Rapid Assessment of Octocoral Diversity and Habitat on Saba Bank
2010

P J Etnoyer
H H Wirshing
J A Sánchez
Rapid Assessment of Octocoral Diversity and Habitat on Saba Bank

PLoS ONE
Netherlands Antilles55106682010Received April 15, 2009; Accepted August 31, 2009;Editor: Martin Solan, University of Aberdeen, United Kingdom This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: Netherlands Antilles Department of Environment (MINA), Harte Research Institute, Conservation International, and Saba Conservation Foundation supported this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: There is a competing interest between the lead author and PLoS editor Dr. Craig McClain, who served together as co-editors in a mutual enterprise, Deep Sea News. Deep Sea News was co-edited by Drs. McClain and Etnoyer from
Saba Bank is a large submerged platform (,2200 km 2 ), average depth 30 m, located 4 km southwest of Saba Island in Netherlands Antilles, Caribbean Sea. Ships traveling to and from oil terminals on nearby St. Eustatius routinely anchor on the Bank, damaging benthic megafauna. Gorgonian octocorals are vulnerable to anchor damage, and they are common and conspicuous in shallow water (15-50 m) around the banks. This prompted a rapid assessment of octocoral habitat and diversity. The primary objectives were to estimate total species richness and to characterize habitats vis a vis gorgonians. Landsat imagery and multibeam bathymetry were employed to identify random sites for quantitative transects. A Seabotix LBV200L remotely operated vehicle (ROV) and SCUBA were used to collect and survey to 130 m. A total of 14 scuba dives and 3 ROV dives were completed in 10 days. During that time, 48 octocoral species were collected, including two likely undescribed species in the genera Pterogorgia and Lytreia. Gorgonian richness was exceptional, but not all species were collected, because the species accumulation curve remained steeply inclined after all surveys. Two shallow-water gorgonian habitat types were identified using multidimensional scaling and hierarchical cluster analyses: 1) a high diversity, high density fore-reef environment characterized by Eunicea spp., Gorgonia spp., and Pseudopterogorgia spp. and 2) a low diversity, low density plateau environment characterized by Pseudopterogorgia acerosa, Pterogorgia guadalupensis, and Gorgonia mariae. The analyses support hypotheses of broad (,15 km) habitat homogeneity (ANOSIM, P.0.05), but a significant difference between fore-reef and plateau environments (ANOSIM, P,0.05). However, there was some indication of habitat heterogeneity along the 15 km study section of the 50 km platform edge along the southeast rim. Our results highlight the complexity and biodiversity of the Saba Bank, and emphasize the need for more scientific exploration.

Introduction

Saba Bank is a large submarine platform 4 km west of Saba Island across a deep-sea channel in the Netherland Antilles, Caribbean Sea. The total surface area above the 200 m isobath is ,2200 km 2 , most of which is shallow water between 20-30 m depth, within the limits of recreational scuba diver depths. The Bank is elliptically shaped, with a 40 km short axis, and a 60 km long axis oriented ENE-WSW (Fig. 1). A linear ridge approximately 50 km in length occurs along the platform edge of the Bank. The ridge feature is raised 10-20 m, encrusted by many hard and soft tropical corals. The walls of Saba Bank are steepsided below the platform edge, dropping off the southern rim to water deeper than 500 m between Saba Bank and the nearby island of St. Eustatius.

Geologists have traditionally referred to Saba Bank as a ''remnant coastal plain'' [1], an ''atoll-lagoon floor, deprived of its original reef'' [2], and/or a ''remnant tidal marsh environment during Pleistocene or post-Pleistocene'' sea level fluctuations [3]. The geological origin of Saba Bank is unclear, but Dutch SCUBA divers and surveyors [4] consider it an actively growing submerged atoll. This would make Saba Bank ''the third largest atoll in the world,'' though the feature never breaks surface.

Saba Bank is arguably the largest continuous shallow water habitat in the eastern Caribbean, isolated from direct terrestrial influence by a 4 km wide deep-sea channel. The platform edge is a linear ridge system, 15-30 m deep on the ESE side, characterized as sparse corals and alcyonarians, with no evidence of an interlocking coral framework [3]. This disqualified the ridge feature as a ''submerged reef'', but spur and groove formations were observed during our surveys. Depth profiles of the platform edge fore-reefs indicate a series of ridges 8-10 m high, adjacent to trough-like features (Fig. 2). A classification scheme was developed to distinguish between habitat types on Overall Bank [5, Fig. 1], but this study recognizes only two classes of habitat; 'fore-reef' and 'plateau'.

Saba Bank may be an important Caribbean reef habitat because it has a large extent, and it is positioned in an upstream position relative to neighboring Puerto Rico (to the northwest) and the Meso-American Barrier Reef (to the far west), in relation to the direction of flow for the Caribbean current. The Caribbean current flows east to west along the southern parts of the Antilles Ridge, turning to the northwest at higher latitudes. Long-lived invertebrate larvae (e.g. spiny lobster Panulis argus, Queen conch Strombus gigas) could theoretically disperse from Saba Bank to downstream habitats. Saba Bank is targeted for conservation because of the presence of endangered species of Acropora corals, and threats to habitat quality by anchor damage. The Bank is also home to small-scale local fisheries for red snapper and lobster [6,7].

Two research expeditions studied corals on Saba Bank [4,7]. The biological collections focused primarily on scleractinian (hard coral) species, a group with moderate richness on Saba Bank-28 different species in 17 genera. The entire Caribbean boasts only ,50 spp. of shallow Scleractinia [8]. Alcyonarian corals (octocorals, soft corals, gorgonians, and sea fans) are common and conspicuous on Saba Bank, but the species list for soft corals was short (14 spp., 9 genera) [4]. Gorgonian habitat types are fairly commonplace, but several groups were classified only to the genus level and these include the most diverse genera (e.g. Pseudopterogorgia spp., Eunicea spp., Muricea spp., Plexaurella spp.). Some lobster fishermen have an appreciation for the octocorals, because they believe their prey is attracted to gorgonian colonies. Gorgonians provide structural complexity for associated species of fish and invertebrates.

One intention of the scientific expedition to Saba Bank in 2007 was to improve existing knowledge of octocorals through a rapid assessment of diversity and habitat using SCUBA diving and a remotely operated vehicle (ROV). Seventeen total dives were conducted -eight dives made quantitative transects, three dives used the ROV, and six dives were dedicated entirely to sampling. The primary objectives of the expedition were (1) to document gorgonian species richness, and (2) to characterize habitat vis a vis gorgonians. Completeness was evaluated using a species accumulation curve with comparisons to other Western Atlantic sites [9][10][11]. Two gorgonian habitats were proposed and demonstrated, a fore-reef assemblage, and a plateau assemblage.

Our research questions asked (1) how well did we sample gorgonian diversity, (2) are the gorgonian assemblages in fore-reef and plateau environments the same or different, and, (3) is the gorgonian assemblage on Saba Bank the same or different from other sites in the Western Atlantic and wider Caribbean? The intention of the study was to encourage better conservation and management of natural resources on the Saba Bank for future generations, with particular regard to the gorgonian coral community.

Results

Gorgonian species were common and conspicuous at all sites surveyed, except for one site, the deep-water (60 m) rubble flats south of Poison Bank, north of site A7 and A12 in Figure 1. The number of gorgonian species collected from shallow water (,40 m) was 43. The number of gorgonian species from deep water (.40 m) was 11. There was some overlap in the zones, with 6 shallow water species (Muriceopsis flavida, Muricea laxa, Eunicea clavigera, Pseudopterogorgia (Ps.) acerosa,Ps. albatrossae, and Ps. bipinnata) occurring below 40 m. A total of 48 species were collected, including two putative new species in the genera Pterogorgia and Lytreia (Table 1).

Gorgonian richness was high, 10-30% more species than other published surveys in Colombia, Puerto Rico, and Florida [9][10][11], the assemblage was not significantly different from other western Atlantic and Caribbean sites (SIMPROF, P.0.05). Published species lists were .70% similar. Southeast Florida had the highest similarity (Fig. 3). The rate of species accrual on Saba Bank remained inclined after all dives (Fig. 4). Discovery rate decreased as effort increased, but the accrual curve showed no clear asymptote, so more species would be expected to occur from further sampling. Not all gorgonian species were likely to be collected, though richness was substantial.

Gorgonian diversity, richness, and abundance were significantly higher on fore-reef sites, when compared to the plateau (Mann-Whitney U-Test, P,0.01). The fore-reef assemblage had high species richness (S = 10-17 min/max), high diversity (Simpson mean = 2.0), and high density of gorgonian colonies (3.5-4.7 colonies m 22 ). The plateau assemblage was lower richness (S = 3-5), lower diversity (Simpson mean = 1.1, and lower density (0.18-1.50 colonies m 22 ) compared to the fore-reef ( Table 2).

