diversity

Article Species Diversity of Epilithon Diatoms and the Quality of the Waters of the Donuzlav Gulf Ecosystem (, the )

Larisa I. Ryabushko, Denis N. Lishaev * and Nelya P. Kovrigina

A.O. Kovalevsky Institute of Biology of the Southern Seas of Russian Academy of Sciences, 2, Nakhimov Av., 299011, Russia * Correspondence: [email protected]; Tel.: +79-787-237-862

 Received: 13 June 2019; Accepted: 10 July 2019; Published: 18 July 2019 

Abstract: In this work, the species diversity of epilithon diatoms of Donuzlav Gulf (Crimean coastal waters of the Black Sea) is presented for the first time. A total of 60 taxa of Bacillariophyta belonging to 3 classes, 18 families and 34 genera were found. The largest number of species (45) of diatoms was observed in summer, and the smallest number of species (12) in winter. A total of 25 saprobity indicator species of diatoms was found on the epilithon, 12 taxa of which belong to a group of betamesosaprobionts—indicators of moderate organic pollution of water. Marine species comprise 55% and brackish-marine ones make up 37% of them, but no freshwater species were identified. The diatom community was dominated by cosmopolites (32%) and arctic–boreal–tropical species (27%). The maximal abundance (186.2 103 cells cm 2) and biomass (0.083 mg cm 2) of diatoms × − − was noted in June 2018 at salinity 16.58% and T = 24.4 ◦C with the dominant cosmopolites species 3 2 of Licmophora abbreviata and Cocconeis scutellum. The minimum values, 14.8 10 cells cm− and 2 × 0.005 mg cm− , were observed in January 2019 at salinity 16.86% and T = 9.0 ◦C with the dominant Berkeleya rutilans and Nitzschia hybrida f. hyalina. The high values of BOD5, oxidizability, silicon, Norg and Porg were noted, indicating a certain degree of water pollution by domestic sewage. It has been shown that the pollution in the southwestern part of the Donuzlav Gulf is greatest since this part of the coast was populated most densely in comparison with the northern coast of the Gulf where there are no coastal settlements except for two mariculture farms.

Keywords: species diversity; diatoms; stones; bioindication; indicators; organic pollution; Crimean coastal waters; Donuzlav Gulf; the Black Sea

1. Introduction Donuzlav Gulf of the Black Sea is a unique semi-enclosed marine with a surface area of 47.5 km2, located off the West coast of the Crimea 150 km from Sevastopol. The length of the sand embankment separating it from the sea is 9 km and its width is about 200–600 m. Most of the depth is less than 4–5 m; in the area of the central basin along the lake, the depth reaches 12–20 m. The first complex studies of biota, including phytoplankton, and monitoring data of hydrochemical and chemical–toxicological characteristics of Donuzlav marine ecosystems were conducted in different years [1–3]. The authors showed that in the ecosystem of Donuzlav marked high trophic waters, productivity of meio- and macrobenthos, as well as the presence of natural populations of mollusks (mussels) and fish make the reservoir very promising for the development of mariculture. The period 2015–2017 showed that the 1 average content of dissolved oxygen in the water changed in the range of 8.77–9.40 mg L− [3]. These values are satisfactory for the life of hydrobionts and the degree of pollution the water environment with heavy metals and a petroleum hydrocarbon contaminated site is quite low. The ecosystem of

Diversity 2019, 11, 114; doi:10.3390/d11070114 www.mdpi.com/journal/diversity Diversity 2019,, 11,, 114x FOR PEER REVIEW 2 2of of 12 site is quite low. The ecosystem of Donuzlav Gulf is of particular interest. On the one hand, it has Donuzlavtwice undergone Gulf is ofits particularformation. interest. At first, On it was the onea se hand,a, then it it has was twice separated undergone from its the formation. sea by a mound, At first, itthen was again, a sea, thenit became it was separateda Gulf. fromCurrently, the sea the by aecosystem mound, then of again,Donuzlav it became is affected a Gulf. Currently,by such theanthropogenic ecosystem ofenvironmental Donuzlav is afffactorsected byas such indust anthropogenicrial sand mining environmental and the factors location as industrial of the sandmussel–oyster mining and farms. the locationTherefore, of the mussel–oysterprimary producers farms. of Therefore,the microphytobenthos the primary producersas an important of the microphytobenthosfood source for all species as an important of aquatic food organisms source for have all speciesnever been of aquatic studied. organisms Diatom haveindices never used been in studied.biomonitoring Diatom of indices water usedquality in biomonitoringof the mostly of waterthe rivers quality [4–10] of the but mostly very poorly of the riversmarked [4– 10on] butthe verymarine poorly benthic marked ecosystems on the marine[11–18]. benthic ecosystems [11–18]. Despite the fact that diversity species of the mi microphytobenthoscrophytobenthos of the BlackBlack Sea is well studied in didifferentfferent ecotopesecotopes [[18–22].18–22]. Ho However,wever, diatoms of microphytobent microphytobenthoshos in in different different seasons of the year have never been studied studied in in the the Donuzlav Donuzlav Gulf. Gulf. In In this this regard, regard, it itbecame became necessary necessary to to conduct conduct a astudy study of of benthic benthic diatoms diatoms as as indicators indicators of of water water quality quality using using the the known environmental saprobity indicators. TheseThese datadata are are poorly poorly understood understood in in the the seas seas of theof the World World Ocean Ocean in general in general and in and the in Black the SeaBlack in Sea particular. in particular. The aimaim ofof thisthis workwork was was the the study study of of species species diversity diversity of benthicof benthic diatoms diatoms and and their their indicator indicator role inrole the in Donuzlav the Donuzlav Gulf Gulf of Crimean of Crimean coastal coastal waters waters of the of Black the Black Sea. Sea.

