Spatial and temporal variability of particulate matter in the benthic boundary layer at the N.W. European Continental Margin (Goban Spur)

Near bottom water samples and sediments were taken during five cruises to 6 stations forming a transect across the N.W. European Continental Margin at Goban Spur. Flow velocity spot measurements in the benthic boundary layer (BBL) always increased from the shelf to the upper slope (1470 m) from 5 to 9 cm s⁻¹ in spring/summer and from 15 to 37 cm s⁻¹ in autumn/winter. Decreasing values were detected at the lower slope (2000 m) and the lowest values of ca. 2 cm s⁻¹ at the continental rise at 4500 m water depth. Long term measurements with a benthic lander at 1470 m show that currents have a tidal component and reach maximum velocities up to 20 cm s⁻¹, sufficiently high periodically to resuspend and transport phytodetritus. During these long-term observations, currents were always weaker in spring/summer than in autumn/winter. Critical shear velocities of shelf/slope sediments increased with depth from 0.5 to 1.7 cm s⁻¹ and major resuspension events and Intermediate Nepheloid Layers (INLs) should occur around 1000 m. Chloroplastic Pigment Equivalents (CPE) ranged from 0.0 to 0.21 μg dm⁻³, Particulate Organic Carbon (POC) from 12 to 141 μg dm⁻³ and Total Particulate Matter (TPM) from 0.2 to 10.0 mg dm⁻³. Aggregates in the BBL occurred with a median diameter of 152 to 468 μm. Data on suspended particulate matter in the near-bottom waters showed that hydrodynamic sorting within the particulate organic fraction occurred. Phytodetritus was packaged in relatively large aggregates and contributed little to the total organic carbon pool in nearbottom waters (CPE/POC ca.0.2%). The main organic fraction has low settling velocities and high residence times within the benthic boundary layer. As POC was not concentrated in the near bed region the degree to which carbon is accessible to the benthic community depends on aggregate formation, subsequent settling and/or biodeposition of the POC. Close to the sea bed downslope transport may dominate. Under flow conditions high enough to resuspend fresh phythodetritus from sediments at the productive shelf edge, this could be transported to 1500 m (Goban Spur) or abyssal depth (Canyon site between Meriadzek and Goban Spur) within 21 days.     

Spatial patterns and environmental drivers of benthic infaunal community structure and ecosystem function on the New Zealand continental margin

To investigate regional drivers of spatial patterns in macro- and meio-faunal community structure (abundance, biomass and taxonomic diversity) and ecosystem function (sediment community oxygen consumption [SCOC]), we sampled two regions in close proximity on New Zealand's continental margin—the Chatham Rise and the Challenger Plateau. Sites (n = 15) were selected in water depths ranging from 266–1212 m to generate a gradient in sedimentary properties and, in particular, surface pelagic productivity. Both macro- and meio-fauna abundance and biomass was 2–3.5 times higher on the Chatham Rise than on the Challenger Plateau, reflecting regional differences in pelagic primary production. We also found significant inter- and intra-regional differences in macro-fauna taxonomic diversity with two distinctive site groupings in each region. Univariate and multivariate measures of macro-fauna community attributes were most strongly correlated with sediment photosynthetic pigment (explaining 24%–59% of the variation). Sediment pigment content was as equally important in explaining meio-fauna community structure (36%–7%). Unlike community structure, SCOC was most strongly correlated with depth (44%), most likely reflecting temperature effects on benthic metabolism. Our results highlight the importance of a benthic labile food supply in structuring infaunal communities on continental margins and emphasise a tight coupling between pelagic and benthic habitats.     

Infaunal benthic communities of the New Zealand continental shelf

Seventeen infaunal benthic communities are recognised from sampling on the New Zealand continental shelf and upper continental slope. These are characterised in terms of geographic distribution, substrate and bathymetric range, and commoner infaunal bivalves and echinoderms.     

Bottom boundary layer in the Black Sea: Experimental data,turbulent diffusion,and fluxes

Measurements of 14 vertical profiles of currents and hydrological parameters in the near-bottom layer with a depth resolution of 0.1 m were carried out in several regions of the Black Sea shelf, at five points over the continental slope, and in three deep water regions. The upper boundary of the benthic boundary layer (BBL) was reliably determined at a point at a distance from 5–7 to 35–40 m from the bottom where the gradients of the density and the velocity of the currents changed. The experimental data obtained were used to determine the coefficient of the bottom friction, the friction velocity, the coefficients of the vertical diffusion of momentum and density, and the vertical fluxes of temperature and salinity in the BBL.     