The Eunicea species complex, Gorgonia spp. sea fans, and Pseudopterogorgia sea plumes were typical in the fore-reef environment (Fig. 5a). Large, sparsely spaced Ps. acerosa and Pterogorgia (Pt.) guadalupensis sea plumes and small G. mariae sea fans comprised the plateau assemblage (Fig. 6).

Two octocoral species were collected that may be new to science. Lytreia n. sp. was collected twice from 50 m depth near the A zone. Pterogorgia n. sp. was collected twice from 20 m depth in the D zone. Specimens were observed with other Pterogorgia species in the same habitat, side-by-side with species exhibiting different colonial morphologies. One species previously reported [4] was Fore-reef assemblages were significantly different from plateau assemblages ( Fig. 7 -ANOSIM, P,0.05), but not significantly different from each other (ANOSIM, P.0.05) using raw, untransformed data. Root transformed data gave the same results. Transects within the same zones (e.g. A zone transects A7, A12) were more similar to each other than to sites in other zones (e.g. Conch Valley transects CV1, CV2). Transects in the plateau (E4, E7) were not significantly different from sites D9 and the ''Void'' (ANOSIM, P.0.05), though these sites were 15 km distant.

The MDS ordination supported two groups less than 20% similar. Stress values in the MDS were low (Fig. 7, stress = 0.07), corresponding to a good ordination with slight prospect of a misleading representation [12]. These results support the hypothesis of two different gorgonian assemblages on Saba Bank -a diverse and abundant assemblage on the fore-reef, and a low diversity, low-density assemblage on the plateau. Plateau transects (D9, E3, E4, Void) with low richness and abundance were at the horizontal opposite extreme of fore-reef transects (A7, A12, CV1, CV2) with high richness and abundance in the MDS plot (Fig. 7). The axis also represents a gradient in Eunicea spp. abundance, descending from left to right. Eunicea spp. were uncommon in the plateau. Fore-reef sites with highest richness (A7, 17 spp.) were vertically opposite fore-reef sites with lowest richness (CV2, 10 spp.) in the MDS plot, and plateau sites with high abundance (D9) were opposite sites with lowest abundance (Fig. 7). This vertical axis could also represent a gradient in abundance of shared species. Ps. acerosa and Pt. guadalupensis (at CV1 and D9) would pull in the downward direction with Plexaurella dichotoma (at E4 and A12) pulling in the upward direction. To summarize for brevity, gradients in richness and abundance contributed to the differences between sites, but rare and cosmopolitan species were suspected to be important interactors. Rare species were defined as species that occurred in only one site. Cosmopolitan species were defined as species that occurred in more than one site.  [9], Colombia [10], and Puerto Rico [11]. doi:10.1371/journal.pone.0010668.g003  [21] of expected species richness with 95% confidence intervals (in light blue). The species accumulation curve is steeply inclined, not approaching asymptote. More gorgonian species remain to be discovered on Saba Bank, though richness is already higher than other study sites in the West Atlantic. doi:10.1371/journal.pone.0010668.g004 Four cosmopolitan species occurring at more than 4 sites accounted for 50% of the difference between habitats: (Gorgonia mariae, Pseudopterogorgia acerosa, Eunicea fleuxosa, and Ps. americana). E. fleuxosa, Ps. acerosa, and Ps. americana explained the community patterns best. No single species dominated the comparison, because 13 species were necessary to account for 90% of the difference. The pattern that emerged from the inverse analysis was a central core of common species, with radiations outward from this center, in 4-5 directions. The periphery of the MDS plot was occupied by rare species that occurred once (e.g. Pterogorgia n. sp.

[D9], Muricea muricata [E4], Pseudopterogorgia elisabethae [A12]). The distinction between rare and cosmopolitan species was consistent with this interpretation. Stress was moderate (0.13), suggesting the two dimensional plot was a satisfactory representation of Bray-Curtis distance.

To evaluate the importance of taxonomic resolution in the habitat classification, abundance values for congeners were summed to produce an average density for each transect by genus. Eunicea (n = 11), Pseudopterogorgia (n = 7), and Pterogorgia (n = 4) were the most speciose genera. Generic assemblages on the fore-reef and plateau were significantly different, consistent with the species-level diagnosis (ANOSIM, P,0.05).

Transects in both habitat types were separated by 15 km, but the clusters indicate some degree of homogeneity within habitats, because sites 15 km away were more similar than adjacent sites in different habitats. Fore-reef transects clustered more closely together than plateau sites, suggesting fore-reef transects were more similar to each other than plateau transects. We found strong evidence for two habitat types, with good evidence for broad homogeneity within zones, but only subtle evidence of heterogeneity between remote stations.

We reject the null hypothesis of no difference between fore-reef and plateau environments with good confidence, based on multivariate (ANOSIM, P,0.05) and univariate techniques (Mann-Whitney U test, P,0.01) with robust distinctions across methods. Further exploration of Saba Bank warranted, based on the continuous climb of the species accrual curve (Fig. 4), the discovery of two potentially new species, and good evidence of habitat homogeneity (and heterogeneity) along a biologically rich 50 linear km fore-reef feature.

Discussion

The October 2007 survey results added 28 species and 7 genera to the list of gorgonian octocorals known to occur on Saba Bank. Surveys resulted in a four-fold increase in knowledge of gorgonian species richness. Richness was higher than other sites in the region, but the species composition was not significantly different from other Western Atlantic [8,10] and Caribbean [9] sites. The use of the Seabotix ROV did not account entirely for the difference in species recovery between Saba Bank and other localities, because we collected more shallow octocorals, as well as more species overall.

Our data indicate we cannot assume we collected all of even the most common species. Most species are rare, so an asymptotic function implies only that the most common species were collected. Our species accrual curve did not approach asymptote, so both rare and common species remain to be discovered. This is remarkable, considering the shallow gorgonian assemblage is already rich when compared to other places. Saba Bank has at least 43 zooxanthellate octocoral species, compared to Florida (S = 39), Providencia Island, Colombia (S = 32), and Puerto Rico (S = 38). It would be useful to compare species accrual curves at each of these localities.

There is good potential for a diverse assemblage of deep-water gorgonians on Saba Bank. Conditions appear to be favorable to octocorals, because richness is high and different habitat types occur. Conditions on Saba Bank support a broad variety of octocorals over large areas, so it is logical to assume these conditions would prevail in deep-water, too. This study collected 11 deep-water species in 3 dives. More deep-water exploration of Saba Bank's is necessary, particularly in light of the presumed habitat association for Caribbean spiny lobster, which frequent deep-water.

The likely new species of Lytreia was exciting because the organism was recovered using the ROV to 130 m depth. The  diagnostic characters of the organism were characteristic of Lytreia but different from the one species in the genus, L. plana. Lytreia is presently monophyletic [13]. The likely new species of Pterogorgia was unanticipated because the discovery occurred in shallower, more accessible water depths on the plateau in the ''D zone''. New species in two depth zones suggest further explorations of the deep southern slope and the interior region would be likely to yield new discoveries.

The morphological novelty of the new Pterogorgia sp. was conspicuous. The genus is presently comprised of three species, P. guadalupensis, P. citrina, and P. anceps; characterized by colonies with branches that are flattened or triangular in cross section. Polyps retract into calyces that form a common groove (P. anceps and P. guadalupensis) or into close-set individual calyces (P. citrina) [13]. Saba's Pterogorgia sp. had flattened blades characteristic of P. gudalupensis, but calyces typical of P. citrina. Microscopic observation of the diagnostic calcareous sclerites identified differences between Saba Bank Pterogorgia sp. and its likely sister species. A morphological and molecular description of the new species will be reported elsewhere. Interestingly, all three Pterogorgia spp. were collected sympatrically from the discovery site (D9).

Two large (30 cm) and healthy bright red Nicella sea fans were collected from 60 m depth with brittlestars attached. Nicella occurs 300-400 m deep throughout the Caribbean. The living Nicella colonies had bright white retractile polyps that lent a pattern to the living colonies, so it was interesting to see the color and pattern faithfully reproduced on the brittlestars. Visual camouflage was expected in the photic zone, but less expected in the mesophotic. The photic zone likely extended to depths of 100 m or more on Saba bank, because visibility was generally very good most days. Naturally, the observations were biased by the fact that dives were conducted only when the sea state would accommodate the long trip to Saba Bank.

The idea of two distinct Saba Bank gorgonian habitats was supported by multivariate analysis techniques (Fig. 7). The gorgonian assemblage on the fore-reef was particularly rich, up to 17 spp. occurred in one transect. The assemblages were characterized by Eunicea spp., Gorgonia spp., Plexaura spp., Pseudoplexaura spp., and Pseudopterogorgia spp. in relatively high densities (Fig. 5). The explanation for the richness of fore-reef assemblages was that the substrate is high relief habitat (,10 m) adjacent to a steep precipice bathed by impinging currents. There was good potential for localized upwelling, which could benefit octocorals. Alcyonarians covered .85% of substrate in some places, many hundreds of colonies were evident.