2. Materials and Methods Sampling of stonesstones waswas carriedcarried outout from from April April 2018 2018 to to April April 2019 2019 at at 9 stations9 stations at at a deptha depth of of 0.5 0.5 to 1.5to 1.5 m inm thein the Donuzlav Donuzlav Gulf Gulf (45 ◦(45°20’00”20’00” N, 33N,◦ 00’00”33°00’00” E) ofE)the of the Crimean Crimean coastal coastal waters waters of the of Blackthe Black Sea (FiguresSea (Figure1 and 1 2and). Among 2). Among them, them, Stations Stations 1, 2, 3, 1, 5, 2, 7 are3, 5, located 7 are located near areas near for areas the cultivation for the cultivation of mussels of andmussels oysters. and Inoysters. this work, In this diff work,erent methodsdifferent ofmeth studyingods of diatoms studying were diatoms used [were16,18 ,used19]. Samples[16,18,19]. of stonesSamples were of takenstones manually, were taken by three manually, stones fromby three each samplingstones from station. each In sampling the laboratory, station. each In stone the waslaboratory, scraped each for suspensionstone was scraped for qualitative for suspension and quantitative for qualitative processing. and quantitative The collected processing. stones were The carefullycollected cleanedstones bywere a scraper carefully to collect cleaned suspensions. by a sc Samplesraper to of collect the suspension suspensions. from liveSamples diatoms of were the usedsuspension for the from taxonomic live diatoms study were and the used samples for the were taxonomic fixed by study 2% formaldehydeand the samples for were the quantitativefixed by 2% assessment.formaldehyde The for sizes the ofquantitative the stones wereassessment. of thefollowing The sizes range:of the lengthstones (x):4–12were of cm;the widthfollowing (y):2–7 range: cm; 2 heightlength (x):4–12 (z):1.5–3.5 cm; cm. width Determination (y):2–7 cm; ofheight the surface(z):1.5–3.5 area cm. (S, Determination cm ) of the stones of the was surface carried area out (S, by cm the2) methodof the stones of Graham was carried et al. [out23]:S by= theπ/3 method (xy + yz of+ Grahamxz), where et al. x, [23]:S y, z are = theπ/3 linear(xy + yz of + the xz), substrate where x, sizes. y, z Theare the error linear in determining of the substrate the surface sizes. The area error of the in stonedetermining was 5%. the Goryaev’ssurface area camera of the with stone a volumewas ± 5%. of 3 ± 0.9Goryaev’s mm was camera used with for counting a volume of of diatom 0.9 mm cells.3 was used for counting of diatom cells.

Figure 1. Crimean area of the studied diatoms— diatoms—DonuzlavDonuzlav Gulf of the Black Sea.

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Figure 2. Northern (A) and southern (B) coasts of the Donuzlav Gulf of the Black Sea. Figure 2. Northern (A) and southern (B) coasts of the Donuzlav Gulf of the Black Sea. The study of diatoms was carried out on a light microscope of C. Zeiss “Axioskop 40” with software The study of diatoms was carried out on a light microscope of C. Zeiss “Axioskop 40” with AxioVision Rel. 4.6 at magnifications of 10 40, 10 100 under oil immersion in living material and software AxioVision Rel. 4.6 at magnifications× of 10× × 40, 10 × 100 under oil immersion in living in permanent slides prepared according [23] and mounted in the El’yashev media (n = 1.67). A total material and in permanent slides prepared according [23] and mounted in the El'yashev media (n = of 64 samples was collected and processed. Analysis of number of species (n), species richness (R), 1.67). A total of 64 samples was collected and processed. Analysis of number of species (n), species abundance (N, cells cm 2) and biomass (B, mg cm 2) of community diatoms in each sample was carried richness (R), abundance− (N, cells cm−2) and biomass− (B, mg cm−2) of community diatoms in each out according to the methods described earlier [16,18,20]. The abundance and biomass of diatoms sample was carried out according to the methods described earlier [16,18,20]. The abundance and N n V SV n werebiomass calculated of diatoms according were calculated to the formulae: according to= the formulae:/ , where N = n isV/S the V, numberwhere n ofis the diatom number cells of in 3 thediatom Goryaev cells countingin the Goryaev chamber counting with volume chamber equal with to 0.9volume mm ;equalV is theto sample0.9 mm volume,3; V is the ml; sampleB = h V b/SV , where b is total V , mm3, h, the unit weight of benthic diatoms, is equal to 1.2 10 9 volume,chamber ml; B = h V b/SVchamberchamber, where b is total Vchamber, mm3, h, the unit weight of benthic diatoms,× is− mg µm 3, and others are 1 10 9 mg µm 3 [24]. equal to− 1.2 × 10−9 mg µm−3,× and −others are− 1 × 10−9 mg µm−3 [24]. TheThe hydrologicalhydrological characteristic characteristic of Donuzlavof Donuzlav Gulf watersGulf waters had the had following the following range: the range: temperature the variedtemperature from 7.0varied◦C(March) from 7.0 to °C 27.0 (March)◦C (July) to 27.0 and °C salinity (July) and from salinity 16.58% from (June) 16.58‰ to 18.62% (June) to(May). 18.62‰ The hydrochemical(May). The hydrochemical parameters of parameters Donuzlav Gulfof Donuzlav waters (pH, Gulf O2 ,waters alkaline (pH, permanganate O2, alkaline oxidizability, permanganate Norg , NOoxidizability,2, NO3, NH N4,Porgorg, NO, PO2, 4,NO Si)3 at, NH the stations4, Porg, PO are4, determined Si) at the accordingstations are to conventional determined methodsaccording [25 ,to26 ]. conventionalIn our work methods we used [25,26]. the method of monitoring the quality water of the environment on saprobity indicatorsIn our developed work we forused diatoms the method and adoptedof monitoring species the names quality to modernwater of system the environment [4–15,18,27 –on29 ]. Thesaprobity species indicators composition developed of diatoms for was diatoms determined and usingadopted the followingspecies names sources to [18 ,modern29–32]. Thesystem maps of[4–15,18,27–29]. distribution of The environmental species composition variables of values diatoms and was indicator determined taxa number using the were following generated sources in the Statistica[18,29–32]. 12.0 The program maps of [ 33distribution]. of environmental variables values and indicator taxa number were generated in the Statistica 12.0 program [33]. 3. Results 3. ResultsSpecies diversity of microphytobenthos of the stone substrates in the Donuzlav Gulf are 60 taxa of BacillariophytaSpecies diversity (Table1 of), belongingmicrophytobenthos to 3 classes, of 18the families stone substrates and 34 genera. in the Donuzlav In the diatom Gulf flora, are 60 marine taxa andof Bacillariophyta cosmopolites dominated,(Table 1), belonging which is typicalto 3 classes, for the 18 phytobenthos families and of34 thegenera. Crimean In the coastal diatom waters flora, of themarine Black and Sea cosmopolites [18,21]. dominated, which is typical for the phytobenthos of the Crimean coastal waters of the Black Sea [18,21]. Table 1. Checklist of the epilithon diatoms in the Donuzlav Gulf of the Black Sea, 2018–2019 and their ecologicalTable 1. Checklist and phytogeographical of the epilithon diat characteristics.oms in the Donuzlav Gulf of the Black Sea, 2018–2019 and their ecological and phytogeographical characteristics. ECD Taxa PhG ECD PhG Taxa S RS S RS Achnanthes brevipes C.A. Agardh 1824 β BM K Achnanthes brevipes C.A. Agardh 1824 β BM К Achnanthes longipes C.A. Agardh 1824 β M ABT Achnanthes longipes C.A. Agardh 1824 β М АBТ Amphora angusta W. Gregory 1857 – BM K Amphora angusta W. Gregory 1857 – BM К Amphora arcus W. Gregory 1857 – M AB AmphoraAmphora arcus W. ovalis Gregory(Kützing) 1857 Kützing 1844 o-β – BKМ АB AmphoraAmphora ovalis (Kützing) proteus W. Kützing Gregory 1844 1857 α-β o-βMK B К AmphoraArdissonea proteus W. crystallina Gregory(C.A. 1857 Agardh) Grunow 1880 β α-BMβ М BT К ArdissoneaBacillaria crystallina paxillifera (C.A.(O.F. Agardh) Müller) Grunow T. Marsson 1880 1901 o-α βBM BM K BТ BacillariaBerkeleya paxillifera micans (O.F.(Lyngbye) Müller) T. Grunow Marsson 1880 1901 o о-BMα BM B not К