Coupling of benthic oxygen uptake and silica release: implications for estimating biogenic particle fluxes to the seafloor

A new approach to predict biogenic particle fluxes to the seafloor is presented, which is based on diffusive oxygen uptake and, in particular, opal fluxes to the seafloor. For this purpose, we used a recently published empirical equation coupling benthic silica to oxygen fluxes, and showing a clear negative correlation between Si and O₂ fluxes. Dissolution of biogenic silica mediated by aerobic microbial activity has been inferred at 24 sites along the African and South American continental margins. Based on the assumption that these findings hold essentially for the entire Southern Atlantic Ocean, we applied the silica to oxygen flux ratio to a basin-wide grid of diffusive oxygen uptake extracted from the literature. Assuming that the silica release across the sediment-water interface equals the particulate flux of biogenic opal to the seafloor, we estimated minimum opal rain rates. We combined these calculations with published relationships between aerobic organic carbon mineralization and dissolution rates of calcite above the hydrographical lysocline, thereby assessing the calcite rain rate and particulate organic carbon flux to the seafloor. The addition of the buried fraction completes our budget of biogenic particulate rain fluxes. The combination of such empirical equations provides a powerful and convenient tool which greatly facilitates future investigations of biogenic particle fluxes to the seafloor.     

Hydro-acoustic remote sensing of benthic biological communities on the shallow South East Australian continental shelf

Information regarding the composition and extent of benthic habitats on the South East Australian continental shelf is limited. In this habitat mapping study, multibeam echosounder (MBES) data are integrated with precisely geo-referenced video ground-truth data to quantify benthic biotic communities at Cape Nelson, Victoria, Australia. Using an automated decision tree classification approach, 5 representative biotic groups defined from video analysis were related to hydro-acoustically derived variables in the Cape Nelson survey area. Using a combination of multibeam bathymetry, backscatter and derivative products produced highest overall accuracy (87%) and kappa statistic (0.83). This study demonstrates that decision tree classifiers are capable of integrating variable data types for mapping distributions of benthic biological assemblages, which are important in maintaining biodiversity and other system services in the marine environment.     

Microbial activities at the benthic boundary layer in the Aegean Sea

During the Aegean Sea component of the EU MTP-MATER project, benthic samples were acquired along a depth gradient from two continental margins in the Aegean Sea. Sampling was undertaken during spring and summer 1997 and the microbial metabolic activities measured (Vmax for aminopeptidase activity, ¹⁴C-glutamate respiration and assimilation) displayed seasonal variability even in deep-sea conditions. The metabolic rates encountered in the North Aegean (average depth 566±234 m), were approximately five-fold higher than in the deeper (1336±140 m) Southern part of the Aegean. The aminopeptidase rates, however, were the exception with higher values recorded in the more oligotrophic sediments of the Southern stations (1383±152 vs. 766±297 nmol MCA cm⁻² h⁻¹). A discrepancy in bacterial metabolism also appeared in the near bottom waters. In the Southern stations, 80% of the glutamate uptake was used for energy yielding processes and only 20% devoted to biomass production, while in the North Aegean, most of the used glutamate was incorporated into bacterial cells. During the early burial stages, bacterial mineralization rates estimated from ¹⁴C-glutamate respiration decreased drastically compared to the rates of biopolymer hydrolysis estimated by aminopeptidase assays. Thus, at the 2-cm depth layer, these rates were only 32 and up to 77% of the corresponding average values, respectively, in the superficial layer. Such a discrepancy between the evolution of these two metabolic activities is possibly due to the rapid removal of readily utilizable monomers in the surface deposits. The correlation between bacterial respiration and total organic carbon, or total organic nitrogen, is higher in the surficial sediment (0-2 and 2-4 cm) than in the underlying layer. Conversely, it is only at 4-cm depth layer that the hydrolysis rates appear correlated with organic carbon and nitrogen concentrations. This pattern confirms the drastic degradation of organic matter during the early burial stages.     