Plateau environments were fundamentally different. The gorgonian assemblage on the plateau was relatively depauperate, characterized by Pseudopterogorgia spp., Gorgonia spp., and Pterogorgia spp. in low densities (Fig. 6). The average sea plume colony on the plateau was probably larger than gorgonian colonies on the forereef. Sea plumes reached .2.5 m height above the surrounding pavement. The current flow was characterized by persistent surge. The assemblage was low density, but biomass and abundance was substantial, because colonies were large, but sparsely spaced. The habitat is potentially vast, and may occur throughout a large part of Saba Bank's interior.

Gorgonian habitats were a mosaic of endemism, richness, and abundance superimposed over a background of broad scale (15 km) homogeneity. Conch Valley was the southernmost forereef site we surveyed, not significantly different from sites in the A zone, 15 km to the northeast, but the fore-reef zones were separated in the ordination space, so differences were present. Conch Valley's fore-reef had high abundance of gorgonians, but richness was likely underestimated, only two transects were performed.

The normal analysis of the ordination helped to illustrate that species richness and colony density were important discriminators between sites, but the inverse analysis showed rare species were also important to consider. We found five rare species in 8 sites (18% of species were rare). These five species ''typified'' the sites where they occurred. In a sense, rare species marked a site's departure from some background level of abundance in a small group of 4-5 cosmopolitan species. The utility of this message is that 28 gorgonian species could be classified between two types, rare and cosmopolitan. Both types were important.

Beta diversity, i.e., number of different assemblages at a local scale, is known to occur along a continuum of environmental conditions [14]. Depth, wave exposure, hard substrate availability, and periodic disturbance are the structuring forces in the octocoral community [see [15][16][17]. The other pattern structuring gorgonian assemblages is the availability of plankton resources for heterotrophic octocorals, i.e., species that lack zooxanthellae. Azooxanthellate species are distributed at the edges of reefs where the resource is available, and not yet been depleted by suspension feeders [10,[18][19].

In general, gorgonian populations on Saba Bank appeared healthy, with only a few instances of fungal infections of Aspergillosis on Gorgonia ventalina. Species associations were rare, or inconspicuous, other than brittlestars on Nicella, and a few basket stars wrapped around Pseudopterogorgia axes. Hard corals on the fore-reef area near Poison Bank appeared to be degraded. Many hard coral colonies were bleached and dead, overgrown by calcareous algae. There is precedent for an inverse relationship between hard coral cover and soft coral cover [20].

Our studies were limited to the southeast corner of the Saba Bank, but the results offer some insights on the probable character of gorgonian habitats throughout the Bank. The high diversity/ high abundance habitat is the fore-reef (i.e. the ''A zone'') of fringing reef habitat along 50 km of the Saba Bank perimeter. A lower diversity plateau habitat underlain by hard carbonate pavement appears to dominate the interior parts of the Bank. The latter would be expected to occupy a larger part of the Bank.

Octocoral colonies were vulnerable to damage. Large toppled gorgonian colonies were observed in the sand channel approaching Conch Valley. The area is heavily fished by lobstermen using pots and traps. No similar damage was observed at other sites. This manuscript is part of a larger effort to assess biodiversity of the benthic habitat and to mitigate anchor damage on Saba Bank through the establishment of a Particularly Sensitive Sea Area. Oil tankers en route to and from St. Eustatius regularly drop anchor on the bank, damaging benthic megafauna.

Based on our findings, scenarios for a zoning scheme to mitigate damage to octocorals would range from: 1) the status quo, no control, to 2) the restriction of tanker anchors from the fore-reef perimeter, or 3) the restriction of tanker anchors to a designated sandy zone in the Bank interior, if one exists, or 4) a complete ban prohibiting anchor damage on the bank. The status quo is likely to impact octocorals adversely, because they are broadly distributed and potentially long-lived. Colonies are vulnerable to overturning. The second option would protect the most diverse assemblages of octocorals. The third option would impact low diversity habitat, but habitat loss in a small area could be mitigated by habitat protection in other parts of the bank. The fourth option, a complete anchor ban, would reduce impacts to all habitats, but octocoral colonies would still be vulnerable to bottom-contact fishing gear.

Recommendations for further study include: 1) expansion of the study area to include more fore-reef sites along the southeast rim of the Bank; 2) increased exploration of the heretofore unknown 500 m vertical escarpment in the southern parts, and deep raised topographies in the northern parts; 3) the incorporation of isolated features (e.g. mounds and ridges) into the research design to test the hypothesis of habitat homogeneity in the plateau; and 4) the continued pursuit of greater sample sizes in all zones (especially B and C zones) to confirm these preliminary findings. It would also be interesting to test lobstermen's claims of affinity between octocorals and spiny lobsters, because both these animals are likely to occur in deep, unexplored parts of Saba Bank.

Methods

One of the primary objectives was to collect and identify as many gorgonian species as possible using oxygen enriched air (nitrox) with scuba and a Seabotix LBV200L remotely operated vehicle (ROV) rated to 200 m depth on separate boats. The scuba boat was a 28 ft. vessel powered by twin 115 HP engines. The ROV boat a 35 ft. lobster fishing boat equipped with a winch for hauling traps from deep water. A total of 14 scuba dives and 3 ROV dives were completed over the course of the 10-day expedition, October 19-31, 2007. The first four scuba dives were dedicated to primarily specimen collection and photography. Two additional opportunistic dives on random sites collected specimens not found on other sites. Eight other dives were used to conduct quantitative transects in two different ''habitat zones'', the forereef and the plateau.

All gorgonian specimens were photographed in situ before 10-15 cm branch segments were cut from the distal tips of branches, and preserved in 95% ethanol. These were identified to species using microscope slide preparations of sclerite morphology, using established octocoral protocols [13]. Permission for sample collection was granted through the Saba Conservation Foundation. Following identification and analysis, preserved specimens were curated at the Department of Invertebrate Zoology in the Smithsonian Institution National Museum of Natural History (NMNH). Records were uploaded to the Smithsonian NMNH database with in-situ images.

The research was primarily concerned with the southeastern quadrant of Saba Bank. A block design was established for ''Overall Bank'' south of Poison Bank, but survey sites in Conch Valley and on the plateau were haphazard. Within Overall Bank, sites were random, zones were labeled A-E, with A representing the ''fore-reef'' environment and E representing the ''lagoon environment''. Zonation was simplified to two zones for this study -the fore-reef (A sites) and the plateau (D, E sites).

A total of 8 quantitative dive transects were conducted in five different zones. Four transects (A7, A12, CV1, CV2) were assigned to the fore-reef, and four other transects (D9, E3, E4, Void) were assigned to the plateau. The Void site was given a unique name, as it is ,15 km from the other plateau sites. Fore-reef transects were random at two sites in the A zone (A7 and A12), and haphazard on the southeast rim called Conch Valley 1 (CV1) and Conch Valley 2 (CV2). Fore-reef sites CV1 and CV2 are ,15 km from the A zone fore-reef sites (Fig. 1). Plateau sites appeared to be clustered on Overall Bank, but sites were random. One transect occurred in the D zone at D9, two in the E zone at E3 and E4, and one in the unmapped region of the Bank interior referred to as ''Void''. No gorgonian transects were conducted in zones B and C.

Quantitative transects were conducted by 2 divers working in quadrats along a 50 m transect tape. Octocoral colonies were enumerated by species in 1 m square quadrats placed every 5 meters on either side of the transect tape (11 quadrats per diver, 22 total quadrats per transect). The numbers of colonies in each quadrat were summed together, pooled from both divers, then divided by 22 m 2 to achieve species density values in units of colonies m 22 . The resulting data matrix listed species density values for 28 species (variables) along eight transects (observations). This is not be confused with the total number of gorgonian species collected on Saba Bank (48 spp.), because species were collected independently of the transects.

Univariate techniques used the Mann-Whitney U Test and Simpson's diversity index in the form (1 -l). Multivariate ordination techniques were used to plot similarity, and to test the null hypothesis of no difference between (a) habitats at the species level, (b) habitats at the genus level, and (c) localities at the species level. The hypothesis of no difference between localities compared the Saba Bank species list to other West Atlantic gorgonian lists from Florida [9], Colombia [10], and Puerto Rico [11]. No a priori difference was assumed between western Atlantic and Caribbean localities. Sample-based rarefaction (species accumulation) curves were plotted with 95% confidence intervals using a moment based indicator of expected species richness called Mao Tau in EstimateS 8.0 software [21]. The input matrix used abundance or incidence values, depending on the dive type.

