MICROBIOLOGICAL STUDY OF THE ANTHROPOGENIC PRESSURE ON THE YANTRA RIVER IN THE REGION OF VELIKO TARNOVO

Abstract

The report discusses the microbiological status of the Yantra River in the region of the town of Veliko Tarnovo. Water samples collected at 11 points of the river stretch were analyzed for quantification of E. coli and coliforms, intestinal enterococci and heterotrophic bacteria. It was found increased levels of fecal indicator bacteria in the river waters, varying over time and along the riverside. The number of viable E. coli and enterococci increased under the influence of untreated wastewater influx and the Belitsa River.

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Ecological Engineering and Environment Protection, No 2, 2022, p. 18-29
doi.org/10.32006/eeep.2022.2.1829
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the Creative Commons Attribution License
MICROBIOLOGICAL STUDY OF THE ANTHROPOGENIC PRESSURE ON
THE YANTRA RIVER IN THE REGION OF VELIKO TARNOVO
Zvezdimira Tsvetanova, Dimitar Dimitrov, Hristo Naidenski
The Stephan Angeloff Institute of Microbiology at the Bulgarian Academy of Sciences
Abstract: The report discusses the microbiological status of the Yantra River in the region of the town of Veliko
Tarnovo. Water samples collected at 11 points of the river stretch were analyzed for quantification of E. coli and
coliforms, intestinal enterococci and heterotrophic bacteria. It was found increased levels of fecal indicator bacteria in
the river waters, varying over time and along the riverside. The number of viable E. coli and enterococci increased
under the influence of untreated wastewater influx and the Belitsa River.
Keywords: river water, E. coli, fecal indicators, intestinal enterococci, P. aeruginosa
INTRODUCTION
The reduction of water resources and the
increasing anthropogenic impact on them are global
socially significant problems. To ensure sustainable
social development and ensure the water needs of
future generations, scientifically based management
and protection of water resources is necessary, an
important part of which are the systems for monitoring
and controlling the quality of waters [9, 20, 22].
According to the Water Framework Directive
2000/60/EC, the ecological status of surface waters
is classified on a 5-point scale, as very good, good,
moderate, bad and very bad, based on 4 groups of
indicators ‒ biological, hydromorphological, chemical
and physicochemical elements, and specific
pollutants [9]. However, the framework directive
does not provide for the monitoring of
microbiological indicators, only the Bathing Water
Directive 2006/7/EU [10] includes the assessment
of the microbial load of this type of waters by the
content of fecal indicator bacteria. There are data
from periodic studies on the ecological status of the
largest European river ‒ the Danube River, in
which the waters along the entire river course are
analyzed not only for the regulated chemical,
physicochemical and biological indicators, but also
for the content of fecal indicator bacteria, as
established an indirect indicator for the assessment
of waterborne pathogens [17, 19, 20].
The presence and degree of fecal pollution of
waters are an important indicator of their quality.
Contamination of surface water with fecal indicator
bacteria may be caused by point sources ‒ discharge
of treated or untreated waste waters, as well as
by diffuse pollution through water runoff from
settlements, agricultural lands and pastures. River
birds are also a possible source of fecal pollution,
[11, 14, 16].
Studies of river water for E. coli and intestinal
enterococci content provide evidence of fecal
contamination and potential ecological and health
risk. For practical purposes, various classification
systems have been developed for the interpretation
of microbiological data on surface water quality,
[17, 18, 19]. For example, during the periodic pan-
European monitoring of the Danube River,
different classification systems were used to assess
the organic and fecal pollution of river waters, and
Table 1 presents the limits for the degree of water
pollution according to two of these systems.
Table 1а. Degree of microbiological pollution of river waters
Analyzed
parameters
Class I
(low)
Class II
(middle)
Class III
(critical)
Class IV
(strong)
Class V
(еxcessive)
Organic pollution, CFU/ml
НРС, 22оС
< 500
> 500-10000
> 10000 - 100000
> 100000 - 750000
> 750000
Fecal pollution, CFU/100 ml
Coliforms (total)
< 500
> 500 - 10000
> 10000 - 100000
> 100000 - 750000
> 1 000000
Fecal coliforms
< 100
> 100 - 1000
> 1000 - 10000
> 10000 - 100000
> 100000
Fecal enterococci
< 50
> 50 - 100
> 100 - 1000
> 1000 - 10000
> 10000

Ecological Engineering and Environment Protection, No 2, 2022, p. 18-29
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An improving trend in water quality has been
reported for many European rivers over the last
decade. The better quality of river waters creates
prerequisites for their use for irrigation, recreation
and sports (fishing, swimming, various water sports).
