Phytoplankton and Bacterioplankton in Three

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Reference Sites According to the Water Framework ... bacterioplabkton abundances were investigated during spring and summer of 2005 in three reservoirs in.
ACTA ZOOLOGICA BULGARICA Acta zool. bulg., Suppl. 2, 2008: 155-164

Phytoplankton and Bacterioplankton in Three Reservoirs (Northeastern Bulgaria) Recommended as Potential Reference Sites According to the Water Framework Directive of EU Michaela B. Beshkova*, Hristina V. Kalcheva, Roumen K. Kalchev Institute of Zoology BAS, 1, Tsar Osvoboditel Blvd., Sofia 1000, Bulgaria; e-mail: [email protected]

Abstract:

Some trophic characteristics as chlorophyll-a, Secchi-disk transparency, turbidity, phytoplankton and bacterioplabkton abundances were investigated during spring and summer of 2005 in three reservoirs in northeastern Bulgaria, suggested by the experts of the Ministry of Environment and Waters (MEW) as potential reference sites. Different characteristics show differences in the assessment of the reservoir trophic status as only Tsonevo seems to be appropriate to reference site on the basis of the most of characteristics studied. Secchi-disk transparency seems to be inappropriate for assessment of the ecological status of the studied water bodies without considering its different components especially at the time of spring high waters. It is necessary to separate clearly different zones (water bodies) of reservoirs, which strongly depend on the morphometry, seasons, retention time and development of their shoreline. Phytoplankton (both abundance and composition) seems to be a good indicator for the differentiation of reservoir zones. Bacterial contribution to some of investigated variables (e.g. turbidity) and bacteria-algae relationships were also studied. Indication for top-down control of mixotrophic algae on bacteria was obtained under conditions of P-limitation.

Key words: reservoirs, phytoplankton, bacterioplankton, relationships, reference sites

Introduction The Water Framework Directive (WFD 00/60/EC) of EU is developed in order to establish the framework for community action in which aquatic environments have to be protected in order to achieve a good water status for all waters by year 2015 (BURASCHI et al. 2005).The directive focuses mainly on ecological status estimated by means of biological quality criteria. First steps in the implementation of WFD are the typologization of all water bodies and the choice of reference sites as a crucial element for determining the reference conditions. The present work is a

part from a pilot, (preliminary, tentative) examination of how much the reservoirs offered by the MEW experts as a potential reference sites within a corresponding category, really seem to correspond to criteria, grounded in definition of reference conditions on the basis of phytoplankton (chlorophyll-a) and some related factors. Biological parameters integrate in themselves all elements (morphometrical, hydrological, chemical, physical, etc.) and interactions in the water ecosystems. The definition of the ecological status of a reservoir is a complicated task because

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Beshkova M., H. Kalcheva, R. Kalchev of interacting of two different water ecosystems (lotic and lentic) characterized by high spatial and temporal variability. The river-lake systems depend on a variety of specific factors as water residence time, development of shoreline, etc., and are additionally complicated by the human activity. We accepted the suggestion of BURASCHI et al. (2005) working on the typification of Italian lakes, that the first condition for a certain water body to be a potential reference site is to have oligotrophic status, as defined by OECD (VOLLENWEIDER 1982). Phytoplankton (or chlorophyll-a) considered an appropriate biological factor together with other parameters as phosphorus and transparency lay in the basis of most frequently used classification systems, like this of OECD or some indices like the trophic state index (TSI) (CARLSON,1977). The relevance of the Secchi depth transparency in estimating the trophic status is quite controversial, because in artificial water bodies the transparency–chlorophyll relationship is not always clear due to non-phytoplankton turbidity (CANFIELD, BACHMANN 1981, LIND 1986). Therefore we examine some of these relations from the point of view of mentioned traditional trophic classifications and used the terms “reference” as a synonym of oligotrophy. The main goal of this study is rather to mark some points, which might be taken into consideration at further work on the definition of ecological status considering its evaluation on the basis of phytoplankton and related variables. The accurate implementation of WFD needs more comprehensive investigation of biological and concomitant chemical variables in sufficient number of reservoirs, sampling sites within each reservoir and sampling frequency. Besides this we draw attention to one usually neglected contributor to the ecological state, namely bacterioplankton. The interactions between phytoplankton and bacteria communities are complicated and scantily studied. They cover a variety of interactions – commenasilm/competition (CURRIE, KALFF 1984; BRATBAK, THINGSTAD 1985, GROVER 2000), predation (BIRD, KALFF 1987, OLRIK 1998) and mutualism (CURRIE 1990). Aquatic ecologists have recently documented the widespread distributions and functional relationships of these various organisms in aquatic ecosys156

