MARINE ECOLOGY PROGRESS SERIES Vol. 292: 23–40, 2005 Published May 12 Mar Ecol Prog Ser

Spatial distribution of modern cysts in polluted estuarine sediments from Buzzards Bay (Massachusetts, USA) embayments

Vera Pospelova1, 2,*, Gail L. Chmura1, Warren S. Boothman3, James S. Latimer3

1Department of Geography and Centre for Climate and Global Change Research, McGill University, 805 Sherbrooke Street West, Montreal, Quebec H3A 2K6, Canada 2School of Earth and Ocean Sciences, University of Victoria, Petch 168, PO Box 3055 STN CSC, Victoria, British Columbia V8W 3P6, Canada 3US Environmental Protection Agency, Office of Research and Development, NHEERL, Atlantic Ecology Division, Narragansett, Rhode Island 02882, USA

ABSTRACT: Analysis of the spatial distribution of the dinoflagellate cyst assemblages in 19 surface sediment samples collected from 3 Buzzards Bay (Massachusetts, USA) embayments revealed the potential applicability of dinoflagellate cysts as biological indicators of environmental conditions in estuarine systems. Sites with the highest levels of toxic pollution and hypertrophic conditions are characterized by the lowest dinoflagellate cyst species-richness and concentrations. Among the abi- otic factors influencing the distribution of dinoflagellate cysts, nutrients and toxic pollution are the major controls, as in these embayments salinity and temperature variability is low. Principal compo- nent analysis, based on the proportions of cyst taxa, indicated that cyst assemblages gradually change when moving away from the sources of nutrient pollution, sewage outfalls in particular.

KEY WORDS: Dinoflagellate cyst · Eutrophication · Heavy metals · Sewage · Wastewater treatment plant · PCBs · Apponagansett Bay · New Bedford Harbor

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INTRODUCTION have proven to satisfy all the above criteria (Fensome et al. 1996). Human activities such as nutrient enrichment and are single-celled organisms that con- toxic pollution cause water-quality degradation and stitute an important part of the phytoplankton population habitat loss. These activities are most intensive in estu- in aquatic ecosystems. During their life cycle some di- aries with highly urbanized and industrialized water- noflagellates produce hypnozygotes, or resting cysts, sheds. Concern about water-quality degradation in which can be preserved in sediments (Fensome et al. estuarine waters has stimulated a demand for develop- 1993). The assemblages of dinoflagellate cysts in sedi- ment of indicators of nutrient enrichment and toxic ments encode information on the dinoflagellates in the contamination to examine paleo- and modern environ- upper water column (Dale 1976, Reid & Harland 1978). mental conditions and trends caused by intensified In turn, the population of dinoflagellates is influenced by anthropogenic activities in watersheds. environmental factors such as temperature and salinity, To be a useful biological indicator of environmental nutrients, turbidity and pollution (Taylor 1987). Dinofla- conditions, an organism has to satisfy certain criteria gellate cysts recovered from coastal sediments have the (Gibson et al. 2000). In general, a biological indicator potential to provide substantial information on biological should be ubiquitous, well preserved in sediments, processes and interactions within aquatic systems. numerous enough for statistical treatment, and reflect Several studies have examined the temporal devel- certain environmental parameters. Dinoflagellate cysts opment of estuarine eutrophication and pollution by

*Present address: Victoria. Email: [email protected] © Inter-Research 2005 · www.int-res.com 24 Mar Ecol Prog Ser 292: 23–40, 2005

analyzing the dinoflagellate cyst records in sediment ° ° N Acushnet River 70 54’ W 70 52’ W cores (Dale & Fjellså 1994, Sætre et al. 1997, Thorsen & Boston

Dale 1997, Dale et al. 1999, Matsuoka 1999, 2001, Dale MA 2001, Pospelova et al. 2002). A recent study of the his- RI Sediment Sites CT torical records of dinoflagellate cysts from shallow un- Sewage Outfall Combined Sewer stratified embayments of Buzzards Bay (New Bedford Overflows NBH146 an Atlantic Oce Flood-tidal Harbor and Apponagansett Bay) found that species NBH154 current direction richness (number of taxa), total cyst concentrations and NBH2c Core sites New Bedford Harbor fluxes, proportions of some heterotrophic species, as NBH204 NBH5c FAIRHAVEN well as abundance of indicator species changed as a re- Inner Harbor Popes Island 41° 38’ N sult of eutrophication and toxic pollution (Pospelova et NBH236 Fairhaven S.O. al. 2002). In oligotrophic systems, such as New Bedford NBH247 Harbor in its early history (Pospelova et al. 2002), nutri- NEW Group I BEDFORD ent enrichment initially increases dinoflagellate cyst hurricane barrier species-richness. However, under highly eutrophic and Outer Harbor polluted conditions, species diversity declines, as seen 41° 36’ N during the 20th century in New Bedford Harbor. These DARTMOUTH Clarks extreme conditions are also characterized by large fluc- Clarks NBH317 Apponagansett Cove point tuations in total cyst concentrations and fluxes. The CPC Bay New Bedford S.O. proportion of cysts of heterotrophic dinoflagellates, AB1 CPB AB2 NBH325 Polykrikaceae and Diplopsalidaceae in particular, AB3 NBH324 CPE AB4 CPG tends to increase with increasing nutrient enrichment. AB5 AB1c NBH331 Group III If the dinoflagellate cyst record reflects the temporal 41° 34’ N 1 km changes of the environmental conditions in an estuary, Group II NBH346 Buzzards Bay it must also show spatial change, as environmental con- ditions in an estuary are non-uniform. If so, dinoflagel- Fig. 1. New Bedford Harbor, Clarks Cove and Apponagansett late cyst assemblages from surface samples can be used Bay showing location of surface sediment samples. Direction to support or discard some of the conclusions previously of tidal-flood current from Howes et al. (1996), locations of 3 made about the ‘eutrophication and pollution signals’ core sites from Pospelova et al. (2002) drawn from analyses of dinoflagellate cysts in the cores. No previous studies have investigated spatial distribu- tidal range is about 1 m; the direction of tidal-flood cur- tions of dinoflagellate cysts in relation to eutrophication rent is shown in Fig. 1. and toxic pollution in estuarine systems. This work rep- Mean summer (June, July and August) water salinity resents the first analysis of this kind. varies from 28 to 31 and summer temperatures from 21 In this study we document the dinoflagellate cyst to 23°C (Howes et al. 1999). Concentrations of nutri- assemblages on a ~1 km scale in 3 side embayments of ents and chlorophyll a for the studied embayments are Buzzards Bay, Massachusetts. Comparison of these provided in Table 1. Ammonium is the major form of assemblages to known gradients of nutrient and toxic inorganic nitrogen available throughout the Bay and pollution allows us to assess the utility of cysts as indi- its concentrations are heavily influenced by the cators of response. sewage outfall (Borkman & Turner 1993). A study of economic growth and environmental change in Buz- zards Bay (Terkla et al. 1990) has identified population MATERIAL AND METHODS growth as the dominant factor currently affecting the environmental health of Buzzards Bay. Study area. New Bedford Harbor, Clarks Cove and New Bedford Harbor: New Bedford Harbor, also Apponagansett Bay are side embayments of the north- known as the Acushnet River estuary, has a moderate western shore of Buzzards Bay, Massa- chusetts, USA (Fig. 1). The embay- Table 1. Comparison of mean summer (June to August) concentrations of nutri- ents and chlorophyll a in embayments of Buzzards Bay ments are shallow, with water depths ranging from 1 to 12 m (mean 6 m), generally well mixed, and unstratified. Location Nitrate Ammonium Phosphate Chlorophyll a (µM) (µM) (µM) (µg l–1) There are no major bottom currents in Buzzards Bay, and most of the environ- Inner New Bedford Harbor 11.00 5.7 1.8 8.5 mental energy is in tidal currents and Clarks Cove 1.5 0.9 1.5 3.7 Apponagansett Bay 1.3 2.2 1.6 4.7 waves (Summerhayes et al. 1985). The Pospelova et al.: Dinoflagellate cysts in polluted estuaries 25