Three matrices were employed. The first was prevalence data with species as variables, and sites as observations. The second was prevalence data with genera as variables. The third matrix was incidence data, with species as variables and localities as observations. A resemblance matrix was produced for each matrix using the Bray-Curtis distance measure on raw, untransformed data in PRIMER 6.1 software [12]. The resemblance matrix informed non-metric multidimensional scaling (MDS) plots and group averaged hierarchical dendrograms of sites, species, and localities.

Hypotheses of no difference between sites were tested using ANOSIM Global-R and the SIMPROF statistics in PRIMER 6.1. ANOSIM was used for a priori comparisons between sites and SIMPROF for a posteriori comparisons between localities. ANO-SIM Global R-values are compared to a null distribution of rank distances generated by the random rearrangement of factors (forereef, plateau) and site labels (A7, A12, CV1, CV2, D9, E4, E7, Void) in order to estimate the probability of the same ordination, or Bray-Curtis distance configuration, occurring as a result of random chance. Similarity Percentage (SIMPER) analysis, BEST analysis, and inverse analysis were used in PRIMER 6.1 to understand which species were driving differences between sites.

Figure 1 .
1A map of Saba Bank, in the Netherlands Antilles. Saba Bank is shown relative to its nearest neighbor, Saba. A pale band of color representing 11-20 m depths represents a ''fore-reef'' extending 50 km along the southeast perimeter of the Bank. Red stars indicate the location of 8 quantitative transects, four on the fore-reef (A sites and CV sites) and four in the plateau (E3, E4, D9, Void) of Saba Bank's interior region. The dashed lines indicate Overall Bank, where a zonation scheme with random sites was in effect. Void was random, Conch Valley was non-random. doi:10.1371/journal.pone.0010668.g001

Figure 2 .
2Depth profiles of the Saba Bank platform edge. Profiles of platform edge ''fore-reef'' dive sites on Overall Bank (A7, A12 inFigure 1) and in the Conch Valley (CV1, CV2 inFigure 1). Each fore-reef is bounded on the interior by a trough of varying depth and character. Troughs were generally sandy, with intermittent patch reefs. doi:10.1371/journal.pone.0010668.g002 not encountered, Gorgonia flabellum. G. flabellum is generally considered endemic to the Bahamas or very shallow fore-reef environments [JAS, pers. obs.].

Figure 3 .
3Ordination plot of Caribbean octocoral localities. A non-metric multidimensional scaling (MDS) plot based on Bray-Curtis dissimilarity measures derived from presence and absence of octocoral species in surveys of Florida

Figure 4 .
4Species accumulation curve for Saba Bank gorgonians. The graph shows a sample-based rarefaction curve (in black) based on 17 shallow and deep dives. The technique employs a Mao Tau estimator

Figure 5 .
5High-diversity gorgonian assemblage on the Saba Bank fore-reef. An image from one of the ''A'' sites on the fore-reef of Saba Bank (,25 m) shows an assemblage characterized by sea fans (Gorgonia ventalina), sea plumes (Pseudopterogorgia americana), and the Eunicea/Plexaura sp. complex, Eunicea fleuxosa, center. The fore-reef environment consists of a series of reef crests near the platform edge on Saba Bank[4] with diverse and abundant octocoral species. doi:10.1371/journal.pone.0010668.g005

Figure 6 .
6Low-diversity gorgonian assemblage on the Saba Bank plateau. An image from one of the ''E'' sites in the Saba Bank plateau region shows a large sea plume (,1.5 m-Pseudopterogorgia acerosa) and scattered sea fans Gorgonia mariae attached to a hard carbonate platform covered with a thin veneer of sediment. The plateau region is distinct from the fore-reef, consisting of reef flat and lagoon bottom environments[4]. doi:10.1371/journal.pone.0010668.g006

Figure 7 .
7Ordination plot of Saba Bank transects. A non-metric multidimensional scaling (MDS) plot based on Bray-Curtis dissimilarity measures derived from species abundance values in 8 quantitative transects. Fore-reef sites (A, CV) are significantly different (ANOSIM, P,0.05) from plateau sites (D, E, and Void), but not significantly different from each other. There is good separation between fore-reef and plateau sites, with slight prospect of a misleading representation (2D stress ,0.10). doi:10.1371/journal.pone.0010668.g007

Table 1 .
1List of octocoral species collected from Saba Bank. more than three other site surveys in the greater West Atlantic. Two octocoral species are undescribed. High apparent richness is not entirely due to use of the ROV, because shallow octocorals on Saba Bank were richer than shallow octocorals in other localities. doi:10.1371/journal.pone.0010668.t001Species
Shallow (,50 m)
Deep (.50 m)
Shallow (,50 m)
Species

Briareum asbestinum
X
X
Ctenocella (Ellisella) cf. elongata*

Erythropodium sp.
X
X
Ctenocella (Ellisella) sp.*

Eunicea asperula
X
X
X
Eunicea clavigera

Eunicea calyculata
X
X
Iciligorgia schrammi*

Eunicea flexuosa
X
X
Lytreia n. sp.*

Eunicea fusca
X
X
X
Muricea laxa

Eunicea knighti
X
X
X
Muriceopsis flavida

Eunicea laciniata
X
X
Nicella sp.*

Eunicea laxispica
X
X
X
Pseudopterogorgia acerosa

Eunicea mammosa
X
X
X
Pseudopterogorgia albatrossae

Eunicea pinta
X
X
X
Pseudopterogorgia bipinnata

Eunicea sp. (tayrona)
X

Eunicea succinea
X
* = azooxanthellate

Eunicea tourneforti
X

Gorgonia mariae
X

Gorgonia ventalina
X

Muricea elongata
X

Muricea muricata
X

Muriceides sp.
X

Plexaura cf. nina
X

Plexaura kukenthali
X

Plexaura kuna
X

Plexaurella dichotoma
X

Plexaurella grisea
X

Plexaurella nutans
X

Pseudoplexaura crucis
X

Pseudoplexaura flagellosa
X

Pseudoplexaura porosa
X

Pseudoplexaura wagenaari
X

Pseudopterogorgia americana
X

Pseudopterogorgia elisabethae
X

Pseudopterogorgia hummelincki
X

Pseudopterogorgia rigida
X

Pterogorgia cf. anceps
X

Pterogorgia citrina
X

Pterogorgia guadalupensis
X

Pterogorgia n. sp.
X

The list shows species collected on Saba Bank using SCUBA for shallow (,40 m) octocorals and a remotely operated vehicle (ROV) for deep (.40 m) octocorals. The
chart lists 48 species,

Table 2 .
2Summary table of gorgonian density and diversity. Transect Richness Density Diversity Longitude Latitude Transects in Saba Bank's fore-reef environment (A12 -CV2) had significantly more gorgonians, and higher diversity (Mann-Whitney U Test, P,0.01) than transects in the plateau environment (D9 -Void). Richness is the number of octocoral species present. Density values are in colonies m 22 . The diversity measure is Simpson's index (in the natural log form). doi:10.1371/journal.pone.0010668.t002Location Fore-reef A12
17
4.188
0.846
263.21925 17.46257

A7
13
3.546
0.853
263.22083 17.45819

CV1
13
4.773
0.826
263.25203 17.35279

CV2
10
4.637
0.753
263.24800 17.34500

Plateau
D9
5
1.500
0.628
263.24387 17.44595

E4
5
0.455
0.680
263.26070 17.46343

E3
4
0.182
0.750
263.27110 17.47018

Void
2
0.182
0.375
263.38047 17.41512

PLoS ONE | www.plosone.org
May 2010 | Volume 5 | Issue 5 | e10668
AcknowledgmentsWe gratefully acknowledge the support of the Netherlands Antilles Department of Environment (MINA), Harte Research Institute, Conservation International (CI), and Saba Conservation Foundation for travel and logistical support. We thank the Royal Dutch Navy and the crew of HMS Snellius for providing hydrographic survey data. Dr. S Cairns and T Coffer graciously curated the gorgonian specimens at Smithsonian NMNH. Dr. P Tarazaga and one anonymous reviewer advised and improved the multivariate analysis techniques. Special thanks to L Allen and P Binfield of PLoS, who were gracious to host the Saba Bank collection. P Hoetjes, S Lundvall, W Toller, and J den Dulk provided invaluable support, spirit, and encouragement on Saba Island. M Smith and S McKenna of CI, and P Hoetjes of MINA are recognized for making the research expeditions possible.Author Contributions
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In review. Reef fishes of Saba Bank, Netherlands Antilles-Assemblage structure across a gradient of habitat types. W Toller, A O Debrot, Mja Vermeij, P C Hoetjes, Toller W, Debrot AO, Vermeij MJA, Hoetjes PC. In review. Reef fishes of Saba Bank, Netherlands Antilles-Assemblage structure across a gradient of habitat types.