NotesBenthic foraminiferal distribution in surface sediments along continental slope of the southern Okinawa Trough: dependance on water masses and food supply

Benthic foraminiferal analysis of 29 samples in surface sediments from the southern Okinawa Trough is carried out. The results indicate that benthic foraminiferal abundance decreases rapidly with increasing water depth. Percentage frequencies of agglutinated foraminifera further confirm the modern shallow carbonate lysocline in the southern Okinawa Trough. From continental shelf edge to the bottom of Okinawa Trough, benthic foraminiferal fauna in the surface sediments can be divided into 5 assemblages: (1) Continental shelf break assemblage, dominated by Cibicides pseudoungerianus, corresponds to subsurface water mass of the Kuroshio Current; (2) upper continental slope assemblage, dominated by Cassidulina carinata , Globocassidulina subglobosa, corresponds to intermediate water mass of the Kuroshio Current; (3) intermediate continental slope assemblage, dominated by Uvigerina hispi-da, corresponds to the Okinawa Trough deep water mass above the carbonate lysocline; (4) lower continental slope- trough b     

Carbon flows in the benthic food web at the deep-sea observatory HAUSGARTEN (Fram Strait)

The HAUSGARTEN observatory is located in the eastern Fram Strait (Arctic Ocean) and used as long-term monitoring site to follow changes in the Arctic benthic ecosystem. Linear inverse modelling was applied to decipher carbon flows among the compartments of the benthic food web at the central HAUSGARTEN station (2500 m) based on an empirical data set consisting of data on biomass, prokaryote production, total carbon deposition and community respiration. The model resolved 99 carbon flows among 4 abiotic and 10 biotic compartments, ranging from prokaryotes up to megafauna. Total carbon input was 3.78±0.31 mmol C m⁻² d⁻¹, which is a comparatively small fraction of total primary production in the area. The community respiration of 3.26±0.20 mmol C m⁻² d⁻¹ is dominated by prokaryotes (93%) and has lower contributions from surface-deposit feeding macro- (1.7%) and suspension feeding megafauna (1.9%), whereas contributions from nematode and other macro- and megabenthic compartments were limited to <1%. The high prokaryotic contribution to carbon processing suggests that functioning of the benthic food web at the central HAUSGARTEN station is comparable to abyssal plain sediments that are characterised by strong energy limitation. Faunal diet compositions suggest that labile detritus is important for deposit-feeding nematodes (24% of their diet) and surface-deposit feeding macrofauna (∼44%), but that semi-labile detritus is more important in the diets of deposit-feeding macro- and megafauna. Dependency indices on these food sources were also calculated as these integrate direct (i.e. direct grazing and predator–prey interactions) and indirect (i.e. longer loops in the food web) pathways in the food web. Projected sea-ice retreats for the Arctic Ocean typically anticipate a decrease in the labile detritus flux to the already food-limited benthic food web. The dependency indices indicate that faunal compartments depend similarly on labile and semi-labile detritus, which suggests that the benthic biota may be more sensitive to changes in labile detritus inputs than when assessed from diet composition alone. Species-specific responses to different types of labile detritus inputs, e.g. pelagic algae versus sympagic algae, however, are presently unknown and are needed to assess the vulnerability of individual components of the benthic food web.     

Biological Responses to Seasonally Varying Fluxes of Organic Matter to the Ocean Floor: A Review

Deep-sea benthic ecosystems are sustained largely by organic matter settling from the euphotic zone. These fluxes usually have a more or less well-defined seasonal component, often with two peaks, one in spring/early summer, the other later in the year. Long time-series datasets suggest that inter-annual variability in the intensity, timing and composition of flux maxima is normal. The settling material may form a deposit of “phytodetritus” on the deep-seafloor. These deposits, which are most common in temperate and high latitude regions, particularly the North Atlantic, evoke a response by the benthic biota. Much of our knowledge of these responses comes from a few time-series programmes, which suggest that the nature of the response varies in different oceanographic settings. In particular, there are contrasts between seasonal processes in oligotrophic, central oceanic areas and those along eutrophic continental margins. In the former, it is mainly “small organisms” (bacteria and protozoans) that respond to pulsed inputs. Initial responses are biochemical (e.g. secretion of bacterial exoenzymes) and any biomass increases are time lagged. Increased metabolic activity of small organisms probably leads to seasonal fluctuations in sediment community oxygen consumption, reported mainly in the North Pacific. Metazoan meiofauna are generally less responsive than protozoans (foraminifera), although seasonal increases in abundance and body size have been reported. Measurable population responses by macrofauna and megafauna are less common and confined largely to continental margins. In addition, seasonally synchronised reproduction and larval settlement occur in some larger animals, again mainly in continental margin settings. Although seasonal benthic responses to pulsed food inputs are apparently widespread on the ocean floor, they are not ubiquitous. Most deep-sea species are not seasonal breeders and there are probably large areas, particularly at abyssal depths, where biological process rates are fairly uniform over time. As with other aspects of deep-sea ecology, temporal processes cannot be encapsulated by a single paradigm. Further long time-series studies are needed to understand better the nature and extent of seasonality in deep-sea benthic ecosystems. This revised version was published online in August 2006 with corrections to the Cover Date.     