Researchers continue to debate, however, whether the
water quality assessment system adopted in Directive
2000/60/EC is sufficient to manage river waters,
especially those used for recreation and sports, and
whether the inclusion of additional sanitary-hygienic
indicators (microbiological, toxicological, etc.) can
improve this assessment, despite the fact that water
bodies intended for recreation, including bathing
waters, are registered as protected areas. There are
opinions that the limit values are too liberal and that
the high content of fecal indicators in the waters for
recreation and sports may create conditions for a
health risk, as confirmed by the cases of leptospirosis
found among swimmers in waters with unsatisfactory
microbiological quality [12, 14, 18].
Table 1b. Degree of microbiological pollution of river waters
Analyzed
parameters
Class I
(low)
Class II
(middle)
Class III
(critical)
Class IV
(strong)
Class V
(excessive)
Organic pollution, CFU/ml
НРС, 22оС
< 500
> 500 - 10000
> 10000 - 100000
> 100000 - 750000
> 750000
Fecal pollution, CFU/100 ml
E. coli
< 100
> 100 - 1000
> 1000 - 10000
> 10000 - 100000
> 100000
Intestinal
enterococci
< 40
> 40 - 100
> 100 - 1000
> 1000 - 10000
> 10000
In the literature, there are reported data on a
discrepancy between the improved ecological status
of river waters and their unsatisfactory quality in
terms of microbiological indicators [19, 20, 21].
Data for the Mur River in Austria reveal a similar
discrepancy, showing that river water in 20 out of
21 sites tested did not meet the requirements for
satisfactory quality in terms of fecal indicator
bacteria, and Salmonella spp. was detected in 45% of
all samples, including 30% of those with good
quality. This has given the authors reason to consider
that the assessment of fecal indicators needs to be
an integral part of research to assess the ecological
status of river waters [18].
Similar data on deviations in the content of
fecal indicator bacteria were reported for the
Danube, Sava and Nišava rivers [23]. During the
microbiological monitoring of the Danube River,
the water in ¼ of the examined points was
classified as critical, highly or excessively
polluted. The river sections between Novi Sad
and Belgrade, after Budapest, after Zimnica and
Arzes, including the river tributaries Rusenski
Lom and Arzes have been identified as "hot
spots" of fecal pollution [17, 19]. A quantitative
difference in the content of fecal indicator
bacteria in the water samples taken from three
points along the section of the same river section
‒ the thalweg of the river and both banks ‒ was
proven. Significantly higher values were observed
in coastal waters, while river waters in the
midstream remained unaffected or less affected
by high concentrations of microbial pollutants in
discharges of untreated waste waters, treated
waste waters from wastewater treatment plants
(WWTPs), or inflowing river tributaries [19, 20].
For this reason, it is considered that sampling
from both banks, in addition to the middle course
of the river, allows a deeper assessment of the
faecal pollution pattern of the Danube River and
the microbiological status of the river waters. It is
likely that the water quantities are of decisive
importance, since according to data for smaller
rivers, the sampling location along the river
section has a negligible influence on the
distribution of the indicator bacteria in the water
volume [13].
At the end of the 20th century, an improvement
in the quality of river waters was observed in
Bulgaria as a result of the changed economic
situation and the reduced activity or closure of a
number of industrial enterprises, emitters of
industrial waste waters. Efforts to build sewerage
networks and WWTPs in smaller settlements also
contribute to limiting the pollution of river waters.
For these reasons, the main goal of the present study

Ecological Engineering and Environment Protection, No 2, 2022, p. 18-29
20
is to study the state of one of the large Bulgarian
rivers from a sanitary-hygienic point of view by
determining the fecal indicator bacteria. The object
of study is the section of the Yantra River in the
area of the town of Veliko Tarnovo and its
surroundings, including the right tributary of the
Belitsa River, flowing in immediately before the
Yantra River enters the city territory. In accordance
with the set objective, the microbiological state of
the river waters was studied and the influence of the
tributaries and two point sources of pollution with
untreated domestic wastewater discharged into the
river section was assessed.
