Emiliania huxleyi Spring Bloom in the Black Sea: A Tentative Investigation  

L.V. Stelmakh , E.Yu. Georgieva
The A.O. Kovalevsky Institute of Biology of the Southern Seas National Academy of Sciences of Ukraine, 2, Nakhimov av., Sevastopol 99011, Crimea, Ukraine
Author    Correspondence author
International Journal of Marine Science, 2014, Vol. 4, No. 17   doi: 10.5376/ijms.2014.04.0017
Received: 20 Dec., 2013    Accepted: 20 Jan., 2014    Published: 24 Feb., 2014
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This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Stelmakh and Georgieva, 2014, Emiliania huxleyi Spring Bloom in the Black Sea: A Tentative Investigation, International Journal of Marine Science, Vol.4, No.17: 160-165 (doi: 10.5376/ijms.2014.04.0017)

Abstract

This investigation was carried out in the surface water layer of some coastal and open-sea zones of the Black Sea in May 2013 when a spring bloom of Emiliania huxleyi was developing. The numbers of E. huxleyi in the blooming sea surface varied from 1.3 to 4.3·106 cell/l amounting on the average to 94 % of the total phytoplankton abundance. The bloom emerged as a response to favourable light, temperature and nutrient conditions which have accelerated the phytoplankton growth to 0.80 – 1.44 d-1 while the predatory pressure of microzooplankton on this coccolithophore remained low. High net growth rate of the phytoplankton (0.40 –1.00 d-1) and low ratios of the specific microzooplankton grazing rate to the specific phytoplankton growth rate (34% on the average) in the seawater area can be interpreted as the evidence of beginning bloom. 

Keywords
Black Sea; Phytoplankton growth rate; Microzooplankton grazing; Chlorophyll-a

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 72nd 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 (µDF) was calculated as:
 
µDF = ln(Chlafinal/ Chlainitial)             (1)
                           
The linear regression equations were calculated to estimate the interrelations between the observed phytoplankton growth rate (µDF) and the dilution factor (DF) as:
 
µDF = –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 ‰.

 
The specific rate of phytoplankton consumption by microzooplankton measured in the sea surface usually ranged from 0.04 to 0.99 d -1 (Table 2). The predatory activity was similarly low (0.15 – 0.24 d-1) in the eastern Black Sea off the Crimean shore and increased to the largest (0.19 – 0.99 d-1) in the deep-water part of the sea. In the shallow-water areas occurring from the NW Black Sea to the western coast of the Crimea estimates of the microzooplankton predatory pressure varied very broadly from 0.04 to 0.50 d-1. At the brackish-water stations 33 and 34 the pertinent values were 0.13 and 0.53 d-1, correspondingly.

 

 

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

 

In the zone of E. huxleyi bloom the rate of microzooplankton grazing on the phytoplankton (g) reliably correlated with the relative percentage of dinoflagellates in the total phytoplankton biovolume (Figure 2). Regression analysis has shown that given p = 0.00007, Fisher criterion for regression line was 33.1, i.e. several times greater than critical value. The standard error of regression equation quotient was estimated 15 % and Student criterion – several times as large as critical. The minimums and maximums of the phytoplankton loss due to microzooplankton grazing concurred with the minimal and maximal share of dinoflagellates in the total phytoplankton biovolume. The dinoflagellates were represented mainly by small forms (< 15 – 20 µm). According to the regression equation, in the absence of dinoflagellates the specific rate of microzooplankton grazing declines to zero.

 

 

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

 

In May 2013, net growth rate of phytoplankton (µ – g) in the sea surface was estimated -0.27 – 1.18 d-1 (Table 2). Greatest values (0.56 – 1.18 d-1; average = 0.93 d-1) were registered off the eastern coast of the Crimea. According to the percent ratio g/µ, the loss of phytoplankton production due to microzooplankton grazing was as moderate as 19 % on the average. Along the shallow-water band in the western Black Sea the net growth rate was usually as large as 0.65-1.0 d-1; the only exception was station 28 at which it was very low (0.04 d-1) and the primary production consumed by microzooplankton amounted to 96%. In the samples taken at station 34 diatoms prevailed by both numbers and biovolume, therefore the high net growth rate of the phytoplankton (0.84 d-1) there. However, at station 33 the total phytoplankton biovolume was also largely owing to diatoms, the net growth rate was below zero (– 0.27 d-1) and the daily loss of phytoplankton due to microzooplankton grazing was twice as large as the primary production (g/ µ = 200 %). High net growth rates ranging 0.44 – 0.84 d-1 (average = 0.56 d-1) were registered in the phytoplankton of the deep-water part of the western Black Sea. The primary production consumed by microzooplankton (g/ µ) fluctuated between 18 – 72 %, 51% on the average.
 