Food web flows through a sub-arctic deep-sea benthic community

The benthic food web of the deep Faroe–Shetland Channel (FSC) was modelled by using the linear inverse modelling methodology. The reconstruction of carbon pathways by inverse analysis was based on benthic oxygen uptake rates, biomass data and transfer of labile carbon through the food web as revealed by a pulse-chase experiment. Carbon deposition was estimated at 2.2 mmol C m⁻² d⁻¹. Approximately 69% of the deposited carbon was respired by the benthic community with bacteria being responsible for 70% of the total respiration. The major fraction of the labile detritus flux was recycled within the microbial loop leaving merely 2% of the deposited labile phytodetritus available for metazoan consumption. Bacteria assimilated carbon at high efficiency (0.55) but only 24% of bacterial production was grazed by metazoans; the remaining returned to the dissolved organic matter pool due to viral lysis. Refractory detritus was the basal food resource for nematodes covering ∼99% of their carbon requirements. On the contrary, macrofauna seemed to obtain the major part of their metabolic needs from bacteria (49% of macrofaunal consumption). Labile detritus transfer was well-constrained, based on the data from the pulse-chase experiment, but appeared to be of limited importance to the diet of the examined benthic organisms (<1% and 5% of carbon requirements of nematodes and macrofauna respectively). Predation on nematodes was generally low with the exception of sub-surface deposit-feeding polychaetes that obtained 35% of their energy requirements from nematode ingestion. Carnivorous polychaetes also covered 35% of their carbon demand through predation although the preferred prey, in this case, was other macrofaunal animals rather than nematodes. Bacteria and detritus contributed 53% and 12% to the total carbon ingestion of carnivorous polychaetes suggesting a high degree of omnivory among higher consumers in the FSC benthic food web. Overall, this study provided a unique insight into the functioning of a deep-sea benthic community and demonstrated how conventional data can be exploited further when combined with state-of-the-art modelling approaches.     

Assemblages of benthic foraminifers in the White Sea

Based upon the composition of the dominant species of benthic foraminifers in the bottom sediments sampled at 50 sampling stations in the White Sea, their six assemblages (communities) were defined and mapped. The ranges of these communities depend on the environmental factors determined by the bathymetric, latitudinal, and circum-continental sea zonalities, the sea currents, the bottom upwellings, the continental runoff, the productivity of the waters, and the CaCO₃ content in the near-bottom water layers and bottom sediments.     

Benthic foraminiferal distribution in surface sediments along continental slope of the southern Okinawa Trough: dependance on water masses and food supply

Benthic foraminiferal analysis of 29 samples in surface sediments from the southern Okinawa Trough is carried out.The results indicate that benthic foraminiferal abundance decreases rapidly with increasing water depth.Percentage frequencies of agglutinated foraminifera further confirm the modern shallow carbonate lysocline in the southern Okinawa Trough.From continental shelf edge to the bottom of Okinawa Trough,benthic foraminiferal fauna in the surface sediments can be divided into 5 assemblages:(1) Continental shelf break assemblage,dominated by Cibicides pseudoungerianus,corresponds to subsurface water mass of the Kuroshio Current;(2) upper continental slope assemblage,dominated by Cassidulina carinata,Globocassidulina subglobosa,corresponds to intermediate water mass of the Kuroshio Current;(3) intermediate continental slope assemblage,dominated by Uvigerina hispida,corresponds to the Okinawa Trough deep water mass above the carbonate lysocline.     

