sm81n4-4523

Hypoxia in deep waters of moderately eutrophic marine lakes, Island of Mljet, eastern Adriatic Sea

Enis Hrustić 1, Svjetlana Bobanović-Ćolić 2

1 Ruđer Bošković Institute, Centre for Marine Research, Giordano Paliaga 5, 52210 Rovinj, Croatia.
(EH) (Corresponding author) E-mail: enis.hrustic@irb.hr. ORCID iD: http://orcid.org/0000-0002-5274-8649
2 Institute for Marine and Coastal Research, University of Dubrovnik, Kneza Damjana Jude 12, PO box 83, 20000 Dubrovnik, Croatia.
(SB-C) E-mail: svjetlana.bobanovic-colic@unidu.hr. ORCID iD: http://orcid.org/0000-0003-2378-0604

Summary: In this study, we explored the impact of eutrophication and stratification on hypoxia in deep waters of moderately warm Croatian marine lakes. Although the Mljet Lakes (MLs) are predominantly oligotrophic, mesotrophic conditions are present at depths below 20 m in the Small Lake (SL) and below 30 m in the Big Lake (BL), along with higher apparent oxygen utilization (AOU). Hypoxia at depths ≥25 m in SL and and ≥40 m in BL was observed between October 2009 and January 2010, and in SL in summer (July and September 2010). Significant differences (p<0.05) in several physical, biological and chemical parameters were detected between the lakes, while AOU, derived oxygen utilization rate (OUR) and organic carbon remineralization rate (OCRR) were not significantly different (p>0.05) between the lakes. An intense and persistent pycnocline throughout the year, comparatively high water temperature, extended water renewal time and summer phytoplankton bloom were identified as physical and biological parameters which might have significantly contributed to increased frequency of hypoxic events in a shallow SL. Significantly (p<0.05) higher ammonium concentration in SL, especially in its deep water, seems to be a long-term chemical feature related to the poor ventilation and higher sediment oxygen demand. At the current level of eutrophication and the present climate change trends, the MLs and similar systems may experience more persistent and intense stratification, which could further prevent mixing between upper and deep waters, likely leading to increasing duration of hypoxia and its negative impacts on the biodiversity of benthic communities.

Keywords: hypoxia; eutrophication, marine lakes, coastal zone, Adriatic Sea, Mediterannean Sea.

Hipoxia en aguas profundas de lagos marinos moderadamente eutróficos, isla de Mljet, mar Adriático oriental

Resumen: En este estudio, exploramos el impacto de la eutrofización y la estratificación sobre la hipoxia, en aguas profundas de los lagos marinos croatas moderadamente cálidos. Aunque los lagos Mljet (LsM) son predominantemente oligotróficos, las condiciones mesotróficas están presentes a profundidades inferiores a 20 m en el Lago Menor (LMe) y a 30 m en el Lago Mayor (LMa), junto con un mayor uso del oxígeno aparente (UOA). Fue observada hipoxia a profundidades ≥25 m en el LMe y ≥40 m en el LMa entre octubre de 2009 y enero de 2010, y en LMe en verano (julio y septiembre de 2010). Fueron detectadas diferencias significativas (p<0.05) en varios parámetros físicos, biológicos y químicos entre los lagos, mientras que el UOA, la tasa de utilización de oxígeno derivado y la tasa de remineralización de carbono orgánico no fueron significativamente diferentes (p>0.05) entre los lagos. La intensa y persistente picnoclina a lo largo del año, la temperatura comparativamente alta del agua, el tiempo prolongado de renovación del agua y la floración estival del fitoplancton se identificaron como parámetros físicos y biológicos que podrían haber contribuido significativamente incrementando la frecuencia de eventos hipóxicos en un LMe superficial. Una concentración de amonio significativamente mayor (p<0.05) en LMe, especialmente en sus aguas profundas, parece ser una característica química a largo plazo relacionada con la escasa ventilación y la mayor demanda de oxígeno en los sedimentos. Al nivel actual de eutrofización con las tendencias actuales del cambio climático, los LsM y sistemas similares podrían experimentar una estratificación más persistente e intensa, lo que podría prevenir aún más la mezcla entre aguas más superficiales y profundas, conduciendo posiblemente a un incremento en la duración de la hipoxia y a los impactos negativos sobre la biodiversidad de las comunidades bentónicas.

Palabras clave: hipoxia; eutrofización; lagos marinos; zona costera; mar Adriático; mar Mediterráneo.

Citation/Como citar este artículo: Hrustić E., Bobanović-Ćolić S. 2017. Hypoxia in deep waters of moderately eutrophic marine lakes, Island of Mljet, eastern Adriatic Sea. Sci. Mar. 81(4): 431-447. doi: http://dx.doi.org/10.3989/scimar.04523.25A

Editor: X.A. Álvarez-Salgado.

Received: July 28, 2016. Accepted: September 19, 2017. Published: November 3, 2017.

Copyright: © 2017 CSIC. This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-by) Spain 3.0 License.

Contents

Summary
Resumen
Introduction
Materials and methods
Results
Discussion
Conclusions
Acknowledgements
References

INTRODUCTIONTop

Eutrophication, of either natural or anthropogenic origin, is closely connected to hypoxia in coastal seas (Nixon 1990Nixon S.W. 1990. Marine eutrophication: a growing international problem. Ambio 19: 101.). The cause of deficiency or absence of oxygen in aquatic systems can be of a physical origin. For example, a) sills and barriers affect circulation of water; b) excessive precipitation over evaporation intensifies the stratification that prevents mixing; and c) weak cooling of surface waters hampers water sinking and decreases water column mixing and aeration of deep waters. In addition, if a pronounced pycnocline develops in such a system, diffusion of oxygen through the density gradient can be further prevented. There are large portions of marine systems that are hypoxic, such as subsurface waters of the East China Sea (Chen et al. 2007Chen C.-C., Gong G.-C., Shiah F.-K. 2007. Hypoxia in the East China Sea: One of the largest coastal low-oxygen areas in the world. Mar. Env. Res. 64: 399-408., Wang et al. 2016Wang H., Dai M., Liu J., et al. 2016. Eutrophication-Driven Hypoxia in the East China Sea off the Changjiang Estuary. Environ. Sci. Technol. 50: 2255-2263.) and deep waters of the Baltic Sea (Conley et al. 2009aConley D.J., Björck S., Bonsdorff E., et al. 2009a. Hypoxia-Related Processes in the Baltic Sea. Environ. Sci. Technol. 43: 3412-3420. , Rolff and Elfwing 2015Rolff C., Elfwing T. 2015. Increasing nitrogen limitation in the Bothnian Sea, potentially caused by inflow of phosphate-rich water from the Baltic Proper. Ambio 44: 601-611.), the Black Sea (Konovalov et al. 1999Konovalov S.K., Eremeev V.N., Suvorov A.M., et al. 1999. Climatic and anthropoghenic variations in the sulfide distribution in the Black Sea. Aquat. Geochem. 5: 13-27.) and the Gulf of Mexico (Justić et al. 1996Justić D., Rabalais N.N., Turner R.E. 1996. Effects of climate change on hypoxia in coastal waters: A doubled CO2 scenario for the northern Gulf of Mexico. Limnol. Oceanogr. 41: 992-1003., 2005Justić D., Rabalais N.N., Turner R.E. 2005. Coupling between climate variability and coastal eutrophication: Evidence and outlook for the northern Gulf of Mexico. Neth. J. Sea Res. 54: 25-35. ). Layers of the open ocean can be hypoxic due to a significantly larger supply of organic matter than oxygen (Kamykowski and Zentara 1990Kamykowski D., Zentara S-J. 1990. Hypoxia in the world ocean as recorded in the historical data set. Deep-Sea Res. Part A 37: 1861-1874.), but it is hypoxia/anoxia in coastal seas (Diaz 2001Diaz J.R. 2001. Overview of Hypoxia around the World. J. Environ. Qual. 30: 275-281.) that causes greater concern regarding damage to the biodiversity of ecosystems (Ciglenečki et al. 2015Ciglenečki I., Marguš M., Bura-Nakić E., et al. 2015. Impacts of extreme weather events on highly eutrophic marine ecosystem (Rogoznica Lake, Adriatic coast). Cont. Shelf. Res. 108: 144-155.), which many human activities depend on, such as fisheries and tourism.

Lakes behave like natural laboratories because of their comparatively prompt responses to environmental changes (Ciglenečki et al. 2015Ciglenečki I., Marguš M., Bura-Nakić E., et al. 2015. Impacts of extreme weather events on highly eutrophic marine ecosystem (Rogoznica Lake, Adriatic coast). Cont. Shelf. Res. 108: 144-155.). Those which contain sea water originated a few thousand years ago by processes related to late postglaciation, i.e. isostasia and elevation of sea level. The marine lakes in Scandinavia were transformed into freshwater lakes due to isostasia, and hence some of them contain the trapped sea water at the bottom (Bøyum 1973Bøyum A. 1973. Salsvatn, a lake with old sea water. Schweiz. Z. Hydrol. 35: 262-277.). In comparison, the marine lakes of the eastern Adriatic coast are incorporated into the system of karst, containing sea water in the entire water column (Žic et al. 2012Žic V., Truesdale V.W., Garnier C., et al. 2012. The distribution of iodine in the Croatian marine lake, Mir - The missing iodate. Estuar. Coast. Shelf Sci. 115: 377-387., Hrustić et al. 2013Hrustić E., Carić M., Čalić M., et al. 2013. Alkaline phosphatase activity and relative importance of picophytoplankton in autumn and early spring (Mljet Lakes, eastern Adriatic Sea). Fresen. Environ. Bull. 22: 636-648., Ciglenečki et al. 2015Ciglenečki I., Marguš M., Bura-Nakić E., et al. 2015. Impacts of extreme weather events on highly eutrophic marine ecosystem (Rogoznica Lake, Adriatic coast). Cont. Shelf. Res. 108: 144-155.). The Mljet Lakes (MLs) were formed during the Mesozoic Era as karstic depressions filled with fresh water (Govorčin et al. 2001Govorčin D.P., Juračić M., Horvatinčić N., et al. 2001. Holocene sedimentation in the Soline Channel (Mljet Lakes, Adriatic Sea). Nat. Croat. 10: 247-258.). The postglacial sea level rose around 4 to 5 thousand years ago and consequently the ingression of sea turned them into a marine environment (Bognar and Curić 1995Bognar A., Curić L. 1995. Geomorphologic Characteristics of the Island of Mljet. In: Durbešić P., Benović A. (eds), Proceedings of the Symposium “The natural features and social valorisation of the Island Mljet”: Ecological monographs 6, Croatian Ecological Society, Zagreb, pp. 73-84.). Unlike the well-oxygenated Jezero Mir (Lake Mir) (43.8864°N 15.1667°E), which is up to 10 m deep (Žic et al. 2012Žic V., Truesdale V.W., Garnier C., et al. 2012. The distribution of iodine in the Croatian marine lake, Mir - The missing iodate. Estuar. Coast. Shelf Sci. 115: 377-387.), and the meromictic lake Zmajevo oko (Dragon’s Eye) (43.5309°N 15.9588°E), which is up to 15 m deep, permanently hypoxic below 13 m and occasionally completely anoxic (Ciglenečki et al. 2015Ciglenečki I., Marguš M., Bura-Nakić E., et al. 2015. Impacts of extreme weather events on highly eutrophic marine ecosystem (Rogoznica Lake, Adriatic coast). Cont. Shelf. Res. 108: 144-155.), episodes of hypoxia are expected in deep waters of the MLs (Buljan and Špan 1976Buljan M., Špan J. 1976. Hydrographic properties of the sea water lakes on the Island of Mljet and the adjoining sea in eastern South Adriatic Sea. Acta Adriat. 6: 1-227.) within the system of coastal karst in Croatia.

