The collection of bottom sediment and hydrologic variable measurements (concentration of oxygen in the sur- face and bottom) were carried out in the periods of 6th to 17th of July 2000 (winter), and 1st to 15th of February 2001 (summer) in 26 stations (Figure 1). The sediment sampling was carried out with a Petersen Grab sampler. Samples were collected from the interface sediment-water for analysis of foraminiferal species. Sampling sta- tions and the location of the area affected by the spill are shown in Figure 1. Sampling began at the entrance of the bay, towards the middle part and finally at the northern part of the bay.

The dissolved oxygen concentration of water was measured with an oximeter Metler Toledo, and for pH mea- surements, the electrodes were coupled to an Eydan portable analyzer, model pH 91. Both measurements were performed in situ.

The collection of sediment for analysis of biological material was performed at 26 points in the winter and re- peated in the summer, totaling 52 samples. For the analysis of microfauna, the top layer (five centimeters) was removed from the sediment collected, and placed in vials with Bengal Rose (1 g/1000ml etilic alcohol). Bengal Rose stains the protoplasm of organisms alive at the time of collection, while the role of the alcohol is to prevent bacterial attack. The sediment containing the foraminifers (50 cc) was washed in two successive sieves of 0.500 mm and 0.062 mm and then dried. The samples were separated by flotation in carbon tetrachloride. After this, the foraminifera were identified at species taxonomic level. The determination of species was done using a bi- nocular stereoscope. Specimens were counted to a maximum of 500. After that number, the sample was divided using a splitter (Geological Green). Foraminifera were counted in one portion and multiplied by the number of fractions which the sample was divided into, obtaining the average absolute frequency of specimens sampled per station.

Ÿ AE-index

To assess the degree of hypoxia in the Bay, we used the ecological AE index to measure hypoxia (O₂ < 2 mg/l) related to coastal eutrophication [14] . This index takes into account the absolute frequency of Elphidium spp., Ammonia tepida and A. parkinsoniana (morphological variations of Ammonia beccarii). Ammonia spp. is ab- undant in polluted sediments rich in organic matter while specimens of Elphidium spp. are not often found in such environments.

The AE index follows the formula below:

where AB = absolute frequency (number of specimens/50cm³).

The AE index is affected by seasonal hypoxia produced by “blooms” of phytoplankton and it is important to infer measures related to the eutrophication of the environment.

From the absolute frequency, we calculated the relative frequency of species of foraminifera in the study area. The data on main foraminiferal relative frequency and AE index of environmental hypoxia was used to generate the contour maps to better visualize patterns. The contour maps (Figures 3-8) were calculated by Surfer 9 from the digitization of nautical chart n. 1501.

The interpretation was based on the kriging technique, using the program Surfer for Windows, which accord- ing to [15] is a statistical technique used to estimate the values of variables distributed in space, with the possi- ble identification of spots when the levels in the different parameters are very disparate. It is known that the kriging technique has been used in studies of geology to estimate the distribution of variables in patches of ma- rine sediments [16] . This methodology was also applied to the chemical variables (oxygen and pH) measured at the time of collection.

The positioning of the stations, the pH of the sediment and dissolved oxygen concentration in surface and bot- tom water, obtained at the time of sampling in winter and summer, can be seen in Table 1. Data from this table were contoured in maps to provide a better view of the chemical parameters measured simultaneously with the collection of bottom sediment.

Figure 3 shows that in winter, the pH values for the northwest region are lower (stations 17 to 26), when compared to other locations (stations 1 - 16). At station 26, near the Duque de Caxias (REDUC) oil refinery, there is the lowest pH value among samples, indicating the highest acidity of the sediment collected (pH 1.81). In the summer, there was an increase of pH in every area.

Regarding the oxygen concentration in both winter and summer, it was observed that higher concentration is found in surface water and the lowest values in bottom waters (Figure 4).

In winter, the entrance of the bay and the central regions are the more oxygenated environments. In general, the less oxygenated sites are located in the northeastern part of the bay, near the Guapimirim APA, and in the northwest, near the REDUC. During this period, the oxygen content near the surface nearby REDUC are very low, potentially indicating that the oil spill and chemical processes involved consumed the oxygen in the water.

Figure 3. The distribution of pH at the collection stations during summer and winter.

Figure 4. The distribution of oxygen concentration at the collection stations during summer and winter.

Still, with respect to the northwestern part of the Bay, the low surface oxygen content in the Iguaçu River is re- lated to existing discharges of pollutants. In summer, bottom oxygen in stations 12 to 24 in the central and nor- theastern Bay were very low (between 0.12 and 0.3) showing the presence of an anoxic zone in this region.