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Report for the Department of Public Health and Environment. E H Meesters, H Nijkamp, L Bijvoet, 96-03. AIDEnvironmentCuracao, Netherlands; Amsterdam, the NetherlandsKNAP ProjectTowards sustainable management of the BankMeesters EH, Nijkamp H, Bijvoet L (1996) Towards sustainable management of the Bank. Report for the Department of Public Health and Environment, Curacao, Netherlands. KNAP Project 96-03. AIDEnvironment. Amsterdam, the Netherlands.

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Patterns of octocoral and black corals distribution in the Oceanic Barrier Reef-complex of Providencia Island, Southwestern Caribbean. J A Sanchez, S Zea, J M Diaz, Caribbean Journal of Marine Science. 343-4Sanchez JA, Zea S, Diaz JM (1998) Patterns of octocoral and black corals distribution in the Oceanic Barrier Reef-complex of Providencia Island, Southwestern Caribbean. Caribbean Journal of Marine Science, vol. 34, no.3-4. pp 250-264.

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Octocoral distributions at Carrie Bow Cay. H Lasker, Coffroth, Marine Ecology Progress Series. 13Lasker H, M Coffroth (1983) Octocoral distributions at Carrie Bow Cay, Belize. Marine Ecology Progress Series 13: 21-28.

Variable effects of hurricane David on the shallow water gorgonians of Puerto Rico. P M Yoshioka, Yoshioka, Bulletin of Marine Science. 40Yoshioka PM, BB Yoshioka (1987) Variable effects of hurricane David on the shallow water gorgonians of Puerto Rico. Bulletin of Marine Science 40: 132-144.

A comparison of the survivorship and growth of shallow-water gorgonian species of Puerto Rico. P M Yoshioka, Yoshioka, Marine Ecology Progress Series. 69Yoshioka PM, BB Yoshioka (1991) A comparison of the survivorship and growth of shallow-water gorgonian species of Puerto Rico. Marine Ecology Progress Series 69: 253-260.

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Assessment of human health risk from organochlorine pesticide residues in Cidade dos Meninos

C I R F Asmus
Carmen Ildes firstname.lastname@example.com
R Fróes Asmus
Núcleo de Estudos em Saúde Coletiva
Universidade Federal do Rio de Janeiro
Rio de JaneiroBrasil

Herling Gregorio
Aguilar Alonzo
Secretaria de Vigilância em Saúde
da Saúde
BrasíliaMinistérioBrasil

Marisa Palácios
Núcleo de Estudos em Saúde Coletiva
Universidade Federal do Rio de Janeiro
Rio de JaneiroBrasil

Alexandre Pessoa Da Silva
AMBIOS Engenharia e Processos Ltda
São PauloBrasil

Maria Isabel De Freitas Filhote
Núcleo de Estudos em Saúde Coletiva
Universidade Federal do Rio de Janeiro
Rio de JaneiroBrasil

Daniela Buosi
Secretaria de Vigilância em Saúde
da Saúde
BrasíliaMinistérioBrasil

Volney De Magalhães Câmara
Núcleo de Estudos em Saúde Coletiva
Universidade Federal do Rio de Janeiro
Rio de JaneiroBrasil

Duque de Caxias
Avaliação de risco à saúde humana por resíduos de pesticidas organoclorados em Cidade dos Meninos
Rio de Janeiro, Duque de Caxias, Rio de JaneiroBrazil, Brasil

Núcleo de Estudos em Saúde Coletiva
Universidade Federal do Rio de Janeiro. Av. Brigadeiro Trompowski s/n
Pça da Prefeitura da Cidade Universitária
21949-900Rio de JaneiroRJBrasil

Assessment of human health risk from organochlorine pesticide residues in Cidade dos Meninos
319808E1CD020DAF85C9B046623DE1A4Organochlorine InsecticidesEnvironmental PollutionEnvimental RisksRisk Assessment
This article presents the results of a heath risk assessment in Cidade dos Meninos, an area contaminated with organochlorine pesticide residues located in the county (municipality) of Duque de Caxias, Rio de Janeiro State, Brazil.The Agency for Toxic Substances and Disease Registry (ATSDR) risk assessment methodology was applied.The following organochlorine compounds exceeded the established limits and were considered target pollutants: HCH and its isomers (in eggs, milk, and soil); DDT and its metabolites; trichlorobenzenes; trichlorophenols; dioxins and furans (eggs and soil).For all the substances studied (with the exception of trichlorophenols), the estimated doses exceeded the minimum risk levels for chronically exposed children and adults.According to the ATSDR classification, the area was considered an "urgent public health hazard" because of its high and serious exposure to substances that are dangerous to human health.

Introduction

One of the most negative consequences of industrial development in the world has been the disposal of residues that are potentially hazardous to human health.The United States produces some 40 million tons of hazardous waste per year 1 .In Brazil, an investigation conducted by the Ministry of Health and covering the years 2001 to 2004 identified 15,237 areas with contaminated soil, of which 11,458 are areas for the final disposal of urban waste, in addition to 3,769 others, including industrial waste and pesticide deposits 2 .Contamination of these areas results in exposure to toxic substances for the resident populations, leading to harmful effects for health.Health institutions and professionals frequently need to characterize the health risk for these populations, and various methodologies have been proposed for this purpose.

The health risk assessment methodology for exposure to hazardous residues developed by the U.S. Agency for Toxic Substances and Disease Registry (ATSDR) 3 defines as objects for assessment: chemical compounds, elements, or combinations which due to their amount, concentration, and physical or toxicological characteristics can pose an immediate or potential hazard to human health or the environment, when inadequately used, treated, stored, transported, or disposed.The principal examples for use in Brazil include residues from industrial processes and urban waste deposits.

Cidade dos Meninos is located in the county (municipality) of Duque de Caxias in the Baixada Fluminense area, Rio de Janeiro State, Brazil, where a factory was set up in 1950 for the production of hexachlorocyclohexane (HCH) and formulation and storage of other pesticides, like trichloro-bis-(chlorophenyl)-ethane (DDT).The factory closed its operations in 1961, and the remaining production was stored in the open air in the immediate vicinity of the production unit 4 .This deposit was estimated at 350 tons of technical HCH, plus other products used in its processing, in direct contact with the soil, in a barren area measuring approximately 13,000m 2 .An attempt at remediation was made in 1995, using calcium oxide (quicklime), mixed with the residues and incorporated into the soil by mechanical processes.The attempt was unsuccessful, leading to a mass of some 29.7 thousand tons of contaminated material and expansion of the principal focus of contamination to an area of 38,000m 2 .

In 2001, the Ministry of Health, through the Pan-American Health Organization, hired a team of specialists and researchers from the company AMBIOS Engenharia e Processos Ltda., in partnership with the Núcleo de Estudos em Saúde Coletiva, Universidade Federal do Rio de Janeiro [Nucleus for Public Health Studies at the Federal University in Rio de Janeiro], to conduct a human health risk assessment based on the ATSDR methodology.The final report from this study was submitted to the Ministry of Health in April 2002 5 .The current article aims to present the results, based on the application of this methodology in the area known as Cidade dos Meninos.

Methodology

Application of the ATSDR risk assessment methodology included all of its proposed stages: survey of the risk area's history, identification of the population's health concerns, characterization of the environmental contamination process, and establishment of health implications.The complete process allowed: classifying the site's public health hazard level; elaborating conclusions and recommendations for health and for future environmental studies; and identifying the necessary measures to mitigate or prevent adverse health effects 6 .

Assessment of the site information included the customary description of the site and its historical aspects, a survey of the demographic data and living, work, and health conditions of the lo-cal population.The above assessment was based on an analysis of all the studies performed as of 2001, made available by the Ministry of Health 5 .The population's health concerns were identified by daily site visits, meetings with representatives of the neighborhood association, and interviews with other community leaders.

Due to the limited amount of data on environmental dispersal of the contaminants outside the focus areas and the lack of data covering all the target contaminants, samples were taken of the topsoil and foods (eggs) for analysis of the contaminant levels.The soil sampling was systematic, using a grid starting from two imaginary straight lines, located respectively 50 and 100m from the shoulders of the Camboaba Road.The sampling points were set at 500m from these straight lines, using as the starting point the principal sentry house for Cidade dos Meninos.In addition to the sampling points on the proposed grid, sampling points were established in the immediate vicinity of the houses along the branch of the Camboaba road leading to the Olaria and Lixão areas, situated inside Cidade dos Meninos.