The strait (depth ~0.8 m, width ~2 m) between SL (~241000 m2, ~3349000 m3) and BL (~1450000 m2, ~58000000 m3) and the strait (depth ~2.5 m, width ~10 m) between BL and Soline Channel (SC) (Fig. 1) hinder the exchange of deep lake waters with the adjacent coastal sea. BL is up to 49 m deep, with an average depth of 13.6 m, while SL is up to 29 m deep, with an average depth of 7.6 m (Cuculić et al. 2012Cuculić V., Cukrov N., Ereš Z., et al. 2012. Report “The impact of the water masses on the spatial and temporal distribution of ecotoxicants in the Small Lake and Big Lake of the National Park Mljet”. Institute Ruđer Bošković, Zagreb, 80 pp.). The advection from the open sea occurrs strictly in the surface layer, while circulation is dominantly driven by tides <25 cm and tidal phase shifts through the straits acting as filters at semidiurnal frequencies (Peharda and Vilibić 2008Peharda M., Vilibić I. 2008. Modelling the recruitment effect in a small marine protected area: the example of saltwater lakes on the Island of Mljet (Adriatic Sea). Acta Adriat. 49: 25-35.). The absence of upwelling, unlike from coastal waters (Batistić et al. 2013Batistić M., Lučić D., Carić M., et al. 2013. Did the alien calycophoran Muggiaea atlantica outcompete its native congeneric M. kochi in the marine lakes of Mljet Island (Croatia)? Mar. Ecol. 34(Suppl. 1): 3-13.), constrains the mixing and supports the development of hypoxia in deep waters of the MLs.

Some instabilities, i.e. internal waves in deep water of BL, were explained by severe cooling and sinking of the surface water to the bottom (Buljan and Špan 1976Buljan M., Špan J. 1976. Hydrographic properties of the sea water lakes on the Island of Mljet and the adjoining sea in eastern South Adriatic Sea. Acta Adriat. 6: 1-227.). Such an event has not been recorded in SL so far, but some water column instability can come from lateral and bottom subaquatic karstic springs (Wunsam et al. 1999Wunsam S., Schmidt R., Müller J. 1999. Holocene lake development of two Dalmatian lagoons (Malo and Veliko Jezero, Isle of Mljet) in respect to changes in Adriatic Sea level and climate. Palaeogeogr. Palaeoclimatol. Palaeoecol. 146: 251-281.). Cuculić et al. (2012)Cuculić V., Cukrov N., Ereš Z., et al. 2012. Report “The impact of the water masses on the spatial and temporal distribution of ecotoxicants in the Small Lake and Big Lake of the National Park Mljet”. Institute Ruđer Bošković, Zagreb, 80 pp. estimated that water renewal times (WRTs) in SL and BL during the mixing period (October-March) were 81 and 70 days and during the period of stratification (April-September) they were 44 and 36 days, respectively. WRT for the stratification period refers only to the upper waters above the depth of 10 m in SL and 17 m in BL. When SL and BL were considered as one system exchanging the water with the open sea, WRTs of the MLs during the mixing and stratifed periods were 82 and 43 days, respectively. These values, being closer to WRT in SL, indicated that the exchange between BL and SL is the limiting factor for the total exchange of water in the system (Cuculić et al. 2012Cuculić V., Cukrov N., Ereš Z., et al. 2012. Report “The impact of the water masses on the spatial and temporal distribution of ecotoxicants in the Small Lake and Big Lake of the National Park Mljet”. Institute Ruđer Bošković, Zagreb, 80 pp.).

In this study, we intended to investigate the link between natural eutrophication, water column stratification and episodes of hypoxia in deep waters of moderately warm marine lakes. The arrangement of data concerning the mixing and stratification periods and water column division was adjusted to be comparable to the recent report on WRT in the MLs (Cuculić et al. 2012Cuculić V., Cukrov N., Ereš Z., et al. 2012. Report “The impact of the water masses on the spatial and temporal distribution of ecotoxicants in the Small Lake and Big Lake of the National Park Mljet”. Institute Ruđer Bošković, Zagreb, 80 pp.), while offering an extension to parts of the research that revealed an anoxic event in deep water of BL more than a decade ago (Benović et al. 2000Benović A., Lučić D., Onofri V., et al. 2000. Ecological characteristics of the Mljet Island seawater lakes (South Adriatic Sea) with special reference to their resident populations of medusae. Sci. Mar. 64 (Suppl. 1): 197-206.). Anoxia in the water of the MLs was not detected after August 1996 and vertical homogenization has been noted to occur regularly since 2003 (Vilibić et al. 2010Vilibić I., Žuljević A., Nikolić V. 2010. The dynamics of a saltwater marine lake (Big Lake, Island of Mljet, Adriatic Sea) as revealed by temperature measurements. Acta Adriat. 51: 119-130.). Having apparent oxygen utilization (AOU) as an important parameter of our study, including the report on the WRT (Cuculić et al. 2012Cuculić V., Cukrov N., Ereš Z., et al. 2012. Report “The impact of the water masses on the spatial and temporal distribution of ecotoxicants in the Small Lake and Big Lake of the National Park Mljet”. Institute Ruđer Bošković, Zagreb, 80 pp.), we were able to determine oxygen utilization rate (OUR) and organic carbon remineralization rate (OCRR) (Feely et al. 2004Feely R.A., Sabine C.L., Schlitzer R., et al. 2004. Oxygen Utilization and Organic Carbon Remineralization in the Upper Water Column of the Pacific Ocean. J. Oceanogr. 60: 45-52.) in the MLs for the first time, and to provide information and conclusions that may be valuable for future studies of similar environments worldwide.

MATERIALS AND METHODSTop

Sampling

The sampling locations and dates are presented in Table 1 and Figure 1. The stations in SL (at its deepest point), BL (Vrbovačka) and Gonoturska Bay (G) are 29, 46 and ~60 m deep, respectively. The sampling was performed between 9 am and 3 pm, repeating the sequence SL, BL and G.

Table 1. – Sampling dates with corresponding seasons and periods. The first three letters of each month (except for Sept.), season and period are used in the abbreviations.

Date 20 Oct. ‘09. 20 Nov. ‘09. 18 Dec. ‘09. 20 Jan. ‘10. 15 Feb. ‘10. 17 Mar. ‘10. 21 Apr. ‘10. 25 May ‘10. 24 June ‘10. 15 July ‘10. 17 Aug. ‘10. 14 Sept. ‘10.
Season Aut Aut Aut Win Win Win Spr Spr Spr Sum Sum Sum
Period Mix Mix Mix Mix Mix Mix Str Str Str Str Str Str

sm4523fig1.jpg

Full size image

Fig. 1. – Sampling stations in the Mljet National Park: Small Lake (SL), Big Lake (BL) and Gonoturska Bay (G).

Thermohaline properties, precipitation, compensation depth

Salinity (S), temperature (t) and density (σT) were determined by an SBE 19plus CTD probe at a depth resolution of 1 m to 27, 45 and 50 m in SL, BL and G respectively. All sensors were regularly calibrated by Sea-Bird Electronics Inc. The difference in t, S and σT for every metre of the water column depth was calculated and named ΔS (m–1), Δt (°C m–1) and ΔσT (kg m–4). The depth visibility of the white Secchi disc (Sec, diameter 30 cm) was multiplied by 3 (Berman et al. 1985Berman T., Walline P.W., Schneller A., et al. 1985. Secchi disk record: A claim for the eastern Mediterranean. Limnol. Oceanogr. 30: 447-448.) to determine the depth of the photic zone (Pz). Data on precipitation at the nearest meteorological station to the MLs in Goveđari (Fig. 1) was ensured by Državni hidrometeorološki zavod (DHMZ, Croatian Meteorological and Hydrological Service, meteo.hr).

Dissolved oxygen concentration, apparent oxygen utilization (AOU), oxygen utilization rate (OUR), organic carbon remineralization rate (OCRR) and nutrients

The samples were collected by 5-L Niskin bottles at the surface (0–1), 5, 10, 15, 20, 25, 30, 35, 40 and 43 m in BL and at the same depths up to 25 m in SL. The samples in G were taken at the same depths up to 20 m plus at 30, 40 and 45 m. Dissolved O2 (DO) was precipitated in Winkler bottles and determined by iodometric titration (Grasshoff et al. 1983Grasshoff K., Ehrhardt M., Kremling K. 1983. Methods of seawater analysis – Second, Revised and extended edition. Verlag Chemie GmbH, Weinheim, 419 pp.). The results (ml O2 l–1) were converted to mg O2 l–1 using the conversion factor 1.42903 (Owens and Millard 1985Owens W.B., Millard R.C.Jr. 1985. A new algorithm for CTD oxygen calibration. J. Phys. Oceanogr. 15: 621-631., Garcia and Gordon 1992Garcia H.E., Gordon L.I. 1992. Oxygen solubility in seawater: Better fitting equations. Limnol. Oceanogr. 37: 1307-1312. , Sea-Bird Electronics Inc.). The threshold for hypoxia was set at <2.8 mg O2 l–1, as proposed by Diaz and Rosenberg (1995)Diaz J.R., Rosenberg R. 1995. Marine benthic hypoxia: a review of its ecological effects and the behavioural responses of benthic macrofauna. Oceanogr. Mar. Biol. Ann. Rev. 33: 245-303., which is equal to <1.96 ml O2 l–1 and <87.5 μmol O2 l–1. Anoxia stands for the environment without DO. AOU was calculated as the difference between DO concentration at saturation of 100% (O2’) at a given S, t and depth (Weiss 1970Weiss R.F. 1970. Solubility of nitrogen, oxygen and argon in water and seawater. Deep-Sea Res. 17: 721-735.) and the associated measured DO concentration, expressed as μmol O2 kg–1 (Feely et al. 2004Feely R.A., Sabine C.L., Schlitzer R., et al. 2004. Oxygen Utilization and Organic Carbon Remineralization in the Upper Water Column of the Pacific Ocean. J. Oceanogr. 60: 45-52.). The unit for DO i.e. O2 (mg l–1) was adjusted to the criterion for hypoxia (Diaz and Rosenberg 1995Diaz J.R., Rosenberg R. 1995. Marine benthic hypoxia: a review of its ecological effects and the behavioural responses of benthic macrofauna. Oceanogr. Mar. Biol. Ann. Rev. 33: 245-303.). We also calculated OUR (μmol kg–1 year–1) as AOU/water age (Feely et al. 2004Feely R.A., Sabine C.L., Schlitzer R., et al. 2004. Oxygen Utilization and Organic Carbon Remineralization in the Upper Water Column of the Pacific Ocean. J. Oceanogr. 60: 45-52.) using corresponding individual WRTs on the basis of “e-folding time” by Cuculić et al. (2012)Cuculić V., Cukrov N., Ereš Z., et al. 2012. Report “The impact of the water masses on the spatial and temporal distribution of ecotoxicants in the Small Lake and Big Lake of the National Park Mljet”. Institute Ruđer Bošković, Zagreb, 80 pp.. OCRR (μmol kg–1 year–1) was calculated as OUR×RC:O (Feely et al. 2004Feely R.A., Sabine C.L., Schlitzer R., et al. 2004. Oxygen Utilization and Organic Carbon Remineralization in the Upper Water Column of the Pacific Ocean. J. Oceanogr. 60: 45-52.), where RC:O represents the molar “Redfield ratio” of C:O. We employed 0.69 for RC:O as it represents an RC:O of 117:170 (Anderson and Sarmiento 1994Anderson L.A., Sarmiento J.L. 1994. Redfield ratios of remineralization determined by nutrient data analysis. Global Biogeochem. Cy. 8: 65-80.) and the average range of RC:O (0.53-0.85) reported by Li and Peng (2002)Li Y.H., Peng T.-H. 2002. Latitudinal change of remineralization ratios in the oceans and its implications for nutrient cycles. Global Biogeochem. Cy. 16: 1130.. The calculation of OCRR represents only the organic carbon mineralized when oxygen is the acceptor of the electron transport system. If another acceptor such as nitrate is also used, there will be an extra OCRR not accounted for by this calculation.