MATERIALS AND METHODS
Description of the studied river section
The studies of the Yantra River were carried
out in a river section covering the passage of the
river through the territory of the town of V. Tarnovo
and ending at the village of Samovodene (Fig. 1).
The studied section also includes the right tributary of
the Belitsa River flowing into the beginning of the
city, studied in its lower course, before and during its
passage through the town of Debelets until its
confluence with the Yantra River, including the
confluence of the Dryanovska River in this section
at the end in the town of Debelec.
Fig. 1. Scheme of the Yantra River and its tributaries in the area of the town of
Veliko Tarnovo and surroundings, including the river water sampling points
The investigated section of the Yantra River
basin falls into the water body described in the
national register of water bodies BG1YN700R1017 ‒
the Yantra River from the confluence of the Belitsa
river near the town of V.Tarnovo to the confluence
of the Lefedja River near the village of Gorski dolen
Trumbesh. The section of the Yantra River before
reaching the town of V. Tarnovo falls into water
body BG1YN900R1015 ‒ the Yantra River from the
confluence of the Kozlyata River near the town of
Gabrovo to the confluence of the Belitsa River near
the town of V. Tarnovo. Part of the studied river
section are the water bodies: BG1YN800R1033 ‒
Belitsa River from the village of Voneshta
Voda to its confluence with the Yantra River
and BG1YN800R1016 ‒ Dryanovska River from
Tryavna to its confluence with the Belica River
near Debelets.
Sampling points
Eleven water sampling points from the Yantra
River and the Belitsa River (Table 2) were selected
to reflect the current state of the water bodies and
the influence of inflowing untreated household-
fecal waste waters or river tributaries.

Ecological Engineering and Environment Protection, No 2, 2022, p. 18-29
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Таble 2. River water sampling points
Point
№
Description of the sampling points
R
Yantra River near the village of Ledenik or the village of Shemshevo
1
Yantra River before confluence with Belitsa River (bridge at ZZU, left side)
2
Yantra River after confluence with Belitsa River (rope bridge, Cholakovtsi quarter, left side)
3
Yantra River near Boruna (under the Asenevtsi monument, right side)
4
Yantra River, Asenev district (in front of the church "St. Peter and Paul", left side)
5
Yantra River near Samovodene village, 4-5 km after WWTP (bridge, right side)
6
Belitsa River before the town of Debelets (bridge on the road to Hainboaz)
7
Belitsa River, town of Debelets, 150 m after discharge of sewage treatment plant (right side)
8
Belitsa River after the confluence of the Dryanovska River (bridge after the town of
Debelets, left side)
9
Yantra River, Asenev district (bridge to the church "St. Sv. 40 Martyrs", left side)
10
Yantra River, 150 m after discharge of the OV (rope bridge, Cholakovtsi quarter, left bank)
Four of these points correspond to points from
the national monitoring network of the Ministry of
Environment and Water (MoEW), namely: point
No. 2 ‒ at point BG1YN00079MS200 ‒ the Yantra
River after the confluence of the Belitsa River near
the town of V. Tarnovo; point No. 5 ‒ at point
BG1YN00079MS190 ‒ Yantra River after the
town of V. Tarnovo, bridge near the village of
Samovodene; point No. 8 and the monitoring point
of the Belitsa River after the confluence of the
Dryanovska River. Point R, located on the Yantra
River near the village of Ledenik (water body
BG1YN900R1015) was chosen to reflect the state
of the river before it passage through the territory
of the town of V. Tarnovo and can be considered
as a reference point.
The selected points are located in coastal areas
commonly used for fishing, without stagnant areas
or near bridges. Two pairs of points were chosen so
as to provide an opportunity to evaluate the influence
of discharge channels with untreated waste waters:
on the Yantra River ‒ points No. 2 and 10; on the
Belitsa River ‒ the pair of points No. 6 and No. 7).
Points No. 1, 2 and 8 allow to compare the
composition of the waters of the Belitsa River and
the Yantra River and to assess the effect of the inflow
of the Belitsa River tributary. The data for points
R, No. 2, 3, 4 and 9 allow tracing the state of the
Yantra River as it passes through the territory of
the town of V. Tarnovo and the potential influence
of diffuse and point sources of fecal pollution. The
location of point No. 5, a few kilometers after the
discharge of the purified organic matter from the
urban sewage treatment plant of V. Tarnovo, allows
to clarify the self-purification ability of the river.