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Table 1. Cont.

ECD Taxa PhG S RS Berkeleya rutilans (Trentepohl ex Roth) Grunow 1880 – BM AB not Caloneis liber (W. Smith) P. Cleve 1894 – MK Carinasigma rectum (Donkin) G. Reid 2012 – M BT not Cocconeis costata W. Gregory1855 – MK Cocconeis scutellum Ehrenberg 1838 β BM K Cylindrotheca closterium (Ehrenberg) Reimann et Lewin 1964 β MK Diploneis bombus (Ehrenberg) Ehrenberg 1894 – M ABT Diploneis chersonensis (Grunow) P. Cleve 1894 – M ABT Diploneis smithii (Brébisson) P. Cleve 1894 – BM K Entomonei spaludosa (W. Smith) Reimer 1975 – BM AB not Falcula media var. subsalina Proschkina-Lavrenko 1963 o MB Grammatophora marina (Lyngbye) Kützing 1844 β MK Gyrosigma prolongatum (W. Smith) Griffith et Henfrey 1856 – M ABT Halamphora coffeiformis (C.A. Agardh) Levkov 2009 – BM ABT Halamphora costata (W. Smith) Levkov 2009 – M BT Halamphora hyalina (Kützing) Rimet et R. Jahn 2018 β M ABT not Haslea ostrearia (Gaillon) Simonsen 1974 – MB Licmophora abbreviata C.A. Agardh 1831 β MK Licmophora dalmatica (Kützing) Grunow 1867 – MB Licmophora flabellata (Greville) C.A. Agardh 1831 β M BTnot Licmophora gracilis (Ehrenberg) Grunow 1867 – M ABT Licmophora hastata Mereschkowsky 1901 – MB Lyrella clavata (W. Gregory) 1990 – M BT Melosira lineata (Dillwyn) C.A. Agardh 1824 α BM ABT Melosira moniliformis (O.F. Müller) C.A. Agardh 1824 o-β BM K Navicula cancellata Donkin 1873 – MK Navicula directa (W. Smith) Ralfs ex Pritchard 1861 – MK Navicula distans (W. Smith) Ralfs ex Pritchard 1861 – MB Navicula pennata var. pontica Mereschkowsky 1902 – BM BT Navicula perrhombus Hustedt ex Simonsen 1962 – M BT Navicula salinarum Grunow 1880 β-o B AB not Nitzschia hybrida f. hyalina Proschkina-Lavrenko 1963 β BM B Nitzschia lanceolata W. Smith 1853 – B BT not Nitzschia sigma (Kützing) W. Smith 1853 – B ABT Nitzschia tenuirostris Mereschkowsky 1902 – BB Parlibellus delognei (Van Heurck) E.J. Cox 1988 – M ABT Petroneis monilifer (Cleve) A.J. Stickle et D.G. Mann 1990* – M AB Plagiotropis lepidoptera (W. Gregory) Kuntze 1898 o M ABT Pleurosigma elongatum (W. Smith) 1852 – BM K Psammodictyon panduriforme (W. Gregory) D.G. Mann 1990 – M BT not Rhaphoneis amphiceros (Ehrenberg) Ehrenberg 1844 – BM ABT Seminavis ventricosa (W. Gregory) M. Garsia-Baptista 1993 β MK Striatella delicatula (Kützing) Grunow ex Van Heurck 1885 – BM ABT Striatella unipunctata (Lyngbye) C.A. Agardh 1832 – M BT Tabularia fasciculata (C.A. Agardh) D.M. Williams et Round 1986 x-o BM K Tabularia parva (Kützing) D.M. Williams et Round 1990 α BM ABT Tabularia tabulata (C.A. Agardh) Snoeijs 1992 β-α BM K Thalassiosira eccentrica (Ehrenberg) P. Cleve 1904 – MK Trachyneis aspera (Ehrenberg) P. Cleve 1894 β M ABT not Tryblionella coarctata (Grunow) D.G. Mann 1990 – BM B Undatella lineolata (Ehrenberg) L.I. Ryabushko 2006 – BM ABT Note: (–)—the absence of the diatoms species, (*)—the species was first recorded in the benthos of the Black Sea; ecological characteristic of diatoms (ECD): S—species-specific index of saprobity according Sládeˇcek[4]: α— alphamesosaprobiont, α-β—alpha-betamesosaprobiont, β—betamesosaprobiont, β-α—beta-alphamesosaprobiont, β-o—beta-oligosaprobiont, o—oligosaprobiont, o-α—oligo-alpha-mesosaprobiont, o-β—oligo-beta-mesosaprobiont, x-o—xeno-oligosaprobiont; RS—the ratio of species to the water salinity: M—marine species, BM—brackish-marine, B—brackish; phytogeographycal elements (PhG): B—boreal species, AB—arcto–boreal, BT—boreal–tropical, ABT—arcto–boreal–tropical, K—cosmopolite, not—notal species, found in the southern hemisphere. Diversity 2019, 11, x FOR PEER REVIEW 5 of 12

