Table 3).
Of the 9 water sampling points analyzed, only
2 points (1, 2) were characterized by the best mi-
crobiological condition in terms of the level of E.
Coli contamination. These are the territories of
the headwaters of the sources of the Siret River,
which are located mainly in the mountain and for-
est part of the protected zone of the Vyzhnytskyi
National Nature Park, which can be compared
to the reference ones. The worst microbiological
quality of water occurs in densely populated areas
(items 3-7), where there is no centralized system
of municipal sewage treatment. Downstream, the
microbiological quality slightly improves (item 9),
which can be explained most likely by the reduc-
tion of anthropogenic pressure on the water eco-
system and the process of its spatial self-renewal.
The minimum physical and chemical indica-
tors of watercourses are also recorded for these
territories. The water pollution index (WPI) was
calculated on the basis of physical and chemical
parameters using six indicators (BOC5, O2, PP-
petroleum products, NH4
+, NO2
-, Cl-) according to
the formula (Snizhko, 2001).
 




(1)
where: Ci – the arithmetic average value of the
water quality indicator, MPC – the maxi-
mum permissible concentration of the in-
dicator. For O2, the MPK is divided by the
average value of its concentration. The
criteria for assessing the quality of sur-
face water are presented in Table 4.
So, according to physical and chemical indi-
cators, the surface waters of the Siret River ba-
sin, in accordance with the existing requirements
Table 3. Classes of microbiological quality of water
Sampling sittes (N0)
1 2 3 4 5 6 7 8 9
Exellent quality Poor quality Sucient

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Ecological Engineering & Environmental Technology 2023, 24(9), 55–63
of Ukrainian standards [24, 25, 27], belong to the
4th class of water quality. The waters of the upper
reaches of the basin and sparsely populated bor-
der areas belong to the rst I and II classes. The
water pollution index varies for these areas in the
range of 0.28–0.78, which corresponds to the na-
tional classication of very clean and clean water
classes. Sampling points (3, 4, 5, 6, 7), located in
the zone of active anthropogenic activity and ur-
ban landscapes, belong to III and II water quality
classes (polluted and dirty water). It should be not-
ed that the existing oodwater monitoring system
in Ukraine does not provide for the determination
of microbiological indicators (Pro zatverdzhennia
Poriadku…, 2018). The above assessment of the
Siret River basin according to microbiological
indicators, in accordance with the requirements
of the EU Water Framework Directive (Directive
2006/7/EC…, 2006), made it possible to compare
the results obtained according to physico-chemical
and microbiological indicators. The results of the
conducted research indicate that water sampling
points 3–8 are characterized by an unsatisfactory
ecological condition, both in terms of physico-
chemical and microbiological indicators.
CONCLUSIONS
For the rst time, the sanitary and ecological
condition of the upper reaches of the Siret River
(Danube basin) on the territory of Ukraine was
analyzed. 6 physico-chemical and 4 microbiologi-
cal indicators were studied in 9 sampling sites.
The conducted studies showed that the main cause
of physical, chemical and microbiological pollu-
tion of the Siret River basin within the territory of
Ukraine is the lack of centralized treatment facili-
ties in the region. The greatest pollution was ob-
served in urbanized regions. The lowest values of
the studied indicators were observed in the upper
reaches of the Siret River basin, located mostly in
the territory of the Vyzhnytskyi National Nature
Park and mountainous sparsely populated areas
with little anthropogenic inuence. The obtained
microbiological indicators indicate signicant
water pollution in a larger number of sampling
sites than according to physical and chemical in-
dicators. A comparative analysis of the obtained
results with the results of research conducted in
the lower part of the Siret River basin in the area
of Galati (Romania) was carried out. Taking into
account the cross-border nature of the studied ter-
ritory, the use of sanitary and microbiological in-
dicators in the monitoring system of the upper part
of the water ecosystem of the Siret River is urgent.
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