River water sampling procedures were
developed based on the current standards BDS EN
ISO 19458 and BDS EN ISO 25667 [1, 3]. Ten
river water sampling campaigns were carried out,
where 250 ml of water were taken from the
respective river points in sterile glass bottles.
Methods of microbiological analysis
Water samples are analyzed to quantify:
- Escherichia coli and coliform bacteria,
according to BDS EN ISO 9308-1 [7];
- Intestinal enterococci, according to BDS
EN ISO 7899 [2];
- Heterotrophic bacteria - on R2A agar, 7-day
incubation at 22oC;
- Pseudomonas aeruginosa, according to
BDS EN ISO 16266 [4];
- Salmonella spp., according to BDS EN
ISO 19250 [5].
Analytical procedures
A certain volume of the analyzed sample or its
corresponding dilution is analyzed by the membrane
filtration method for determination of E. coli and
coliform bacteria, intestinal enterococci and Ps.
aeruginosa according to the above standards. The
assay for the detection of Salmonella spp. in the

Ecological Engineering and Environment Protection, No 2, 2022, p. 18-29
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sampled waters involves pre-enrichment of the
medium with target bacteria, after which suspected
Salmonella spp. are detected by plating on a
selective nutrient medium. The number of viable
heterotrophic bacteria was determined by plating the
appropriate dilution in triplicate on R2A agar and
culturing for 7 days at 22°C.
The counting of the target bacteria and the
presentation of the results is carried out in accordance
with BDS EN ISO 8199 [6]. Results are presented
as means, with 95% confidence intervals calculated.
RESULTS AND DISCUSSION
Content of coliform bacteria and E. coli in
river waters
Forty six river water samples were analyzed
and the results obtained for the content of coliform
bacteria are presented in Fig. 2, and for E. coli
content ‒ in Fig. 3
Fig. 2. Content of coliform bacteria in river water samples during the period October 2018 – March,2019;
dark circles - river waters; grayt circles – river waters after mixing with inflowing untreated waste waters;
white circles - data from comparison point R, Yantra River before the town of V. Tarnovo.
The comparative analysis of the data on the
content of coliform bacteria in the waters of the
Yantra River passing through the territory of the
town of V. Tarnovo show from 1 to 3 orders of
magnitude higher values compared to those
established in the river section before reaching the
town (point R). Data presented on Fig. 2 reveal
significant fluctuations in the amount of indicator
coliform bacteria in individual points during the
studied autumn-winter period. At the same time,
the data show that before the research period, the
pollution of the Yantra River waters and those of the
Belitsa River in the studied section according to this
indicator was from critical to excessive, according
to the 5-level classification scale (Table 1a).
Based on the data concerning the number of
coliform bacteria (Fig. 2), the river waters of the
investigated section of the Yantra River can be
distinguished into those with less pollution (points
№. 4, 5, 6, 8 and 9) and those with a significant
content of coliform bacteria (points № 1, 7 and 10).
Similar results are also observed regarding the content
of E. coli in the river waters (Fig. 3) ‒ with the
lowest content are the waters originating from
points No. 4, 6 and 9, followed by those in points
№ 1, 5 and 8.
The data presented also show that the
discharge of untreated waste waters in the vicinity
of site point 10 caused 1 ‒ 2 orders of magnitude
higher E. coli and coliform bacteria content in the
river waters compared to the previous point
pollution site № 2. As a result of mixing of waste
waters with the river waters and their dilution, as
well as a result of the self-purification ability of the
river, the content of E. coli and coliform bacteria in
the water decreased and in point № 3 (at a distance
of several km from the pollution point) it was
similar to point № 2 values. When the river passes

Ecological Engineering and Environment Protection, No 2, 2022, p. 18-29
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through the territory of the town of V. Tarnovo (at
points № 2, 3, 4 and 9), no other significant
negative impact was registered, as the content of E.
coli and coliform bacteria continued to decrease at
points № 4 and 9 where the lowest values were
found.
Fig. 3. Content of E. coli in river water samples during the period October 2018 - March 2019; dark circles -
river water samples; grey circles – river water samples after their mixing with inflowing untreated waste
waters; white circles - data from comparison point R, Yantra River before the town of V. Tarnovo.