3 Discussions
It is known that phytoplankton bloom is generated by concurrent favourable environmental factors such as light, seawater temperature and the availability of nutrients. However, phytoplankton abundance and biomass are rapidly increasing to bloom values only when specific growth rate of the phytoplankton is far greater than the rate of microzooplankton grazing.
 
Some recent investigations point out that in the Black Sea E. huxleyi most frequently increases its abundance to bloom in summer, namely in June – July (Oguz and Merico, 2006, Pautova et al., 2007). During these months light intensity enhances to near-maximum (40 – 50 E/m2·d) and the upper quasi-homogeneous (mixed) layer (UML) warms up to 20℃ and above. Noteworthily, studies on E. huxleyi culture have shown that culture-specific temperature optimum for this coccolithophore is exactly 20℃, and high light intensities do not inhibit microalgal photosynthesis and growth (Tyrrell and Merico, 2004). Therefore the assumption that the high light and warm sea can be the drivers of summer E. huxleyi blooms. However, in the Black Sea E. huxleyi blooms are not necessarily a response to high light intensity and warm sea water. In 2010, the western part of the sea was blooming in October when, compared to summer estimates, the intensity of light has decreased 2 – 3 times to 25 E/m2·d, and the UML has cooled to 15-17℃ (Stelmakh et al., 2013). N.V. Morozova-Vodyanitskaya (Morozova-Vodyanitskaya, 1957) described a spring bloom she observed in deep- and shallow-water areas of the Black Sea in April 1952; at some locations the abundance of Pontosphaera huxleyi (E. huxleyi) amounted to 1 million (and more) cells per litre. Presumably, the intensive autumn and spring coccolithophore blooms were provoked not only by abiotic factors but by biotic interactions in the plankton, too.
 
In May 2013, the temperature and light in the sea surface favoured intensive multiplication of the coccolithophore; nutrients were sufficient and therefore could not have been a growth-limiting factor. High values of phytoplankton specific growth rate measured in the bloom are not casual; they rather represent the actual tempo of E. huxleyi growth. The investigations conducted on E. huxleyi culture (Tyrrell and Merico, 2004) and our own data allow concluding that the recent estimates of specific growth rate agree with the maximum admitted for this coccolithophore. The relatively low microzooplankton predatory pressure on the phytoplankton in the blooming Black Sea water (g/µ ratio 34 % on the average) can indicate beginning of the bloom. Earlier it was stated that in marine ecosystems under food deficiency herbivorous protists are not likely to be able to prevent the initiation and development of mass blooms when conditions are favorable for rapid phytoplankton growth (Sher and Sher, 2009). It is very probable that in the Black Sea in May 2013 phytoplankton mortality due to grazing has been limited by both quantity and quality of the prey organisms. Extremely low chlorophyll-а estimates measured in the blooming sea water suggest prey deficiency, and the taxonomic structure of phytoplankton evidence poor quality of the prey. Usually, E. huxleyi are a prey of minor interest to microzooplankton grazers (Tyrrell and Merico, 2004) that is, probably, due to the ability of this coccolithophore to generate DMPS (dimethylsulfoniopropionate). For example, laboratory investigations point out that the rate of microzooplankton grazing decreased when strains of E. huxleyi with high DMPS lyase activity were used (Strom et al, 2003). Five species (including ciliates and heterotrophic dinoflagellates) showed lower feeding rates on E. huxleyi strains with high DMPS lyase activity than on low-lyase strains. However, the heterotrophic dinoflagellate Oxyrrhis marina eagerly grazed on all the tested strains without showing a decrease in the consumption rate, i.e., the grazing rate did not depend on DMPS lyase activity. As DMSP (20 µM) was added to laboratory cultures of ciliates, Strombidinopsis acuminatum and Favella sp., and the dinoflagellate Noctiluca scintillans it resulted in a 28–75% decrease in the feeding rates on prey dinoflagellates Gymnodinium simplex, Heterocapsa triquetra and Prorocentrum micans (Fredrickson and Strom, 2009).
 
The evidence given in this publication and previously (Stelmakh, 2013) suggests that high phytoplankton net growth rates and low g/µ percent ratios can warn of the beginning E. huxleyi bloom. The pertinent records show that in a dying out diatom bloom net growth rate of the phytoplankton usually dropped to zero or even below, and g/µ ratio was not lesser than 80% and more. 
 
4 Conclusions
The bloom of coccolithophore E. huxleyi that was observed in the Black Sea in May 2013 has emerged primarily owing to favorable environmental factors. Therefore a specific growth rate of the phytoplankton was the maximal or near-maximal. We suppose that high phytoplankton net growth rate and low values of g/µ ratio indicate that E. huxleyi bloom is beginning and will, probably, only enhance.
 
Acknowledgements
We are grateful to our associates from IBSS – I.I. Babich, engineer, who measured phytoplankton biomass and identified species and N.Yu. Rodionova, who measured the content of nutrients.
 
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