BM—brackish-marine, B—brackish; phytogeographycal elements (PhG): B—boreal species, DiversityАB2019—arcto–boreal,, 11, 114 BТ—boreal–tropical, АBT—arcto–boreal–tropical, K—cosmopolite, not—notal5 of 12 species, found in the southern hemisphere.

From thethe total total species species of diatoms of diatoms on stone on substrates stone insubstrates the Donuzlav in the Gulf Donuzlav the Bacillariophyceae Gulf the classBacillariophyceae dominated. Massclass dominated. species of pennate Mass spec diatomsies of pennate presented diatoms of genera presentedLicmophora, of genera Grammatophora, Licmophora, Cylindrotheca,Grammatophora, Trachyneis, Cylindrotheca, Cocconeis, Trachyneis,and Striatella Cocconeis,, etc. and Striatella, etc. The geographical characteristic of the diatoms of Gulf was carriedcarried out. It is shown that in the floraflora diatom,diatom, predominantpredominantwere were cosmopolites cosmopolites (32%) (32%) and and arctic–boreal–tropical arctic–boreal–tropical species species (27%), (27%), which which is commonis common species species for for the the microphytobenthos microphytobenthos of of the the Crimean Crimean coastal coastal waters waters of of the the Black Black Sea Sea (Table (Table1). The1). The number number of species of species (40; 12;(40; 29; 12; 30; 29; 21; 30; 37; 21; 38) 37; was 38) distributed was distributed by stations by accordingstations according to the values to the of watervalues salinity of water (16.58; salinity 16.86; (16.58; 17.06; 16.86; 17.81; 17.06; 18.52; 17.81; 18.54; 18.52; 18.62% 18.54;). From 18.62‰). all 24 From species all found 24 species throughout, found thethroughout, range of investigatedthe range of waterinvestigated salinity water values salinity was from values 16.58 was to 18.62%from 16.58 (June). to 18.62‰ Only two (June). species Only of Amphoratwo species arcus of andAmphoraBerkeleya arcus rutilans and Berkeleyawere found rutilans at allwere stations found in at the allDonuzlav stations in Gulf. the Donuzlav Gulf. The 18 species found once or twice in the rangerange ofof thethe wholewhole waterwater salinitysalinity spectrum were as follows: Berkeleya micans, Carinasigma rectum, Gyrosigma prolongatum, Halamphora cocoffeiformis,ffeiformis, HasleaHaslea ostrearia, Licmophora dalmatica, L. flabellata,flabellata, L. gracilis,gracilis, LyrellaLyrella clavata,clavata, MelosiraMelosira moniliformis,moniliformis, NaviculaNavicula perrhombus, N. salinarum, Nitzschia sigma, N. tenuirostris,tenuirostris, Parlibellus delognei, Pleurosigma elongatum, TabulariaTabularia tabulata,tabulata, TryblionellaTryblionella coarctata coarctata(Table (Table1). 1). The largest number of speciesspecies (45)(45) waswas observedobserved inin JulyJuly atat salinitysalinity ofof 16.58%16.58‰ and temperaturetemperature 24.4 ◦°C.C. TheThe smallest smallest number number of speciesof species (12) (12) was was in January in January at salinity at salinity of 16.86% of 16.86‰ and T = and9.0 ◦TC. = Despite9.0 °C. theDespite low salinitythe low of salinity the Gulf of waters, the Gulf marine waters, species marine dominated species (55% dominated of all species) (55% and of all brackish-marine species) and speciesbrackish-marine were at 37% species (Table 1were), which at are37% generally (Table characteristic 1), which ofare the generally microphytobenthos characteristic of the of Black the Seamicrophytobenthos [16,18,19]. No freshwater of the Black species Sea [16,18,19]. have been No found, freshwater although species in the have past been the Gulf found, was although separated in fromthe past the the sea Gulf and waswas calledseparated a lake from or liman.the sea However, and was called according a lake to or our liman. data, theHowever, studied according area can beto consideredour data, the a marinestudied Gulf.area can be considered a marine Gulf. Other data on the species diversity is the dynamic of their quantitative values (species richness, abundance and biomass of thethe community),community), which togethertogether with thethe speciesspecies composition,composition, and environmental and phytogeographical characteristics can be used to estimate the productivityproductivity and waterwater qualityquality ofof the studied reservoir. Therefore, thethe followingfollowing valuesvalues werewere obtained.obtained. The richness of species varied fromfrom 1111 toto 2424 (Figure(Figure3 3).).