The quantitative comparison of the E. coli or
coliform bacteria content of the water samples
from the pair of points № 6 and 7 revealed a similar
polluting effect caused by the domestic sewage
discharged into the Belitsa River near point № 7.
The negative effect significantly decreased after
the inflow of the Dryanovska River (registered in the
point № 8), but the content of E. coli remains at least
one order of magnitude higher compared to point
№. 6. In both cases of point pollution with sewage
waste water - on the Yantra River (registered in point
№ 10) and on the Belitsa River (registered in point №
7), the dilution and self-purification potential of the
river reduced the amount of E. coli to a certain extent,
but the microbial pollution of the water remained
strong according to the 5-level classification scale
(Table 1b).
The comparison of the microbiological quality
of the water in points № 1, 2 and 8 allows to
evaluate the effect of the flow of the Belitsa River
(№ 8) into the Yantra River (№. 1), without,
however, taking into account the exact flow rate of
the individual water bodies. The dilution effect from
the inflow of the Belitsa River is more pronounced
with regard to coliform bacteria. If we take into
account the statistical data on the average annual
outflow of the Yantra River (4 m3/s at the town of
Gabrovo) and its tributary the Belitsa River (2.2 m3/s
at the village of Vaglevtsi, which subsequently
doubles from the inflow of 2.1 m3/s runoff of the
Dryanovska River) it can be seen that the two rivers
contribute equally to the average annual runoff of
11.9 m3/s of the Yantra River near the town of V.
Tarnovo (point № 2).
The data for points № 2, 3, 4 and 9 reveal a
minor change in the microbial load of the Yantra
River as it passes through the territory of the town
of V. Tarnovo, occurring as a result of diffuse or
unidentified point sources of fecal pollution. The
data for site № 5, located a few km after the
discharge of treated waste waters from the urban
WWTP, show slightly higher values compared to
sites № 4 and 9, but lower than sites № 2 and 3,
suggesting a good self-purification ability of the
river and the absence of a negative effect from the
discharged treated waste waters from the WWTP.
The graphically presented data reveal significant
fluctuations in the amount of E. coli and coliform
bacteria along the Yantra River and over time, which
can be related to the anthropogenic pressure of waste
water discharge, the inflow of river tributaries and
the self-purification ability of the water body. In
addition to point sources of fecal pollution, a number
of factors that lead to bacterial reduction can

Ecological Engineering and Environment Protection, No 2, 2022, p. 18-29
24
contribute to the fluctuations of bacterial indicators:
biotic, such as the rate of die-off in natural waters and
destruction by predatory protozoa; abiotic, such as
temperature, sunlight and sedimentation. The
individual contribution of these factors cannot be
distinguished and evaluated without additional,
specially designed experiments, but remains reflected
in the final score of the indicators. A significant
contribution to the increase in the proportion of fecal
indicator bacteria in the river body can be made by
surface and underground water runoff as a result of
heavy rainfall and floods. The amount and intensity
of rainfall and the resulting runoff can significantly
increase the content of fecal indicators in river
waters [12, 15, 22, 24].
According to the E. coli indicator, river water
pollution in the investigated section of the Yantra
River can be classified as:
- weak ( < 102 CFU/100 ml) ‒ mainly in point R;
- average (> 102 to 103 CFU/100 ml) ‒ in
separate samples from points № 6, 9 and R;
- critical (> 103 to 104 CFU/100 ml) in points
No. 1, 4, 5, 6 and 9;
- strong (> 104 to 105 CFU/100 ml)
predominantly in points № 2, 3 and 8, and in
individual samples from point №. 5;
- excessive (> 105 CFU/100 ml) – in points №. 7
and №. 10 under the influence of the inflowing
sewage sludge.
