original scientific paper UD C 574:628.427(262.3 Piranski zaliv) ECOLOGICAL CHARACTERISTICS OF SEAWATER INFLUENCED BY SEWAGE OUTFALL Patricija MOZETIČ, Vlado MALAČIČ & Valentina TURK National institute of Biology, Marine Biological Station, S1-6330 Piran, Fornače 41 ABSTRACT The impact of sewage discharge from the treatment plant at Piran through submarine outfalls approx. 3500 m off the coast was studied. During the years 1998-1999 six surveys of oceanographic parameters, nutrient concentrations and faecal coliforms were carried out at the central station between the two outfalls and at the reference station. The spreading of the sewage in the water column was detected through observing small vertical salinity oscillations (~0.1 PSU) in layers of thickness less than 1 m, a few meters above the bottom. Sewage dispersal was confirmed by the presence of faecal coliforms. Their number increased (max. 1160/100 ml) in the layers with highest salinity oscilla­tions. The ecological impact of submarine outfalls on the surrounding environment was highest in the case of am­monium, while for other nutrients the impact was almost negligible. This was confirmed by the relatively high corre­lation between faecal coliforms and ammonium (p-0.58) in the near-field sewage plume, while there was no corre­lation (p~-0.05) between these properties in the sewage at the treatment plant's outlet. The reference station proved to be unsuitable for comparison due to periodic bacterial contamination. Key words: sewage, submarine outfall, faecal bacteria, nutrients, coastal sea, Gulf of Trieste INTRODUCTION Near-shore marine areas are susceptible to many different land-based sources of pollution, among which direct, untreated wastewater discharge seems to be one of the main causes of increased eutrophication. This problem is even more evident in coastal areas where economically important activities, such as tourism and aquaculture, set high standards of seawater quality. Many legislative acts have been successfully imple­mented in order to control the quality of the water body receiving municipal wastewaters and for the protection of human health. Slovenia started to monitor some pol­lution parameters within the frame of UNEP/MED-POL Program Phase II (Tusnik eta!., 1989) in 1983. Among several "hot spots", referred as land-based sources of pollution, sewage effluents at treatment plants were ana­lysed from the very beginning. In comparison to this well-developed net of pollution control on the land, the seawater in the vicinity of sewage discharges along the Slovenian coast did not gain such attention. Up to now, the majority of research work concerning the effect of sewage dispersal in the marine environment was focused on the Piran sewage outfall (Gulf of Trieste, Adriatic Sea). The municipality of Piran, which is one of the most touristically developed Slove­nian areas, solved the problem of sewage disposal in the late 70's by constructing a submarine sewage outfall 3450-m off the coast. Eleven years later, in 1987, another submarine outfall was constructed parallel to the old one at a horizontal distance of less than 200 m and 3250 m off the coast (Vukovtg & MalaCii, 1997). A mechanical treat­ment plant produces a sludge that is periodically removed from digestion tanks to an onshore dumping-ground, and an effluent, that is discharged by gravity through the outfalls into a depth of 21 m (Malej, 1980; Malacie, 1997). The old and new outfalls terminate with diffusers that are 108 m and 185 m long, respectively. They are placed 1 m above the bottom and are drilled alternately with 11 to 17 lateral holes on both sides of the pipes. Up to now these diffusers are the only ones in Slovenian waters. Average effluent flow is about 10360 m3 per day and may increase during the season due to increased population (D. Kleva - Svagelj, pers. comm.). ANNALES • Ser. hist.