Figure 3.3. SpeciesSpecies richness,richness, abundance abundance of of diatoms diatoms and and water wa temperatureter temperature in Donuzlav in Donuzlav Gulf ofGulf the of Black the SeaBlack (2018–2019). Sea (2018–2019).

From these, 10 species dominated in abundance and biomass: Berkeleya rutilans, L. abbreviata in

April, maximum values of species cosmopolites L. abbreviata and C. scutellum in June at T = 24.4 ◦C, Diversity 2019, 11, x FOR PEER REVIEW 6 of 12 Diversity 2019, 11, 114 6 of 12 From these, 10 species dominated in abundance and biomass: Berkeleya rutilans, L. abbreviata in April, maximum values of species cosmopolites L. abbreviata and C. scutellum in June at T = 24.4 °C, A. hyalina ,, Th.Th. eccentrica eccentrica inin July, July, N.N. perrhombus perrhombus andand A.A. longipes longipes inin October, October, and and minimum minimum values values of B. of rutilansB. rutilans, N., N.hybrida hybrida var.var. hyalinahyalina in inJanuary, January, G. G.marina marina andand C. C.closterium closterium in inMarch. March. 3 2 The values of abundanceabundance ((NN)) diatomsdiatoms changedchanged (14.8–186.2)(14.8–186.2) × 10103 cellscells cmcm−−2 with average N == 3 2 × 3 2 100.5 × 10103 cellscells cm cm−2− (Figure(Figure 3).3 ).The The minimum minimum abundance abundance was was N =N 14.8= 14.8 × 103 cells10 cells cm−2 cmwith− Bwith = 0.005B = × 2 × mg·cm0.005 mg−2 werecm− inwere January in January at salinity at salinity16.86‰ 16.86%and T = 9.0and °C T with= 9.0 dominant◦C withdominant of B. rutilans of B.and rutilans N. hybridaand · 3 2 2 N. hybrida f. hyalina. The maximum abundance 186.23 10 cells−2 cm with a biomass of 0.083 mg−2 cm f. hyalina. The maximum abundance 186.2 × 10 cells× cm with −a biomass of 0.083 mg·cm ·were− foundwere found in June in 2018 June with 2018 dominant with dominant L. abbreviataL. abbreviata and C. andscutellumC. scutellum. The biomass. The biomass (B) varied (B )from varied 0.006 from to 2 2 0.006 to 0.083−2 mg cm with an average B = 0.044−2 mg cm (Figure4). 0.083 mg·cm with· an− average B = 0.044 mg·cm (Figure· −4).

Figure 4. SpeciesSpecies biomass biomass diatoms diatoms in in the the Donu Donuzlavzlav Gulf of the Black Sea (2018–2019).

Another environmental characteristic is the ratio of species to organic water pollution. Diatoms are indicators of water quality; this is known in pa particularrticular for freshwater bodies. The study of marine waters on this issue is still lagging behind. Bioindication of of organic pollution in the coastal waters of the Black Sea microphytobenthos communities was was gathered for the first first time; therefore, the materials for comparison are are not yet available. With With the the help help of of ecological characterist characteristicsics of of these species, the proportion of saprobiont species was studied. A A total total of of 25 25 species-indicators of of saprobity saprobity were were found found in in the epilithon of the Donuzlav Gulf from which 12 taxa belong to a group of betamesosaprobiontes species of moderate organic pollution of water (Table1 1).). AnalysisAnalysis ofof thethe distributiondistribution ofof species-indicatorsspecies-indicators ofof organicorganic pollution in the epilithon showed that class 2, 4, and 5 indicators of water quality have been also presented. WeWe choosechoose indicators indicators of classof class 2 (low 2 (low organic organic pollution) pollution) and class and 5 (highclass organic5 (high pollution) organic pollution)of water quality of water to demonstrate quality to demonstrate the contrast of the its distributioncontrast of its in Donuzlavdistribution stations in Donuzlav (Figure5 A,B).stations (FigureFor 5A,B). the development of bottom-diatoms important are not only hydrological values, but also the hydrochemical parameters of the environment (Table2).