The number of specimens observed during the winter was higher than the number of specimens observed in

Figure 5. Distribution of the relative frequency of Ammonia tepida.

summer. In the winter, 295,113 specimens were found and in summer, 116,241 specimens were found. These specimens are distributed in 51 species of foraminifera belonging to the suborders Rotaliina (vast majority), Textulariina and Miliolina. The main species found in winter were Ammonia tepida, Bolivina striatula, Bulimmina elongata, Buliminella elegantissima, Cassidulina subglobosa, and Quinqueloculina seminulum and in the summer, the dominant species were also A. tepida, B. striatula and B. elegantissima. Both in winter (Table 2) and summer (Table 3), the highest total relative frequencies were found at stations 6, 7, 8, 9, located in the south of Ilha do Governador, 13 and 14 located in the central portion of the Bay and at station 22, which is located in the northeast portion.

The most frequent species found in these regions belong to the suborder Rotaliina, Ammonia tepida, Bolivina striatula, Buliminella elegantissima, Fursenkoina pontoni, and Bulimmina elongata. Specimens of suborder Textulariina (Gaudryina exillis) were found in a lower percentage and concentrated only in the middle part of the bay. In winter, the species Quinqueloculina seminulum (Miliolina) was fairly representative in the northern regions of the Bay.

Contour maps of relative frequency of the main species of foraminifera show that calcareous hyaline species Ammonia tepida was dominant at almost all stations, indicating its cosmopolitan characteristic (Figure 5). However, note that the highest relative abundance of this species occurs in the north and northeast of the Bay, near the APA Guapimirim. In summer, one year after the accident, there is the absence of this species near the site of the oil spill, especially in station 26. The shells of calcium carbonate (CaCO₃) were dissolved due to the increased acidity of the sediment. This acidity which began during the oil spill continues gradually dissolving shells, demonstrating ecological effects after the accident.

The species belonging to the genus Elphidium are mixohaline, characteristic of environments with lower sa- linity. In Figure 6, we observe the occurrence of this species in stations 22 to 26, where there is greater influ- ence of river drainage. This genus had a high relative abundance in the REDUC region only in winter, while in summer; the region was a barren zone.

In winter, station 26, near REDUC presents very acidic pH of the sediment. This acidity negatively influences the diversity of species, limiting the occurrence of species with thinner shells and thereby increasing the relative dominance of Elphidium spp. This was the only sterile station in the summer. This fact can be explained by the continuous dissolution of these shells; being thicker, they are the last ones to dissolve, culminating in the total disappearance of these organisms one year after the accident.

Through the AE index, we can verify that in the summer, compared with data obtained in winter, there was an increase of hypoxia due to decreased oxygen concentration in all environments (Table 4). In winter, highest values of this index were observed closer to the margin of the Bay indicating high productivity related to hy- poxia and eutrophication. Hypoxia has been found especially in the central regions.

In summer, the southern and central portions of the Ilha do Governador, near the São J. do Meriti, have higher values indicating high environmental hypoxia. These data corroborate oxygen concentration of an anoxic zone in this region.

Figure 6. Distribution of the relative frequency of Elphidium spp.

Figure 7. AE index for the samples collected in the winter and summer.

Figure 8. Distribution of the relative frequency of Buliminella elegantissima.

Buliminella elegantissima is also typical of environments with low oxygen and high concentration of organic matter and was present in abundance stations in winter, at stations 6 to 10 (south of Ilha do Governador) and in summer, at stations 13 and 14 (the central part of the Bay) in the anoxic zone (Figure 8). B. elegantissima do- minates anoxic regions, near São J. do Meriti River, south of Ilha do Governador and the central region. How- ever, in the anoxic station 26, near REDUC, this species is absent due to the fragility of its thin test that does not tolerate low pH.

The low pH, derived from the oil spill, caused a relative increase of specimens of Elphidium spp. in winter (6 months after the accident in January 2000). It is believed that this relative increase is mainly due to the absence of other species with thinner shells that were quickly dissolved. Next to REDUC, the acidity of the sediment suggests that the dissolution of carbonates in the region, culminated in the complete sterility of the sample one year after the accident (station 26, single station sterile). For example, B. elegantissima was found in the area of environmental preservation (APA Guapimirim) in winter, but not close to REDUC, indicating that this species does not tolerate acidic pH environments, given the fragility of its thin shell. The damage assessment 6 months and 1 year after the accident was interesting since it was still evident through mortality and other symptoms in the population for more than one year.

It is noteworthy that the oil spill reached an already stressed and altered ecosystem since the Guanabara Bay is highly urbanized. The dumping of sewage from the city of Rio de Janeiro takes place mainly in the waters of the Rio S. J. Meriti located west of the Ilha do Governador. Our results show that the south of Ilha do Governador is seriously compromised because low efficiency of water renewal is unable to export the pollutants from the São J. do Meriti River.

According to [9] , the west margin and the inner parts of the bay show high concentrations of nutrients (nitro- gen, phosphate and sulfur) due to the enrichment from domestic discharge and low efficiency in the renewal of the waters near Ilha do Governador and Ilha do Fundão. Our study indicated these locations as seriously im- pacted because the São J. do Meriti River influences the environments also at the north of the bay. This happens because in the west of the Ilha do Governador, which would be the “natural” way of water flow, is stagnated and closed due to silting and littering in the region. Consequently, the passage of water tends to occur to the north, preventing renewal of waters.