tems and their relation to the organic and inorganic supply conditions, which however are still far to be fully uncovered (GROVER 2000). Description of the Reservoirs The studied Tsonevo, Ticha and Kamchia reservoirs are located in the region of northeastern Stara Planina Mountain (Balkan Range), in the valley of the Kamchia River (Ticha Reservoir) and the Luda Kamchia River (Tsonevo and Kamchia reservoirs). The surrounding landscape is hilly and the main rocks are of calcareous nature. Ticha and Tsonevo are used for irrigation, while Kamchia and partly Ticha – for a drinking water supply. The main morphometric characteristics and the nutrient data for average total phosphorus and nitrogen of the year 2005 (taken from the annual report of the Ministry of Environment and Waters and the Ministry of Health, published in Internet) are presented in Table 1. According the average theoretical retention time the reservoirs are of lake-river type. Table 1. Morphometric characteristics, total phosphorous (TP)*, total nitrogen (TN)* and trophic evaluation after OECD (by TP) and after Likens (by TN) classification system Ticha Altitude , m a.s.l Area, ha Volume,106 m3 Length, km Маx depth,. m TP, mg l-1 ** TN, mg l -1 ** Trophy (by TN) N:P rato (by atoms.) Trophy (by TP)

186 1870 311.8 16 50 0.02 1.34 meso 134.6 oligo

Tsonevo 67 1730 330 25 39 0.03 0.495 oligo 36.47 oligo

Kamchiya 253 900 234 26,4 40 0.07 1.00 meso 32.09 meso

*Data taken from the annual report of MEW and MH ** Annual average value for the year 2005

Materials and Methods The Tsonevo and Ticha reservoirs were sampled twice within 2005 – in spring (March-April) and in summer (August), while Kamchia was visited only in summer. In spring one integrated sample was

Phytoplankton and Bacterioplankton in Three Reservoirs (Northeastern Bulgaria)... taken from each of the 7 to 8 sampling sites along the reservoir from the river part to the dam. The integrated samples were obtained by mixing equal volumes of bathometric samples from several layers within euphotic zone (Secchi-depth multiplied by 2.7). Different sub samples were taken from this mixed sample in order to analyse the phytoplankton, bacterioplankton and chlorophyll-a. Water temperature (ºC) was also measured in situ from several depth layers. In the summer the samples were collected at the deepest part near the dam from several depths between 0 and 15 m depending on the vertical temperature profile and the thermocline localization. The samples for phytoplankton and bacterioplankton were preserved with formalin (to 4% for the phytoplankton and to 2% for the bacterioplankton). Phytoplankton was counted on a light microscope in the chamber of Bürker (200x) and numbers were expressed as individuals l-1. The phytoplankton biomass (fresh weight, mg l-1) was obtained after estimating the individual cell volumes (of at least 30 organisms) using simple geometric formula (ROTT 1981). Bacterioplankton samples were filtered under vacuum through membrane filters (Sartorius, Germany) with a pore size of 0.2 μm and stained with erythrosin (RASOUMOV 1932, in its contemporary modification, NAUMOVA 1999). Numbers were obtained by direct count of bacteria (at least 600 cells in average on 20 random fields) using immersion, under a phasecontrast microscope (magnification 1600x). For the biomass estimation up to 400 cells were measured. The bacterial biomass was calculated in the similar way like phytoplankton biovolume and the obtained volume was converted in carbon content, according the allometric relationship of NORLAND (1993) as cited by STRAŠKRABOVÁ (1999). Multiple and simple linear regression (general linear model) was used to examine the relation between total turbidity, chlorophyll-a, numbers and biomass of phytoplankton, numbers and biomass of bacterioplankton, and concentration of phaeopigments. Correlations were estimated by means of Spearman nonparametric coefficient. The transparency (SD, m) was measured by Secchi-disk. The total turbidity (TT, m-1) was ob-

tained as a reciprocal value of the Secchi-disk depth. The ratio of non-phytoplankton (algal) turbidity (NAT) was calculated for each reservoir separately by derived from the regression between the total turbidity and vital chlorophyll-a. The presented equation 1/S=0.339 + 0.547Chl-a, Р=0.003 is for Tsonevo Reservoir.