freshwater input (median flow 0.54 m3 s–1) from the Clarks Cove: In contrast to New Bedford Harbor, the river flowing into the northern part of the harbor. This neighboring Clarks Cove (Fig. 1) has relatively good harbor is heavily urbanized, as it is surrounded by 35% water quality because of the absence of major freshwa- of the population of the entire Buzzards Bay watershed ter sources and a sewered watershed, with discharges (Howes et al. 1999). As a result, ~20 combined sewer located at Clarks Point (New Bedford Wastewater overflows (CSO) discharge into the inner part of New Treatment Plant). Clarks Cove water quality is main- Bedford Harbor, in addition to the outfall of the tained primarily by its tidal dynamics (Howes et al. Fairhaven Wastewater Treatment Plant (Fig. 1). From 1999). Despite the fact that Clarks Cove is a compara- 1974 to 1996, the Fairhaven facility has continuously tively deep (~9 m) and well-flushed embayment, the discharged primary-treated sewage to the harbor, total nitrogen level here is higher than in the waters of resulting in a load of 140 t N yr–1 (SAIC 1991). A hurri- outer Buzzards Bay. This enrichment of Clarks Cove cane barrier greatly reduces water circulation relative waters comes from the Clarks Point outfall when its to the rest of Buzzards Bay and affects water quality waters enter the system during periodic shifts in the (Costa et al. 1996). Based on Nixon’s (1995) classifica- outer harbor circulation, and from the tidal waters of tion, the inner New Bedford Harbor is a ‘hypertrophic’ Apponagansett Bay to the west (Howes et al. 1999). or highly eutrophic system, and is one of the most Sample collection. Surface sediments were collected eutrophic embayments in Buzzards Bay (Howes et al. from 19 locations throughout New Bedford Harbor, 1999). Clarks Cove and Apponagansett Bay (Fig. 1, Table 2). New Bedford Harbor also is one of the most contam- For the purpose of this study, we divide all samples into inated sites in the United States (Nelson et al. 1996) 3 groups (Table 2), based on their location: Group I due to polychlorinated biphenyls (PCBs) used in the comprised 5 samples from the inner part of New Bed- manufacture of electrical capacitors. Past discharges of ford Harbor, Group II comprised 9 samples from the PCBs and other pollutants in the upper harbor have outer harbor and Clarks Cove, and Group III comprises resulted in modern sediment concentrations as high as 5 samples from the outer part of Apponagansett Bay. 35 000 ng g–1 PCBs, 1500 µg g–1 Cu, 600 µg g–1 Pb, and Sediments were collected with a van Veen or petite 1200 µg g–1 Zn. New Bedford Harbor is listed in the US Ponar grab sampler during the US Environmental Pro- Environmental Protection Agency's National Priorities tection Agency (EPA) cruises in October 1996. All sam- List for cleanup because of PCBs contamination ples were stored at ~4°C in the dark until processing in (Bergen et al. 1998). 1999. We assume that the top 2 cm of collected sedi- New Bedford’s outer harbor, south of the hurricane ments represent ~10 yr deposition, based on the esti- barrier (Fig. 1), is expansive and well-flushed. The mates of accumulation rates previously reported for primary difference between the outer and inner harbor is the embayments (Summerhayes et al. 1985, Latimer et a lower sedimentary concentration of PCBs, heavy al. 2003). Sediments are generally characterized as metals and other industrial pollutants in the former. fine sand and mud (Table 2). However, the surface plume of effluent from the New Sediment chemistry. Sedimentary metal concentra- Bedford Wastewater Treatment Facility, located off tions were measured by inductively-coupled plasma Clarks Point, influences local water-quality conditions. atomic-emission and graphite furnace atomic-absorp- The New Bedford Wastewater Treatment Plant dis- tion spectrophotometry after digestion by microwave charges up to 962 t N yr–1 into the system, mostly in the heating in pressurized digestion vessels with a mixture form of ammonium (SAIC 1991). Elevated concentrations of concentrated acids (for details see Latimer et al. of nitrate (1.2 µM), ammonium (16.4 µM), and phospho- 2003). PCBs were extracted using a microwave proce- rus (3.0 µM) were measured near the outfall by Borkman dure and analyzed using gas chromatography with & Turner (1993). Despite the high sewage-effluent input, electron-capture detector (for details see Latimer et effects of the discharge on water quality are detectable al. 2003). Percent organic carbon was measured by only within 5 km of the outfall (Costa et al. 1999). continuous-flow elemental analysis/isotope-ratio mass- Apponagansett Bay: Similar to New Bedford Harbor, spectrometry as described by Latimer et al. (2003). inner Apponagansett Bay is one of the most eutrophic Dinoflagellate cysts. For dinoflagellate cyst analyses, embayments of Buzzards Bay because of its restricted sediment samples of known volume and dry weight geomorphology and nutrient loading from the water- were processed using standard palynological prepara- shed (Howes et al. 1999). The high nutrient load to the tion methods (Rochon et al. 1999). Sediment was dried at estuary originates mostly from its densely populated 40°C, weighed, sieved through 125 µm mesh and re- watershed, followed by commercial and industrial tained on a 10 µm mesh (to eliminate coarse and fine ma- development. In contrast to the inner bay, the outer terial), treated with warm HF (40%) to dissolve silicates, Apponagansett Bay is better flushed, with moderate and HCl (10%) to remove carbonates and silicofluorides. nutrient levels (Howes et al. 1999). Calibrated tablets of Lycopodium clavatum spores 26 Mar Ecol Prog Ser 292: 23–40, 2005

Table 2. Surface sediment samples. Collection data

Group Location Latitude Longitude Water depth Sediment Sampling Station ID (°N) (°W) (m) type method

Group I NBH146 New Bedford Harbor (inner) 41.660 70.917 4.6 Sandy silt Petite Ponar NBH154 New Bedford Harbor (inner) 41.657 70.918 7.3 Sandy silt Petite Ponar NBH204 New Bedford Harbor (inner) 41.652 70.920 4.0 Sandy silt Petite Ponar NBH236 New Bedford Harbor (inner) 41.634 70.913 9.8 Mud Petite Ponar NBH247 New Bedford Harbor (inner) 41.627 70.905 3.0 Sandy silt Petite Ponar Group II NBH324 Clarks Cove/New Bedford Harbor (outer) 41.582 70.899 ~9.0~ Sandy silt Petite Ponar CPE Clarks Cove/New Bedford Harbor (outer) 41.582 70.890 9.0 Mud Petite Ponar CPC Clarks Cove/New Bedford Harbor (outer) 41.588 70.895 9.0 Fine sand Petite Ponar CPB Clarks Cove/New Bedford Harbor (outer) 41.582 70.907 9.6 Mud Petite Ponar CPG Clarks Cove/New Bedford Harbor (outer) 41.576 70.886 8.8 Mud Petite Ponar NBH317 New Bedford Harbor (outer) 41.594 70.890 9.8 Sandy silt Petite Ponar NBH325 New Bedford Harbor (outer) 41.582 70.881 ~10.0~0 Sandy silt Petite Ponar NBH346 Buzzards Bay 41.545 70.891 ~10.000 Mud Petite Ponar NBH331 Clarks Cove/Apponagansett Bay 41.570 70.928 7.5 Mud Petite Ponar Group III AB1 Apponagansett Bay 41.584 70.948 2.4 Sandy silt Van Veen AB2 Apponagansett Bay 41.581 70.947 4.6 Sandy silt Van Veen AB3 Apponagansett Bay 41.580 70.947 5.2 Silt Van Veen AB4 Apponagansett Bay 41.578 70.947 5.2 Sandy silt Van Veen AB5 Apponagansett Bay 41.576 70.945 4.9 Sandy silt Van Veen

(Stockmarr 1977), added during processing, allowed cal- scured the archeopyle characteristics, thus preventing culation of dinoflagellate cyst concentrations. In this identification to species level. study, we express dinoflagellate cyst concentrations in 2 Species richness is used in this study as a measure of units: cysts per volume of wet sediments, and cysts per dinoflagellate cyst diversity (see discussion in Pospe- dry weight of sediments. Cyst concentrations based on lova et al. 2002). To correct for non-equal cyst counts in dry weight of sediments facilitate comparison with core different samples, Fisher’s alpha index is often intro- studies, whereas concentrations based on volume are duced (Fisher et al. 1943). This is a diversity index, often applied to study spatial cyst distributions. defined for each sample implicitly by the formula: Aliquots were mounted on microscope slides with S = α · ln(1 + n/α) glycerine jelly. Dinoflagellate cysts were counted with a transmitted light microscope (63× and 100× objectives). where S is number of cyst taxa, n is the cyst count and α Identification of dinoflagellate cysts was made on the ba- is Fisher’s alpha. All statistical calculations for this work sis of published descriptions in accordance with taxon- were made using SPSS 10.1 for Windows software. omy given by Lentin & Williams (1993) and Rochon et al. (1999). However, when species-level identification was not possible, identification was to level. There are RESULTS different taxonomies for cysts and thecal stages of di- noflagellates because paleontological studies of cysts Metals, PCBs and organic carbon originally were carried out independently from biologi- cal studies of the motile forms. Here we use the paleon- Samples from the inner part of New Bedford Harbor tological nomenclature according to Head (1996), Head (Group I) had the highest concentrations of all metals et al. (2001), and Pospelova & Head (2002). A list of the (Table 4, Fig. 2), with average concentrations of Zn of dinoflagellate cysts counted and their known biological 372 µg g–1, Cu of 516 µg g–1, and Pb of 156 µg g–1. The name or thecal equivalent is provided in Table 3. concentrations of metals in the outer harbor and Clarks is presented in Appendix 1 (available at: Cove (Group II) are lower, on average, by a factor of 3, www.int-res.com/journals/suppl/Pospelova_appendix.pdf). with Zn 144 µg g–1, Cu 55 µg g–1, and Pb 49 µg g–1. Spiniferites spp. includes all Spiniferites cysts except Apponagansett Bay (Group III) was characterized by S. bentorii, S. elongatus and S. membranaceus. For sta- low levels of metals pollution: Zn 92 µg g–1, Cu 40 µg tistical treatment, species of the genus Brigantedinium g–1, and Pb 23 µg g–1. The concentration of PCBs fol- (B. cariacoense and B. simplex) were grouped together lowed the same trend (Table 4), the highest concentra- because cyst-folding or orientation sometimes ob- tions being in Group I (~17 000 ng g–1), moderate con- Pospelova et al.: Dinoflagellate cysts in polluted estuaries 27