Data on the microbiological state of the Yantra
River and its tributaries can supplement the
ecological assessment of river waters by chemical
and biological elements of quality [8]. According
to data from the national monitoring system, the
condition of the water body YNRWB1017, Yantra
River in the area of V. Tarnovo, in terms of
chemical and physico-chemical indicators,
biological quality elements and specific pollutants
is moderate (due to increased values of nitrite and
total nitrogen, orthophosphates and total
phosphorus), and for priority substances
(pesticides, polycyclic aromatic hydrocarbons,
etc.) it is good. Before the confluence of the
Belitsa River, the condition of the Yantra River
near the village of Ledenik (YNRWB1015) was
assessed in a similar way ‒ moderate in terms of
chemical indicators, specific pollutants (aluminum)
and biological quality elements, and good in terms
of priority substances. The state of the Belitsa
River (YNRWB1033) before its confluence with
the Yantra River is moderate in terms of chemical
indicators, specific pollutants (iron) and biological
elements, and is influenced by the confluence of
the Dryanovska River (YNRWB1016), which has
a moderate state in terms of chemical indicators
and specific pollutants, and good status by priority
substances and biological elements.
Content of intestinal enterococci in river waters
The data presented in Fig. 4 for intestinal
enterococci show similar trends to those already
established for E. coli.
Fig. 4. Content of intestinal enterococci in river waters.; dark circles – river water samples; grey
circles – river water samples after their mixing with the inflowing untreated OM; white circles -
data from comparison point R, Yantra River before the town of V. Tarnovo.

Ecological Engineering and Environment Protection, No 2, 2022, p. 18-29
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The amount of intestinal enterococci in the
river waters is the highest in stations № 7 and 10,
which are influenced by the inflowing untreated
waste waters. The values are at least one order of
magnitude higher than the other points. With the
lowest content of enterococci are the waters of the
Yantra River at points 4 and 9, and of the Belitsa
River ‒ at point № 6.
The data on the number of intestinal
enterococci in the river waters allow the fecal
pollution of the Yantra River in the studied section
to be classified as predominantly strong and
excessive. None of the examined samples has
pollution better than average (< 102 CFU/100 ml)
and only in individual samples the content of
enterococci in the water is within the values
limited for the level of critical pollution. In the
studied river section, heavy pollution (> 103 to 104
CFU/100 ml) was predominantly recorded, and in
individual samples from sites № 3 and 8, and
predominantly in samples from site № 2, pollution
could be classified as excessive. From the
discussed data, it can be summarized that the level
of fecal water pollution in the studied river section
with intestinal enterococci is greater compared to
E. coli. By river points, the degree of enterococci
contamination is classified as severe and
predominantly excessive, while the E. coli
contamination is defined as predominantly critical
and severe.
In the investigated river water samples, the
presence of opportunistic pathogens Pseudomonas
aeruginosa (Table 3) and suspected Salmonella
spp. were proved.
Table 3. Content of P. aeruginosa in river water samples, CFU/100 ml
Date
Point №
2
3
4
5
6
7
9
10
29.01.2019
850
-
-
-
80
2000
-
10000
05.02.2019
5200
-
-
-
500
1000
-
30000
19.02.2019
-
730
1550
50
-
-
450
-
The obtained experimental data support the
efforts of legislative, executive and control institutions
in Republic of Bulgaria to maintain a good ecological
status of water resources through policies to limit
point and diffuse sources of pollution and wastewater
treatment. The results presented herewith show
that microbiological indicators complement the
information on the level of pollution of surface
waters with information important from a sanitary
and hygienic point of view, which reveals the
potential health risk in their use for irrigation,
recreation, fishing, water sports and swimming.
The data support the trends established for other
water bodies for significant anthropogenic pressure
on different microbiological indicators under the
influence of point sources of sewage water pollution
[11, 21, 24]. The ones depicted in Fig. 5 data reveal
the already discussed negative effect of the discharge
of untreated domestic sewage into the Belitsa River
and the effect of the inflow of its tributary, the
Dryanovska River. The discharge of untreated waste
waters before point № 7 leads from 2 to 3 orders of
magnitude higher values of E. coli content in river
waters compared to the unaffected by pollution
point № 6 (Fig. 5a), as well as up to 2 orders of
magnitude higher values for enterococci (Fig. 5b).

Ecological Engineering and Environment Protection, No 2, 2022, p. 18-29
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(a)
(b)
Fig. 5. Content of E. coli (a) and intestinal enterococci (b) in the waters of the Belitsa
River, before (point № 6) and after the discharge of untreated sewage (point № 7), and
after the inflow of the Dryanovska River (point № 8).
As a result of the self-purifying ability of the river
and after mixing with the waters of the Dryanovska
River, the content of indicator bacteria is significantly
reduced in the waters at point № 8.