Results Horizontal Distribution of Different Parameters in Tsonevo and Ticha Reservoirs In spring almost complete homothermy and holomixis were registered in Tsonevo with temperature difference along the vertical axis within 0.5 1 оС interval and horizontal fluctuations of surface water temperature from 9 оС (river part) to 7 оС (near the dam). Ticha was thermally more heterogeneous, with a weak stratification (about 3 оС) at sampling sites 4 and 6. The average theoretical retention time (RT) was low as a whole (Fig. 1, A) and remained within the lower limits for the Bulgarian reservoirs. The Secchi-disk transparency (SD) was unexpectedly low for not anthropogenically impacted water bodies, which they are assumed to be (Fig. 1, B) Conversely, the total turbidity (TT) was high in both reservoirs (Fig. 2, C, D). In Tsonevo it sharply decreased to the dam synchronously with chlorophyll concentration, as the slope was more abrupt between 5 and 4 stations (Fig. 2, A). Non-algal turbidity (NAT) first decreases from the river part to point 5 and thereafter increases to station 1 (Fig. 2, C). The relationship between turbidity, biomass of phytoplankton (BMph), biomass of bacterioplankton (BMB), and phaeopigment concentration (Pheo) was studied by means of multiple regressions method. The turbidity was found to be related to bacterioplankton and phaeopigments by following equation: (E 1) TT = – 0,47 + 1.74. BM B – 0.44. Pheo; (R= 0.99, P = 0.003) It is obvious that the main contributor to the turbidity is bacterioplankton (positive relation). In this month bacterioplankton ranged between 0.81 157

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and 1.82 x 105 cell ml-1 in numbers and from 1.036 to 2.588 μg C l-1 in biomass decreasing fluently from river to the dam (Fig. 3). The change of bacteria biomass was concurrent with that of chlorophyll-a and TT but inversely related to those of NAT (Fig. 2, Fig. 3). Phytoplankton ranged from 99.103 to 952.103 ind. l-1 in numbers and from 0.05 to 1.59 mg l-1 in biomass. Phytoplankton biomass reached a pronounced peak at station 5 (about 20 km from the dam, 9 m of depth) (Fig. 3). Phytoplankton composition also varied in different parts of the reservoir (Fig. 4, A). In the river part (stations 6 and 7) the main part of phy158

toplankton biomass was formed mostly by diatoms (Nitzshia acicularis W. SMITH, Stephanodiscus sp.). In the transitional part an increase of flagellated algae from chrysophyceae (Synura sp.) at station 5 and cryptomonads (Rhodomonas sp., Cryptomonas sp.) at stations 4 and 2 and (Fig.4, A) was observed. In the deepest station the diatoms prevailed again and were presented by small centric species (Stephanodiscus sp.). No correlation between bacteria and phytoplankton abundances was observed. However the correspondence between bacterioplankton and chlorophyll-a was stronger than between chlorophyll-a

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and phytoplankton. In Ticha neither TT, nor NAT changed particularly along the reservoir (Fig. 2, D). It may be said regarding these variables that the whole reservoir seems like the lake part of Tsonevo. Better expressed was the gradient of the chlorophyll, which reached two peaks in station 6 and 8 respectively and sharply decreased down to the dam (Fig. 2, B). The total turbidity, which was almost completely non-algal (Fig. 2, D) was found to be related mainly to phaeopigments by the equation: (E 2) TT = 0.70 + 0.05. Pheo; (R= 0.82, P = 0.046) Bacterioplankton numbers ranged from 2.18 to