Table 3. Taxonomic designation of dinoflagellate cysts counted in this study. Thecal equivalents from Head (1996), except for Islandinium brevispinosum (from Pospelova & Head 2002) and I. cezare and I. minutum (from Head et al. 2001)

Cyst species Dinoflagellate thecate name (paleontological name) or affinity (biological name)

Autotrophic Gonyaulacaceae – Alexandrium tamarense Impagidinium spp. ? Gonyaulax sp. indet. Lingulodinium machaerophorum Lingulodinium polyedrum Nematosphaeropsis spp. Gonyaulax spinifera complex Operculodinium centrocarpum Protoceratium reticulatum sensu Wall & Dale 1996 Operculodinium israelianum ? Protoceratium reticulatum Spiniferites bentorii Gonyaulax digitalis Spiniferites elongatus Gonyaulax spinifera complex Spiniferites membranaceus Gonyaulax spinifera complex Spiniferites spp. Gonyaulax complex Tectatodinium pellitum Gonyaulax spinifera complex Peridiniaceae – Pentapharsodinium dalei Pyrophacaceae Tuberculodinium vancampoae Pyrophacus steinii Heterotrophic Diplopsalidaceae Dubridinium spp. Diplopsalid group Fig. 2. Distribution of sedimentary concentrations of Zn, Cu, – Gymnodinium spp. Pb (µg g–1) and organic carbon (OC, %) content in New Bed- Polykrikaceae ford Harbor, Clarks Cove and Apponagansett Bay – schwartzii – centrations (~1300 ng g–1) in Group II, and the low- Peridiniaceae Peridinium wisconsinense Peridinium wisconsinense est (500 ng g–1) in Group III. Organic carbon con- Protoperidiniaceae tent (Table 4, Fig. 2) was highest in the inner New Brigantedinium cariacoense Protoperidinium avellanum Bedford Harbor sediments (3.0 to 5.3%). Sediment Brigantedinium simplex Protoperidinium conicoides samples from Apponagansett Bay, the outer harbor Brigantedinium spp. ? Protoperidinium spp. Islandinium brevispinosum Protoperidinium sp. indet. and Clarks Cove had comparable organic carbon Islandinium? cezare Protoperidinium sp. indet. content ranging from 0.4 to 2.7%. Distribution of Islandinium minutum Protoperidinium sp. indet. Ag and Cd did not follow the same pattern, as the Lejeunecysta oliva Protoperidinium sp. indet. highest values were found in the outer harbor, near Lejeunecysta sabrina Protoperidinium leonis Protoperidinium minutum Protoperidinium minutum the location of the New Bedford sewage outfall. Protoperidinium spp. Protoperidinium sp. indet. We calculated Pearson’s correlations to deter- Quinquecuspis concreta Protoperidinium leonis mine the degree of covariance between concen- Selenopemphix nephroides Protoperidinium subinerme Selenopemphix quanta Protoperidinium conicum trations of contaminants and content of organic Stelladinium stellatum Protoperidinium stellatum carbon (Table 5). Most correlation coefficients Trinovantedinium applanatum Protoperidinium pentagonum were higher than 0.50 (p ≤ 0.02). Thus, sedimen- Votadinium calvum Protoperidinium oblongum tary concentrations of PCBs, Zn, Cu, Cr, Pb, Ni, Ag Votadinium spinosun Protoperidinium claudicans and organic carbon content were highly covariant and the effect of individual pollutants on the system could not be separated. (Table 6). Microphotographs of cyst taxa are shown in Figs. 3 to 7. Dinoflagellate cysts were recovered from all the Distribution of dinoflagellate cysts sediment samples. Total dinoflagellate cyst concentra- tions varied by an order of magnitude, from ~300 to An average of 317 cysts and a minimum of 102 were 4200 cysts cm–3 and ~500 to 9400 cysts g–1, averaging counted in each sample. We identified and counted ~2000 cysts cm–3 and ~4300 cysts g–1 (Tables 7 & 8, 35 dinoflagellate cyst taxa in sediments from 19 sites Fig. 8). In general, the total cyst concentrations in inner 28 Mar Ecol Prog Ser 292: 23–40, 2005