-nat. • 9 • 199 9 • 2 (17) Pairicija MOZETI Č et a/.: ECOLOGICA L CHARACTERISTIC S O F SEA WATE R INFLUENCE D B Y SEWAC E OUTFALL , 1 77-130 ^ f Trieste/ JEE Slimni a -I. ­ H I 1 6 I 0mgfl^ •iii^^^lllliill I'iriin I m ^llllllplll •i s N. ,' Fig. 1: Location of the Piran submarine outfallstation grid and sampling stations in the southern part of the Gulf of Trieste. Sampling station PI-16 is located in the centre of the station grid. SI. 1: Lokacija piranskega podvodnega izpusta, mreže postaj in vzorčevalnih postaj v južnem delu Tržaškega zaliva. Postaja PI-16 leži v sredini mreže postaj. Early studies comprised an overview of environ­mental conditions in the water column and at the sea floor before (1974/75) and after (1978/79) the construc­tion of the first submarine outfall (Avčin ef a!., 1978; Male], 1980; FaganelS, 1982). The authors reported a minimal effect of the outfall upon the surrounding ma­rine ecosystem, suggesting a very rapid dispersal of the effluent and thus the high effectiveness of the Piran treatment plant. No studies were done after the con­struction of the second outfall untif recently. In the last three years an extensive project has been undertaken in order to analyse the dispersion of the sewage plume from the Piran submarine outfall (Malačič, 1997, 1998; Vukovič & Malačič, 1997). The dynamics of sewage plume dispersion is controlled by stratification, shear currents, turbulence and the Coriolis force. Nutrients and faecal bacteria were occasionally monitored along with oceanographic parameters at the centra! station located between the two diffusers. The purpose of this paper is to estimate the extent of sewage pollution in the water column based upon eco­logical parameters, and to extract the most representative parameters (i.e. indicators) of such pollution. Further­more, we'll try to compare these new data with the data collected 20 years ago in order to assess possible dete­rioration of the marine environment and the effectiveness of the sampling strategy designed for such studies. MATERIALS AND METHODS Sampling strategy and field survey Sampling was carried out at station Pi-16 which is located between the submarine outfalls of Piran and at station F (Fig. 1) which is defined as the reference station. The central station PI-16 was chosen from a grid of 31 stations (MalaCiE, 1999a). During the years 1996-1998 several oceanographic surveys of the water column were undertaken. However, only four samplings from 1998 are presented in this paper. Biological and chemical para­meters were sampled in addition to the CT D casts (conductivity, temperature, depth). An additional two samplings were performed in 1999. Surveys, labelled from 1 to 6, correspond to the following days: 2 April 1998 (1), 18 june 1998 (2), 31 August 1998 (3), 12 October 1998 (4), 24 May 1999 (5), and 21 July 1999 (6). Samplings were performed in calm weather and during low tidal currents - the tidal range of sea-surface elevation (peak-to-peak) was less than 0,6 m in the port of Koper. The bottom at stations PI-16 and F is 21 m deep. The number and position of sampling depths varied from one sampling to another, and were defined each time separately with regard to the vertical salinity profile of station PI-16. The spreading of the sewage plume was detected from slightly lower salinity (for about 0.1 PSU) in thin layers within the water column. The number of sampling depths varied from 9 to 14, while the sampling depth interval was smaller than 2 m in the layers with slightly lower salinity. The reference station F was sam­pled only at a few depths, the number of which did not exceed five - the number of standard oceanographic depths (0.5, 5, 10, 15 m and just above the bottom). The exception was Survey 1 when sampling depths at station F corresponded to those at station Pl-16. Information about the