Concentration data for each contaminant detected in the soil samples in the principal emission foci in Cidade dos Meninos were compared to the reference values for Dutch legislation for residential soils (MRT -maximum risk tolerance -and intervention value) 7 .The tests were performed by a private laboratory from Rio de Janeiro (method's detection limit: 0.01µg/kg for all the compounds analyzed; o,p'DDE, o,p'DDD, and o,p'DDT were quantified by the semi-quantitative method).

In relation to foods, eggs were chosen because they are large bioconcentrators of organochlorine pesticides, and together with cow's milk constitute a basic source of animal protein for local residents.One egg each was collected from all the houses where chickens were raised, totaling 22 eggs.Figure 1 shows a spatial distribution map of the samples collected in Cidade dos Meninos.The pesticide levels in milk samples from cows raised in Cidade dos Meninos and used to calculate the exposure dose in foods were determined by Mello 8 in her Master's thesis.Sample collection was done with the dairy farmers located at the time of the studies.Each sample was a composite of the daily production for each farmer.The samples thus came from the dairy herds that grazed freely throughout the entire Cidade dos Meninos area.

The pesticide analyses of the food samples were conducted by a private laboratory from Rio de Janeiro (method's detection limit: 0.01µg/kg for all the compounds analyzed; o,p'DDE, o,p'DDD, and o,p'DDT were quantified by the semi-quanti- tative method).The pesticide residue levels in the foods were compared to the maximum residue levels set by the European Food Safety Authority (EFSA) 7 , considered the most complete and rigorous international standard.

Selection of the target contaminants was based on comparison with the maximum concentrations detected for each contaminant in each environmental compartment analyzed, that is, the worst scenario for each, considering the toxicity of the respective compounds and the precautionary criterion.

The exposure doses were estimated based on the concentrations of contaminants found in the environmental compartments.All the environmental media would have to be considered for Cad.Saúde Pública, Rio de Janeiro, 24(4):755-766, abr, 2008 this purpose (water, soil, air, and foods), as well as all the exposure routes (inhalatory, oral, dermal, and mucosal).However, estimation of the exposure doses in Cidade dos Meninos did not consider exposure through water and air.At the time, there were no conclusive studies on either the groundwater contamination plume or reliable information on consumption of well water in the area or contamination of the 31 existing wells in Cidade dos Meninos.Meanwhile, respiratory exposure due to inhalation of particulate matter (suspended dust from the road, with the movement of pedestrians and various forms of transportation) was frequent due to the circulation especially of women and children on the road (surface soil), where secondary foci were identified.The Department of Science and Technology under the Ministry of Health 9 conducted an analysis of samples of household dust and identified human exposure to the organochlorine compounds found in this material, even in households farther from the principal focus.However, the qualitative characteristics of the methodology employed 10 , although efficient in the identification of the contaminated households, did not allow estimating the exposure of residents.Therefore, estimation of population exposure used only the results of the residue analyses for soil and food samples.

The entire population was considered potentially exposed, because everyone has contact with the soil and eats the food produced in the area.Two age brackets were determined to estimate the exposure dose: 11 years or younger, considering a mean body weight of 30kg, and 12 years and older, considering a mean body weight of 70kg.This also required calculating the food consumption and frequency and duration of exposure.Intake was estimated assuming one egg a day and 100g of milk for adults and 200g for children (one glass).Exposure was considered to be continuous, throughout life.

The exposure doses were estimated for the contaminants present in the soil (ingestion) and foods (ingestion) using the following formulas 6 .

Estimated dose from soil ingestion (EDsi)

where, C = concentration of contaminant in topsoil (mg/kg); IR = soil ingestion rate; EF = exposure factor; BW = body weight.Standard values (ATSDR): adults -50mg/day; children -50 to 200mg/day (normal children, without appetite disorders common in children up to three years of age and from low-income families).

Estimated ingestion dose from contaminated foods (EDf)

where, C = concentration of contaminant in food group i (mg/g); Ri = intake rate for food group i (g/day); EF = exposure factor; BW = body weight.The estimated doses were then compared to parameters indicating the level of health risk.For the risk of non-carcinogenic effects, the ATSDR minimum risk level 11 and Environmental Protection Agency reference dose 12 were used as indicators

The minimum risk level is based on the highest dose at which no adverse health effect is observed, obtained through experimental studies in animals (NOAEL, no observed adverse effect level) for chronic exposure.Taking alpha-HCH as an example for the occurrence of hepatic nodules in male rats fed orally, a NOAEL of 0.8mg/ kg-day and an uncertainty factor of 100 (10 for interspecies extrapolation and 10 for intraspecies variations) yield a minimum risk level of 0.008mg/kg-day.

Calculation of the risk of carcinogenic effects used the slope factor 13 to estimate the excess cancer risk for each substance, based on estimated exposure.This calculation was obtained by adding all the risks for individual contaminants, based on calculation of lifetime exposure for each contaminant.Taking alpha-HCH as an example, assuming an estimated dose of 0.541µg/kg/day up to 11 years and 0.12µg/kg/day from 12 to 70 years (taking 70 years as the mean lifespan) yields an estimated lifetime exposure of 0.188µg/kg/ day (total daily childhood dose x 12 years plus total daily adult dose x 58 years, divided by 70 to obtain the weighted mean).The resulting dose is converted to mg/kg and multiplied by the corresponding slope factor 14 .

Results

Socio-demographic characterization

Cidade dos Meninos is characterized by poverty and lack of urban infrastructure, with large landholdings for growing sugar cane, manioc, and vegetables and livestock operations like swine and beef and dairy cattle.There is electricity, but no sewage or garbage disposal, and the local residents have the habit of burning their household waste.An analysis of the Family Health Program registry 15 showed a total of 1,199 individuals (594 females and 605 males).Considering the existence of additional risks due to the presence of pesticide residues, the most relevant age groups were the following: < 1 year, 19 (1.6%); 1-4 years, 115 (9.6%); 5-14 years, 212 (18.5%); women 15-34 years of age, 190 (33.4%); and > 65 years, 83 (7%).

Environmental data

The vast majority of residents in Cidade dos Meninos live alongside the Camboaba Road and its local branches.Camboaba Road has long stretches with secondary foci of pesticide residue contamination, especially the stretch from the main entrance on Avenida Presidente Kennedy to the proximities of the principal focus.This stretch crosses Vila Malária, an area with numerous households.Table 1 shows the concentrations of target contaminants in the principal contamination focus and the most important secondary foci.

During the visits and interviews with local residents, the team observed livestock grazing in the surrounding pastures and pigs rooting and chickens pecking in the backyards.Table 2 shows the target contaminants found in eggs from Cidade dos Meninos.

The following organochlorine compounds exceeded the established standards and were considered target contaminants to be observed in the monitoring and remediation processes and in the health follow-up studies on the affected populations: HCH and its isomers (eggs, milk, soil); DDT and its metabolites (eggs, milk, soil); trichlorobenzenes (soil); trichlorophenols (soil); dioxins and furans (eggs, soil).

Toxicological assessment

Tables 3 and 4 show the estimated exposure doses of contaminants via ingestion of soil and eggs.Similar calculations were done for milk.

In Cidade dos Meninos, exposures to DDT and dioxin exceed the acute, intermediate, and chronic minimum risk levels.For HCH isomers, the acute and chronic minimum risk levels are exceeded by the estimated exposure dose in children.

Table 5 shows the oral exposure doses for the target population groups that exceed the minimum risk level and the calculated excess risk for non-carcinogenic effects.

Table 6 shows the calculated excess cancer risk for children and adults in Cidade dos Meninos, using the estimated exposure doses (µg/kg-day) and the dose-response slope factor for cancer, the result of which was 0.024 for the set of target contaminants.

Health data

There are no records on overall and specific mortality, infant mortality (neonatal and late), maternal mortality, cancer incidence, birth defects, or the principal causes of hospitalization for the population in Cidade dos Meninos 16 .

In the health survey done as part of the risk assessment, 21% (59/281) of interviewees reported some health problem, but no specific pattern was found.Importantly, the miscarriage rate in females over 12 years of age was 31% (28/90).No alterations were found in mean birth weight (3.1kg) or mean gestational age (8.867 months) in the last pregnancy.

Discussion

This study identified the main active ingredients, the magnitude of environmental contamination, and the exposure routes for the population in Cidade dos Meninos.Based on their presence in the topsoil, foods, wells, and air, these compounds can be absorbed by ingestion, skin contact, or inhalation.In addition to the principal focus, the study found secondary foci and inadequate use of residues (as household insecticide), leading to exposure to the target contaminants among the population in the study area.

The reference values come from animal studies extrapolated to humans, and in this case to the specific population in Cidade dos Meninos, which generates uncertainties.These uncertainties are of a general nature, present in any extrapolation, but in this case the specific uncertainties should also be analyzed.Estimated exposure based on environmental concentration data raised doubts because of the limited number of samples from the contaminated site, and the highest concentration found in the samples was used.