New Bedford Harbor (Group I) were 2 to 3 times lower 4.9, with an average of 2.0 (Table 6). Cysts of auto- compared to the outer harbor and Clarks Cove (Group trophic dinoflagellates usually comprised more than II) and Apponagansett Bay (Group III). The highest 50% of the assemblages (Fig. 9), with the exception of concentrations were found in Group II, which was also 3 sites—CPG, CPB and AB3 (46 to 47%). Cysts of het- characterized by the largest range of variation within erotrophic dinoflagellates comprised an average of the group; the total cyst concentrations ranged from 36% of each cyst assemblage, ranging from 17 to 53% ~500 to 4200 cysts cm–3 and ~500 to 9400 cysts g–1. We (Fig. 9). The proportion of Diplopsalidaceae and Poly- divided the highest by the lowest value for the total krikaceae was ~12% at all sites (Fig. 9). cyst concentration within each group to characterize A total of 37 dinoflagellate cyst taxa were identified fluctuations in cyst concentrations. These fluctuations (Table 6). The number of taxa (species richness) in can be expressed as a ratio of 6:8:4 for Groups I, II and samples ranged from 12 to 26, with an average of 22, III if concentrations are expressed in units of cysts per and Fisher’s index changed from 3.5 to 6.4 (Table 6, volume of sediments, and as 12:20:7 if cysts per dry Fig. 8). Cyst assemblages in Group I, the inner part of weight are used. New Bedford Harbor, were characterized by lower The ratio between cysts produced by autotrophic diversity (an average species richness of 17, α = 4.5) and heterotrophic dinoflagellates ranged from 0.9 to compared to assemblages in Groups II (24 taxa, α = 5.7) and III (21 taxa, α = 5.2). There were significant (p < 0.01) negative correla- Table 4. Concentrations of organic carbon (OC, %), PCBs (ng g–1) and metals (µg g–1) in sediments tions between dinoflagellate cyst spe- cies-richness, Fisher’s index and sedi- Group OC PCBs Zn Cu Cr Pb Cd Ni Ag mentary concentrations of Cu, Zn, Pb, Site PCBs and organic carbon content (Table 7, Fig. 10). A highly significant Group I correlation (R = 0.91, p < 0.01) between NBH 146 5.3 33525 564.9 706.8 204.1 222.10.73 0.01 39.9 1.850.02 NBH154 4.7 22532 339.1 480.0 249.7 194.91.06 0.01 38.3 4.630.05 species richness and Fisher’s alpha NBH204 3.4 15052 362.7 647.0 296.2 160.21.39 0.01 41.5 3.020.03 showed that the correction for non- NBH236 4.0 10495 349.1 373.0 210.7 132.20.85 0.01 27.6 3.470.03 uniform cyst counts in different NBH247 3.0 4520 245.7 374.4 122.4 71.50.54 0.01 11.1 2.650.03 samples was not significant. Group II NBH324 2.1 2757 153.4 161.2 121.9 64.80.75 02.9 29.4 2.9 2.9001.4 The composition of the dinoflagel- CPE 2.3 1039 317.7 74.8 197.4 70.60.20 000. 40.6 4.640.05 late cyst assemblages is described with CPC 0.8 938 85.7 22.8 39.0 27.30.20 000. 7.9 7.9 0.730.01 respect to proportions (Table 6) and CPB 1.8 1481 205.0 55.5 92.1 60.00.42 000. 24.8 2.020.02 CPG 0.4 138 80.2 6.9 23.4 19.40.06 000 5.8 5.8 0.22000. concentration by volume and dry NBH317 2.5 3885 183.5 83.3 111.8 75.50.78 0.01 20.9 4.420.04 weight, respectively (Appendices 2 NBH325 1.6 1205 108.7 50.6 85.3 51.00.24 000. 19.4 1.360.01 & 3; www.int-res.com/journals/suppl/ NBH346 0.4 61 37.3 4.2 23.4 19.20.10 000. 6.6 6.6 0.14000. Pospelova_appendix.pdf). The domi- NBH331 1.6 729 123.7 37.7 78.4 50.40.41 000. 23.9 1.150.01 nant taxa belonged to Gonyaula- Group III AB1 0.8 134 47.4 19.7 34.3 5.80.09 000 3.0 3.0 0.28000. caceae, Spiniferites spp. and Opercu- AB2 2.5 726 112.2 48.7 60.5 28.10.30 000. 7.4 7.4 0.790.01 lodinium centrocarpum sensu Wall & AB3 2.7 537 112.5 52.4 63.5 32.60.18 000. 11.2 1.030.01 Dale (1966) (Table 6, Fig. 11). Cyst AB4 1.8 398 90.8 39.1 54.2 18.50.17 000. 5.0 5.0 0.860.01 AB5 2.2 579 96.9 38.0 57.4 30.50.21 000. 7.6 7.6 0.890.01 taxa that comprised >5% of the assem- blages include Protoperidinium spp., Dubridinium spp., Pentapharsodinium Table 5. Correlation matrix of organic carbon, PCBs and metal sedimentary dalei, Islandinium brevispinosum, Bri- concentrations. Coefficients with R > 0.5 and significance at 0.01 level in bold gantedinium spp., Polykrikos kofoidii plus P. schwartzii, Islandinium minu- OC PCBs Zn Cu Cr Pb Cd Ni Ag tum and Spiniferites elongatus. At 4 sites, cysts of the toxic bloom-form- OC 0.849 0.878 0.849 0.811 0.894 0.730 0.687 0.617 PCBs 0.849 0.879 0.911 0.742 0.952 0.686 0.678 0.411 ing Alexandrium tamarense were Zn 0.878 0.879 0.891 0.877 0.936 0.705 0.837 0.639 found in low proportions (<2%). Cu 0.849 0.911 0.891 0.853 0.930 0.820 0.694 0.482 Principal component analysis (PCA) Cr 0.811 0.742 0.877 0.853 0.893 0.868 0.900 0.788 was performed on taxa percentages Pb 0.894 0.952 0.936 0.930 0.893 0.824 0.833 0.627 Cd 0.730 0.686 0.705 0.820 0.868 0.824 0.745 0.693 using Subprogram 10.1 of the Statisti- As 0.306 –0.0110 0.259 –0.0060 0.245 0.143 –0.4660 0.497 –0.1960 cal Package for the Social Sciences Ni 0.687 0.678 0.837 0.694 0.900 0.833 0.745 0.766 (SPSS 10.1). The first principal com- Ag 0.617 0.411 0.639 0.482 0.788 0.627 0.693 0.766 ponent (PC1) represented 90% of the Pospelova et al.: Dinoflagellate cysts in polluted estuaries 29

variance, whereas the second and the third compo- the sewage outfalls (Fig. 13) for sites from Groups I and nents explained less than 5 and 2%, respectively. II, for which the distance from the point-sources of Thus, only PC1 was considered for further analyses. sewage discharge could be determined. Sites from The first principal component (PC1) was distributed Group III were not considered, as there is no identified in 2 contrasting patterns. It increased from the upper point source of sewage discharge in Apponagansett part of New Bedford Harbor seaward towards the hur- Bay. Thus, this is a strong indication that PC1 is related ricane barrier, but decreased from Clarks Cove Point to the discharge of sewage enriched with nutrients. and inner Apponagansett Bay in the seaward direction Fig. 11 shows relative abundances of the dinoflagel- (Fig. 12). The highest values of PC1 were near the late cysts taxa that constituted >1% of the assemblages. sewage outfalls of Fairhaven and New Bedford Waste- Amongst these taxa, the proportions of Islandinium water Treatment Plants, as well as at Site AB1, the brevispinosum and Protoperidinium spp. decreased sample from the uppermost reach of Apponagansett as PC1 increased, i.e. towards the sewage outfalls Bay included in our study. There was a significant (Fig. 11), whereas the proportions of cysts of Quinque- (R2 = 0.71 and p < 0.001) linear correlation between cuspis concreta, Protoperidinium minutum, Spiniferites PC1 scores and the distance to the nearest locations of elongatus and Cyst Type E tended to increase with

Fig. 3. Bright-field photomicrographs. 1. Gymnodinium spp. indet., NBH325, Slide x, W53/2, lateral surface; 2. Polykrikos schwartzii, NBH346, Slide 1, O29/3, equatorial view; 3. Lingulodinium machaerophorum, NBH247, Slide 1, L391/2, orientation uncertain; 4,5. Operculodinium centrocarpum sensu Wall & Dale (1966), NBH325, Slide 1, M51/4, dorsal surface (4), and NBH325, Slide 1, dorsal surface (5); 6. Operculodinium centrocarpum var. truncatum, NBH204, Slide 2, S58/3, orientation uncertain; 7. Operculo- dinium israelianum, AB2, Slide 1, R41/1, dorsal surface; 8,9. Nematosphaeropsis spp. indet, CPE, Slide 1, V38/0, optical section (8), ventral surface (9). Scale bars = 10 µm 30 Mar Ecol Prog Ser 292: 23–40, 2005

increasing PC1. The abundance of Nematosphaeropsis the complexity of the suggested connection between spp. was greater at low (<0.91) and high values of PC1 the dinoflagellate cyst record in the sediments and (>0.97) than at the intermediate values. environmental parameters. Many factors affect the multi-step process from dinoflagellate distribution in estuarine waters to cyst accumulation in sediments. DISCUSSION Distribution of dinoflagellate cysts depends on the dis- tribution of dinoflagellates, biological and ecological Our work documents the composition of dinoflagel- controls over cyst formation, and hydrographic condi- late cyst assemblages in surface sediments and studies tions affecting the cyst deposition in sediments. the cyst distribution on small spatial scales in 3 Buz- Spatial distribution of the motile stage of dinoflagel- zards Bay embayments. Spatial variability in cyst con- lates is controlled by biotic and abiotic factors. These centrations, species diversity and species composition factors may substantially differ for different groups of of the assemblages suggest that the cyst assemblages dinoflagellates. About half of the dinoflagellates are reflect gradients of nutrients and toxic pollution in the heterotrophic and half are autotrophic, but some di- embayments. However, prior to discussing the details noflagellates can feed both ways, termed mixotrophic of these cyst signals, we would like to highlight (Jacobson & Anderson 1994, Dale 1996). The distribu-

Fig. 4. Bright-field photomicrographs. 1–3. Spiniferites bentorii, CPC, Slide 1, V38/0, lateral surface (1), NBH317, Slide 2, optical section (2), and ventral surface (3); 4. Spiniferites elongatus, NBH325, Slide 1, X36/1, ventral surface; 5,6. Spiniferites membranaceus, NBH236, Slide 1, optical section (5) and AB5, Slide 3, optical section; 7. Spiniferites cf. delicatus, NBH324, Slide 2, optical section; 8,9. Spiniferites spp. indet, NBH325, Slide 1, lateral surface (8) and NBH204, Slide 1, optical section (9). Scale bars = 10 µm Pospelova et al.: Dinoflagellate cysts in polluted estuaries 31

tion of heterotrophic dinoflagellates is controlled, in ments, because not all species of dinoflagellates are cyst- part, by availability of prey (diatoms and small flagel- producing, and the process of cyst formation or encyst- lates), whereas the distribution of autotrophic species ment can also be influenced by environmental factors. depends on the availability of light and dissolved nutri- Encystment may happen spontaneously, but also can ents. Both types of dinoflagellates are common in New be enhanced by external factors such as depletion Bedford Harbor and Apponagansett Bay. of nutrients—phosphorus and nitrogen in particular Observations of dinoflagellates in Buzzards Bay by (Anderson et al. 1984, Ellegaard et al. 1998), light limi- Pierce & Turner (1994) over a 2 yr period revealed that tation (Anderson et al. 1985) and other factors. There- the stations near the New Bedford Harbor sewage out- fore, high rates of cyst production can reflect high falls had significantly (by a factor of 2 to 10) higher dino- abundances of dinoflagellates and/or enhanced rates flagellate abundance than the other parts of Buzzards of encystment (e.g. caused by depletion of nutrients). Bay. In the same study, significant temporal variability in However, it is important to note that even in the case of dinoflagellate abundances caused by seasonal blooms the surface sediments, the cyst record represents many was recorded in New Bedford Harbor. However, high years of deposition, which is approximately 10 yr in the abundances of dinoflagellates may not necessarily be di- studied embayments. Thus, much of the temporal vari- rectly reflected in high concentrations of cysts in sedi- ability in dinoflagellate abundance and the rate of