Samples (50 ml) for analysis of ammonium (NH4) were stabilized by addition of 2 ml of phenol (1 mol l–1) dissolved in 95% vol/vol ethanol (Ivančić and Degobbis 1984Ivančić I., Degobbis D. 1984. An optimal manual procedure for ammonia analysis in natural waters by the indophenol blue method. Water Res. 18: 1143-1147.) and stored in the dark at 4°C. The samples (500 ml) for other nutrients were stored at –22°C. The concentrations of nitrate (NO3), nitrite (NO2), NH4, reactive silicates (SiO4), orthophosphate (PO4), total phosphorus (TP) and total nitrogen (TN) were determined according to Strickland and Parsons (1972)Strickland J.D.H., Parsons T.R. 1972. A Practical Handbook of Sea Water Analysis (second edition). B. Fish. Res. Board Can. 167: 1-310. without filtration. Difference between TP and PO4 was designated as “other P” i.e. Poth. Difference between TN and dissolved inorganic nitrogen (DIN) (NO3+NO2+NH4) was designated as “other N” i.e. Noth. The persulfate oxidation of unfiltered samples in autoclave (127°C, 25 min) enabled us to measure TP (Menzel and Corwin 1965Menzel D.W., Corwin N. 1965. The measurement of total phosphorus in seawater based on the liberation of organically bound fractions by persulfate oxidation. Limnol. Oceanogr. 10: 280-282.) and TN (D’Elia and Steudler 1977D’Elia C.F., Steudler P.A. 1977. Determination of total nitrogen in aqueous samples using persulfate digestion. Limnol. Oceanogr. 22: 760-764.), which meant that Poth and Noth consisted of dissolved organic P (DOP) and N (DON), and non-dissolved (colloidal and particulate) organic and non-dissolved inorganic P and N species, respectively.

Chlorophyll a and trophic index

DIN (μg l–1), TP (μg l–1), chlorophyll a (Chl a) (μg l–1) detected by the CTD probe and O2 absolute deviation (%) from saturation (O2’) were used to calculate TRIX:

TRIX=[Log10(TP×DIN×Chl a│O2(%)-O2’(%)│)+1.5]/1.2.

TRIX is the trophic index first developed for the Italian northwestern Adriatic coastal waters, and it has a range of values from 0 to 10, where 0-4, 4-5, 5-6 and 6-10 indicate the high, good, moderate and poor quality of the water, respectively (Vollenweider et al. 1998Vollenweider R.A., Giovanardi F., Montanari G., et al. 1998. Characterization of the trophic conditions of marine coastal waters with special reference to the NW Adriatic Sea: proposal for a trophic scale, turbidity and generalized water quality index. Environmetrics 9: 329-357.). The detector of fluorescence was calibrated by the Chl a measurements from the culture of a diatom Thalassiosira weissflogii at the Sea-Bird Electronics Inc. laboratories. The same concept was employed by Viličić et al. (2009)Viličić D., Kuzmić M., Bosak S., et al. 2009. Distribution of phytoplankton along the thermohaline gradient in the north-eastern Adriatic channel; winter aspect. Oceanologia 51: 495-513.. The data on Chl a in October and November 2009 were lost, so TRIX could not be calculated for October and November 2009.

Picoplankton

The samples (50 ml) were preserved in neutralized solution of formaldehyde (~4% vol/vol) on board and stored in the dark at 4°C until analysis. Abundance and sizes of identified picoplankton groups were determined by epifluorescent microscopy (Zeiss Jenalumar, magnification 1500). Heterotrophic bacteria (HBa) were detected by direct epifluorescent microscopy (Hobbie et al. 1977Hobbie J.E., Daley R.J.S., Jasper S. 1977. Use of Nucleopore filters for counting bacteria by fluorescence microscopy. Appl. Environ. Microb. 33: 1225-1228.). Heterotrophic picoflagellates (HPF), autotrophic picoflagellates (APF) and picocyanobacteria (Cyano) were detected using proflavine (Haas 1982Haas L.W. 1982. Improved epifluorescence microscopy for observing planktonic micro-organisms. Ann. Inst. Oceanogr. Paris 58(Supplement S): 261-266.). Biomass of HBa was calculated by the conversion factor 20 fg C cell–1 (Lee and Fuhrman 1987Lee S., Fuhrman J.A. 1987. Relationships between biovolume and biomass of naturally derived marine bacterioplankton. Appl. Environ. Microb. 53: 1298-1303.). Biomass of Cyano was calculated by the conversion factor 250 fg C cell–1 (Kana and Gilbert 1987Kana T.M., Gilbert P.M. 1987. Effect of irradiance up to 2000 μE m–1 s–1 on marine Synechococcus WH7803: I. Growth, pigmentation and cell composition. Deep-Sea Res. 34: 479-495.). The volume (μm3) of picoplankton cell categories was calculated using the equations according to morphological classes: ellipsoid (V=πLw2/6) and sphere (V=4πr3/3), where L is the length (μm), w is the width (μm) and r is the radius (μm) of the cell. Biomass of HPF was calculated as B=nVFC, where n is abundance, V is average cell volume (μm3) and FC is the conversion factor 0.22 pg C μm-3 (Borsheim and Bratbak 1987Borsheim K.Y., Bratbak G. 1987. Cell volume to cell carbon conversion factors for a bacterivorous Monas sp. enriched from seawater. Mar. Ecol. Prog. Ser. 36: 171-175.). The equation 0.433(μm3)0.863=pg C cell–1 (Verity et al. 1992Verity P.G., Robertson C.Y., Tronzo C.R., et al. 1992. Relationships between cell volume and the carbon and nitrogen content of marine photosynthetic nanoplankton. Limnol. Oceanogr. 3: 1434-1446. ) was employed as a conversion factor for APF by taking into account average cell volumes (μm3).

Statistics

Statistica for Windows (Statsoft Inc.) was used to determine the distribution of variables by Kolmogorov-Smirnov and Lilliefors criteria and to calculate Spearman Rank (non-normal distribution) and Pearson’s correlation coefficients (r) at p<0.05 after log(x+1) transformation of the data. One-way analysis of variance and Student-Newman-Keuls tests were applied to determine the significant differences (p<0.05) between SL, BL and G, representing the coastal sea. T tests were employed to determine significant differences (p<0.05) between upper and deep waters at each location, using the depths 10 and 17 m (Cuculić et al. 2012Cuculić V., Cukrov N., Ereš Z., et al. 2012. Report “The impact of the water masses on the spatial and temporal distribution of ecotoxicants in the Small Lake and Big Lake of the National Park Mljet”. Institute Ruđer Bošković, Zagreb, 80 pp.) as distributors for SL and BL, respectively, while 20 m was employed as a distributor for G. Furthermore, t tests were employed to detect significant differences (p<0.05) between periods of mixing (October-March) and stratification (April-September) at each location.

RESULTSTop

The water column stratification

We found that salinity (S) was significantly (p<0.05) lower in the MLs than in G (Supplementary material Tables S1, S2). Higher surface water S in early autumn (October) (Fig. 2), also found by Miloslavić et al. (2015)Miloslavić M., Lučić D., Žarić M., et al. 2015. The importance of vertical habitat gradients on zooplankton distribution in an enclosed marine environment (South Adriatic Sea). Mar. Biol. Res. 11: 462-474., was probably related to the faster NW surface water currents (Leder et al. 1995Leder N., Smirčić A., Gržetić Z. 1995. Seasonal changes of the sea currents in the western aquatory of the Island Mljet. In: Durbešić P., Benović A. (eds), Symposium “The natural features and social valorisation of the Island Mljet”: Ecological monographs 6, Croatian Ecological Society, Zagreb, pp. 415-436.), which introduced the open sea water into the MLs. SL was exposed to the most intense mainland influence in terms of the widest ranges of t (8.55-27.74°C) and S (31.61-38.26) (Fig. 2A). At SL, the coldest water (8.55°C) had the lowest S (31.61) (depth 1 m, December) (Fig. 2A). Also, SL had a significantly (p<0.05) higher ΔσT than other locations (Table S1, S2, Figs 2A and 3A). ΔSmax at G (0.42 m–1) was detected in the 29-30 m layer (37.08-37.50, respectively) in January (Figs 2C and 3C). ΔSmax in BL (0.31 m–1) was detected in the subsurface waters (2-3 m, S 35.70-36.01, respectively) in February (Figs 2B and 3B). The situation of ΔS in SL was significantly amplified: ΔSmax in SL (2.41 m–1) was detected in the subsurface water (2-3 m, S 33.17-35.58, respectively) in December, which is ~6 and 8 folds higher than the ΔSmax in G and BL, respectively (Figs 2 and 3). An average ΔS (1-4 m, S 31.61-36.81) in SL in December equalled 1.73 m–1, almost matching the ΔS in surface waters of lower parts of highly stratified estuaries on the eastern Adriatic coast (Carić et al. 2012Carić M., Jasprica N., Kršinić F., et al. 2012. Hydrography, nutrients and plankton along the longitudinal section of the Ombla Estuary (south-eastern Adriatic). J. Mar. Biol. Assoc. U.K. 92: 1227-1242. , Cindrić et al. 2015Cindrić A.-M., Garnier C., Oursel B., et al. 2015. Evidencing the natural and anthropogenic processes controlling trace metals dynamic in a highly stratified estuary: The Krka River estuary (Adriatic, Croatia). Mar. Poll. Bull. 94: 199-216.). A sharp halocline (1-7 m) was observed in SL (December-April) (Figs 2A and 3A). Minimum monthly ΔSmax (0.95 m–1) in SL (6-7 m) developed in April.