Content of heterotrophic bacteria in river waters
The content of heterotrophic bacteria in river
waters is a measure of their pollution with
biodegradable organic substances. The data presented
in Fig. 6 show significant temporal and spatial
fluctuations in the number of viable heterotrophic
bacteria in river waters. Their quantitative content in
the river waters when the Yantra River passes through
the town of V. Tarnovo (points № 1, 2, 3, and 4) has
similar average values and varies within similar
limits. The water at the reference point R did not
differ significantly in heterotrophic bacteria counts
from the points in the urban section, in contrast to
the significant differences in the content of fecal
indicator bacteria. In points № 3, 5 and 9, the water
of the Yantra River has the lowest content of viable
heterotrophic bacteria, also in points № 6 and 8 of its
tributary, the Belitsa River.

Ecological Engineering and Environment Protection, No 2, 2022, p. 18-29
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Fig. 6. Number of heterotrophic bacteria in river water samples during the period October 2018-
March 2019; dark circles - river waters; grey circles – river waters after mixing with the
inflowing untreated waste waters; white circles - data from comparison point R, Yantra River
before the town of V. Tarnovo.
Similar to the fecal pollution data from point
sources of untreated waste water, the number of
heterotrophic bacteria was highest at sites № 2 and
10, where the nutrient content was highest. The
data are consistent with the rule of higher
microbial load in waters with a higher degree of
organic pollution. Based on the results presented, it
can be summarized that the quantitative data on
heterotrophic bacteria reveal the presence of
organic pollution, but cannot identify its origin,
unlike the data on fecal indicator bacteria, which
reveal the type and degree of pollution.
CONCLUSIONS
The waters of the Yantra River in the area of
V.Tarnovo are in a deteriorated state in terms of
microbiological indicators compared to the previous
river section.
The content of E. coli and intestinal
enterococci in the waters of the Yantra River in the
studied section:
- varies over time and along the river course;
- shows high values;
- proves pollution with untreated waste waters
from point sources;
- is influenced by the inflow of the Belitsa
River.
Opportunistic pathogens P. aeruginosa and
suspected Salmonella spp are found in river waters.
The content of heterotrophic bacteria reveals the
presence of significant organic pollution of river
waters.
REFERENCES
1. BDS EN ISO 25667-2:2000. Water quality
– Sampling. Part 2: Guide to sampling techniques.
2. BDS EN ISO 7899-2:2000. Water quality.
Identification and enumeration of intestinal
enterococci. Part 2: Membrane filtration method.
3. BDS EN ISO 19458:2006. Water quality.
Sampling for microbiological analysis.
4. BDS EN ISO 16266:2008. Water quality.
Detection and enumeration of Pseudomonas
aeruginosa. Membrane filtration method.
5. BDS EN 19250:2010. Water quality.
Detection of Salmonella spp.
6. BDS EN ISO 8199:2012. Water quality -
General guidelines for the enumeration of
microorganisms by cultivation/growth.
7. BDS EN ISO 9308-1:2014. Water quality.
Determination of the number of bacteria
Escherichia coli and coliform bacteria. Part 1:
Method by membrane filtration of waters with a

Ecological Engineering and Environment Protection, No 2, 2022, p. 18-29
28
low bacterial flora background.
8. Annual report on the state of the
environment for 2017, RIOSV-V. Tarnovo.
9. The Water Framework Directive
2000/60/EC (Directive 2000/60/EU of the
European Parliament and of the Council of 23
October 2000 establishing the framework for
Community action in the field of water policy).
10. Bathing Water Directive 2006/7/EU.
11. Naumova S.M., T.P. Todorov, 1984.
Microbiological study of the influence of the
Blagoevgradska Bistrica River on the quality of the
Struma River. Hydrobiology, 22, 25-37.
12. Abia A.L.K., E. Ubomba-Jaswa, B.
Genthe, M.N.B. Momba, Quantitative microbial
risk assessment (QMRA) shows increased public
health risk associated with exposure to river water
under conditions of riverbed sediment
resuspension, Sci Total Environ, 566–567, 2016,
1143-1151.
13. Baleux B., M. Troussellier, 1989,
Optimization of a sampling design and significance
of bacterial indicators application to the
bacteriological survey of the Ardeche River,
France, Wat Res, 23 (9), 1183-1190.