input load was gained from the bacteria and nutrient analysis of sewage samples that were collected at the outflow of the treatment plant before each survey. The samples were collected about three to four hours before the sampling at station PI-16. )t takes about this amount of time for the sewage to reach the sea (station PI-16). On the basis of several oceanographic surveys of the water column near the sewage plume during different stratified conditions, a station grid was designed (Malacic, 1997). The grid with a diameter of approx. FiltrioM MOZETIČ et .il.: ECOLOGICA L CHARACTERISTIC S O F SF A WATE R INFLUENCE D BY 5LWAG L OUTFALL , 1 ?',M 90 900-m covers 31 stations around both outfalls and en­ables a fast dynamic survey (within one hour) of the plume extent. At grid stations vertical profiles of tem­perature, salinity, oxygen and fluorescence were ob­tained using the fine-scale multiparameter CT D probe, designed at the Centre for Water Research, University of Western Australia. The vertical resolution of the probe is of about 2.5 cm for a conventional drop speed of about 1 m/s. Analyse s Nutrients. Concentrations of nitrate (NO3*), nitrite (NO2"), ammonium (NH4+), phosphate (PO43"} and sili­cate (Si04 4-) were measured in unfiltered seawater and filtered sewage samples using standard colorimetric pro­cedures (Grasshoff, 1983). Sewage samples were filtered through glass fibre filters (Whatman GF/F). Total nitro­gen (N-tot) and total phosphorus (P-tot) were analysed in unfiltered samples. Inorganic nitrogen (SNi n J was cal­culated as NH 4 + + NO3- + N02". Faecal coiiform bacteria. The number of faecal con­forms was determined following the recommendations of UNEP/WHO (1994). Water samples were filtered through the 0.45 pm pore-size MiSJipore filters and in­cubated on m-FC agar medium at 44.5±0.2°C for 24 hours. Statistical analyses. Data from nutrient concentra­tions and faecal bacteria counts measured in the sewage at the outlet of the treatment plant and at the station Pl­16 were statistically elaborated. Cross-correlation of standardised data was used to calculate correlation co­efficients (p) in order to examine the linear relationship between properties (i.e. nutrients and faecal bacteria). Data were standardised using the following equation: (-Xj)/SD where Xj represents measured value, mean value and SD is the standard deviation of the specific parameter. RESULTS Sewage composition Nutrient analyses and bacterial counts were per­formed on sewage effluent that was treated mechani­cally (Tab- 1). Almost all inorganic nitrogen was present as ammonium. Concentrations of ammonium, nitrite and nitrate varied substantially: from below the detec­tion limit up to 243 pmol I"1 in the case of nitrate. Ex­cept for samples collected on 12 October 1998 and 21 July 1999 the inorganic nitrogen (51-98% of total nitro­gen) prevailed over organic forms. More than 50% of total phosphorus was in the form of phosphate (PO43"), while other forms that are nor­mally present in sewage, e.g. polyphosphate and or­ganic phosphate, were not analytically separated. The highest concentrations of ammonium, phosphate, total nitrogen and phosphorus were measured during the summer months (31 August 1998 and 21 July 1999), while in the case of nitrate and nitrite it was just the op­posite: during the summer they were at their minimum. Average count of faecal bacteria was around 7.3x106/100 ml with a standard deviation of 2.7x106 . Tab, 1: Composition of sewage water at the outflow of the Piran treatment plant: concentrations of nitrate (NO3), nitrite (N021, ammonium (NH4+), inorganic nitrogen (ZNjnJ, phosphate (PO43'), silicate (Si044'), total nitrogen (N-tot) and phosphorus (P-tot) and number of faecal coiiform bacteria (FC). Mean values of the parameters (< X >) and standard deviations (SD) are shown. Tab. 1: Sestava odpadne vode na iztoku iz piranske čistilne naprave: koncentracije nitrata (NOf), nitrita (NOf), amonija (NH4+), anorganskega dušika (T,NjnJ, fosfata (PO43'), silikata (Si044'), celotnega dušika (N-tot) in fosforja (P-tot) ter število fekalnih koliformnih bakterij (FC). Podane so srednje vrednosti (< X >) in standardne deviacije (SD) merjenih parametrov. date N 54.60 6.82 1112.12 1173.54 51.75 243.53 2111.05 79.55 7.26E+06 [SD 93.33 7.19 634.44 613.27 27.13 830.02 1619.34 30.80 2.68F.