Most of the bays are characterized by high concentration of oxygen in the sediment, which enables the main- tenance of high biological activity [18] . However, large amounts of waste of organic material (sewage) can create areas where oxygen consumption exceeds supply, especially during the summer months, making the anoxic environment, precisely what was observed in this study to the south of the Ilha do Governador. The cen- tral region of the Bay has the lowest background concentration of oxygen. This fact is also related to the in- crease in temperature this time of year and the “bloom” of phytoplankton, which results in a greater area of eu- trophication and consequent hypoxia. The study of seasonality in the Bay shows seasonal periods of hypoxia similar to those observed in coastal areas: Chesapeake Bay [19] , Gulf of Mexico [20] , and the Continental Shelf of Louisiana [14] .

In an assessment of cellular stress in mollusks, [21] observed that animals from the south of Ilha do Gover- nador were more clearly affected by local pollution. Our study clearly reflects the loss of biological communities in relation to the rest of the Bay and the south of Ilha do Governador requires further attention since they are re- gions where there is a high organic dump across the S. J. do Meriti River. Our results also agree with [22] , who observed that the concentration of ammonia, chlorophyll and turbidity observed in regions close to the Ilha do Governador and Ilha do Fundão are high relative to the rest of the Bay, and far above the standard set by CONAMA (National Environmental Council), evidencing the process of degradation in the Bay.

The influence of contamination (sewage) on the population of foraminifera has been widely studied and a thorough review is cited in [23] . In Guanabara Bay, benthic foraminifera have been looked at by [24] -[26] as bioindicators in the determination of human impacts in coastal and paralic environments as well as in the Ro- drigo de Freitas Lagoon, RJ [27] . In the inner continental shelf of southern Brazil, in Laguna [28] foraminifera benthic fauna has undergone degradation in the central sector, because the accumulation of sand and the absence of fine material here are conducive to the development of a barren zone. Populations of coliform bacteria, how- ever, exist all over; and their development does not seem to be related to the amount of silt and clay in the sedi- ment. The accumulation of silt and clay in Laguna [28] and in the present study is linked to both a “natural” source, influenced by the river run off, and an anthropogenic source, i.e., the sewage outfall.

A study done at the Olaria River (Cananéia, SP), by [29] found that organic pollution mainly associated with regular renewal of the water on each tide, favored the establishment of opportunistic species; the same could be observed in Guanabara Bay.

The analysis of the levels of hypoxia, through the AE index, suggests that Guanabara Bay is a highly anthro- pogenic altered environment on the margins, reaching their maximum values in these regions. It is known that this index considers the genera Ammonia and Elphidium as indicators of pollution levels, Ammonia as an indi- cator of polluted environments, and Elphidium characterized as less tolerant to pollution [14] . This hypothesis was corroborated by [30] in Guanabara Bay and by Pereira [31] in Cananéia and Santos estuarine systems, where both authors observed that Ammonia tepida was resistant to low concentration of dissolved oxygen (anoxic environment) and high sulfur values characteristic of toxic sediments. Larger population of A. tepida re- lates to the reduction of dissolved oxygen concentration in the bottom sediments (related to 3 mg/l, [32] ), while smaller populations are found where the pH is acidic [33] .

Our study revealed that B. elegantissima is more abundant in the anoxic regions south of Ilha do Governador and central bay. This finding reinforces that B. elegantissima is typical of organic matter enriched sediments, with low oxygen, occurring in sediments contaminated by domestic sewage [14] [29] [34] , and that excessive nutrient enrichment favors the proliferation of opportunistic foraminiferal species with high growth rates, in- creasing the local dominance [11] .

Environments with high organic matter concentration are directly related to higher concentrations of fine se- diment [35] . This type of sediment is characteristic of low energy environment which facilitates the organic matter deposition. Moreover, in these regions, the oxygen concentration is very low. Note that B. elegantissima presented its highest relative frequency exactly where hypoxia is enhanced in the region south of Ilha do Go- vernador in winter and in the central region near the anoxic zone in the summer. Anoxic zones are usually lo- cated where the increase of bacteria from extreme concentrations of pollutants decreases oxygen concentration [36] .

The hypoxic region of REDUC suggests that this environment, although its oxygen concentration is similar to the APA, has greater limitation in productivity, compared to the preserved region. The dominance index values are high both in the region closer to REDUC and in the central part, anoxic zones, reflecting the establishment of a few species adapted to those polluted and contaminated conditions.

Our study indicates that Guanabara Bay is highly impacted, reflecting a low diversity of fauna, dominated by a few species. The number of species per sample increases when environmental conditions are more typically saline. Therefore, one would expect that if compared to other less saline lagoons and estuaries in Brazil, for ex- ample, the Estuarine Lagoon System Cananéia-Iguape, SP [29] [37] , Bertioga Channel, SP [11] [38] [39] , the estuaries of Estação Ecológica Juréia-Itatins, SP [40] , Lagoa da Conceição, SC [41] , Guanabara Bay would have more foraminiferal species.