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Fig. 2. – Temperature, salinity, density in Small Lake (A), Big Lake (B) and Gonoturska Bay (C).

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Fig. 3. – Gradients of temperature (Δt), salinity (ΔS) and density (ΔσT) in Small Lake (A), Big Lake (B) and Gonoturska Bay (C).

The thermocline in SL appeared in March, but a deeper and more pronounced thermocline developed around the depth of 10 m between April and June (Figs 2A and 3A). Further, the thermocline advanced deeper during the summer, being the strongest (–2.716°C m–1) between 10 and 11 m (24.685-21.969°C, respectively) in August and the deepest in September (14-15 m, –2.22°C m–1). These results supported the division of the water column of SL at a depth of 10 m, as proposed by Cuculić et al. (2012)Cuculić V., Cukrov N., Ereš Z., et al. 2012. Report “The impact of the water masses on the spatial and temporal distribution of ecotoxicants in the Small Lake and Big Lake of the National Park Mljet”. Institute Ruđer Bošković, Zagreb, 80 pp.. The thermocline in BL and G developed with a small time lag after that in SL (Fig. 2). The most intense Δt in BL stretched from 13-16 m in April to 22-25 m in September (Figs 2B and 3B). Δtmax (–2.107°C m–1) in BL developed in the 23-24 m layer (18.476-16.369°C, respectively) in September (Figs 2B and 3B). Δtmax did not ideally match the division of the water column in BL at a depth of 17 m, but the most intense ΔσT were detected in the 16-19 m layer, except in September (23-24 m, ΔσT 0.29 kg m–4) (Figs 2B and 3B), which supported the division at a depth of 17 m, as proposed by Cuculić et al. (2012)Cuculić V., Cukrov N., Ereš Z., et al. 2012. Report “The impact of the water masses on the spatial and temporal distribution of ecotoxicants in the Small Lake and Big Lake of the National Park Mljet”. Institute Ruđer Bošković, Zagreb, 80 pp.. Δtmax in G (–0.843°C m–1) developed in the 3-4 m layer (21.210-20.367°C, respectively) in August (Figs 2C and 3C). The water column of G had a notable ΔσT in May-September, being occasionally pronounced both above and below 20 m (Figs 2C and 3C).

Deep water of BL was persistently colder than coastal sea at comparable depths (Fig. 2B, C). A similar finding was reported by Benović et al. (2000)Benović A., Lučić D., Onofri V., et al. 2000. Ecological characteristics of the Mljet Island seawater lakes (South Adriatic Sea) with special reference to their resident populations of medusae. Sci. Mar. 64 (Suppl. 1): 197-206.. The only water that is denser than the near-bottom water of BL in October (σT 28.81 kg m–3, depth ≥43 m) was situated below the depth of 44 m in G (σTmax 28.83 kg m–3, depth of 49 m) in March (Figs 2C). ΔσTmax in G (0.34 kg m–4) was found in May (16-17 m) (Fig. 2C). ΔσTmax in BL (0.32 kg m–4, 18-19 m, July) (Figs 2B) was similar to that in G, while ΔσTmax in SL (1.44 kg m–4, 2-3 m, December) exceeded the values of the highest ΔσT in BL and G more than fourfold. In addition, all samplings in SL had greater ΔσTmax (the lowest monthly ΔσTmax 0.36 kg m–4, 14-15 m, September) than overall ΔσTmax in G and BL. It was noted that ΔσTmax in SL in July was almost double (0.62 kg m–4, 8-9 m) the overall values, 10 m deeper ΔσTmax in BL in July. The pycnocline in the MLs plunged deeper towards the end of summer (Fig. 2A, B).

Because of the extraordinary low S in the surface water of SL in December 2009, data on precipitation before and during samplings were analysed (Fig. 4). Intense precipitation from 14 to 17 December (average, ~30 mm m–2 day–1) significantly impacted on the lowest S in the surface water of SL on 18 December. By applying the partial differential equation

ΔσT = (∂ρ/∂S)t × ΔS + (∂ρ/∂t)S × Δt,

 

while keeping the S and t constant (http://www.csgnetwork.com/h2odenscalc.html) in two separate tests between the depths of 2 and 3 m, we concluded that ΔS (2.411 m–1) and Δt (2.319°C m–1) contributed circa 130% and -30%, respectively, to the detected ΔσTmax in the 2-3 m layer in SL in December.

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Fig. 4. – Precipitation in Goveđari, close to the Mljet Lakes (October 2009-September 2010).

The depth of the photic zone

Mutually different Secchi disc (Sec) values decreased from the outer station towards SL (Table 2). Minimum Sec in SL (June-July) indicated a comparatively high concentration of suspended matter in late spring-summer, most likely due to increased biological activity. The average depth of the photic zone (Pz) reached the bottom of the sampled water columns in SL (25 m) and BL (43 m), while at G an average depth of the Pz (56 m) was 11 m below the last sampled depth by a Niskin sampler (45 m). At all stations the depth of the Pz was on average 3-4 m above the surface sediments.

Table 2. – The estimation of the photic zone depth in Small Lake (SL), Big Lake (BL) and Gonoturska Bay (G). Pz, the depth of the photic zone; Sec, the depth of the Secchi disc visibility.

Parameter/ Location Sec: Min-Max (m) Pz: Min-Max (m) Month/Year of the Min Sec detection Month/Year of the Max Sec detection Avg±sd (m)
SL 7-12
21-bottom (29)
June-July ‘10. Mar. ‘10. Sec: 8.77±1.56 Pz: 25.35±3.57
BL 11-19
33-bottom (46)
June and Aug. ‘10. Dec. ‘09. Sec: 14.5±2.84 Pz: 43.5±8.51
G 14.5-22
43.5-bottom (60)
Jan. ‘10 Dec. ‘09 and
July-Aug. ‘10
Sec: 18.77±2.69 Pz: 56.25±8.49
t tests for Sec differences (p<0.01) SL vs. BL
t=4.36×10–5
SL vs. G
t=1.78×107
BL vs. G
t=3.54×10–3

DO, AOU, OUR and OCRR

The maximum (193.90 μmol kg–1) and minimum (–71.89 μmol kg–1) AOU were detected at 43 m in BL (October) and 10 m in SL (August), respectively (Fig. 5A, B). There were only two samplings in SL (April, August), when O2 at 25 m exceeded 5 mg l–1 (5.16, 5.63 mg l–1) (Fig. 5A) accompanied by AOU 93.42 and 75.60 μmol kg–1 (Fig. 5A), respectively. In February, March, May and June, O2 ranged from 3.13 to 4.16 mg l–1 (Fig. 5A), with AOU from 124.00 to 155.22 μmol kg–1 at 25 m in SL (Fig. 5A). The near-bottom waters of SL at 25 m were hypoxic from October to January and in July and September (Fig. 5A). In comparison, the near-bottom waters of BL (depths of 40 and 43 m) were hypoxic only in the period October-January, having a range of 2.06-2.76 mg O2 l–1 (Fig. 5B). In spite of having higher DO than samples from 25 m depth in SL (October-January, AOU 182.62–189.83 μmol kg–1, Fig. 5A), the deepest samples from BL (43 m) had a similar or higher AOU (184.04–193.90 μmol kg–1) (Fig. 5B). We found that AOU in the water column of G was mostly negative, being negligibly positive only occasionally (three samples in December and two samples in February) (Fig. 5C), whereas AOU was positive in the whole water columns of SL and BL in December (Fig. 5A, B). Significantly greater consumption than production of O2 was detected below 20 m in SL and 30 m in BL throughout the whole study period (Fig. 5A, B. The minimum of O2 (1.70 mg l–1) was detected at 25 m in SL in December (Fig. 5A), when ΔσTmax occurred in the subsurface water (2-3 m). Using individual WRTs (Cuculić et al. 2012Cuculić V., Cukrov N., Ereš Z., et al. 2012. Report “The impact of the water masses on the spatial and temporal distribution of ecotoxicants in the Small Lake and Big Lake of the National Park Mljet”. Institute Ruđer Bošković, Zagreb, 80 pp.) as the water age in SL and BL, we calculated OUR and OCRR (Table S3, S6, S9, S10).

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Fig. 5. – Dissolved oxygen concentration, apparent oxygen utilization and ammonium concentration in Small Lake (A), Big Lake (B) and Gonoturska Bay (C).

Nutrients and estimate of phytoplankton growth

The negative correlations (p<0.05) between O2 and inorganic nutrients in the MLs (Table 3) were most likely associated with regeneration after decomposition of the sinking organic matter excreted by Aurelia sp. (Alvarez-Colombo et al. 2009Alvarez-Colombo G., Benović A., Malej A., et al. 2009. Acoustic survey of a jellyfish-dominated ecosystem (Mljet Island, Croatia). Hydrobiologia 616: 99-111., Tinta et al. 2016Tinta T., Kogovšek T., Turk V., et al. 2016. Microbial transformation of jellyfish organic matter affects the nitrogen cycle in the marine water column – A Black Sea case study. J. Exp. Mar. Biol. Ecol. 475: 19-30. ) and other zooplankton and phytoplankton. The positive correlations between S and NO3 in the MLs are in accordance with oxidation (i.e. remineralization) of organic matter during its sinking towards deep waters of greater S (Fig. 2A, B). SL had significantly (p<0.05) higher NH4 than BL and G, especially in deep waters (Fig. 5), while G had significantly (p<0.05) lower SiO4 and SiO4/DIN than the MLs (Table S1, S2). In April, SiO4 and NO3 were notably depleted in the water columns and upper waters of the MLs, respectively (Fig. 6A, B). SiO4 was significantly (p<0.05) correlated with O2, Chl a and t in BL. The nutricline (NO3 and SiO4) in the MLs (Fig. 6A, B), except for SiO4 in April and NO3 in SL in the stratification period, was accompanied by the rise of AOU under ~20 m in SL and 30 m in BL (Fig. 5A, B). Concentration of PO4 had the maxima of 0.39, 0.50 and 0.71 μmol l–1 (not presented) in G, BL and SL at 0–1 m in February, at 0–1 m and at 5 m in January, respectively. Maximum NO2 concentrations of 0.28, 0.24 and 3.17 μmol l–1 (not presented) in G, BL and SL were detected at 45 m in March, 30 m in March and 25 m in October, respectively. Maximum NH4, NO3, and SiO4 concentrations (4.10, 6.55 and 52.50 μmol l–1) were detected at 25 m in SL in November (Fig. 5A) and 43 m in BL in October and in December (Fig. 6B), respectively.

Table 3. – Significant (p<0.05) correlations (r) at explored locations in Mljet waters. SL, Small Lake; BL, Big Lake; G, Gonoturska Bay; Poth, total phosphorus concentration – PO4; t, temperature; S, salinity; HPF, biomass of heterotrophic picoflagellates; APF, biomass of autotrophic picoflagellates; HBa, biomass of heterotrophic bacteria; Cyano, biomass of picocyanobacteria; Chl a, chlorophyll a concentration.