-t-Q6 ANNALES • Ser. hist nal. • 9 • 1999 • 2 (17) Palricija MOZFFIC etui: ECOLOGICAL CHARACTERISTICS OF SEA WATER INFLUENCED 8Y SEWAGE OUTFALL, ! 77-190 1 . 0 0.8 0.6 0.4 0.2 0.0 P04 J" P-tot NO!- NOj-NH)"1' N-int SifV " pcy- P-tot NO;- NOj- Nt V N-tot SiOi4' -0.2 Fig. 2: Coefficients of cross-correlation analyses SI. 2: Koeficienti korelacijske analize med fekalnimi between faecal bacteria and nutrients. bakterijami in hranilnimi snovmi. a) data from the sewage at the outlet of the treatment a) podatki iz odpadne vode na iztoku iz čistilne na~ plant prave b) data from station Pl-16 b) podatki s postaje Pl-16 Tab. 2: Coefficients of cross-correlation analyses performed on nutrient concentrations and faecal bacteria counts, which were measured in the sewage at the outlet of the Piran treatment plant (N ~ 6). Tab. 2: Korelacijski koeficienti, izračunani za koncentracije hranilnih snovi in števila koliformnth bakterij (N = 6). Analize so bile opravljene v odpadni vodi na iztoku iz piranske čistilne naprave. PO43- P-tot NO2 ­ N oy P04 3 ­ 1 0.80 -0.60 0.03 P-tot 0.80 1 -0.81 0.05 NO2 ­ -0.60 -0.81 1 0.11 NC V 0.03 0.05 0.11 1 NH4+ 0.71 0.95 -0.79 -0.24 N-tot 0.96 0.76 -0.68 -0.17 SÍO44- 0.22 0.64 -0.33 -0.21 FC 0.42 -0.01 0.45 0.01 Cross-correlation analyses (Tab. 2) of nutrient con­centrations and faecal bacteria in sewage show variable relationships among parameters. Some nutrients are highly correlated among each other (e.g. PO43", P-tot, NH4+, and N-tot), while faecal bacteria are only slightly correlated to phosphate and nitrite (Fig. 2a). There is no meaningful correlation between the bacteria and am­monium in the sewage. Vertical structure of the water column CT D casts were performed at every survey and sampling depths at station Pi-16 were determined using the salinity vertical profile. In this work only one vertical profile of temperature, salinity, fluorescence and dis­solved oxygen will be presented as an example (24 May 1999) of the fine vertical structure of the water column that is affected by the sewage plume (Fig. 3). Warming of the atmosphere in late spring contributed to the rise of seawater temperature in the upper layers. Consequently, a weak stratification of the water column with a ther­mocline at approx. 12 m was re-established, separating the upper warmer layers (17.0-18.8°C) from the colder bottom ones (12.4-13.4^). Salinity gradually increased with depth from the value at the surface (36.27 PSU) towards the value at the depth of the thermocline (37.04 PSU). tn the layer between 11 and 16 m slight oscilla­tions of salinity ranged from 0.03 to 0.12 PSU. They in­dicate an intrusion of less saline water presumably originating from the outfalls. Below 16 m depth the sa­linity increased again and reached the maximum (37.69 PSU) at the bottom. The fluorescence peak and highest oxygen concentration were detected at a depth of around 16 m. The salinity vertical profile was crucial for NH4+ N-iot SÍO44- FC 0.71 0.96 0.22 0.42 0.95 0.76 0.64 -0.01 -0.79 -0.68 -0.33 0.45 -0.24 -0.17 -0.21 0.01 1 0.71 0.74 -0.05 0.71 1 0.21 0.33 0.74 0.21 1 -0.01 -0.05 0.33 -0.01 1 Patrici a MOZETI Č eí si : ECOLOGICA L CHARACTERISTIC S O f SEAWATE R INFLUENCE D BY SEWAG E OUTEALL , 177-190 Temp (°C) Sal (PSU) CM a {mg/m3) D.O.(m!/i) S I 16 3« 36.S 3/.