Fig. 5. Bright-field photomicrographs. 1. Spiniferites spp. indet, CPG, Slide 1, optical section; 2. Tectatodinium pellitum, CPE, Slide 1, X48/1, dorsal surface; 3,4. Alexandrium tamarense, NBH247, Slide 1, optical section (3) and NBH247, Slide 2, optical sec- tion (4); 5. Tuberculodinium vancampoae, NBH317, Slide 1, T43/1, apical surface; 6. Pentapharsodinium dalei, CPE, Slide 1, F613/4, optical section; 7–9. Dubridinium spp. indet, NBH324, Slide 1, D38/1, apical view (7), CPB, Slide 2, apical surface (8), and NBH236, Slide 4, apical surface (9). Scale bars = 10 µm 32 Mar Ecol Prog Ser 292: 23–40, 2005

encystment average out. At the same time, those envi- Hydrographic regimes in the estuary also affect spa- ronmental factors that are present on the scale of the tial distribution of dinoflagellate cysts in sediments. last 10 yr may be reflected in the ‘integrated’ sedimen- When produced, dinoflagellate cysts behave as parti- tary cyst record. The correlations between dinoflagel- cles in the water column, and there are several known late cyst assemblages and environmental parameters mechanisms of cyst accumulation in the sediments (see found in this study cannot reveal particular mecha- e.g. Harland & Pudsey 1999). The presence of strong nisms by which environmental parameters influence currents may lead to systematic transport of cysts away the distribution of dinoflagellates and the rates of from the place where they were produced and/or encystment. To address these mechanisms in details, resuspension. In the studied embayments, there are one would have to tie together the studies of the motile no strong bottom currents, and the influence of the stage of dinoflagellates in the water column, the sedi- Acushnet River is negligible. Moreover, the inner New ment-trap studies of cyst accumulation, and simultane- Bedford Harbor is almost completely enclosed by a ous measurements of water-quality parameters (see hurricane barrier that restricts water circulation with e.g. Godhe et al. 2001) over an extended period of the rest of the bay. There is no reason to expect that time. At present, such studies in Buzzards Bay do not tidal currents in the harbor would transport cysts over appear to be feasible. significant distances. Hydrographic conditions can be

Fig. 6. Bright-field photomicrographs. 1. Brigantedinium cariacoense, CPG, Slide 1, F55/4, lateral surface; 2. Brigantedinium simplex, NBH325, Slide 1, L51/1, dorsal surface; 3. Islandinium brevispinosum, CPG, Slide 1, D48/4, orientation uncertain; 4. Islandinium? cezare, NBH204, Slide 1, orientation uncertain; 5. Islandinium minutum, NBH317, Slide 1, F41/4, optical section; 6. Lejeunecysta oliva, CPC, Slide 1, V56/4, dorsal surface; 7. Protoperidinium minutum, CPE, Slide 1, T46/2, orientation uncertain; 8. Protoperidinium oblongum, CPG, Slide 2, dorsal surface; 9. Protoperidinium spp. indet, NBH325, Slide 2, orientation uncertain. Scale bars = 10 µm Pospelova et al.: Dinoflagellate cysts in polluted estuaries 33

also assessed by studying the spatial distribution of Total dinoflagellate cyst concentrations pollutant concentrations in the sediments. High con- centrations of metals and other pollutants are found in Despite a number of factors affecting dinoflagellate much greater concentrations near the sources of conta- cyst concentrations in sediments, the total cyst concen- mination (in the upper New Bedford Harbor and near tration has been regarded as a proxy of dinoflagellate the sewage outfalls), suggesting that the sediment production (e.g. Dale 2001). A potential problem with transport is not substantial. However, we cannot this approach is that the change in sediment accumu- discount the possibility that effects of short-distance lation rate affects dinoflagellate cyst concentrations. In transport are also encoded in our data. this study, we observe large fluctuations in the total Below we discuss the particular characteristics of the cyst concentrations in the surface sediment samples dinoflagellate cyst assemblages in the studied embay- that could be attributable to varying dinoflagellate ments and their relation to environmental conditions, production and encystment rates and/or sedimentation and compare spatial cyst distribution with the temporal rate. However, it is difficult to determine the sedimen- cyst records from the sediment cores taken in New tation rates station by station. Estimates show that they Bedford Harbor and Apponagansett Bay (Pospelova et typically vary between 0.2 and 0.6 cm yr–1 in the inner al. 2002). New Bedford Harbor and between 0.1 and 0.5 cm yr–1

Fig. 7. Bright-field photomicrographs. 1. Quinquecuspis concreta, NBH324, Slide 2, N51/1, ventral surface; 2. Selenopemphix nephroides, CPE, Slide 1, C49/0, apical surface; 3. Selenopemphix quanta, NBH346, Slide 1, X58/4, apical surface; 4. Stella- dinium stellatum, CPC, Slide 1, Y59/2, dorsal surface; 5. Trinovantedinium applanatum, CPG, Slide 1, O62/3, dorsal surface; 6. Votadinium calvum, NBH317, Slide 1, dorsal surface; 7. Votadinium spinosum, CPG, Slide 1, O62/3, dorsal surface; 8,9. Cyst type E, CPC, Slide 2, dorsal surface (8), and ventral surface (9). Scale bars = 10 µm 34 Mar Ecol Prog Ser 292: 23–40, 2005 8 2 .8

Unknown cysts* 0.8 0.5 0.2 0.9 0.9 5 7.3 2 1.6 7 0.3 .0 1.0

Cyst Type E 1.0 2.0

Votadinium spinosum

Votadinium calvum

Tuberculodinium vancampoae

Trinovantedinium applanatum .

Tectatodinium pellitum

Stelladinium stellatum

Spiniferites spp.

Spiniferites membranaceus

Spiniferites elongatus

Spiniferites bentorii sediments of New Bedford Harbor, Clarks Cove sediments of New Bedford Harbor,

Peridinium wisconsinense Selenopemphix quanta

Selenopemphix nephroides

Quinquecuspis concreta

Peridinium wisconsinense*

Protoperidinium minutum

Protoperidinium spp.

Polykrikos kofoidii plus P. schwartzii

Pentapharsodinium dalei

Operculodinium israelianum

Operculodinium centrocarpum var. truncatum

Operculodinium centrocarpum sensu Wall & Dale (1966)

Nematosphaeropsis spp.

Lingulodinium machaerophorum

Lejeunecysta sabrina

Lejeunecysta oliva

Islandinium minutum

Islandinium? cezare

Islandinium brevispinosum

Impagidinium spp.

Gymnodinium spp. indet.

Dubridinium spp.

Brigantedinium spp.