Location Parameter NO3 NO2 NH4 PO4 Poth SiO4 t Depth
SL O2 –0.45 –0.51 –0.30
S 0.42 0.40 0.38
t –0.32
HPF 0.38
APF 0.47
BL O2 –0.42 –0.33 –0.28 –0.24 –0.39
Chl a 0.34 0.42 0.30 0.36 –0.37 0.58
S 0.33 0.48
t –0.39 –0.62 –0.47
HPF –0.31 0.43
G O2 0.36 –0.53
Chl a 0.42 0.47 –0.66 0.59
t –0.63
HBa 0.37
Cyano 0.46 0.37 0.43 0.40 –0.41

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Fig. 6. – Nitrate concentration, silicate concentration, chlorophyll a concentration and TRIX in Small Lake (A), Big Lake (B) and Gonoturska Bay (C).

Chl a and TRIX

Chl a had the highest (p<0.05) concentration in SL (Table S1, S2). The trend of rising Chl a concentration from G towards the more enclosed waters in SL was observed. Chl a was positively correlated with depth at deeper locations (BL, G) (Table 3). Chl a max (1.76 μg l–1) was detected in SL at 27 m in March (Fig. 6A). Chl a max in BL (1.09 μg l–1) at 40 m in July (Fig. 6B) might have been connected to the rich pool of nutrients (Fig. 6B) and photoadaptation of phytoplankton (Geider et al. 1997Geider R.J., MacIntyre H.L., Kana T.M. 1997. Dynamic model of phytoplankton growth and acclimation: responses of the balanced growth rate and the chlorophyll a:carbon ratio to light, nutrient-limitation and temperature. Mar. Ecol. Prog. Ser. 148: 187-200.) in the colder (11.78°C) deep water of BL (Fig. 2B). Seasonal development of Chl a was similar for the MLs. While SL had relatively high Chl a in the whole water column in April and July, BL had a notable concentration of Chl a (Fig. 6A, B) only below the thermocline in the same months. Chl a in BL had also positive correlations (p<0.05) with SiO4, Poth, NO3 and NO2 and a negative correlation (p<0.05) with t. A deep chlorophyll maximum (Zavatarelli et al. 1998Zavatarelli M., Raicich F., Bregant D., et al. 1998. Climatological biogeochemical characteristics of the Adriatic Sea. J. Mar. Syst. 18: 227-263., Viličić et al. 2008Viličić D., Orlić M., Jasprica N. 2008. The deep chlorophyll maximum in the coastal north eastern Adriatic Sea, July 2007. Acta Bot. Croat. 67: 33-43.) of 0.67 μg l–1 was detected at 49 m in G in July (Fig. 6C). The spring (April) and the summer (July) blooms were evident because of the higher and more homogenous Chl a concentrations (1.04±0.05 and 0.99±0.03 μg l–1, respectively) throughout the water column of SL. The bloom in July was accompanied by weaker hypoxia than in September, although the largest difference in σT between upper and deep waters was detected in SL in July (Fig. 2A). Hypoxia was not detected in the less eutrophicated deep water of SL in August (Figs 5A and 6A). The mesotrophic conditions (TRIX>4) dominantly developed below 20 m in SL and below 30 m in BL (Fig. 6A, B), while the investigated locations were prevailingly oligotrophic (TRIX<4). TRIXmax (4.83) was found at 35 m in BL in September (Fig. 6B).

Differences between the mixing and stratification period

Although October-March was considered as the mixing period, the upper water of SL up to 7 m was highly stratified except in October-November because of the pronounced halocline (Fig. 2A). In the mixing period, SL had 5 and 10-fold higher ΔS than BL and G, respectively (Supplementary material Table S3). Unlike SL, where ΔS and Δt exchanged the leading role in defining ΔσT during the mixing and stratification period, respectively, Δt dominantly impacted on ΔσT in BL and G in April-September (Tables S3, S4). The mixing period for SL corresponds to the fact that, except in October and November, the upper waters of SL (≤7 m) were notably stratified on the sampling days in the mixing period (October-March). There was no significant difference in parameters related to O2 between the mixing and stratification period in SL (Tables S3, S4, S5). AOU was significantly higher (p<0.05) in BL and G in the mixing period than in the stratification period (Tables S3, S4, S5).

HPF had significantly larger biomass (p<0.05) in SL than in G (Tables S1, S2). There was no significant difference in picoplankton between the specified periods in SL (Table S3, S4, S5). APF and HPF reacted positively to t increase in SL (Table 3), but other impacts (probably grazing was the most important) did not support stronger development of the picoplankton in the warmer period (Tables S3, S4, S5). An increase in t was significantly (p<0.05) correlated with HPF in BL (Table 3), resulting in larger HPF in BL in the stratification period (Tables S3, S4, S5). Also, an increase in t resulted in significantly (p<0.05) higher HBa and lower Cyano in G in the stratification period. Cyano had almost unchanged averages in the MLs between the specified periods (Tables S3, S4).

Potentially limiting nutrients, according to Redfield et al. (1963), had a uniform succession: PO4, DIN, SiO4 at all locations in the contrasting periods (Table S3, S4). DIN at G in the stratification period was within the range (0.1-0.6 μmol l–1) of half saturation constant (Km) for NO3 and NH4 uptake by the phytoplankton community in the oligotrophic environment (MacIsaac and Dugdale 1969MacIsaac J.J., Dugdale R.C. 1969. The kinetics of nitrate and ammonia uptake by natural populations of marine phytoplankton. Deep-Sea Res. 16: 45-57.). Taking into account the comparatively low PO4 (Table S4), it seems that phytoplankton growth at G during stratification was potentially N and P co-limited. PO4 concentrations at all stations were significantly (p<0.05) higher in the mixing period than in the stratification period (Table S5).

In the stratification period, Chl a was significantly (p<0.05) higher in SL, not significantly different in BL and significantly (p<0.05) lower in G than in the mixing period. The contrast was evident between notable Chl a and O2 concentrations in the deep water of the MLs in early spring and poor Chl a concentration in the same, but hypoxic water in late autumn (Figs 5A, B and 6A, B). TRIX was significantly (p<0.05) lower at all locations in the stratification period than in the mixing period. HPF in BL, Cyano and HBa in G had significantly different (p<0.05) values between the mixing and stratification periods (Tables S3, S4, S5).

Upper and deep waters in different periods

There was no significant difference in parameters related to S between specified layers in each lake within the stratification period (Supplementary material Tables S6, S7, S8). All investigated locations above the thermocline had similar O2 and, according to AOU, significantly greater production than consumption of O2 (Table S6). SL had persistently higher averages of O2 sat. than BL, even at lower O2 (Table S4, S6, S7), due to significantly higher t than in BL (Table S2). All locations, above the thermocline, had low PO4 accompanied by NO3 and NH4 means within the range (0.1-0.6 μmol l–1) of Km of their uptake by the phytoplankton community in the oligotrophic environment (MacIsaac and Dugdale 1969MacIsaac J.J., Dugdale R.C. 1969. The kinetics of nitrate and ammonia uptake by natural populations of marine phytoplankton. Deep-Sea Res. 16: 45-57.), indicating a potential N and P co-limitation (Table S6). The most pronounced oligotrophy was detected in G above the thermocline. The significant difference in SiO4 between specified layers during stratification in the MLs (Tables S6, S7, S8) might have been caused by the sinking detritus, which was notably composed of diatoms. PO4 was significantly (p<0.05) higher only in deep water of BL during the stratification period (Tables S6, S7, S8).

Chl a had three and six-fold higher concentrations below the thermocline at deeper locations, BL and G, respectively, while Chl a rise in deep water of SL was not dramatic (Table S7). NO3 was three- and nine-fold higher below the thermocline in SL and BL, respectively (Table S7). All locations had low DIN above the thermocline, while G had low DIN even below the thermocline. NO2, especially in SL, and TRIX were significantly (p<0.05) higher below the thermocline and accompanied by higher AOU (Tables S6, S7, S8). DIN/PO4 below the thermocline in the MLs was notably higher than Redfield’s 16. Two specified layers at G did not show significant differences in the ratios of analysed nutrients (Table S8). APF and Cyano had significantly (p<0.05) higher values above the thermocline than in deep waters in SL (Tables S6, S7, S8).

Since division on mixing and stratification period did not match the reality in SL, at least for our samplings, it was preferred to determine averages and standard deviations of the study parameters in the mixing period (Tables S9, S10) associated with the same layers in the stratification period, including the test of the potentially significant (p<0.05) differences between the layers (Table S11) and within the same layers in different periods (Table S12). SiO4 in deep water of G was an order of magnitude lower than in the MLs (Table S10), perhaps as a consequence of the decomposition of detritus in the stagnant deep waters of the more productive MLs. The upper waters at all locations had DIN/PO4 close to Redfield’s 16 (Table S9), but only deep water of G retained similar DIN/PO4 (Table S10). Although DIN in deep waters of the MLs did not differ notably, NH4 and NO2 were more important constituents of DIN in deep water of SL, whereas NO3 was more important in deep waters of BL and G (Table S10). There were no significant changes in picoplankton constituents at all stations among upper and deep waters during the mixing period (Table S11). The largest number of significant changes between the layers occurred in SL (Table S11). This is in line with stratification of SL even in the mixing period.

Same layers in different periods

SL did not have significantly different ΔσT between the mixing and stratification periods (Supplementary material Table S12), indicating a notable pycnocline throughout the year. Deep water of SL did not show significant differences in parameters related to O2 in different periods, while in BL only O2 was significantly (p<0.05) lower in deep water in the mixing period (Tables S7, S10, S12). The stratification period, at least in its upper waters, seemed to be more productive according to differences (p<0.05) in O2 sat. and parameters derived from O2 sat. (Tables S6, S9, S12). Deep waters of the MLs were moderately eutrophic in both periods (Tables S7, S10, S12). Upper water of SL did not differ significantly in PO4 between the periods (Table S12), despite an extremely high concentration of PO4 at 5 m in January (not presented) that lifted the average of PO4 (Table S9) notably above the average in that layer in the stratification period (Table S6). Unlike in the MLs, the Chl a concentration in deep water of G was not significantly different between the periods (Table S12). Also, there were no significant differences in parameters related to picoplankton and nutrients other than inorganic ones in water layers between the specified periods in SL (Table S12). Significant differences (p<0.05) in constituents of picoplankton (HPF in BL, Cyano and HBa in G) and Noth (BL and G) were detected in the upper waters, while concentration of analysed organic matter differed significantly (p<0.05) in deep waters of BL (Noth and Poth) and G (Noth) between the specified periods (Table S12).

DISCUSSIONTop

The lack of significant differences in any of the parameters related to O2 between the mixing and stratification periods in SL was most likely related to persistent stratification (no significant difference in ΔσT between the mixing and stratification period), which constrained the mixing between its upper and deep water. In addition, we found the lowest numbers of parameters with significant (p<0.05) changes between the periods in upper and deep water of SL (Supplementary material Table S12), which are most likely related to the same reason. In comparison, the significant (p<0.05) differences in the upper water of BL (HPF) and G (Cyano and HBa) between the periods (Table S12) are most likely a consequence of changes in the food web, which is a topic beyond the scope of this study. Of note, Noth in BL and G was significantly (p<0.05) higher in the mixing period, but we cannot offer a plausible explanation for these observations.