(J 37.S O.tr 2.0 í. a S.O 5.0 6.0 Fig. 3: Temperature, salinity (Sal), in situ fluorescence (Chi a) and dissolved oxygen (O.O.) vertical profiles recorded on May 24, 1999 using CTD fine-scale probe. A detail of vertical profiles of temperature and salinity between 10 and 16 m depth is shown in the lower figure. Si. 3: Vertikalni profili temperature, slanosti (Sal), in situ fluorescence (Chi a) in raztopljenega kisika (D.O,), posneti s CTD sondo 24. maja 1999. Na spodnji sliki je prikazan izsek vertikalnih profilov temperature in slanosti na globini 10 do 16 m. determination of 12 sampling depths. Between the depth range 11.5-16.5 m the samples were collected at depths with an interval smaller than 1 m. Water quality of the recipient Nutrient concentrations and the sanitary quality of the seawater were measured in the near-field of the sewage plume (station Pf-16), as well as at the reference station (station F), in order to examine the impact of sewage on the surrounding media. Fine-scale vertical distributions of chemical and microbiological parame­ters during six surveys at station PI-16 are shown in fig­ures 4a and 4b. Faecal col i forms are the fundamental indicators of sewage pollution. Therefore, we first followed the distri­bution of bacteria in the water column. Secondly, we compared nutrients to bacterial distribution in order to determine the most representative nutrient(s) of sewage dispersion. The number and vertical distribution of fae­cal coliforms differed substantially among the six sur­veys (Fig. 4a, upper panel). O n 18 june 1998 we ob­served no sewage at station Pl-16, while on other occa­sions the number of bacteria varied from less than 1 to 1160/100 ml. A peak value of 1160 counts/100 ml was counted on 2 April 1998 at depth of 12 m, with the sec­ond highest 890 counts/100 ml at depth of 14 m on 24 May 1999. Low values, apart from the situation of zero counts on 18 june 1998, were counted during late summer (31 August 1998). Then, the highest count of 215/100 ml was reached at the bottom. The vertical distribution of faecal coliforms also var­ied substantially. During spring surveys (2 April 1998 and 24 May 1999) the bacteria were present only in a narrow layer of a thickness of 3 m between depths of 11 and 14 m. An approx. 15-fold increase was detected at depths of 12 m and 14 m with respect to counts at other depths. During other periods faecal coliforms were more evenly distributed throughout the water column. Bacte­ria were found in a thicker layer between 7 and 13 m, with peak values at the bottom (31 August 1998) and at depths between 11 and 16 m (12 October 1998 and 21 july 1999). With a few exceptions vertical profiles of ammonium followed faecal vertical distributions (Fig. 4a, upper panel). Peaks of ammonium (4.08 - 7.33 pmol I"1) were measured at the same depths as bacterial maximums, and also at the bottom (3.23 pmol H) . Concentrations of other nitrogen forms, especially nitrate, were generally higher in the upper iayers of the water column (1.83 ­ 6.01 pmol I"1) and again at the bottom (7.72 pmol I"1; Fig. 4b, lower panel). Phosphate and total phosphorus (Fig. 4b, upper panel) showed a similar vertical pattern. Extremely high phosphate concentrations were meas­ured on 31 August 1998 at the surface (0.30 pmol I"1), at the. bottom (0.92 pmol h1) and at 15 m depth (0.41 pmol I"1), in other cases the concentrations of phosphate were below 0.10 pmol H . increased phosphate and total phosphorus values were only occasionally observed in the layers where faecal coliforms were present. Silicate concentrations varied from 1.85 to 34.72 pmol I"1 (Fig. 4a, bottom panel). Similarly to nitrate, the highest values of silicate concentrations were generally measured in the bottom layers (except for 2 April and 31 August 1998). AN N ALES • Ser. hist, nat. • 9 • 199 9 • 1 (15) Patricija MOZETIČ el sh ECOLOGICAL CHARACTERISTICS OF SEAWATER INFLUENCED BY SEWAGE OUTEALL, 177-S SO ON 3 Lcs o o r—! Hi Q