Introduction

Blooms of Emiliania huxleyi (Lohmann) Hay & Mohler 1967 have been repeatedly registered all over the global ocean including the Black Sea (Morozova-Vodyanitskaya, 1957; Tyrrell and Merico, 2004; Pautova et al., 2007; Stelmakh et al., 2013). It was proposed that as the abundance of this coccolithophore is estimated 1×10⁶ cell/L and more, it is a bloom and the plentiful microalgae change optical properties of the sea water so that satellite imagery reports of “white water” (Balch et al., 1991; Tyrrell and Merico, 2004). The triggering mechanism of this phenomenon has not been clearly understood as yet. The known factors causing outbreaks of this coccolithophore species are primarily light, temperature and nutrients; biotic relations in the plankton are also of key importance. For instance, the surveys made in the Bering Sea during E. huxleyi bloom (July – August 1999) provided evidence that in the blooming sea water average specific rate of microzooplankton grazing on the phytoplankton was three times lower than the specific growth rate of the phytoplankton (Olson and Strom, 2002). The authors supposed that the low predatory impact has been the key to formation of the summer E. huxleyi bloom.

 

By carrying out this investigation we intended to gain insight firstly into the spatial variability of specific growth rate of the phytoplankton and the rate of phytoplankton consumption by microzooplankton in relation to the spring bloom in the surface of the Black Sea, and secondly into the role these processes in formation of E. huxleyi bloom.

 

1 Data and Methods

Samples of plankton were taken from the surface water layer of the Black Sea at some near-shore and open-sea areas from 20 to 29 May 2013 during the 72^nd expedition of the RV “Professor Vodyanitsky” (Figure 1). The surveys were carried out in the western (to 34° E) and eastern (34 – 37º E) parts of the sea.

 

Figure 1 Location of sampling stations

 

The rates of phytoplankton growth and loss as result of microzooplankton grazing were determined using dilution procedure (Landry and Hasset, 1982). The major advantage of this method is that it assesses the rate of total phytoplankton growth along with the rate of microzooplankton grazing on the phytoplankton. Samples of seawater (12~15 L) were taken from the sea surface (~ 0.5 m depth) early in the morning using Niskin bottle and gently poured through a 200 µm Nitex mesh. To have filtrate, а water volume of 6–8 L was sieved through a glass fiber filter (Whatman GF/F;low pressure (<0.1 atm) to avoid the breakage of the phytoplankton cells and thus to minimize their intrusion into the filtrate. The native sample was then diluted with the filtrate freshly obtained by a factor of dilution of 1.0, 0.80, 0.60, 0.40 and 0.20 in duplicates. The factor 1.0 means an undiluted sample, while the factor of 0.20 means the sample was diluted five times with filtrate. The prepared solutions were then placed into polycarbonate bottles of 1 L volume, which were prewashed with 10% HCl and then with distilled water. The bottles were incubated for 24 hours on board, opened for the solar impact, and cooled down to 20℃ by running pumped surface water of the same temperature. As the experiment ended, the water was filtered through Whatman GF/F filters. After filtration filters were placed in 90% acetone (5 mL) and chlorophyll was extracted for 24 h at 4 ºC in the dark (Protocols JGOFS, 1994). The acetone extracts were centrifuged, and their fluorescence determined before and after acidification in a fluorometer (excitation 440 to 480 nm, emission > 665 nm), which was calibrated with pure chlorophyll-a (Sigma Chemical Co). The precision of these measurements was high, with a relative standard deviation of 5%.

 

The phytoplankton growth rate was calculated under the daily increase of Chl a in the experimental bottles. The initial concentration of Chl a was determined only for the undiluted samples, while, for the diluted samples it was recalculated according to the dilution factor (DF). The observed daily phytoplankton growth rate for each of the 5 dilution treatments (µ_DF) was calculated as:

 

µ_DF = ln(Chla_final/ Chla_initial)             (1)

                           

The linear regression equations were calculated to estimate the interrelations between the observed phytoplankton growth rate (µ_DF) and the dilution factor (DF) as:

 

µ_DF = –g·DF + µ                  (2)

                                            

where µ ­is true phytoplankton growth rate (d^-1) and g – the microzooplankton grazing rate (d^-1).