Brigantedinium simplex

Brigantedinium cariacoense

Alexandrium tamarense

and Apponagansett Bay. Asterisks denote taxa not counted for cyst richness, including freshwater and Apponagansett Bay. Ratio autotrophs/heterotrophs

Fisher’s diversity index

Species richness

Nos. of counted cysts NBH 146 102 12 3.5 1.3 0.0 0.0 1.0 10.8 13.7 0.0 0.0 2.0 1.0 0.0 0.0 0.0 0.0 2.9 8.8 0.0 0.0 1.0 0.0 12.7 0.0 0.0 0.0 0.0 0.0 1.0 0.0 2.0 40.2 0.0 0.0 0.0 0.0 0.0 0.0 NBH154 137NBH204 15 194 4.3NBH236 18 1.8 356 4.8 0.0NBH247 22 1.7 0.0 250 5.2 0.0 0.0 19 1.5 0.0 6.6 4.8 0.6 13.1 0.5 1.5 0.0 0.0 6.2 1.6 10.8 1.7 0.0 0.0CPE 0.0 9.0 3.6 2.0 0.0 0.7 7.0CPC 3.6 2.1 0.0 0.0 13.2 428 0.5 0.0 0.3CPB 0.0 3.1 24 0.0 0.0 1.4 266 0.0 5.5CPG 1.5 0.0 2.0 23 0.0 2.8 1.5 268 3.7 0.0 6.0 10.9NBH317 1.5 0.0 0.0 3.2 23 1.8 0.0 446 371 1.5 0.0 0.0 10.8 0.0 6.0 0.0 2.2NBH325 1.2 25 26 0.3 0.5 0.0 344 0.9 0.0 0.0 5.7 3.5 6.4 2.5 1.0NBH346 1.2 0.0 1.5 25 0.0 14.9 1.4 0.9 336 2.3 2.0 0.0 1.5 6.2 4.5 0.0 0.0 6.6 14.8 0.5NBH331 0.0 1.5 21 0.5 2.4 0.0 4.9 0.0 0.0 0.0 331 0.5 11.9 0.2 5.0 9.0 0.0 0.0 1.1 0.0 0.0 2.5 3.5 0.0 22 2.1 2.5 0.9 8.6 0.0 0.0 9.3 6.7 0.0 2.8 4.6 5.3 0.0 0.0 1.5 0.0 0.0 1.5 0.5 4.4 3.4 6.3 2.9 3.3 5.1 0.0 0.0 5.2 0.0 0.4 0.5 0.3 0.0 0.9 4.4 0.0 0.0 1.5 0.7 2.6 2.3 3.8 0.0 0.0 0.2 0.0 0.8 0.0 0.0 5.7 0.0 1.5 0.3 0.4 1.5 1.1 0.6 0.0 0.7 3.6 2.7 4.5 0.7 8.2 0.0 0.4 0.0 0.0 1.6 2.1 0.0 4.2 41.6 1.1 0.0 18.0 0.0 1.1 0.5 0.6 3.5 0.4 4.3 0.0 6.5 4.2 43.3 0.0 1.2 0.4 1.5 1.7 0.6 0.0 0.0 0.7 0.0 19.9 0.0 0.0 0.2 0.7 0.6 1.1 4.9 0.8 0.0 0.0 0.3 0.0 0.0 0.0 0.7 0.3 0.8 8.2 0.0 2.0 0.7 0.0 13.8 1.3 0.0 0.0 34.3 1.8 6.8 5.4 0.0 0.8 2.5 0.0 0.0 0.8 0.0 0.3 3.0 0.3 33.6 16.1 0.0 16.4 1.2 2.6 0.7 0.4 0.0 0.6 6.8 1.8 0.0 0.9 0.0 0.0 0.9 0.0 1. 0.0 0.0 0.7 19.2 0.0 0.0 2.3 0.0 1.2 0.0 0.0 0 0.0 9.3 0.3 0.0 0.0 1.5 0.0 4.0 0.9 0.8 29.2 0.0 0.3 1.2 0.0 6.7 0.0 8.7 0.2 6.7 12.5 0.0 0.0 0.6 0.0 0.4 16.2 1.5 0.5 4.1 3.6 34.4 0.0 2.0 0.0 0.0 0.8 0.4 1.4 0.9 1.5 1.7 7.7 3. 0.0 0.0 1.1 1.2 0.0 0.0 0.6 3.0 0.4 0.5 1.9 0.2 0.7 0.0 3.9 39.9 0.7 0.3 1.2 1.1 0.0 0.6 5.1 0.7 1.9 1.1 0.3 0.4 0.9 0.3 34.6 3.6 1.9 0.7 1.6 0.0 1.8 0.3 0.8 0.6 0.2 0.0 0.3 0.9 1.8 28.0 0.6 0.0 0.0 0.0 0.3 0.3 0.2 1.1 24.3 2.3 0.0 0.0 0.0 29.4 0.6 0.4 0.0 0.3 0.0 0.3 0.0 0.7 1.8 32.8 0.0 0.5 0.0 0.9 1.2 0.2 1.2 0.0 0.0 0.3 0.0 2.1 0.7 0.0 0.6 0.6 0.7 0.0 29.2 3.8 0.3 0.4 0.0 0.0 0.8 0.6 1.1 0.0 0.7 0.0 28.7 1.1 0.5 0.0 0.7 0.0 0.0 0.4 0.8 0.3 2.7 0.3 0.3 0. 0.0 1. 0.6 0.0 0.0 0.0 0.0 1.8 0.0 0.9 NBH324 621 26 5.5 1.6 0.0 0.3 1.1 4.5 4.2 0.2 0.3 3.1 0.6 4.5 0.0 0.2 0.2 1.3 21.4 0.8 0.2 3.4 6.9 8.4 1.4 0.2 0.6 0.0 0.2 0.8 1.0 0.2 32.4 0.3 0.0 0.0 0.0 0.0 0.2 0.8 AB4AB5 274 22 488 5.6 24 1.9 5.3 0.0 2.3 0.0 0.0 1.5 0.2 3.6 1.0 2.9 3.5 0.0 4.7 0.0 0.0 9.5 0.0 0.0 7.6 0.4 0.2 0.0 0.6 0.0 0.0 0.4 0.2 2.2 31.4 1.0 0.0 2.5 0.4 5.5 2.9 0.4 3.3 0.8 10.2 11.9 0.4 5.3 0.0 5.3 0.4 0.4 0.0 0.0 0.0 0.0 0.7 0.0 1.5 0.2 0.7 1.0 23.0 2.3 0.7 1.2 0.4 42.6 0.4 0.4 0.4 0.4 0.0 0.0 0.0 0.0 0.7 0.0 2. 0.2 0.2 0.2 AB1AB2 284AB3 21 285 5.2 20 2.3 245 4.9 0.0 20 4.9 0.0 5.2 0.0 0.4 0.9 0.0 5.6 0.0 0.0 5.3 0.0 3.2 0.0 0.0 4.9 0.0 7.3 0.0 4.2 9.0 0.0 1.1 0.0 1.4 1.4 0.0 0.7 12.2 0.0 0.4 0.4 0.0 0.0 4.1 0.7 0.0 0.0 2.1 24.3 0.4 0.0 0.0 1.1 0.8 28.8 0.7 1.2 0.0 18.0 3.2 1.1 0.0 9.2 0.4 3.9 1.4 2.5 3.7 0.4 6.5 1.8 0.0 10.2 0.0 0.4 0.4 0.0 0.0 0.0 0.4 0.4 0.0 0.0 0.7 0.0 1.1 2.1 0.0 2.5 0.7 0.8 34.2 5.3 0.4 0.0 0.7 0.0 39.3 0.0 22.0 0.0 0.0 0.4 0.4 0.0 0.0 0.0 0.0 0.4 0.0 0.4 0.0 0.0 0.4 0.0 0.0 1.1 0.4 0.4 0.4 0.4 0 Group I Group II Group III Group Sample Table 6. Relative abundance, total counts, Fisher’s diversity index and species richness of dinoflagellate cyst taxa in surface 6. Relative abundance, total counts, Fisher’s Table Pospelova et al.: Dinoflagellate cysts in polluted estuaries 35

70° 54' W 70° 52' W 70° 54' W 70° 52' W Sewage Outfall Autotrophic 16 # Cyst Taxa N Heterotrophic N Protoperidiniaceae Cysts cm-1 Polykrikaceae & Diplopsalidaceae 300 ° 41 40' N Unknown 41° 40' N 1500 12 NBH146 NBH146 3000 15 NBH154 NBH154

New Bedford Harbor New Bedford Harbor NBH204 18 NBH204 Popes Island Popes Island Inner Harbor 41° 38' N Inner Harbor 41° 38' N NBH236 22 Fairhaven S.O. NBH236 Fairhaven S.O.