The thermocline was crucial for stratification at all explored stations in the stratification period, while a relatively shallow (up to 7 m) and persistent halocline was crucial for stratification of SL in the mixing period, to the point that difference in t in the 2-3 m layer in December, which impacted negatively on the pycnocline by ~30%, was compensated by an 130% positive influence of ΔS on ΔσT in that layer. High range (>10°C) between low and high t in the upper water of SL during less and more productive periods (Table S9, S6), respectively, played a role in balancing between different levels of O2 production and solubility. These include an exponentially positive relation between the rate of metabolic processes and O2 requirements of marine organisms at increasing t (Brown et al. 2004Brown J.H., Gillooly J.F., Allen A.P., et al. 2004. Toward a metabolic theory of ecology. Ecology 85: 1771-1789.), along with decreasing O2 solubility in seawater with the rise of t (Garcia and Gordon 1992Garcia H.E., Gordon L.I. 1992. Oxygen solubility in seawater: Better fitting equations. Limnol. Oceanogr. 37: 1307-1312. ). Ciglenečki et al. (2015)Ciglenečki I., Marguš M., Bura-Nakić E., et al. 2015. Impacts of extreme weather events on highly eutrophic marine ecosystem (Rogoznica Lake, Adriatic coast). Cont. Shelf. Res. 108: 144-155. detected an increase in t of 1.1°C in the surface layer (0-2 m) and 0.64°C at 12 m in Dragon’s Eye for 2001-2012 in comparison with 1996-2000. Therefore, we speculate that the presented trend will have greater influence on SL than on BL concerning the diminished solubility of O2 with rising t. Decreased O2 solubility in the upper water of SL in both periods due to higher t is mitigated by lower S in comparison with BL (Table S6, S9). An important difference between the MLs was the notably warmer (2.12 and 2°C) and somewhat saltier (0.32 and 0.38; negative effect on solubility of O2) deep water of SL during periods of stratification and mixing (Tables S7, S10), respectively. Although phytoplankton blooms produced organic matter and DO throughout the water column of SL in April and July, saltier (on average 0.198 and 0.167) and warmer (on average 2.242°C and 2.933°C) deep water of SL in April and July, respectively, caused reduced solubility of O2 in comparison with deep water of BL, while having higher O2 requirements due to organisms at higher t at the same time (Brown et al. 2004Brown J.H., Gillooly J.F., Allen A.P., et al. 2004. Toward a metabolic theory of ecology. Ecology 85: 1771-1789.). It was predicted by Vaquer-Sunyer and Duarte (2011)Vaquer-Sunyer R., Duarte C.M. 2011. Temperature effects on oxygen thresholds for hypoxia in marine benthic organisms. Glob. Change Biol. 17: 1788-1797. that survival times of benthic macrofauna would decrease by a mean of 35.6% under hypoxia with the average 25.5% increase of DO concentration threshold for high mortality in a 4°C warmer ocean, representing the average increase in t expected at the end of the 21st century under moderate CO2 emission scenarios. Along these lines, Lojen et al. (2010)Lojen S., Juračić M., Sondi I. 2010. Geochemical conditions for the preservation of recent aragonite-rich sediments in Mediterranean karstic marine lakes (Mljet Island, Adriatic Sea, Croatia). Mar. Freshw. Res. 61: 119-128. have detected 4-5°C higher t (12°C in May and 13°C in October) in the pore water of SL than in the pore water of BL (8°C). Therefore, the difference of ≥2°C between deep waters of SL and BL should be monitored in the future.

Detailed discussion on the impact of p(CO2) and potential synergistic effects of hypoxia and increased p(CO2) on benthic invertebrates and fish can be found in Steckbauer et al. (2015)Steckbauer A., Ramajo L., Hendriks I.E., et al. 2015. Synergistic effects of hypoxia and increasing CO2 on benthic invertebrates of the central Chilean coast. Front. Mar. Sci. 2: Article number 49. , where measurement of calcification rates are stressed as an important additional tool to provide more prediction on the future outcome of marine invertebrates. Influence of hypoxia on benthic communities of the MLs (Kružić 2002Kružić P. 2002. Marine fauna of the Mljet National Park (Adriatic Sea, Croatia). 1. Anthozoa. Nat. Croat. 11: 265-292., Mikac 2003Mikac B. 2003. Echinoderrnata in the littoral area of Mljet National Park. Period. Biol. 105: 405-412.) has not been investigated. BL is a home for the scleractinian coral Cladocora caespitosa, which forms a reef (650 m2) between depths of 6 and 18 m near the Vrbovačka sampling station (Hrustić et al. 2013Hrustić E., Carić M., Čalić M., et al. 2013. Alkaline phosphatase activity and relative importance of picophytoplankton in autumn and early spring (Mljet Lakes, eastern Adriatic Sea). Fresen. Environ. Bull. 22: 636-648.). While experimenting on another scleractinian coral reef, Reynaud et al. (2003)Reynaud S., Leclercq N., Romaine-Lioud S., et al. 2003. Interacting effects of CO2 partial pressure and temperature on photosynthesis and calcification in a scleractinian coral. Glob. Change Biol. 9: 1660-1668. found out that calficification was decreased by 50% when t and p(CO2) were both elevated, with no significant change in response to increased p(CO2) under normal t. Their findings indicated a need to re-evaluate the projected decrease of marine calcification by the year 2100. Taking into account their findings, McNeil et al. (2004)McNeil B.I., Matear R.J., Barnes D.J. 2004. Coral reef calcification and climate change: The effect of ocean warming. Geophys. Res. Lett. 31: L22309. reported that the annual average coral reef calcification rate will increase with future ocean warming and eventually exceed pre-industrial rates by about 35% by the year 2100.

Although notably stratified throughout the year, anoxia did not develop in deep waters of SL. Overturn of the water column under influence of strong cooling of the surface water has not been documented in SL so far, but such events were documented in the meromictic lake Dragon’s Eye a few hundred kilometres farther north, causing the mass mortality of benthic and planktonic organisms in the early autumns of 1997 (Kršinić et al. 2000Kršinić F., Carić M., Viličić D., et al. 2000. The calanoid copepod Acartia italica Steuer, phenomenon in the small saline Lake Rogoznica (EasternAdriatic coast). J. Plankton Res. 22: 1441-1464., Barić et al. 2003Barić A., Grbec B., Kušpilić G., et al. 2003. Mass mortality event in a small saline lake (Lake Rogoznica) caused by unusual holomictic conditions. Sci. Mar. 67: 129-141.) and 2011 (Ciglenečki et al. 2015Ciglenečki I., Marguš M., Bura-Nakić E., et al. 2015. Impacts of extreme weather events on highly eutrophic marine ecosystem (Rogoznica Lake, Adriatic coast). Cont. Shelf. Res. 108: 144-155.). These events revealed that the copepod Acartia italica has been well adapted to the extreme conditions of anoxia, while some benthic Pennatae diatoms also survived anoxia in Dragon’s Eye in 1997 (Barić et al. 2003Barić A., Grbec B., Kušpilić G., et al. 2003. Mass mortality event in a small saline lake (Lake Rogoznica) caused by unusual holomictic conditions. Sci. Mar. 67: 129-141.). In addition, the research in the northern Adriatic Sea at 24 m conducted by inducing anoxia revealed that meiobenthic harpacticoid copepods from the family Cletodidae were well adapted to anoxia (Grego et al. 2014Grego M., Riedel B., Stachowitsch M., et al. 2014. Meiofauna winners and losers of coastal hypoxia: case study harpacticoid copepods. Biogeosciences 11: 281-292.).

The absence of upwelling in the MLs indicates that diffusion is the most important mechanism of transport of dissolved matter from deep to upper waters. If upwelling were a normal feature of the MLs, PO4 released from iron-bound P in oxygen-poor sediments would reach the upper water very fast, alleviating potential P limitation according to DIN:PO4>16 (Table S1) and acting as a positive feedback to increase hypoxia by stimulating phytoplankton production (Conley et al. 2009bConley D.J., Carstensen J., Vaquer-Sunyer R., et al. 2009b. Ecosystem thresholds with hypoxia. Hydrobiologia 629: 21-29.). Deep water of SL was >100% and ~150% richer in total Fe than deep water of BL and G (Buljan and Špan 1976Buljan M., Špan J. 1976. Hydrographic properties of the sea water lakes on the Island of Mljet and the adjoining sea in eastern South Adriatic Sea. Acta Adriat. 6: 1-227.), respectively, while iron-phosphorus still remains unexplored in sediments of this system.

The experiment of fertilization of the MLs was started via addition of 21.5 tonnes of “superphosphate” in eight instalments in BL in 1954 (Buljan and Špan 1976Buljan M., Špan J. 1976. Hydrographic properties of the sea water lakes on the Island of Mljet and the adjoining sea in eastern South Adriatic Sea. Acta Adriat. 6: 1-227.). In total, an average of 1.15 μmol PO4 l–1 was introduced in SC and the MLs. The system reacted by multiplying the primary productivity more than six-fold in comparison with 1953 (Buljan and Špan 1976Buljan M., Špan J. 1976. Hydrographic properties of the sea water lakes on the Island of Mljet and the adjoining sea in eastern South Adriatic Sea. Acta Adriat. 6: 1-227.). Hydrogen sulfide (H2S) was almost persistently present at 25 m in SL between 1951 and 1956 (Buljan and Špan 1976Buljan M., Špan J. 1976. Hydrographic properties of the sea water lakes on the Island of Mljet and the adjoining sea in eastern South Adriatic Sea. Acta Adriat. 6: 1-227.). It was occasionally present at notable concentrations at 20 m (e.g. 4.84 mg l–1; overall maximum 4.95 mg l–1 was detected in SL at 25 m on the same day, 11 December 1952). The fertilization experiment caused formation of H2S (2.93 mg l–1) at 13 m at Pospile Donje in BL on 20 July 1954 (Buljan and Špan 1976Buljan M., Špan J. 1976. Hydrographic properties of the sea water lakes on the Island of Mljet and the adjoining sea in eastern South Adriatic Sea. Acta Adriat. 6: 1-227.). These authors noted “pink and red water” at 20 m in SL in May and June 1953, which was most likely connected to the presence of anoxygenic phototrophic purple sulfur bacteria, commonly found at the chemocline and in the hypolimnion (Pjevac et al. 2015Pjevac P., Korlević M., Berg J.S., et al. 2015. Community shift from phototrophic to chemotrophic sulfide oxidation following anoxic holomixis in a stratified seawater lake. Appl. Environ. Microbiol. 81: 298-308. ). Consequently, the MLs were recognized as a highly vulnerable ecosystem and became the protected area of the Mljet National Park (NP) in 1960. It has been shown that the widening and deepening of SC and its entrance to BL in 1960 mitigated anoxic conditions in the near-bottom water of SL, according to historical profiles of the redox-sensitive elements in sediment cores of the MLs (Sondi et al. 2017Sondi I., Mikac N., Vdović N., et al. 2017. Geochemistry of recent aragonite-rich sediments in Mediterranean karstic marine lakes: Trace elements as pollution and palaeoredox proxies and indicators of authigenic mineral formation. Chemosphere 168: 786-797.). WRT in the MLs before 1960 was most likely longer than the WRT estimated recently by Cuculić et al. (2012)Cuculić V., Cukrov N., Ereš Z., et al. 2012. Report “The impact of the water masses on the spatial and temporal distribution of ecotoxicants in the Small Lake and Big Lake of the National Park Mljet”. Institute Ruđer Bošković, Zagreb, 80 pp.. Buljan and Špan (1976)Buljan M., Špan J. 1976. Hydrographic properties of the sea water lakes on the Island of Mljet and the adjoining sea in eastern South Adriatic Sea. Acta Adriat. 6: 1-227. did not detect H2S in deep water of SL in 1961, which is in line with the statement of Sondi et al. (2017)Sondi I., Mikac N., Vdović N., et al. 2017. Geochemistry of recent aragonite-rich sediments in Mediterranean karstic marine lakes: Trace elements as pollution and palaeoredox proxies and indicators of authigenic mineral formation. Chemosphere 168: 786-797., indicating poor ventilation as one of the most important parameters linked to estimated persistent anoxia in the near-bottom water of SL before 1960.