1.56 x 105 cell ml-1 and biomass from 3.849 to 2.496 μg C l-1. Bacteria abundance at first rose from river to station 4 (about 7.5 km from the dam) and later slightly decreased to the dam (Fig. 3). Phytoplankton numbers ranged from 685 to 4632. 103 ind.l-1 and biomass from 0.21 to 2.95 mg l-1. The peak of phytoplankton coincides with one of the peaks of chlorophyll registered at station 8 (about 16 km from the dam) (Fig. 3, Fig. 2 B). Downstream the changes of phytoplankton biomass were not significant. The phytoplankton structure is presented at Fig. 4, B. The small centric diatoms (Stephanodiscus sp.) formed a high part of the phytoplankton biomass especially in lacustrine zone (the deepest station). In 159

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Fig. 3. Horizontal distribution of bacterioplankton biomass (μg l-1), phytoplankton biomass (mg l-1) and total turbidity (m-1) in Tsonevo and Ticha reservoirs in March 2005.

the other points (from 4 to 8) the ratio of flagellated algae (Rhodomonas sp., small Chlamydomonas-like species and unidentified flagellates) was high (Fig. 4, B). Bacteria numbers (NB) and biomass (BMB) were found to correlate negatively with phytoplankton numbers (NPh) and biomass (BPh) as well as with flagellates numbers (NFl) and biomass (BFl). Stronger correlations were observed between (NB x NPh, : (R= – 0.96; P= 0.0005) and BMB x BFl: (R= – 0.96, P= 0.0005). Vertical Distribution of Different Parameters in Tsonevo, Ticha and Kamchia Reservoirs in Summer In August the reservoirs were well stratified and the stratification pattern was closely related to the reservoir’s altitude (Fig. 5). Thus the widest epilimnion was observed in the lowest situated Tsonevo, while in the uppermost Kamchia the epilimnion was very short and the thermocline began immediately after the first 1-2 m of depth. The SD was low, comparable to that in March

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(Fig. 1, B), but the ratio of non-algal turbidity was lower. Not typically, the flashing rate in Ticha was higher in summer than in spring (Fig. 1, A) which may be due to intensive irrigation. Both phyto- and bacterioplankton were considerably higher in Ticha than it the other two reservoir (Fig. 6). They also were more abundant in summer than in spring, as the differences between spring and summer values were higher as regards the bacterioplankton. Thus the ratio between summer and spring values (on average) of the phytoplankton biomass was 2.2 in Tsonevo and 4.7 times in Ticha, while in terms of bacterioplankton they were 8.8 and 9.8 respectively. This shows that bacterioplankton increases in abundance faster than phytoplankton does. The vertical distribution of phytoplankton and bacterioplankton showed similar pattern in Tsonevo and Ticha (Fig. 5). Bacterial numbers and biomass increased with the depth and attained maximum val-

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The assessment of trophic status showed differences depending on different parameters used. By toPhyto ,mg l-1 ; Bacterio ,μg l -1 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 tal phosphorus and nitrogen only Tsonevo fall into 0,3 the oligotrophic range after VOLLENWEIDER, KEREKAS 5 10 (1982) and LIKENS (1975). Ticha was in the upper 15 limits of mesotrophy (by TN) and Kamchia showed 20 BM phyto 25 mesotrophic stage. In all the three reservoirs the SD BM bacterio 30 showed worse conditions than supposed for the refTemperature 35 erence sites. This is especially true in Tsonevo in 0 5 10 15 20 25 30 0 Temperature, ɋ spring where the TSI index by SD (CARLSON 1977) showed hypertrophic to eutrophic conditions from Fig. 5.Vertical distribution of bacterioplankton biomass (μg l-1), phytoplankton biomass (mg l-1) and temperature riverine to lacustrine part. At the same time the in(ºC) in A – Tsonevo, B – Ticha, C – Kamchia in August dex calculated by chl-a showed oligotrophy along 2005. the whole reservoir which indicated that the light ues at 15 m. The maxima of phytoplankton and chlo- limitation on the phytoplankton is very probable in rophyll-a were in the upper layers of the epilimnion this season. In Ticha SD showed eutrophy in all of (0.3-3 m) where phytoplankton consisted mainly of the sampling sites, while chl-a indicated eutrophy in green chlorococcal algae. At the depth of 15 m the riverine, mesotrophy in transitional and oligotrophy centric diatoms (Cyclotella) prevailed. In Ticha the in lacustrine parts. In summer Tsonevo and Kamchia bacterioplankton showed a second lower peak at 3 were close to the reference (oligotrophic) conditions m, which coincided with the maximum of phyto- according chl-a, but showed meso or meso-eutrophy by SD (after CARLSON 1977). These discrepancies plankton and chlorophyll-a. In Kamchia Reservoir the peaks of phytoplank- were caused by high share of non-algal turbidity ton (numbers, biomass, vital chlorophyll-a) and in spring (Fig. 2). Our results support the observa161