 

For determination of phytoplankton biovolume and species composition, 3~4 L samples of sea water were concentrated  under the nucleopore membranes (1 μm pore size; the product of the Institute of Nuclear Researches, Dubna, Russia) in the inverse filtering plexiglass funnel, (Sorokin et al., 1975). After the samples were condensed to 50 ml, they were fixed with neutralized 1% formaldehyde (final concentration in the sample) and immediately processed. The numbers and dimensions of microalgae were measured in a 0.1 ml drop in 3~5 replications under the light microscope ZEISS Primo Star (x400). The precision of these measurements was with a relative standard deviation of 25%. Phytoplankton biovolume was calculated from cell counts and dimension measurements assuming simple geometric shapes. The abundance and biomass of microzooplankton were not determined.

 

Nitrate, ammonium, phosphate and silicon contents were measured using the previously described technique (Stelmakh et al., 2013).

 

Mathematical treatment of all data involved using Microsoft Office Excel 2007 and Sigma Plot 2001 software for Windows.

 

2 Results

By the end of May 2013, the sea surface has warmed to about 20° C, and diurnal photosynthetic active radiation (PAR) was 44 E/m²·d on the average. The depth of the upper mixed seawater layer (UML) varied from 4 to 19 m, being about 10 m on the average. Nitrate content was relatively low (0.10 – 0.30 mmol/m³) both in the shallow- and deep-water areas of the sea (Table 1).

 

Table 1 The phytoplankton growth rate (µ), the concentration of chlorophyll-a (Chl a), specific Primnesiophyta biovolume (Bprim.), Bacillariophyta biovolume (Bbacil.) and Dinophyta biovolume (Bdinoph.) algae, nutrients in the studied waters of the Black Sea in May 2013

 

Nitrogen was present as ammonium, varying from 0.50 to 1.90 mmol/m³. The content of phosphates was as large as 0.20 – 0.40 mmol/m³, therefore N:P ratio averaged for the entire seawater area of the investigation was considerably lesser than Redfield ratio (5.1 (±1.8) vs. 16:1, correspondingly).

Nearly all of the seawater areas were blooming. The numbers of E. huxleyi in the sea surface varied from 1.3 to 4.3·10⁶ cell/l amounting on the average to 94 % of the total phytoplankton abundance. The specific bio- volume generated by this coccolithophore which domin- ated among Primnesiophyta ranged between 60-90% of the total phytoplankton biovolume (Table 1). Chlorophyll-а concentration in the blooming sea water was not large (0.10 – 0.18 mg/m³) and specific growth rate of the phytoplankton varied inconsiderably (0.80 – 1.44 d^-1). In the seawater areas receiving the Dnieper input (st. 33 and 34) diatoms, primarily Cerataulina pelagica, Cyclotella caspia and large Pseudosolenia calcar-avis, were the major contributor to the total phytoplankton abundance and biovolume. The chlorophyll-а content measured in samples collected in the brackish-water locations (st. 33 and 34) was several times as large as in other areas – 1.10 and 0.33 mg/m³, correspondingly. Specific growth rate of the total phytoplankton was relatively high (0.97 d^-1) at st. 34 where seawater salinity was estimated 16.30 ‰, and about 3-fold lesser at station 33 where the salinity decreased to 15.50 ‰.

Table 2 The microzooplankton grazing of phytoplankton (g), the net phytoplankton growth rate (µ – g) and ratio g/µ in the studied waters of the Black Sea in May 2013

Figure 2 Relationship between specific biovolume of Dinophyta and microzooplankton grazing of the phytoplankton in the area of E. huxleyi bloom