The negative correlation (p<0.05) between PO4 and O2 in the water columns of the MLs is common in hypoxic waters, e.g. bottom waters of the Baltic Sea (Conley et al. 2002Conley D.J., Humborg C., Rahm L., et al. 2002. Hypoxia in the Baltic Sea and basin-scale changes in phosphorus biogeochemistry. Environ. Sci. Technol. 36: 5315-5320.). The periods (Table S5) rather than water layers (Tables S8, S11), except for BL in the period of stratification, differed significantly (p<0.05) in concentration of PO4 at all stations, having higher values during the mixing period (Tables S3, S4, S5). Because of the relatively high DIN/PO4 at high productivity in a P-enriched environment, Buljan and Špan (1976)Buljan M., Špan J. 1976. Hydrographic properties of the sea water lakes on the Island of Mljet and the adjoining sea in eastern South Adriatic Sea. Acta Adriat. 6: 1-227. assumed N2 fixation in the MLs. A similar hypothesis, derived from the excess of NO3 in SL in early spring, was proposed by Hrustić et al. (2013)Hrustić E., Carić M., Čalić M., et al. 2013. Alkaline phosphatase activity and relative importance of picophytoplankton in autumn and early spring (Mljet Lakes, eastern Adriatic Sea). Fresen. Environ. Bull. 22: 636-648.. N2 fixation can increase eutrophication, especially if coupled with a flux of PO4 from deep water or sediments (Jensen et al. 1995Jensen H.S., Mortensen P.B., Andersen F.Ø., et al. 1995. Phosphorus cycling in a coastal marine sediment, Aarhus Bay, Denmark. Limnol. Oceanogr. 40: 908-917.) to the photic zone (Conley et al. 2009aConley D.J., Björck S., Bonsdorff E., et al. 2009a. Hypoxia-Related Processes in the Baltic Sea. Environ. Sci. Technol. 43: 3412-3420. ). In oligotrophic P-starved regions, N2 fixers can increase their competitiveness by using excessive N and energy to produce exoenzymes involved in utilization of DOP (Landolfi et al. 2015Landolfi A., Koeve W., Dietze H., et al. 2015. A new perspective on environmental controls of marine nitrogen fixation. Geophys. Res. Lett. 42: 4482-4489.). The problem of eutrophication in the Baltic Sea was a good reason for the development of a new bioassay to test the distance between N and P limitation in mesotrophic surface waters (Hrustić et al. 2017Hrustić E., Lignell R., Riebesell U., et al. 2017. Exploring the distance between nitrogen and phosphorus limitation in mesotrophic surface waters using a sensitive bioassay. Biogeosciences 14: 379-387. ). The integrated (depth of 0–10 m) samples from the ocean acidification experiment in the Tvärminne field station (Baltic Sea) in August 2012, without a bloom of diazotrophic cyanobacteria, showed the surplus of P, P+=0.30±0.10 μmol l–1, but the effect of ocean acidification was not detected by this method (Hrustić et al. 2017Hrustić E., Lignell R., Riebesell U., et al. 2017. Exploring the distance between nitrogen and phosphorus limitation in mesotrophic surface waters using a sensitive bioassay. Biogeosciences 14: 379-387. ). It would be interesting to use this new bioassay in the future to explore the distance between N and P limitation in the protected mesotrophic MLs, where the food web is controlled by Aurelia sp., and the particulate feeding of Cladocora caespitosa on phytoplankton from the surface currents in BL (Hrustić et al. 2013Hrustić E., Carić M., Čalić M., et al. 2013. Alkaline phosphatase activity and relative importance of picophytoplankton in autumn and early spring (Mljet Lakes, eastern Adriatic Sea). Fresen. Environ. Bull. 22: 636-648.) may supply missing nutrients in the coral reef (Wyatt et al. 2010Wyatt A.S.J., Lowe R.J., Humphries S., et al. 2010. Particulate nutrient fluxes over a fringing coral reef: relevant scales of phytoplankton production and mechanisms of supply. Mar. Ecol. Prog. Ser. 405: 113-130.).

Anoxia below 39 m in BL in the period 26-28 August 1996 (Benović et al. 2000Benović A., Lučić D., Onofri V., et al. 2000. Ecological characteristics of the Mljet Island seawater lakes (South Adriatic Sea) with special reference to their resident populations of medusae. Sci. Mar. 64 (Suppl. 1): 197-206.) did not last for long and wintertime cooling homogenized the entire water column. Quiescent or slowly moving speciemens within a swarm of Aurelia sp. were noted just above the anoxic layer. Aurelia sp. were repeatedly observed in swarms with very large numbers of individuals in BL except in September 1997. The quiescent individuals above the layer of anoxia were most likely affected by hypoxia since decreasing O2 sat. from the maximum of 101% at 18.5 m to depleted O2 at 39 m (Benović et al. 2000Benović A., Lučić D., Onofri V., et al. 2000. Ecological characteristics of the Mljet Island seawater lakes (South Adriatic Sea) with special reference to their resident populations of medusae. Sci. Mar. 64 (Suppl. 1): 197-206.) probably caused death to many individuals of Aurelia sp., which might have been a significant source of NH4 in BL (Tinta et al. 2010Tinta T., Malej A., Kos M., et al. 2010. Degradation of the Adriatic medusa Aurelia sp. by ambient bacteria. Hydrobiologia 645: 179-191. , 2016Tinta T., Kogovšek T., Turk V., et al. 2016. Microbial transformation of jellyfish organic matter affects the nitrogen cycle in the marine water column – A Black Sea case study. J. Exp. Mar. Biol. Ecol. 475: 19-30. ). However, deep water of SL had significantly (p<0.01) higher NH4 than deep water of BL, which is, together with the gradient of NH4 concentration below the depth of 20 m in SL (Benović et al. 2000Benović A., Lučić D., Onofri V., et al. 2000. Ecological characteristics of the Mljet Island seawater lakes (South Adriatic Sea) with special reference to their resident populations of medusae. Sci. Mar. 64 (Suppl. 1): 197-206.), in line with our results (Fig. 5A, B; Tables S7, S10).

These findings point out other reasons why NH4 in deep water of SL is significantly higher than NH4 in deep water of BL. Higher t and Chl a are positively related to phytoplankton growth (Agawin et al. 2000Agawin S.R.N., Duarte C.M., Agusti S. 2000. Nutrient and temperature control contribution of picoplankton to phytoplankton biomass and production. Limnol. Oceanogr. 45: 591-600.). Increased NO2 in the photic zone might be notably related to excretion during phytoplankton growth (Al-Qutob et al. 2002Al-Qutob M., Häse C., Tilzer M.M., et al. 2002. Phytoplankton drives nitrite dynamics in the Gulf of Aqaba, Red Sea. Mar. Ecol. Prog. Ser. 239: 233-239.). This fits with the detection of maximum NO2 concentrations in the photic zone of BL and G in March, but is less likely to be relevant for the maximum value of NO2 in deep water of SL in October. According to the highest yearly means of t and Chl a (p<0.05) (Tables S1, S2), a comparatively intense phytoplankton growth was estimated for SL. However, the difference in NH4 between the MLs was markedly higher during lower production, i.e. in the mixing period when the difference in Chl a was lower (Tables S3, S4). Similar findings can be seen throughout the layers in different periods, epecially in upper waters during the mixing period when BL had higher Chl a but lower NH4 than SL (Table S9). This leads us to conclude that phytoplankton is probably not responsible for significantly higher NH4 in SL.

Because of its smaller size, SL receives more terrestrial organic and mineral detritus relative to the amount of authigenic sediment than BL (Lojen et al. 2010Lojen S., Juračić M., Sondi I. 2010. Geochemical conditions for the preservation of recent aragonite-rich sediments in Mediterranean karstic marine lakes (Mljet Island, Adriatic Sea, Croatia). Mar. Freshw. Res. 61: 119-128. ). Particulate organic matter (POM) undergoes remineralization through the water column. A negligible portion of POM (~1%) escapes remineralization, being passively buried in oceanic sediments (Delaney 1998Delaney M.L. 1998. Phosphorus accumulation in marine sediments and the oceanic phosphorus cycle. Global Biogeochem. Cy. 12: 563-572. Article number 98GB02263), but the percentage of POM received by sediments in the shallower SL is expected to be notably higher. Sondi et al. (2017)Sondi I., Mikac N., Vdović N., et al. 2017. Geochemistry of recent aragonite-rich sediments in Mediterranean karstic marine lakes: Trace elements as pollution and palaeoredox proxies and indicators of authigenic mineral formation. Chemosphere 168: 786-797. determined oxic-anoxic boundaries in the sediment cores of SL, BL and G at depths of 2, 5 and 15 cm, respectively, followed by the profile of dissolved iron. These results led us to conclude that surface sediments of SL have significantly higher oxygen demand than sediments of BL and G, producing notably more NH4 from the increased amount of POM. That would be an explanation for the negative correlation (p<0.05) between NH4 and O2 in SL (Table 3). Wang et al. (2017)Wang B., Hu J., Li S., et al. 2017. A numerical analysis of biogeochemical controls with physical modulation on hypoxia during summer in the Pearl River estuary. Biogeosciences 14: 2979-2999. reported that re-aeration and sediment oxygen demand were more important than photosynthesis and water column respiration as counterbalanced coupled processes affecting the near-bottom DO concentration in the shallow estuary. Re-aeration of deep waters in the MLs is not expected in summer (Vilibić et al. 2010Vilibić I., Žuljević A., Nikolić V. 2010. The dynamics of a saltwater marine lake (Big Lake, Island of Mljet, Adriatic Sea) as revealed by temperature measurements. Acta Adriat. 51: 119-130.). Intermittent summer (July and September) hypoxia in the warmer deep water of SL can be attributed to presumably higher organic matter supply to the surface sediments, which have an increased oxygen demand. The plunge of the intense pycnocline might have contributed to hypoxia in the deep water of SL in summer.