NBH24719 NBH247

hurricane barrier hurricane barrier Outer Harbor Outer Harbor

° ° Clarks 41 36' N Clarks 41 36' N NBH317 Apponagansett Cove Cove NBH317 25 Apponagansett Bay CPE AB1 CPC AB2 CPC Bay AB1 23 CPB New Bedford S.O. 21 20 AB3 CPB 23 New Bedford S.O. AB3 24 25 NBH325 20 22 26 AB2 NBH331 26 NBH325 NBH324 CPE AB4 24 CPG AB4 NBH331 CPG AB5 NBH324 22 AB5 41° 34' N 41° 34' N 1 km 1 km NBH346 NBH346 21 Buzzards Bay Buzzards Bay

Fig. 8. Distribution of dinoflagellate cyst species-richness and Fig. 9. Relative abundance (%) of cysts of heterotrophic (Pro- total dinoflagellate cyst concentration in surface sediments of toperidiniaceae, Polykrikaceae and Diplopsalidaceae) and New Bedford Harbor, Clarks Cove and Apponagansett Bay autotrophic dinoflagellates in assemblages from New Bedford Harbor, Clarks Cove and Apponagansett Bay in the outer harbor, and Apponagansett Bay (Summer- hayes et al. 1985, Latimer et al. 2003). Low cyst concen- value of 0.2 m (Howes et al. 1999). The reduction of the trations (~400 cysts cm–3; ~900 cysts g–1) in the inner photic zone may result from high levels of organic car- part of New Bedford Harbor, north of the Popes Islands bon in the water column. (Sites NBH 146, 154 and 204), are one-fifth those at the other sites, which is probably too large a difference to be explained by variation in the sedimentation rate. Species diversity Thus the low dinoflagellate cyst concentration in the upper part of inner New Bedford Harbor is likely to be A decline in species richness of phytoplankton has the result of reduced dinoflagellate production. We been noted as a possible response to eutrophication suggest that the suppressed production is caused by (Sommer 1995, Tsirtsis & Karydis 1998). A decrease in high levels of toxic pollutants in the upper part of inner richness of dinoflagellate cyst taxa has been sug- New Bedford Harbor and possibly by the reduction of gested as a general indicator of polluted and highly the photic zone. Extremely high concentrations of met- eutrophic estuarine systems (Pospelova et al. 2002). als and other pollutants found in the sediments indi- Sediment samples from the upper part of the inner cate toxic water conditions that may affect the dino- harbor (Group I) that had lower cyst concentrations flagellate population. Limited light penetration in this also had lower species richness and a lower Fisher part of the harbor was supported by 1 measurement of index. Species diversity is independent of variation in Secchi depth in August 1993, which showed a very low sedimentation rates and is a more robust indicator

Table 7. Correlation matrix of dinoflagellate cyst species richness (Richness) and the Fisher’s diversity index (Fisher’s) versus organic carbon, PCBs and metal sedimentary concentrations. Coefficients with R > 0.5 and significance at 0.01 level in bold

OC PCBs Zn Cu Cr Pb Cd Ni Ag Richness Fisher’s

Richness –0.713 –0.813 –0.649 –0.761 –0.509 –0.704 0.264 –0.492 –0.190 0.911 Fisher's –0.755 –0.783 –0.661 –0.750 –0.537 –0.696 0.036 –0.440 –0.041 0.911 36 Mar Ecol Prog Ser 292: 23–40, 2005

30 30 dinoflagellate cysts may be regarded as R = 0.713 R = 0.761 a combined signal of organic and toxic p = 0.001 p < 0.000 pollution in the harbor. Further support of 25 25 this explanation is found in the negative linear relationship between dinoflagellate 20 20 cyst species-richness and sedimentary con- centrations of Cu, PCBs, Zn, and Pb, as well as % organic carbon (OC). Fisher’s 15 15 index shows similar correlations. Although the effects of each constituent may differ, 10 10 their covariances prevent us from deter- 0123456 0 100 200 300 400 500 600 700 800 % OC Cu (μg g–1) mining more detailed relationships. 30 30 R = 0.813 R = 0.649 p < 0.000 p = 0.003 Spatial distribution of PCA 25 25 At our study sites, the gradients of salinity 20 20 and temperature are short compared to

# taxa # taxa those examined in marine and oceanic stud- ies (Wall et al. 1977, Dale 1996, Mudie & 15 15 Harland 1996, de Vernal et al. 1997, Rochon et al. 1999). They are also smaller than gra- 10 10 dients of temperature and salinity found in 0 7000 14000 21000 28000 35000 0 100 200 300 400 500 600 Total PCBs (μg g–1) Zn (μg g–1) the coastal lagoons from the same region 30 30 (Pospelova et al. 2004). Water temperature R = 0.109 R = 0.704 and salinity, commonly identified as 2 major p < 0.435 p = 0.001 factors controlling distribution of dinoflagel- 25 25 late cysts, did not vary with the first princi- pal component in this study. New Bedford 20 20 Harbor and Apponagansett Bay showed

# taxa similar patterns, with minor increases in salinity and slight decreases in temperature 15 15 in the seaward direction, but with opposite trends in the PC1 scores (Fig. 12). 10 10 The availability of nutrients has been 03691215 0 50 100 150 200 250 –1 –1 identified as an important factor controlling Ag (μg g ) Pb (μg g ) the distribution of phytoplankton, and Fig. 10. Relationship between dinoflagellate cyst species-richness and sedi- dinoflagellates in particular (Taylor 1987). mentary concentrations of Cu, Zn, Ag, Pb, PCBs and % organic carbon (OC) Unfortunately, there are no data on the con- centrations of nitrogen or other nutrients on that the environmental conditions north of Popes the scale of our sample stations. Nevertheless, we can Island are less suitable for dinoflagellate population assume that nutrient concentrations decrease with compared to conditions south of the island. In particu- increasing distance from the sewage outfalls. lar, we found that in the upper part of inner New Bed- Previous studies have identified Ag as a marker of ford Harbor the following cyst taxa were absent or sewage effluents (MacKay et al. 1972, Abu-Hilal & highly suppressed in proportions relative to propor- Badran 1990, Bothner et al. 1994). In our study, those tions of the same taxa in the rest of the samples: sites with high sediment silver-content (≥3.5 µg g–1) were Spiniferites elongatus, Pentapharsodinium dalei, Poly- NBH324, CPE, NBH317, NBH236, and NBH154. Indeed, krikos schwartzii plus P. kofoidii, Islandinium minu- all these sites except NBH154 are in close proximity to tum, Protoperidinium minutum. The absence of these the sewage outfalls and also had the highest PC1 scores species from this part of the harbor is probably due to based on the proportions of dinoflagellate cysts in the the same adverse conditions that are responsible for assemblages. the suppression of the total cyst concentrations e.g. The correlation between PC1 scores and proximity to high levels of toxins and hypertrophic conditions. The sewage outfalls (Fig. 13) suggests that PC1 mainly re- decrease in Fisher’s index and the species richness of flects nutrient gradients, and that cyst assemblages Pospelova et al.: Dinoflagellate cysts in polluted estuaries 37

0.88Component 1 0.99 of the cores from the same embay- ments analyzed by Pospelova et al. 4 Cyst Type E (2002). Surface and core assemblages 0 2 have the same ranges for cyst concen- Protoperidinium minutum 0 tration and diversity, and are domi- 2 nated by the same taxa. Quinquecuspis concreta 0 6 Large fluctuations in the total cyst Spiniferites elongatus 0 concentration have been proposed 10 (Pospelova et al. 2002) as a signature Islandinium minutum 0 of stressed environments. These fluc- 10 tuations can be seen both in the core Polykrikos schwartzii & kofoidii 0 4 and surface assemblages, and the Spiniferites bentorii 0 range of the fluctuations in New 30 10 Bedford Harbor is larger than in Dubridinium spp. 0 4 Apponagansett Bay. We consider this Nematosphaeropsis spp. 0 as a signal of stressed environmental 15 conditions in New Bedford Harbor. Pentapharsodinium dalei 0 However, we understand that a large 2 Selenopemphix quanta 0 range of fluctuations in surface sedi- 50 ments is also a reflection of rather Spiniferites spp. 0 large differences in the sediment 40 O. centrocarpum sensu Wall & Dale (1966) accumulation rates from site to site 0 15 as a result of different hydrological Brigantedinium spp. 0 conditions. 2 Islandinium cezare 0 Another important characteristic of 4 the cyst assemblages in both the tem- Spiniferites membranaceus 0 poral and spatial records is the cyst 4 diversity that can be expressed as Operculodinium israelianum 0 15 species richness or Fisher’s index. Islandinium brevispinosim 0 Species richness in the surface sam- 20 ples and in top samples from the cores Protoperidinium spp. 0 4 had similar values. Species richness in Lingulodinium machaerophorum 0 the top samples of Core NBH2c from New Bedford Harbor had 15 to 18 AB4AB5146AB3 CPG CPB AB2 CPE AB1CPC taxa (Pospelova et al. 2002) and the NBH NBH331NBH154NBH346NBH204NBH325NBH247NBH236 NBH317NBH324 closest surface sites (NBH154 and Fig. 11. Proportions of selected dinoflagellate cysts in assemblages from surface NBH204) in this study had corre- sediment samples ordered by first principal component (PC1) spondingly 15 and 18 taxa. Top sam- ples from Core AB1c (Apponagansett change with distance from point-sources of nutrient Bay) had 21 to 23 taxa, while the surface sites AB4 and pollution. We infer that dinoflagellate cyst assemblages AB1 had 22 and 24 taxa. Thus, both in surface and core in Apponagansett Bay also reflect a nutrient signal, be- sediments, species richness in inner New Bedford Har- cause nutrient gradients decrease in the seaward direc- bor is lower than in Apponagansett Bay. Comparison of tion (Fig. 12). Since ammonia is the main form of nitro- the Fisher index values reveals the same trend. Similar gen from sewage effluent (Borkman & Turner 1993), it to temporal records, species richness and the Fisher is likely to be a critical parameter affecting the distribu- index were negatively correlated to sedimentary con- tion of the dinoflagellate cyst assemblages in the stud- centrations of toxic metals and organic carbon content. ied embayments. The decrease in species diversity is a response to hy- pertrophic conditions and/or high levels of inorganic pollutants in the inner part of New Bedford Harbor. The Comparison of spatial and temporal cyst records determination of the relative importance of each of these 2 factors on the decline in cyst diversity does not Dinoflagellate cyst assemblages in surface sediments appear to be feasible with the current data set. The in- from New Bedford Harbor and Apponagansett Bay can crease in species richness in outer Apponagansett Bay be compared to the assemblages from the upper parts was noted as a possible response to eutrophication 38 Mar Ecol Prog Ser 292: 23–40, 2005