Other uncertainties were related to the consumption of contaminated foods in Cidade dos Meninos.There were doubts on this information, because during the interviews the residents appeared afraid to lose their place of living.Estimated consumption was based on information about farm animals (HCH concentrates in adipose tissue, and 33% of the families raised chickens).

The ideal situation would be to calculate individual food consumption, but since this was impossible, the study worked with possible exposure scenarios.Exposure estimation considered only milk and eggs.This is certainly not the worst scenario, but animal fat (e.g., lard) should also be considered; although it was not quantified, the concentration is probably also high.Importantly, Cad.Saúde Pública, Rio de Janeiro, 24(4):755-766, abr, 2008 these calculations underestimate exposure to the target contaminants in Cidade dos Meninos, since the exposure doses were not measured for all the foods produced and consumed or for all the contaminated environmental compartments.

Dermal exposure doses (human body contact with contaminated soil and dust) were not included in the final exposure calculation for two reasons: (1) the international literature review only yielded the bioavailability factor for HCH, which served as the basis for calculating the oth- ### Standard body weight for children from 1 to 11 years 3 ; § Standard adult body weight (50th percentile) 3 ; § § Exposure dose from ingestion of contaminated soil, assuming a child with body weight of 10kg (µg/kg-day); § § § Exposure dose from ingestion of contaminated soil, assuming a child with body weight of 30kg (µg/kg-day); † Exposure dose from ingestion of contaminated soil, assuming an adult with body weight of 70kg living in Cidade dos Meninos (µg/kg-day); † † Concentration of total dioxins plus furans in toxic equivalents (TEQ).

er contaminants, thus leaving a large uncertainty factor and (2) because the international literature also failed to provide either risk indicators for dermal exposure or, consequently, parameters to compare the results.

In order to consider the worst scenario, since the compounds are interrelated and have synergic effects (although with different degrees of toxicity), it is possible to compare the sum of all the isomers with the minimum chronic exposure risk level.Importantly, the estimated oral exposure doses exceed the minimum risk level for short-term exposure (for gamma-HCH, but not for beta-HCH), intermediate exposure, and longterm (chronic) exposure.

For DDT, DDD, and DDE, the NOAEL parameter was also used for intermediate exposure to DDT.A NOAEL of 0.05mg/kg-day and uncertainty factor of 100 (10 for interspecies extrapolation and 10 for intraspecies variation) yields a minimum risk level of 0.00005mg/kg-day.In order to consider the worst situation, the sum of all the metabolites was compared with the minimum intermediate risk level, given that they have synergic effects, even with different toxicities.

Importantly, the estimated exposure doses for dioxins exceed the minimum risk level for children by more than 100 thousand times.The reference values pertain to experiments with low doses and long duration (chronic), similar to the situation in Cidade dos Meninos.In addition, the exposure exceeded the acute and intermediate minimum risk levels, thus demonstrating the need for urgent intervention.

Cad. Saúde Pública, Rio de Janeiro, 24(4):755-766, abr, 2008 ### Exposure dose from ingestion of contaminated food (eggs), assuming a child with 30kg body weight (µg/kg-day); § Exposure dose from ingestion of contaminated food (eggs), assuming an adult weighing 70kg and living in Cidade dos Meninos (µg/kg-day); § § TEQ, sample collected, 5 eggs.

Importantly, for all the compounds studied except for trichlorophenols (chronic exposure), the estimated doses exceed the minimum risk levels for children and adults, thus demonstrating the magnitude of exposure in Cidade dos Meninos.

The chemical compounds found are highly fat-soluble, i.e., easily absorbed by the body and concentrated in adipose tissues.This important characteristic allows the confirmation of high concentration in animal food sources (eggs and milk), consumed at relatively high proportions in the community.The fact that they are fat-soluble facilitates their absorption by the stomach and especially the intestine.Likewise, the results in water and soil suggest the possible absorption of these compounds through other environmental compartments, facilitated by their lipophilic characteristics.

According to the international literature 17,18,19,20,21,22 , the principal non-carcinogenic effects of these compounds are observed in the liver and central nervous and reproductive systems.A study on seven families in Cidade dos Meninos 23 showed liver enzyme alterations, but the clinical history and laboratory tests did not rule out interference by other factors (like consumption of alcohol and other drugs) in these results.The miscarriage rate of 31% (28/90) in women over 12 years of age also called attention, but this figure requires further investigation, since data collection on this type of information is always difficult.

As for classification of the target contaminants' carcinogenicity, HCH and its isomers and DDT and its metabolites belong to the category of "possibly carcinogenic to humans", since 2,3,7,8-TCDD (from the dioxins group) is classified as "carcinogenic to humans" 24 .For every carcinogenic substance, there is a risk of developing cancer at any dose different from zero.Thus, the 2.4% excess cancer shown in Table 6 generally means that one can expect that part of this population, namely 40 of the 1,660 inhabitants, could develop cancer due to exposure to these contaminants.Two cross-sectional studies 5,23 pointed to eight cancer cases without a specific pattern.Meanwhile, one of the major difficulties in studying the carcinogenicity of chemical substances is the scarcity of data in humans.For most of the agents, the existing information is extracted from data on animal experiments.To extrapolate these data to humans, it is necessary to observe that in addition to the differences between species, the experiments are done with doses of a single substance, with a specific absorption route, while most situations of human exposure actually involve multiple substances with different absorption routes 25 .Finally, the illness process is peculiar to each person, resulting from collective factors like the environment and the social, economic, historical, and cultural context in a given society.It is also determined by other factors of an individual nature, like each person's genetic map, the genetic legacy we inherit from our ancestors, and our body's nutritional status, development, and degree of maturity.The combination of these two orders of factors determines the relationship between health and disease in a person and explains why some fall ill and others do not, when exposed to chemical substances, and why different diseases can occur in persons exposed to the same compound.

Beyond the considerations on the need for research on the occurrence of health effects, there is the need for care for these populations, through training of technical personnel, development of specific protocols to treat cases of chemical

Conclusion

The human health risk assessment methodology for areas with hazardous residues is a powerful instrument to aid health managers in decisionmaking.After all the calculations, the results showed an estimate of the danger for the resident population in Cidade dos Meninos, both for the risk of cancer and the risk of systemic toxic effects.However, assessment of the effects of these chemical compounds on this population's health is still quite inconclusive.The results reflect the need to structure a health monitoring and in-formation system capable of generating reliable data on the affected population's health, progressively providing the elements for discussing the harm to human health caused by environmental contaminants.

To follow up on this population and offer the best treatment for all its health problems is an ethical commitment by the Brazilian Federal government.Finally, it is also important to highlight the need for legislation to support a national policy to identify and correct similar situations throughout Brazil's territory.

Resumo

Cidade dos Meninos é uma área localizada no Município de Duque de Caxias, Rio de Janeiro, Brasil, onde ocorreu contaminação ambiental por deposição de pesticidas organoclorados.Este artigo apresenta os resultados de um estudo de avaliação de risco para a saúde da população residente, utilizando-se a metodologia de avaliação de risco à saúde da Agency for Toxic Substances and Disease Registry.Os seguintes compostos organoclorados superaram as normas estabelecidas e foram considerados contaminantes de interesse: hexaclorociclohexano (HCH) e seus isômeros (ovo, leite, solo); tricloro-bis-(clorofenil)-etano (DDT) e seus metabólitos (ovo, leite, solo); triclorobenzenos (solo); triclorofenóis (solo); dioxinas e furanos (ovo, solo).Para todos os compostos estudados, exceto triclorofenóis, na situação de exposição crônica, as doses estimadas estavam acima dos níveis mínimos de risco à saúde, para crianças e adultos.A área foi classificada como de perigo urgente para a saúde pública por exposição de alta intensidade e grave a substâncias nocivas à saúde humana.Os autores observam que a realização de estudos de avaliação de risco para a saúde humana é uma importante ferramenta para a tomada de decisões para ações de saúde pública.

Inseticidas Organoclorados; Poluição Ambiental; Riscos Ambientais; Medição de Risco Cad.Saúde Pública, Rio de Janeiro, 24(4):755-766, abr, 2008 Contributors C. I. R. F. Asmus wrote the manuscript and participated in all phases of the fieldwork and was responsible, in the corresponding report, for the section on implications for human health.V. M. Câmara and H. G. A. Alonzo participated in the critical analysis and correction of the manuscript.A. P. Silva coordinated the fieldwork and was responsible, in the corresponding report, for the section on environmental data.D. Buosi participated in the fieldwork and conducted the final reading of the text.M. I. F. Filhote and M. Palácios participated in the fieldwork and were responsible, in the corresponding report, for the sections on general community information and human health implications, respectively.