Diversity patterns along and across the Chilean margin: a continental slope encompassing oxygen gradients and methane seep benthic habitats

In the present study we review datasets available for the Chilean margin to assess the relationship between environmental (or habitat) heterogeneity and benthic diversity. Several factors, such as the presence of different water masses, including the oxygen-deficient Equatorial Sub-surface Waters (ESSW) at the continental shelf and upper slope, and the Antarctic Intermediate Waters (AIW) at mid slope depths appear to control the bathymetric distribution of benthic communities. The presence of methane seeps and an extended oxygen minimum zone (OMZ) add complexity to the benthic distribution patterns observed. All these factors generate environmental heterogeneity, which is predicted to affect the diversity patterns both along and across the Chilean continental margin. The response to these factors differs among different faunal size groups: meio-, macro-, and megafauna. Physiological adaptations to oxygen deficiency and constraints related to body size of each group seem to explain the larger-scale patterns observed, while sediment/habitat heterogeneity ( e.g. at water mass boundaries, hardgrounds, biogeochemical patchiness, sediment organic content, grain size) may influence the local fauna diversity patterns.     

我国典型潮间带底栖硅藻群落空间分布特征

底栖硅藻是河口、泥质海滩生态系统的重要生物类群,其数量与分布变化直接或间接地影响到多种海洋生物的饵料供给、海鸟的生存与迁徙。通过对我国从南到北14个典型潮间带表层沉积物中底栖硅藻的分析,共鉴定出硅藻153种,其物种多样性（Shannon-Weaver 指数范围:1.61~4.39）与生物量（范围:0.09×103 ~ 10×103个/g,干质量）在空间上存在显著差异;聚类分析与相似性分析检验（r=0.738,P=0.1%< 0.01）表明,14个调查区域的硅藻群落在空间上可分为4个大的组合,分别对应着渤、黄、东、南海4个区域,吻合了我国近海浮游植物的地理分布特征。与环境因素的相关性分析表明,底栖硅藻群落的物种多样性与生物量受沉积物类型与盐度的影响显著,泥质区域更有利于硅藻的保存,而生物多样性在泥砂质低盐区域相对较高。此外,底栖硅藻生物量与表层沉积物中TOC含量表现出显著正相关性;δ13C数值的空间变化范围为-25‰~-20‰,指示了有机质陆海混合的特征,其中,涧河河口、黄河口、大沽河口、苏北浅滩区域,δ13C数值明显偏正,表明底栖硅藻可能是这些区域沉积物中有机碳的重要贡献者。     

Fatty acid profiles of marine benthic microorganisms isolated from the continental slope of bay of bengal: a possible implications in the benthic Food web

Marine bacteria, actinomycetes and fungal strains were isolated from continental slope sediment of the Bay of Bengal and studied for fatty acid profile to investigate their involvement in the benthic food-web. Fifteen different saturated and unsaturated fatty acids from bacterial isolates, 14 from actinomycetes and fungal isolates were detected. The total unsaturated fatty acids in bacterial isolates ranged from 11.85 to 37.26%, while the saturated fatty acid ranged between 42.34 and 80.74%. In actinomycetes isolates, total unsaturated fatty acids varied from 27.86 to 38.85% and saturated fatty acids ranged from 35.29 to 51.25%. In fungal isolates unsaturated fatty acids ranged between 44.62 and 65.52% while saturated FA ranged from 20.80 to 46.30%. The higher percentages of unsaturated fatty acids from the microbial isolates are helpful in anticipating the active participation in the benthic food-web of Bay of Bengal.     

Biological structures as a source of habitat heterogeneity and biodiversity on the deep ocean margins