70° 54' W 70° 52' W 3.0 Sample sites 2 Sewage Outfall N R = 0.712 p < 0.000 Component 1 2.5

0.88-0.90 41° 40' N 0.91-0.93 2.0 0.94-0.97 NBH146 0.98-0.99 NBH154 1.5 New Bedford Harbor NBH204 Popes Island 1.0

Inner Harbor 41° 38' N NBH236 Fairhaven S.O. 0.5 NBH247 Distance (km)outfalls fromsewage 0.0 hurricane barrier 0.900 0.925 0.950 0.975 1.000 Outer Harbor Principal Component 1

41° 36' N Clarks Fig. 13. Relationship between Principal Component 1 and dis- Apponagansett Cove NBH317 tance from municipal sewage outfalls Bay CPC AB2 CPB New Bedford S.O. AB1 AB3 NBH324 CPE NBH325 AB4 Spiniferites spp. seem to be the most tolerant taxa, AB5 CPGCPG NBH331 and they were encountered in high abundances in

41° 34' N both studies in the core and surface sediments. Tempo- 1 km NBH346 Buzzards Bay ral cyst records show that certain rare and common cyst taxa, Dubridinium spp., Islandinium minutum plus Fig. 12. Spatial distributions of first principal component (PC1) I. cezare, Spiniferites bentorii and cysts of Polykrikos in surface sediments from New Bedford Harbor, Clarks Cove schwartzii plus P. kofoidii, increased in abundance and Apponagansett Bay with increasing nutrient enrichment as the system shifted from mesotrophic to eutrophic or highly when a system changes from oligotrophic/mesotrophic eutrophic conditions (Pospelova et al. 2002). In most to eutrophic conditions by Pospelova et al. (2002). In surface sediments of New Bedford Harbor, these taxa surface samples, the highest species richness was are present in proportions similar to those observed in recorded in the outer New Bedford Harbor and Appon- sediments concurrent with periods of eutrophication agansett Bay, which have mesotrophic to eutrophic and toxic pollution (~7% of Dubridinium spp., ~3% of conditions (Howes et al. 1999). I. minutum plus I. cezare, ~1.5% of S. bentorii, and The increased abundance of cysts of heterotrophic ~5% of P. schwartzii plus P. kofoidii). The spatial distri- dinoflagellates, particularly Polykrikaceae and Diplop- bution of cysts of P. schwartzii plus P. kofoidii showed salidaceae, in temporal records, was seen as a signal of that these taxa are absent from the most polluted sites nutrient enrichment of embayment waters by Mat- in the inner part of New Bedford Harbor. This may suoka (1999) and Pospelova et al. (2002). Cysts of het- indicate that P. schwartzii & kofoidii is suppressed erotrophic dinoflagellates constitute a large proportion in hypertrophic and highly polluted environments. of the cyst assemblages (~36%) in surface sediments, Temporal studies have shown that Lingulodinium indicating a substantial role of dinoflagellates in mod- machaerophorum, Operculodinium israelianum and ern secondary production. These proportions are twice Selenopemphix quanta decline when conditions in the as high as the maximum abundances of cysts of het- embayments change from oligotrophic to mesotrophic erotrophic dinoflagellates that existed in the pre- (Pospelova et al. 2002, Chmura et al. 2004); these spe- settlement period (Pospelova et al. 2002). The abun- cies were absent or contributed ≤1% to most of the cyst dance of cysts of Polykrikaceae and Diplopsalidaceae assemblages in surface sites. This does not contradict remained below 6% throughout the cores in the study our temporal studies, since all modern sites have of Pospelova et al. (2002) but exceeded this value in mesotrophic to eutrophic conditions. the core sediments accumulated during nutrient- To conclude, this study has demonstrated that spatial enrichment periods. Dinoflagellate cyst assemblages distribution of dinoflagellate cysts reflects environ- in the surface sediments also contain high (6 to 18%) mental conditions in polluted estuaries. Sites with proportions of Polykrikaceae and Diplopsalidaceae hypertrophic conditions and the highest levels of toxic cysts, indicating nutrient-rich environments. pollution were characterized by the lowest dinoflagel- Pospelova et al.: Dinoflagellate cysts in polluted estuaries 39

late cyst diversity (species richness and Fisher’s diver- Bothner MH, Takada H, Knight I, Hill RT, Butman B, Farring- sity index), confirming patterns observed in the tempo- ton JW, Colwell RR, Grassle JF (1994) Sewage contamina- ral records. We also found that at small spatial scales, tion in sediments beneath a deep-ocean dumpsite off New York. Mar Environ Res 38:43–59 where salinity and temperature variability were low, Chmura GL, Santos A, Pospelova V, Spasojevic Z, Lam R, the distribution of dinoflagellate cysts reflected the Latimer JS (2004) Response of three palaeo-primary pro- proximity to the major sources of nutrient enrichment duction proxy measures to development of an urban such as sewage outfalls. estuary. Sci Tot Environ 320:225–243 Costa JE, Howes BL, Gunn E (1996) Report of the Buzzards In addition, our study of dinoflagellate cysts from Bay citizens’ water quality monitoring program 1992– surface sediments revealed dinoflagellate species pre- 1995. Buzzards Bay Project, National Estuary Program, viously not recorded in the phytoplankton population Marion, MA of Buzzards Bay waters. In this study, we found Costa JE, Howes BL, Janik D, Aubrey D, Gunn E, Giblin AE Gonyaulax digitalis, G. spinifera, Lingulodinium poly- (1999) Managing anthropogenic nitrogen inputs to coastal embayments: technical basis and evaluation of a manage- edrum, Protoceratium reticulatum, Pentapharsodinium ment strategy adopted for Buzzards Bay. Buzzards Bay dalei, Polykrikos schwartzii, P. kofoidii, Diplopsali- Project Technical Report, Buzzards Bay Project, National daceae and multiple species of the Protoperidiniaceae Estuary Program, Marion, MA group that have never been reported in previous Dale B (1976) Cyst formation, sedimentation, and preserva- tion: factors affecting dinoflagellate assemblages in recent phytoplankton surveys conducted in Buzzards Bay sediments from Trondheimfjord, Norway. Rev Paleobot (Pierce & Turner 1994, Turner et al. 2000). 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King (University of Total Environ 264:235–240 Rhode Island) for help in collecting sediment samples. V. Dale B, Fjellså A (1994) Dinoflagellate cysts as productivity Pospelova also thanks M. J. Head and A. de Vernal for useful indicators: state of the art, potential and limits. In: Zahn R discussions on the taxonomy of dinoflagellate cysts. R. (ed) Carbon cycling in the glacial ocean: constraints in the Harland, B. Dale and 3 anonymous reviewers are greatly ocean’s role in global change. Springer-Verlag, Berlin, acknowledged for critically reading the manuscript and giv- p 521–537 ing many valuable suggestions. We thank the Centre de Dale B, Thorsen TA, Fjellså A (1999) Dinoflagellate cysts as Recherche en Géochimie Isotopique et en Géochronologie indicators of cultural eutrophication in the Oslofjord, Nor- (GEOTOP), Université du Québec à Montréal, for technical way. Estuar Coast Shelf Sci 48(3):371–382 support. We are grateful to R. Lam and L. 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Editorial responsibility: Gareth Harding (Contributing Editor), Submitted: June 13, 2003; Accepted: November 10, 2004 Dartmouth, Canada Proofs received from author(s): March 23, 2005