14. Berthe T., G. Gargala, M. Fournier, M.
Ratajczak, P. Servais, F. Petit, Assessment of
faecal contamination and the relationship between
pathogens and faecal bacterial indicators in an
estuarine environment (Seine, France), Marine
Pollution Bulletin, 54 (9), 2007, 1441-1450.
15. Davies C.M., N.J. Ashbolt, J.A.H. Long, M.
Donald, 1995, Survival of fecal microorganisms in
marine and freshwater sediments, Appl Environ
Microbiol, 61 (5), 1883-1896.
16. Mascher F., W. Mascher, F. Pichler-
Semmelrock, F.F. Reinthaler, G. Zarfel, C.
Kittinger, 2017, Impact of Combined Sewer
Overflow on Wastewater Treatment and
Microbiological Quality of Rivers for Recreation,
Water, 9 (11), 906, doi:10.3390/w9110906.
17. Kavka G., E. Poetsch, 2002,
Microbiology, In: Joint Danube Survey. Technical
report of the International Commission for the
protection of the Danube River (Eds: P. Literathy,
V. Koller-Kreimel, I. Liška,), 138-150.
18. Kittinger C., E. Marth, F.F. Reinthaler, G.
Zarfel, F. Pichler-Semmelrock, W. Mascher W., G.
Mascher, F. Mascher, Water quality assessment of
a Central European River - Does the Directive
2000/60/EC cover all the needs for a
comprehensive classification? Sci Total Environ,
2013, 447(2), 424-429.
19. Kirschner A., S. Jakwerth, S. Kolarevic,
R. Sommer, A.P. Blaschke, G. Kavka, G.
Reischner, A. Farnleitner, 2015, Bacterial faecal
indicators, In: Joint Danube Survey 3. A
comprehensive analysis of Danube Water Quality
(Eds: I. Liška, F. Wagner, M. Sengl, K. Deutsch,
J. Slobodník), 155-161.
20. Reischner G., A. Kirschner, G. Schnitzer,
D. Savio, R.L. Mach, A. Bahlmann, T. Schulze,
W.Brack, A. Farnleitner, Microbial faecal source
traking, In: Joint Danube Survey 3. A
comprehensive analysis of Danube Water Quality
(Eds: I. Liška, F. Wagner, M. Sengl, K. Deutsch,
J. Slobodník), 2015, 161-168.
21. Strathmann M., M. Horstkott, C. Koch,
U. Gayer, J. Wingender, The River Ruhr – an
urban river under particular interest for
recreational use and as a raw water source for
drinking water: The collaborative research project
“Safe Ruhr” – microbiological aspects., Int J
Hygiene Environ Health, 219 (7B), 2016, 643-
661.
22. Servais P., T. Garcia-Armisen, I. George,
G. Billen, Fecal bacteria in the rivers of the Seine
drainage network (France): Sources, fate and
modelling, Sci Total Environ, 375 (1–3), 2007,
152-167.
23. Stevanović S., A. Savić, Biological and
microbiological monitoring of the quality of the
river Nisava, Facta Universitatis, Series: Working
and Living Environmental Protection, 2018, 15
(3), 203 – 208.
24. Salman J.M., A.S.N. Al-Azawey, F.M.
Hassan, 2013, Study of bacterial indicators in
water and sediments from Al- Hilla river, Iraq.
Hydrol Current Res, S13: 001.

Ecological Engineering and Environment Protection, No 2, 2022, p. 18-29
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Assist. Prof. Zvezdimira Tsvetanova
Corresponding author
Department of Infectious Microbiology
The Stephan Angeloff Institute of Microbiology
Nikola Gabrovski Str. 78, fl.4
Veliko Tarnovo, Bulgaria
e-mail: zvezdimira@yahoo.com
Assist. Prof. Dimitar Dimitrov
Department of Infectious Microbiology
The Stephan Angeloff Institute of Microbiology
Nikola Gabrovski Str. 78, fl.4
Veliko Tarnovo, Bulgaria
Prof. Hristo Najdenski, DVM, DSc,
Corresponding Member of BAS
Department of Infectious Microbiology
The Stephan Angeloff Institute of Microbiology
Acad. G. Bonchev Str., Bl. 26
1113 Sofia, Bulgaria
e-mail: hnajdenski@abv.bg