Figure 1 Graphic
1
Figure 1 Graphic representation of households where sample chicken eggs were collected, and concentrations of target pollutants.Cidade dos Meninos, Duque de Caxias, Rio de Janeiro, Brazil, 2002

Table 1
1
Pesticide concentrations in soils in principal pollutant emission foci and reference values for soils (µg/kg).Cidade dos Meninos, Duque de Caxias, Rio de Janeiro State, Brazil, 2002.
CompoundPrincipal focusCamboada RoadReference valuesMaximumMeanMaximumMeanMTRIntervention *EMEG (mg/kg)1,2,4-TCB4,0015643909530,000202,4,6-TCF220511,742174,91502,4,5-TCF195,3512018,7150Alpha-HCH89,46715,4561,104,293224,3442902,000400HCH0000Beta-HCH45,4291,0249242,14924,3029202,00030Gamma-HCH32,24410,080109,56410,9832302,0005Delta-HCH22,270400832,8713,3122,000PCF70615,00050o,p-DDE1733194,000p,p-DDE39389951694,000o,p-DDD1242410294,000p,p-DDD627105521094,000o,p-DDT242582353894,000p,p-DDT1,09524693214394,00025Dioxinsmaximum: 13,900 ppt.1,000 ppt.50,000 ppt.CompoundMaximum concentration in chicken eggsReference valuesTrichlorobenzenes1.356µg/kgTrichlorophenolsNDAlpha-HCH66µg/kg20µg/kg *Beta-HCH4.913µg/kg101µg/kg *Gamma-HCHND10µg/kg *PentachlorophenolNDHexachlorobenzeneND20µg/kg *DDE13.689µg/kg50µg/kg *DDD1.097µg/kg50µg/kg *DDT5.728µg/kg50µg/kg *Dioxins and furans30ng WHO-PCDD/F-TEQ/kg fat3ng WHO-PCDD/F-TEQ/kg fat* Maximum residue levels 7 .
7TR: maximum tolerable risk7; EMEG: Environmental Media Evaluation Guides 3 ; ppt.: parts per trillion.*Interventionvalue7.Table 2 Maximum concentrations of organochlorine compounds measured in eggs, and reference values used.Cidade dos Meninos, Duque de Caxias, Rio de Janeiro State, Brazil, 2002.

Table 3
3
Estimated exposure dose from ingestion of soil contaminated with organochlorines.Cidade dos Meninos, Duque de Caxias, Rio de Janeiro State, Brazil, 2002.
CompoundConcen-IR (chil-IR (adul-EF (chil-EF (adul-BW (chil-BW (chil-BW (adul-ED (chil-ED (chil-ED (adul-tration * dren) **ts) ***dren) #ts) #dren) ## dren) ###ts)  §dren)  § § dren)  § § §ts)  †Alpha-HCH290.20.05111030700.580.190.02Beta-HCH910.20.05111030701.820.610.07Gamma-HCH30.20.05111030700.060.020.00Delta-HCH30.20.05111030700.060.020.00o,p-DDE70.20.05111030700.140.050.01p,p-DDE640.20.05111030701.280.430.05o,p-DDD30.20.05111030700.060.020.00p,p-DDD170.20.05111030700.340.110.01o,p-DDT60.20.05111030700.120.040.00p,p-DDT190.20.05111030700.380.130.012,4,6-trichlorophenol370.20.05111030700.740.250.032,4,5-trichlorophenol00.20.05111030700.000.000.003,4,5-trichlorophenol40.20.05111030700.080.030.002,3,5,6-tetrachlorophenol270.20.05111030700.540.180.022,3,4,5-tetrachlorophenol170.20.05111030700.340.110.01Pentachlorophenol260.20.05111030700.520.170.02Hexachlorobenzene20.20.05111030700.040.010.00Total dioxins + furans5.4296  † †0.20.05111030700.10860.03620.0039
IR: ingestion rate; EF: exposure factor; BW: body weight; ED: exposure dose.* Pesticide concentration in ppb (µg/Kg) in topsoil samples 5 ; ** Soil ingestion rate for children = 200mg/day (upper standard value for age bracket up to 11 years 3 ; *** Soil ingestion rate for adults = 50mg/day 3 ; # Exposure factor, assuming continuous exposure; ## Standard body weight for children up to 1 year of age 3 ;

Table 4
4
Estimated exposure dose from ingestion of eggs contaminated with organochlorines.Cidade dos Meninos, Duque de Caxias, Rio de Janeiro State, Brazil, 2002.
CompoundsConcen-IR (chil-IR (adul-EF (chil-EF (adults)BW (chil-BW (adul-ED (chil-ED (adults)  §tration *dren) **ts) ***dren)dren) #ts) ##dren) ###Alpha-HCH66.690.0550.0551130700.12230.0524Beta-HCH8557.40.0550.05511307015.68866.7237p,p-DDE136890.0550.05511307025.096810.7558o.p-DDD16.760.0550.0551130700.03070.0132p,p-DDD1096.90.0550.0551130702.01110.8619o,p-DDT5728060.0550.05511307010.50254.50111,2,4-trichlorobenzene13560.0550.0551130702.48601.0654Total dioxins + furans36.2849  § §0.0550.0551130700.06650.0285IR: ingestion; EF: exposure factor; BW: body weight; ED: exposure dose.* Pesticide concentration in ppb (µg/Kg) in topsoil samples 5 ;** Egg ingestion rate for children = 0.055kg/day (estimated ingestion of 1 egg per day);*** Egg ingestion rate for adults = 0.055 kg/day (estimated ingestion of 1 egg per day);
# Standard body weight (50th percentile) for children up to 11 years 3 ; ## Standard adult body weight (50th percentile) 3 ;

Table 5
5
Oral exposure doses for target sub-populations that exceed the minimum risk levels.Cidade dos Meninos, Duque de Caxias, Rio de Janeiro State, Brazil, 2002.
CompoundExposureIngestion by all routesReference valueExceeded byDemonstration ofroutefor ingestionestimatedexcess risk forED (children)ED (adults)ValueSourceexposurenon-carcinogenic(µg/kg-day)doseeffects (ED/ Reference) *ED/ED/ReferenceReference(children)(adults)Alpha-HCHTopsoil, foods0.5411950.121978.0MRL-CNo--Beta-HCHTopsoil, foods16.414226.814324200.0MRL-ANo0.6MRL-IYes--Gamma-HCHTopsoil0.0200090.00214910.0MRL-ANo0.01MRL-IYes/No--Delta-HCHTopsoil0.0200090.002149-----Σ HCH*Topsoil, foods16.995436.9405938.0MRL-AYes/No10.0MRL-IYes/No0.01MRL-CYes1,699.5694.1Σ ** (DDT+DDD+DDE)Topsoil, foods40.3458217.675820.5MRL-AYes0.5MRL-IYes80.735.45.0IDA-WHOYesTrichlorophenols **Topsoil0.2467730.0265110MRL-ANo3MRL-INo0.10.0DioxinsTopsoil, foods0.1183030.0443450.0002MRL-AYes2x10-5MRL-IYes1x10-6MRL-CYes118,303.044,345.0ED: exposure dose; MRL-A: acute minimum risk level; MRL-C: chronic minimum risk level; MRL-I: intermediate minimum risk level.* Used MRL derived from chronic exposure studies (> 1 year);
** Sum of concentrations of contaminants, and respective MRL.

contamination, continuing education programs with the community, and other measures to be conducted under the Sistema Único de Saúde [SUS; Unified National Health System].Based on this premise, the Ministry of Health, through the Health Surveillance Secretariat, published the National Health Surveillance Program for Populations Exposed to Contaminated Soils 26 and the text Guidelines for the Elaboration of Surveillance and Health Care Protocols for Populations Exposed to Contaminated Soils 27 , the objective of which is orient health actions to be developed with the affected populations.The two documents propose the organization of work groups with participation by the relevant Municipal, State, and Federal agencies, universities, and the community, to build a joint program for orienting health care activities for these populations.

Table 6
6
Calculated excess cancer risk based on estimated exposure doses for adults and children in µg/kg-day.Cidade dos Meninos, Duque de Caxias, Rio de Janeiro State, Brazil, 2002.
CompoundSlope factorEstimated total daily dose (µg/kg-day)Lifetime exposureLifetime excess riskChildrenAdultsdose (µg/kg-day)Alpha-HCH6.30.5411950.121970.1878480.001183Beta-HCH1.816.414226.8143248.3228780.014981Sum0.016165DDT0.3411.48285.143286.1394910.002087DDD0.243.1339311.6250791.8621840.000447DDE0.3425.7290910.9074613.236570.0045Sum0.007035Trichlorophenols0.0110.2467730.026510.0611236.72E-07Dioxins + furans62000.1183030.0443450.0559670.000347Total excess risk0.023547
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