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Figure 5. Maps of saprobity indicators of class 2 (A) and 5 (B) of water quality in the Donuzlav Gulf ofFigure the Black 5. Maps Sea of(2018–2019). saprobitysaprobity indicators indicators ofof class class 2 2 ( A(A)) and and 5 5 (B (B) of) of water water quality quality in in the the Donuzlav Donuzlav Gulf Gulf of theof the Black Black Sea Sea (2018–2019). (2018–2019). For the development of bottom-diatoms important are not only hydrological values, but also Table 2. The hydrochemical parameters of Donuzlav Gulf waters of the Black Sea. the hydrochemicalFor the development parameters of bottom-diatoms of the environment important (Table are2). not only hydrological values, but also the hydrochemicalO parameters of the environmentNO NO (TableNH 2). Norg PO Porg Si 2 BOD5, 2 3 4 4 Oxid., № st. Table 2. The hydrochemical1 parameters of Donuzlav Gulf waters of the Black Sea. 1 mg L 1 % mg O2 L− µg L 1 mg O2 L− Table− 2. The hydrochemical parameters of Donuzlav Gulf− waters of the Black Sea. 1 10.22О2 103.3 BOD0.555, NO 0.32 NO 12.43 N 10.6Н4 Norg 875 РО 0.64 Рorg 7.6 66.6Si Oxid., 2.98 № st. −1О2 BOD5, −1 NO2 NO3 NН4 Norg−1 РО4 Рorg Si Oxid., −1 № st. mg L % mg O2 L µg L mg O2 L 2 10.42−1 105.1 0.56−1 0.6 17.6 8.3 955−1 1.4 6.1 52.3 3.37 −1 1 mg10.22 L 103.3% mg0.55 O2 L 0.3 12.4 10.6 µg875 L 0.6 7.6 66.6 mg 2.98 O2 L 13 10.32 103.3103.8 0.550.61 0.30.8 12.4 13.7 10.6 14.2 875 1072 0.6 0.9 7.6 6.9 66.6 49.7 2.682.98 2 10.4210.22 105.1 0.56 0.6 17.6 8.3 955 1.4 6.1 52.3 3.37 25 10.30 105.1103.6 0.560.34 0.6 17.6 17.8 8.3 2.7 955 1104 1.4 1.0 6.1 7.8 52.3 75.1 2.693.37 3 10.3210.42 103.8 0.61 0.8 13.7 14.2 1072 0.9 6.9 49.7 2.68 3 103.8 0.61 0.8 13.7 14.2 1072 0.9 6.9 49.7 2.68 56 10.3010.32 10.35 103.6104.1 0.340.57 0.60.8 17.8 11.6 2.7 0.6 1104 972 1.0 1.7 7.8 6.7 75.1124.4 3.40 2.69 5 103.6 0.34 0.6 17.8 2.7 1104 1.0 7.8 75.1 2.69 67 10.3510.30 10.32 104.1103.8 0.570.71 0.80.9 11.6 15.4 0.6 7.7 972 1093 1.7 1.1 6.7 8.1 124.4 51.7 3.573.40 6 10.35 104.1 0.57 0.8 11.6 0.6 972 1.7 6.7 124.4 3.40 78 10.32 10.14 103.8106.7 0.710.82 0.90.5 15.4 9.2 7.7 7.7 1093 1094 1.1 1.1 8.1 9.5 51.7137.4 3.67 3.57 7 103.8 0.71 0.9 15.4 7.7 1093 1.1 8.1 51.7 3.57 8 10.1410.32 106.7 0.82 0.5 9.2 7.7 1094 1.1 9.5 137.4 3.67 8 10.14 106.7 0.82 0.5 9.2 7.7 1094 1.1 9.5 137.4 3.67 1 The analysis showed that the dissolved oxygen contentcontent in thethe rangerange fromfrom 10.1410.14 toto 10.4210.42 mgmg LL−−1 1 −1 with The anan average averageanalysis value valueshowed of of 10.29 that10.29 mg the mg L dissolved− Lin−1 2018–2019in 2018–2019 oxygen (Table co (Tablentent2, Figure in2, theFigure6A). range The 6A). from minimum The 10.14 minimum ofto these10.42 of valuesmg these L −1 wasvalueswith observedan was average observed in thevalue winter in ofthe 10.29 atwinter station mg at L 8, station the in maximum2018–2019 8, the maximum was(Table in June2, was Figure in in the June 6A). middle in The the part minimummiddle of the part Donuzlav of ofthese the DonuzlavGulfvalues (station was Gulf observed 2). (station in 2). the winter at station 8, the maximum was in June in the middle part of the Donuzlav Gulf (station 2).

Figure 6. The average annual concentrations of dissolved oxygen (A) andand BODBOD55 ((BB)) in in the water Figure 6. The average annual concentrations of dissolved oxygen (A) and BOD5 (B) in the water column at the sampling stations in the Donuzlav Gulf of thethe BlackBlack Sea.Sea. column at the sampling stations in the Donuzlav Gulf of the Black Sea. 1 The values values of of BOD BOD5 5werewere low, low, ranging ranging from from 0.34 0.34 to to0.82 0.82 mg mg O2 L O−12 (FigureL− (Figure 6B). 6TheyB). They were were2.5–6 −1 The values of BOD5 were low, ranging from 0.34 to 0.82 mg O2 L−1 (Figure1 6B). They were 2.5–6 times2.5–6 timeslower lowerthan the than minimum the minimum permissible permissible concentration concentration (2.0 mg (2.0 O2 mgL ), Owhich2 L− ), indicates which indicates the relative the times lower than the minimum permissible concentration (2.0 mg O2 L−1), which indicates the relative cleanness of the waters. The magnitude of the oxidizability changed in the range of 2.68 to 3.67 mg O2 cleanness of the waters. The magnitude of the oxidizability changed in the range of 2.68 to 3.67 mg O2

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−−11 −−11 LDiversity andand 2019 werewere, 11 belowbelow, 114 thethe minimumminimum permissiblepermissible levellevel (4.0(4.0 mg O22 LL ) confirming no water pollution8 of in 12 thisthis area.area. The values of organic nitrogen varied from 875 to 1104 µg L−−11 andand waswas highesthighest atat stationstation 77 (Figurerelative 7A); cleanness inorganic of the forms waters. of nitrogen The magnitude varied ofwithin the oxidizability the following changed limits: innitrites the range (NO22 of) ranged 2.68 to 1 1 −1 −1 from3.67 mg 0.3 Oto2 0.9L− µgand L− were1,, nitratesnitrates below (NO(NO the33)minimum from 9.2 to permissible 17.8 µg L−1 level andand (4.0ammoniumammonium mg O2 L nitrogennitrogen− ) confirming (NH(NH44) nofrom water 0.6 topollutionto 14.214.2 µgµg in LL this−−11 (Table(Table area. 2,2, FigureFigure 7B7B andand 8A,8A, B).B). TheThe maximummaximum concentrationconcentration ofof nitritenitrite nitrogennitrogen waswas 1 belowThe the values maximum of organic permissible nitrogen concentration varied from (20 875µg L to−−11). 1104 µg L− and was highest at station 7 −1 (FigureThe7A); values inorganic of organic forms phosphorus of nitrogen varied (Porg) varied within from the following 6.1 to 9.5 limits: µg L−1 nitrites andand thethe (NO concentrationsconcentrations2) ranged from ofof 1 1 µ µ −1 mineral0.3 to 0.9 phosphorusg L− , nitrates (PO44) (NO varied3) from in a narrow 9.2 to 17.8 rangeg L(from− and 0.9 ammonium to 1.7 µg L−1 nitrogen) (Figure (NH9). In4) general, from 0.6 the to 1 14.2contentµg Lof− nutrients(Table2, Figuresin the Donuzlav7B and8A,B). Gulf The increase maximumd from concentration April to June of and nitrite decreased nitrogen from was October below 1 totheto January.January. maximum permissible concentration (20 µg L− ).