Beshkova M., H. Kalcheva, R. Kalchev tions of other authors (Lind 1986) who found that the Secchi depth data are inappropriate in estimating of trophic states of waters containing even moderate amounts of non-algal turbidity. Therefore the Secchidepths – chlorophyll-a relationship has to be drawn and “calibrated” for each reservoir separately. If the non-algal turbidity is high and variable, such calibrations are meaningless because of the great variation in turbidity both by sampling station and by time. Furthermore it is appropriate (especially for the Tsonevo Reservoir) to distinguish at least three water bodies with different trophic status which were well differentiated by all variables. The river part was characterized with higher turbidity, chlorophyll and bacteria abundance obviously brought or developed shortly after the river inflow. In this part the phytoplankton consisted of diatoms. In the transitional zone as a consequence from the intensive processes of sedimentation and mineralization the TT, bacterioplankton and chlorophyll declined, but the algal abundance sharply increases probably as a result of the ecotone effect. In this part the proportion of flagellates, which are known as good competitors at the transitional conditions (OLRIK 1994) increased. Thereby, it may be said that the phytoplankton abundance and composition well differentiated the separate reservoir zones. The lacustrine part was characterized by low trophy (low TT, bacterio- and phytoplankton), but higher proportion of non-algal turbidity and share of phaeopigments. The results from the equation (E1), the high correspondence between bacteria and chl-a and the worse correspondence between phytoplankton biomass and chl-a suggested that the TT might be caused by autotrophic bacteria which normally decreases towards the dam. However, it is not clear which is the reason and character for the rise of non-algal turbidity in the same direction. It is possible that other factors like zooplankton grazing or other trophic interactions might have an influence. The differentiation of distinct zones in respect to SD was not so clear in Ticha, where the transitional zone was not well manifested. No substantial gradient was observed in respect to TT and NAT (Fig. 2, D), but chlorophyll concentration and phytoplankton 162

biomass were much higher at the riverine part (Fig. 2 B, Fig. 3). One possible reason for absence of clear differences between different parts of reservoir might be the more developed shore line of Ticha reservoir, namely the influence of some additional rivers with discharges similar to that of the main feeding river. Perhaps they cause an overlap of several transitional zones which mask the main one. It was found that in this reservoir the main contributor to the total turbidity were phaeopigments (E2), while in Tsonevo the bacterial biomass seemed to be the main contributor to the total turbidity. Therefore, it is appropriate to use the already known classification of OECD (1982) to distinguish the different parts of reservoirs. A good example for such differentiation by the trophic assessment of different parts of the reservoir on the base of Carlson indices is the investigation of Kardzhali Reservoir (TRAYKOV at al. 2003). Bacterioplankton and Phytoplankton Similarly to other data reported (STRASKRABOVA, 1979, PERRY at al. 1990) the spatial and seasonal changes of the bacteria abundance are better expressed than the vertical gradient during the time of summer stratification. Furthermore the seasonal changes (differences between spring and summer abundances) of bacterioplankton biomass were better expressed than the algal ones. As was reported by CURRIE (1990), the nature of the relationship between algae and bacteria may change with trophic status. In the most oligotrophic lakes, as TP increases bacteria increase in abundance more rapidly than the algae reaching a maximum at TP concentration near to 57 μg liter-l. As TP increases above this level, algal abundance increases more rapidly than the bacterial abundance does. Other authors pointed out the fact that bacteria are better competitors for P than algae, especially at more oligotrophic conditions (BRATBAK, THINGSTAD 1985, GROVER 2000). The high N:P ratio (Table 1) suggests that the phytoplankton may be phosphorus limited. This especially refers to Ticha where the ratio is very high. This may be an explanation for the negative correlation between algae (and flagellates) and bacteria observed in this reservoir. If