Biological structures exert a major influence on species diversity at both local and regional scales on deep continental margins. Some organisms use other species as substrates for attachment, shelter, feeding or parasitism, but there may also be mutual benefits from the association. Here, we highlight the structural attributes and biotic effects of the habitats that corals, sea pens, sponges and xenophyophores offer other organisms. The environmental setting of the biological structures influences their species composition. The importance of benthic species as substrates seems to increase with depth as the complexity of the surrounding geological substrate and food supply decline. There are marked differences in the degree of mutualistic relationships between habitat-forming taxa. This is especially evident for scleractinian corals, which have high numbers of facultative associates (commensals) and few obligate associates (mutualists), and gorgonians, with their few commensals and many obligate associates. Size, flexibility and architectural complexity of the habitat-forming organism are positively related to species diversity for both sessile and mobile species. This is mainly evident for commensal species sharing a facultative relationship with their host. Habitat complexity is enhanced by the architecture of biological structures, as well as by biological interactions. Colony morphology has a great influence on feeding efficiency for suspension feeders. Suspension feeding, habitat-forming organisms modify the environment to optimize their food uptake. This environmental advantage is also passed on to associated filter-feeding species. These effects are poorly understood but represent key points for understanding ecosystems and biodiversity on continental margins. In this paper we explore the contributions of organisms and the biotic structures they create (rather than physical modifications) to habitat heterogeneity and diversity on the deep continental margins.     

Benthic response of Munnopsurus atlanticus (Crustacea Isopoda) to the carbon content of the near-bottom sedimentary environment on the southern margin of the Cap-Ferret Canyon (Bay of Biscay,northeastern Atlantic Ocean)

The response of benthic organisms to organic carbon fluxes in a continental margin region was studied by investigating the diet of the suprabenthic isopod Munnopsurus atlanticus, which is well represented on the southern margin of the Cap-Ferret Canyon (Bay of Biscay). The grain-size distribution, foraminiferal assemblages, particulate organic carbon and pigments found in the sediment and in the gut of the isopods were analyzed. These results suggest that M. atlanticus feeds on benthic agglutinated foraminifers which are in a high “nourishment state” and represent a link between primary and secondary producers.     

Organic carbon content in surface sediments—defining regional provinces

Approaches to quantify the organic carbon accumulation on a global scale generally do not consider the small-scale variability of sedimentary and oceanographic boundary conditions along continental margins. In this study, we present a new approach to regionalize the total organic carbon (TOC) content in surface sediments (<5 cm sediment depth). It is based on a compilation of more than 5500 single measurements from various sources. Global TOC distribution was determined by the application of a combined qualitative and quantitative-geostatistical method. Overall, 33 benthic TOC-based provinces were defined and used to process the global distribution pattern of the TOC content in surface sediments in a 1°×1° grid resolution. Regional dependencies of data points within each single province are expressed by modeled semi-variograms. Measured and estimated TOC values show good correlation, emphasizing the reasonable applicability of the method. The accumulation of organic carbon in marine surface sediments is a key parameter in the control of mineralization processes and the material exchange between the sediment and the ocean water. Our approach will help to improve global budgets of nutrient and carbon cycles.     

Comparative biomass structure and estimated carbon flow in food webs in the deep Gulf of Mexico

A budget of the standing stocks and cycling of organic carbon associated with the sea floor has been generated for seven sites across a 3-km depth gradient in the NE Gulf of Mexico, based on a series of reports by co-authors on specific biotic groups or processes. The standing stocks measured at each site were bacteria, Foraminifera, metazoan meiofauna, macrofauna, invertebrate megafauna, and demersal fishes. Sediment community oxygen consumption (SCOC) by the sediment-dwelling organisms was measured at each site using a remotely deployed benthic lander, profiles of oxygen concentration in the sediment pore water of recovered cores and ship-board core incubations. The long-term incorporation and burial of organic carbon into the sediments has been estimated using profiles of a combination of stable and radiocarbon isotopes. The total stock estimates, carbon burial, and the SCOC allowed estimates of living and detrital carbon residence time within the sediments, illustrating that the total biota turns over on time scales of months on the upper continental slope but this is extended to years on the abyssal plain at 3.6 km depth. The detrital carbon turnover is many times longer, however, over the same depths. A composite carbon budget illustrates that total carbon biomass and associated fluxes declined precipitously with increasing depth. Imbalances in the carbon budgets suggest that organic detritus is exported from the upper continental slope to greater depths offshore.The respiration of each individual “size” or functional group within the community has been estimated from allometric models, supplemented by direct measurements in the laboratory. The respiration and standing stocks were incorporated into budgets of carbon flow through and between the different size groups in hypothetical food webs. The decline in stocks and respiration with depth were more abrupt in the larger forms (fishes and megafauna), resulting in an increase in the relative predominance of smaller sizes (bacteria and meiofauna) at depth. Rates and stocks in the deep northern GoM appeared to be comparable to other continental margins where similar comparisons have been made.