Figure 7.7. AverageAverage annual annual concentrations concentrations of of organic organic nitrogen nitrogen (A) ( andA)) andand dissolved dissolveddissolved nitrites nitritesnitrites (B) in ((B the)) inin water thethe watercolumn column at the samplingat the sampling stations stations in the Donuzlavin thethe DonuzlavDonuzlav Gulf of GulfGulf the Blackofof thethe Sea. BlackBlack Sea.Sea.

Figure 8. ConcentrationsConcentrations of of di dissolvedssolved nitrates nitrates ( (AA))) andand ammoniumammonium (( (BB))) inin thethe water water column column at at the the sampling stations in the Donuzlav GulfGulf ofof thethe BlackBlack Sea.Sea.

1 The values of organic phosphorus (Porg) varied from 6.1 to 9.5 µg L− and the concentrations of 1 mineral phosphorus (PO4) varied in a narrow range (from 0.9 to 1.7 µg L− ) (Figure9). In general, the content of nutrients in the Donuzlav Gulf increased from April to June and decreased from October to January. 1 The concentrations of dissolved silicon varied from 49.7 to 137.4 µg L− in the waters of the Donuzlav Gulf in January 2019 (Figure 10). The maximum was marked at station 8 in autumn and winter. The minimum was near the farm at station 3, most likely due to the development of diatoms.

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Figure 9. Dissolved organic phosphorus (A) and orthophosphate (B) concentrations in the water column at the sampling stations in the Donuzlav Gulf of the Black Sea.

The concentrations of dissolved silicon varied from 49.7 to 137.4 µg L−1 in the waters of the DonuzlavFigure Gulf 9.9. Dissolved in January organic 2019 phosphorus (Figure 10). ( AThe) and maximum orthophosphate was marked ( B) concentrations at station 8 inin the autumn waterwater and winter.column The minimum atat thethe samplingsampling was stationsstationsnear the inin farm thethe DonuzlavDonuzlav at station GulfGulf 3, most ofof thethe likely BlackBlack dueSea.Sea. to the development of diatoms.

The concentrations of dissolved silicon varied from 49.7 to 137.4 µg L−1 in the waters of the Donuzlav Gulf in January 2019 (Figure 10). The maximum was marked at station 8 in autumn and winter. The minimum was near the farm at station 3, most likely due to the development of diatoms.

Figure 10. ConcentrationsConcentrations of of dissolved dissolved silicon silicon in in the the water water column at the sampling stations in the Donuzlav Gulf of the Black Sea.

4. Discussion Discussion Figure 10. Concentrations of dissolved silicon in the water column at the sampling stations in the A comparison ofof thethe quantitative quantitative data data obtained obtained for for benthic benthic diatoms diatoms from from other other areas areas of the of Black the Donuzlav Gulf of the Black Sea. BlackSea are Sea recorded are recorded for diatom for communities diatom communities as follows. Inas the follows. epilithon In ofthe stones epilithon from Karantinnaya of stones from Bay 3 Karantinnaya(Sevastopol) the Bay species (Sevastopol) richness (Rthe= species16) and richness abundance (R diatoms= 16) and (average abundanceN= 338.6 diatoms160.0 (average10 cells N= 4. Discussion2 3 2 ± × 338.6cm− )± at 160.0 maximum x 103 Ncells= 641.4 cm−2) 10at maximumcells cm− withN = dominant641.4 × 10 of3 diatomcells cmNitzschia−2 with dominant tenuirostris of(N diatom= 477.2 3 2 × 10 Acells comparison cm ) was of in the April quantitative 19883 at T =data13.0− 2obtainedC[18], whichfor benthic was also diatoms marked from in theother Donuzlav areas of Gulf. the Nitzschia× tenuirostris− (N = 477.2 × 10 cells cm )◦ was in April 1988 at T = 13.0 °C [18], which was also markedInBlack the epiphytonSea in theare Donuzlav recorded of the brown Gulf. for diatomIn alga theCystoseira epiphyton communities crinita of the Dubyas brown follows. 1830 alga in InCystoseira the the Donuzlav epilithon crinita Gulf Duby of 35stones 1830 species infrom the of 3 2 DonuzlavdiatomsKarantinnaya were Gulf foundBay 35 species (Sevastopol) with anof diatoms average the specieswere abundance found richness ofwith 104 (Ran =average10 16)cells and abundance cm abundance− in June of 2018diatoms104 × at 10 T3 (average=cells25 ◦cmC[− 222N in=]. × 3 2 JuneThe338.6maximum 2018 ± 160.0 at T x= values 2510 3°C cells of[22]. the cm The species−2) maximumat maximum richness values (26) N and =of 641.4 the abundance species × 103 cellsrichness (74.78 cm− (26)210 with cellsand dominant cmabundance− ) of algaeof (74.78diatom were × × 3 10observedNitzschia3 cells cm tenuirostris in−2 epizoon) of algae of(N were Karantinnaya= 477.2 observed × 103 cells Bayin epizoon incm February−2) was of Karantinnayain 2016 April at 1988 T = 9.7atBay T◦C in= and13.0 February in°C April [18], 2016 2015which at (62.0T was = 9.7 also 10°С 2 3 2 × andcellsmarked in cm April −in,T the 2015= Donuzlav10.3 (62.0◦C), × as10 Gulf.3 well cells In as cm the in−2 January,epiphyton T = 10.3 2016 °С of), (65.1asthe well brown 10as in cellsalga January cmCystoseira− 2016,T = (65.19.5crinita◦ C)× 10Duby [213 cells]. The1830 cm largest −in2, Tthe = 2× 9.5biomassDonuzlav °С) [21]. was Gulf The registered 35 largest speciesin biomass Augustof diatoms was of 2016 wereregister (0.272 founded mg in with cmAugust− an,T average =of25.5 2016◦ abundance C).(0.272 Near mg the cmof area 104−2, T for × = 10 25.5 the3 cells cultivation °С ).cm Near−2 in theofJune mussels, area 2018 for at 24the T species-indicators= cultivation 25 °C [22]. ofThe mussels, maximum have been24 specie values detected,s-indicators of the among species have which richness been 16 aredetected, (26) beta-mesosaprobionts and among abundance which (74.78 16 [are21 ×]. beta-mesosaprobiontsIt10 should3 cells cm be−2 noted) of algae that [21].were in the It observed should microphytobenthos be in noted epizoon that of of in Karantinnaya the the Azov, microphytobenthos Black Bay and in JapaneseFebruary of the seas2016 Azov, this at BlackT group = 9.7 and ° ofС Japanesesaprobityand in April seas also 2015 this dominated (62.0group × 10of [11 3saprobity cells,13,16 cm,18−2 ,,also 19T =]. 10.3dominated °С), as well[11,13,16,18,19]. as in January 2016 (65.1 × 103 cells cm−2, T = 9.5 °СFor) [21]. comparison, The largest we biomass present was data register on theed structure in August and of functioning 2016 (0.272 of mg the cm plankton−2, T = 25.5 and °С benthic). Near communitiesthe area for the of cultivation Donuzlav of in mussels, the conditions 24 specie ofs-indicators anthropogenic have pollution been detected, of 2000 among [2]. Fromwhich a 16 total are numberbeta-mesosaprobionts of 82 taxa in phytoplankton [21]. It should of be Donuzlav, noted that including in the microphytobenthos 45 species of diatoms, of the 12 species Azov, ofBlack benthic and Japanese seas this group of saprobity also dominated [11,13,16,18,19].