Phytoplankton and Bacterioplankton in Three Reservoirs (Northeastern Bulgaria)... we suppose that the observed correlation is an indicator for some top-down effect of algae on bacteria, it might be due to the strong P limitation. At such condition the mixotrophic flagellates might switch to a phagotrophic manner of nutrition. Unfortunately the quantity of data is rather insufficient for a correct interpretation of the complex interaction between bacterioplanton and phytoplankton in the reservoirs.

Conclusions 1. The characteristics like Secchi-disk transparency, phytoplankton biomass and chlorophyll-a show differences in the assessment of the reservoir trophic status. Phytoplankton biomass and chlorophyll-a indicated comparatively low trophy only for Tsonevo and Kamchiya which therefore seems appropriate for the reference sites. The results confirm the already established fact that in the contemporary trophic classification systems for stagnant waters the phytoplankton biomass values draw the waters closer while the chlorophyll-a contributes to their differentiation. 2. The Secchi-disk transparency of the studied water bodies seems to be inappropriate for assessment of their ecological status without considering of its different components. It seems useless during spring at times of high waters. It should be applied with caution for evaluation of ecological status taking into account different types of reservoirs. For its successful application it is necessary to obtain the

regression equation chlorophyll-a-transparency on sufficient number of reservoirs with Tsonevo as a representative for reference conditions. It may serve as a basis for adaptation of some widespread indices as TSI index of CARLSON (1977). 3. It is necessary to separate clearly different zones (water bodies) of reservoirs which strongly depend on the morphometry, seasons, retention time and the development of shoreline, as the last should be taken into account for typology of reservoirs. Phytoplankton (both abundance and composition) is a good indicator for the differentiation of different zones of reservoirs. According to chlorophyll-a values measured during the investigation only Tsonevo together with all of its three zones may be considered a reference site. Since data are not mean, but rather isolated values, the answer of the question whether the reservoirs may be chosen for reference site could not be answered definitely. It needs additional measurements during the four seasons and on many other reservoirs of the same type. 4. There are some indications that bacteria-algae relationship depends on the trophic conditions and that mixotrophic flagellates might exert a topdown effect upon bacterioplankton under conditions of severe P limitation. Acknowledgements: The investigation is financed by the BMEW within the project “Setting the system of bio monitoring in correspondence to the application V of the Water Framework Directive of EU (2000/60/ЕU)”

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Фитопланктон и бактериопланктон в три язовира (Североизточна България), предложени като потенциални референтни пунктове според Рамковата директива за водите на ЕС М. Бешкова, Хр. Калчева, Р. Калчев

(Резюме) Изследвани са хлорофил-а, прозрачност, мътност, количество на фитопланктона и бактериопланктона през пролетта и лятото на 2005 г. в три язовира в североизточна България, предложени от МОСВ като потенциални референтни места за съответната категория водни басейни. Отделните характеристики показват различия при оценката на трофичния статус, като само язовир Цонево изглежда по-близък до критериите за референтен пункт на базата на повечето изследвани параметри. Прозрачността по Секи изглежда неподходяща за оценка на екологичния статус на изследваните водни тела без отчитане на отделните и компоненти особено по време на пролетното пълноводие. Необходимо е ясно да се диференцират отделните зони (водни тела) в язовирите които до голяма степен зависят от морфометрията, сезоните, времето на престой на водата и развитието на бреговата линия. Фитопланктонът (както количеството, така и съставът му) е добър индикатор за диференциацията на отделните зони на язовирите. Изследвани са също отношенията между фитопланктона и бактериопланктона. Установени са индикации за top-down контрол от миксоторофните водорасли спрямо бактериопланктона в условията на фосфорно лимитиране. 164