Positive AOU in the whole water columns of SL and BL in December (not presented), as well as higher AOU at all investigated locations in autumn-winter, were most likely related to the lower photosynthetic activity. The critical depths of dominantly higher consumption than production of O2, i.e. where AOU>0 μmol kg–1, involving a notable regeneration of nutrients by organic carbon remineralization, were situated ~10 m below the water columns’ division points of 10 m for SL and 17 m for BL (Fig. 5A, B). There were no significant differences (p>0.05) between MLs concerning O2, AOU, OUR and OCRR. The larger volume of water in BL was characterized by a net utilization of O2 (AOU>0 μmol kg–1) (Fig. 5A, B) at a higher average OUR than in SL. A larger amount of organic carbon was remineralized in deep water of BL in the mixing period, due to a higher OCRR and a larger volume of water with positive values of OCRR than in deep water of SL in the same period (results not presented, derived from AOU). This finding is in line with a presumably higher % of POM that escapes remineralization during sinking in SL. Since we did not have data on WRT for deep waters of the MLs in the stratification period or any data on WRT in G, we performed statistical analyses for OUR and OCRR to a lesser extent.

According to the vertical profiles of SiO4 and NO3 in the MLs in April (Fig. 6A, B), we assume that diatoms were a major player in a spring bloom. It is interesting that Chl a had high concentrations only at depths ≥30 m in BL during the whole water column blooms in SL in April and July. Since a major player in a mesozooplankton community of BL, the calanoid copepod Calanus helgolandicus, cannot reproduce in SL (Miloslavić 2012Miloslavić M. 2012. Zooplankton dynamics in an enclosed marine ecosystem (Mljet Lakes, NP “Mljet”): seasonal and long-term changes. Ph.D. Thesis, Univ. Split, 150 pp.), we speculate that the higher grazing on diatoms in the upper water of BL, at least in April, may explain this result. In the warmer and more oligotrophic July, when C. helgolandicus is expected to reside below the thermocline in BL, it is possible that Aurelia sp. feeding on copepods had a positive effect on autotrophic biomass below the thermocline (Turk et al. 2008Turk V., Lučić D., Flander-Putrle V., et al. 2008. Feeding of Aurelia sp. (Scyphozoa) and links to the microbial food web. Mar. Ecol. 29: 495-505.). In our study, maximum concentrations of nutrients (Figs 5A, B and 6A, B) in the MLs were higher than those in SL in 1997 and BL in 1997-1998, except for PO4 in SL and TN in BL, which were similar to the values detected in 1997-1998 (Benović et al. 2000Benović A., Lučić D., Onofri V., et al. 2000. Ecological characteristics of the Mljet Island seawater lakes (South Adriatic Sea) with special reference to their resident populations of medusae. Sci. Mar. 64 (Suppl. 1): 197-206.). We could not compare TP as it was not reported by Benović et al. (2000)Benović A., Lučić D., Onofri V., et al. 2000. Ecological characteristics of the Mljet Island seawater lakes (South Adriatic Sea) with special reference to their resident populations of medusae. Sci. Mar. 64 (Suppl. 1): 197-206.. Average nutrient concentrations in our study seem to be similar to those reported in Benović et al. (2000)Benović A., Lučić D., Onofri V., et al. 2000. Ecological characteristics of the Mljet Island seawater lakes (South Adriatic Sea) with special reference to their resident populations of medusae. Sci. Mar. 64 (Suppl. 1): 197-206. and we cannot infer more about any change in the trophic status of the study area. Under the circumstances, we could not calculate the trophic status of the MLs from the data collected two decades ago, but we present TRIX for the period October 2009-September 2010.

Steckbauer et al. (2011)Steckbauer A., Duarte C.M., Carstensen J., et al. 2011. Ecosystem impacts of hypoxia: thresholds of hypoxia and pathways to recovery. Environ. Res. Lett. 6: 12 pp. Article number 025003. proposed 3.5 mg O2 l–1 as a new operational threshold to designate hypoxic waters, representing 88% of the distribution of mean lethal DO concentrations in warm coastal waters, except for those of the 12% most sensitive species. That threshold (~52% DO saturation) is less conservative than 4.6 mg O2 l–1 (~70% DO saturation) for warm coastal waters, representing 90% of the distribution of mean lethal DO concentrations (Vaquer-Sunyer and Duarte 2008Vaquer-Sunyer R., Duarte C.M. 2008. Thresholds of hypoxia for marine biodiversity. Proc. Natl Acad. Sci. USA. 105: 15452-15457.). Therefore, it is a scientific effort of the highest priority to re-evaluate DO thresholds that define hypoxic waters, taking into account climate change trends. In this context, the MLs seem to be an excellent laboratory for studying differences in benthic communities related to a future potential t increase.

Detailed research on the thermohaline properties of SL in the mixing period should be conducted to review whether the upper water tends to be markedly stratified year after year. Persistent stratification along with the present trends in climate change, e.g. higher precipitation (Justić et al. 2005Justić D., Rabalais N.N., Turner R.E. 2005. Coupling between climate variability and coastal eutrophication: Evidence and outlook for the northern Gulf of Mexico. Neth. J. Sea Res. 54: 25-35. ), have a potential to extend the duration and impact of hypoxia. Since small systems like marine lakes respond fast to environmental changes (Ciglenečki et al. 2015Ciglenečki I., Marguš M., Bura-Nakić E., et al. 2015. Impacts of extreme weather events on highly eutrophic marine ecosystem (Rogoznica Lake, Adriatic coast). Cont. Shelf. Res. 108: 144-155., Miloslavić and Lučić 2015Miloslavić M., Lučić D. 2015. Temporal patterns of the calanoid copepod community in Veliko Jezero, an isolated marine lake (South Adriatic Sea): links to larger-scale climate changes. J. Nat. Hist. 49: 2783-2798. ), research on these lakes and similar systems should be encouraged in order to gain more insights into the impacts of driving factors of hypoxia on biodiversity in moderately warm marine systems worldwide.

CONCLUSIONSTop

In the present study, we have demonstrated that two moderately eutrophic Mediterranean marine lakes that communicate and depend on each other, with only a few significant differences, were affected by hypoxia in different frequencies throughout the year. A long-term chemical feature of significantly higher ammonium concentration in a smaller, shallower lake more isolated from the coastal sea has been shown by comparison with data collected from the period more than a decade ago. A notable difference in temperatures of deep waters in these lakes might be one of the key parameters to focus on in future studies, for example, on the impact of driving factors of hypoxia on the benthic communities in the Mljet NP. For the first time, an apparent oxygen utilization and the OUR as well as OCRR have been presented for this study area. Although not significantly (p>0.05) different between the lakes, AOU, OUR and OCRR indicated that a larger volume of deep water in BL in the mixing period was affected by net oxygen utilization and organic carbon remineralization. These findings are in line with the expectation that more POM in the shallower water escapes remineralization, and is then subsequently oxidized in the surface sediments of SL.

ACKNOWLEDGEMENTSTop

We thank Zoran Jurić, Božo Grmoljez, Ana Car and Marko Žarić for their help during the sampling and Rade Garić for his help in developing Figure 1. The public institution Mljet NP lent the boat for the sampling, while the Croatian Meteorological and Hydrological Service (meteo.hr) provided the data on precipitation in Goveđari, Mljet. We thank Vlado Cuculić (Ruđer Bošković Institute, Division for Marine and Environmental Research) and Mljet NP for allowing us to use the data on the water renewal time in the MLs. We would like to thank the anonymous referee for their thorough evaluation of our manuscript and insightful comments. We also thank Nan Chiang and Eliana Ruiz Martinez for their help during the preparation of the final version of the manuscript. The study was conducted within the project 275-0000000-3186 supported by the Ministry of Science, Education and Sports (Republic of Croatia).

REFERENCESTop

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SUPPLEMENTARY MATERIAL

The following supplementary material is available through the online version of this article and at the following link:
http://scimar.icm.csic.es/scimar/supplm/sm04523esm.pdf

Table S1. – The yearly Avg±sd of the investigated parameters in Small Lake (SL), Big Lake (BL) and Gonoturska Bay (G); t, temperature; Δt, temperature change with depth; S, salinity; ΔS, salinity change with depth; σT, density; ΔσT, density change with depth; AOU, apparent oxygen utilization; Chl a, chlorophyll a; DIN, dissolved inorganic nitrogen; Poth, total phosphorus – PO4, Noth, total nitrogen – DIN; TRIX, index of eutrophication (Vollenweider et al. 1998); APF, biomass of autotrophic picoflagellates; Cyano, biomass of picocyanobacteria; HBa, biomass of heterotrophic bacteria; HPF, biomass of heterotrophic picoflagellates.

Table S2. – Significant (p<0.05) differences among the explored locations in Mljet waters (SL, Small Lake; BL, Big Lake; G, Gonoturska Bay). See the list of abbreviations in Table S1.

Table S3. – Mean±sd of the analysed parameters in the mixing period (October 2009-March 2010); n/a, not analysed; OUR, oxygen utilization rate; OCRR, organic carbon remineralization rate. See the list of abbreviations in Table S1.

Table S4. – Mean±sd of the analysed parameters in the stratification period (April 2010-September 2010). See the list of abbreviations in Table S1.

Table S5. – Significant (p<0.05) differences in the study locations between the periods of mixing (October 2009-March 2010) and stratification (April 2010-September 2010). See the list of abbreviations in Table S1.

Table S6. – Mean±sd of the analysed parameters in the upper waters of the study locations within the stratification period (April 2010-September 2010); n/a, not analysed; OUR, oxygen utilization rate; OCRR, organic carbon remineralization rate. See the list of abbreviations in Table S1.

Table S7. – Mean±sd of the analysed parameters in the deep waters of the study locations within the stratification period (April 2010-September 2010). See the list of abbreviations in Table S1.

Table S8. – Significant (p<0.05) differences at the study locations above and below the thermocline during the stratification period (April 2010-September 2010). See the list of abbreviations in Table S1.

Table S9. – Mean±sd of the analysed parameters in the upper waters of the study locations within the mixing period (October 2009-March 2010); n/a, not analysed; OUR, oxygen utilization rate; OCRR, organic carbon remineralization rate. See the list of abbreviations in Table S1.

Table S10. – Mean±sd of the analysed parameters in the deep waters of the study locations within the mixing period (October 2009-March 2010); n/a, not analysed; OUR, oxygen utilization rate; OCRR, organic carbon remineralization rate. See the list of abbreviations in Table S1.

Table S11. – Significant (p<0.05) differences between upper and deep waters at the study locations during the mixing period (October 2009-March 2010). OUR, oxygen utilization rate; OCRR, organic carbon remineralization rate; OUR and OCRR were not estimated for Gonoturska Bay. See the list of abbreviations in Table S1.

Table S12. – Significant (p<0.05) differences within the specified water layers between the periods of mixing and stratification at the study locations. OUR, oxygen utilization rate; OCRR, organic carbon remineralization rate; OUR and OCRR were not estimated for Gonoturska Bay. See the list of abbreviations in Table S1.



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