Diversity 2019, 11, 114 10 of 12 diatoms were found that we observed in the microphytobenthos of this area. The largest abundance 3 3 of 5823 million cells m− with biomass of 1932.4 mg m− of phytoplankton was found in May [2]. However, the authors noted that these values were lower in the Black Sea as a whole. Currently phytoplankton is developing worse than before. The abundance and biomass of diatoms in Donuzlav benthos were also lower than in the Sevastopol region. Over the study period, microphytobenthos in the value of pH = 8.41 in the environment was constant and was close to that found in Kazach’ya Bay (Sevastopol) = 8.14 [18]. The range of oxygen saturation variability (103–105%) was small. The maximum value of saturation corresponds to the maximum value of the absolute oxygen content, and the minimum value of oxygen saturation was marked at Station 1 in Donuzlav. In general, there was a slight super saturating of oxygen from 3–5%. 1 Here the average content of dissolved oxygen in the water changed in the range of 8.77–9.40 mg L− in the period 2015–2017 [3]. There was a greater range of variation and seasonal variation than in the open waterbody [1]. In Karantinnaya Bay, as a comparison, the corresponding range was 93–125% [21]. The seasonal variability of concentrations of organic nitrogen and phosphorus was not observed. The maximum values of BOD5, oxidizability, silicon and a high content of Norg were noted, which indicates a certain degree of contamination by domestic sewage.

5. Conclusions The species diversity of benthic diatoms on stony substrates in the Donuzlav Gulf was presented for the first time. A total of 60 taxa of Bacillariophyta, belonging to 3 classes, 18 families, and 34 genera were found. The largest number of diatom species (45) was observed in summer and the smallest (12) was detected in winter. Marine species comprise 55% and brackish-marine ones make up 37% of them, but no freshwater species was identified. The diatom community is dominated by cosmopolites (32%) and arctic–boreal–tropical species (27%). 3 2 2 The maximal abundance (186.2 10 cells cm− ) and biomass (0.083 mgcm− ) of diatoms were noted in June 2018 at salinity 16.58% and T = 24.4 ◦C with the dominant species of L. abbreviata and C. scutellum. The minimum values, 14.8 103 cells cm 2 and 0.005 mg cm 2, were observed in January 2019 · − − at salinity 16.86% and T = 9.0 ◦C with the dominant species of B. rutilans and N. hybrida f. hyalina. A total number of 25 saprobity indicator species of diatoms were found on the epilithon, 12 taxa of which belong to a group of betamesosaprobionts—indicators of moderate organic pollution of water. The high values of BOD5, oxidizability, silicon, Norg and Porg were noted, indicating a certain degree of water pollution by domestic sewage. It has been shown that the pollution in the southwestern part of the Donuzlav Gulf was greatest since this part of the coast is populated most densely in comparison with the northern coast of Donuzlav where there are no coastal settlements except for two mariculture farms. Thus, the species diversity, environmental and quantitative characteristics of diatomic benthos and hydrochemical parameters of the Donuzlav Gulf indicate a high supply of oxygen-enriched waters and a sufficient supply of nutrients for the development of biota. The nutrient levels in the water of the gulf are typical for semi-enclosed waters of the Crimean coastal shelf.

Author Contributions: Methodology, R.L.I.; resources, K.N.P., L.D.N.; writing—original draft preparation, R.L.I.; writing—review and editing, R.L.I., L.D.N. Funding: This research received no external funding. Acknowledgments: This work was carried out within the state assignment AAAA-A18-118021350003-6 from A.O. Kovalevsky Institute of Biology of the Southern Seas of Russian Academy of Sciences. We would like to thank S.S. Barinova from University of Haifa (Israel) for their help and advice on the preparation of drawings on bioindication maps of the Donuzlav Gulf. Conflicts of Interest: The authors declare that they have no conflict of interest. Diversity 2019, 11, 114 11 of 12

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