The seasonality of phytoplankton in the North American Great Lakes,a comparative synthesis

The phytoplankton and productivity of the North American Great Lakes has been studied extensively by Fisheries and Oceans Canada during the past 15 years to monitor the impact of nutrient and contaminant loading on the plankton of the ecosystem. Lakewide cruises were conducted at monthly intervals mainly during the spring to fall period. This provided extensive biomass, species, size, productivity and nutrient concentration data for the Great Lakes. These data were collected using the Utermöhl inverted microscope technique together with standardized taxonomic, productivity and data-handling procedures. These standardized methodologies were applied to all the Great Lakes which resulted in a comprehensive phycological and ecological data base for the first time. These data form the basis for the evaluation of the complex phenomenon of seasonality.The eutrophic/mesotrophic Lower Great Lakes exhibited well-developed seasonal peaks of high biomass, with inshore-offshore differentiation and spring maxima most pronounced in the inshore region. However, the oligotrophic Upper Great Lakes had low biomass and generally lacked well-developed seasonal patterns. No marked seasonal trends were observed in the ultra-oligotrophic Lake Superior. The seasonality of biomass and various taxonomic groups of phytoplankton showed differentiation between individual lakes and is discussed in detail. The seasonal succession of species provided interesting comparisons between the Lower Great Lakes, which harbour eutrophic and mesotrophic species, and the Upper Great Lakes, which harbour oligotrophic species.Due to the voluminous nature of our data, a general overview has been given for all the Great Lakes with Lake Ontario treated in detail as a case study. The Lake Ontario case study provides the state-of-the-art status ranging from the lakewide surveys of 1970 to the current research with minute organisms such as ultraplankton and picoplankton.     

The response of zooplankton and phytoplankton from the North American Great Lakes to filtration

Filtration of ballast water was investigated as a means of minimizing the introduction of nonindigenous zooplankton and phytoplankton by ships visiting the North American Great Lakes-St. Lawrence Seaway system (GLSLSS). An automatic backwash screen filtration (ABSF) system with nominal filtration options of 25, 50 or 100 μm was mounted on the deck of an operating Seaway-sized dry bulk carrier, the MV Algonorth. Water was pumped through the ABSF with a deck mounted pump at 341 m³ hr⁻¹ during routine ship operations in the GLSLSS, and effectiveness of the various screen pore sizes at removing taxonomic categories of zooplankton and phytoplankton was measured using matched treatment and control ballast tanks. The smallest pore sizes (25 and 50 μm) performed better than the 100 μm pore size at removing biological material. There was no difference in the filtration efficiency of the 25 and 50 μm screens relative to macro- or microzooplankton in these tests, but this result was probably due to low densities of macrozooplankton, and soft-bodied (aloricate) characteristics of the microzooplankton present. The 25 and 50 μm pore sizes were subjected to more controlled tests on board a stationary barge platform equipped with triplicate 700 L catchment bins moored in Duluth Harbor of Lake Superior. In these tests, filter pore size, organism size and rigidity influenced zooplankton removal efficiency by the ABSF. The 25 μm screen reduced both macrozooplankton and microzooplankton significantly more than the 50 μm screen. Zooplankton width was more determinative of filtration performance than length, and both filters removed loricate species of rotifers significantly more efficiently than aloricate species of the same length and width size classes. The 25 and 50 μm ABSF also significantly reduced algal densities, with the exception of colonial and filamentous green algae (50 μm only). Filter efficiency relative to algal particles was influenced by filter pore size, organism morphology and structure, and intake density, while algal particle size was not determinative. This research provides compelling evidence that 25 or 50 μm filtration is a potentially powerful means of reducing densities of organisms discharged by ships operating in the Great Lakes but an additional treatment step would be necessary to effectively minimize risk and meet the International Maritime Organization's discharge standards associated with organisms of all sizes in the water column.     

Ecosystem health as measured from the molecular to the community level of organization,with reference to sediment bioassessment

The current recognition that chemical measurements are uncertain indicators of biological consequences of pollution has shifted the emphasis away from assessing environmental chemistry alone toward the inclusion of measurements of the health of organisms. Effects of pollutants begin with the individual, have subsequent repercussions on population level processes, and ramifications for community structure and functions. Pollutants act at a molecular level and the biochemical lesions is the first step in the manifestation of effects. Technologies that operate at the cellular level assist in elucidating toxicity. Higher levels of integration include an organism's capacity for growth. Laboratory bioassays andin situ research can monitor physiological incapacities and assist in predicting population level effects. A yet higher level of organization is that of the ecological community.     

Sediment toxicity testing in two areas of concern of the Laurentian Great Lakes: Toronto (Ontario) and Toledo (Ohio) Harbours

The impact of elutriated sediment-associated contaminants from Toronto and Toledo Harbours on ultraplankton (5–20 µm) and microplankton/netplankton (> 20 µm) carbon assimilation rates was determined using Algal Fractionation Bioassays (AFBs). All of the Toronto elutriate caused significant inhibition of ultraplankton carbon assimilation. The Toronto Site 2 elutriate caused the greatest significant inhibition (38 percent, p < 0.001) with a 20 percent dose of standard elutriate. Similarly, all Toledo elutriates caused significant inhibition of ultraplankton productivity. Toledo Site 2 elutriate was the most toxic with the 20 percent elutriate dose (35 percent, p < 0.001).The treatment of elutriates with Chelex-100 resin was used to remove dissolved free metal ions which, in some samples, resulted in the recovery of ¹⁴C assimilation. This was attributed to the elimination of the toxic effects of dissolved metals removed by the Chelex treatment. Residual toxicity after the Chelex treatment was ascribed to the high PCB levels observed in the sediment samples taken from both harbours and possibly to other organic contaminants. Due to the extreme sensitivity of the technique, an EC25 is proposed as an early warning indicator for applied use by regulatory agencies. Our procedure has been included amongst a battery of tests recommended by the International Joint Commission for monitoring areas of concern in the Laurentian Great Lakes.     

Regulation of phytoplankton dynamics in a Laurentian Great Lakes estuary

The composition and dynamics of phytoplankton populations were examined in Old Woman Creek estuary, Lake Erie (USA). The centric bacillariophytes,Cyclotella atomus Hust.,Cyclotella meneghiniana Kütz., andAulacoseira alpigena (Grun.) Krammer, and the cryptophytes,Cryptomonas erosa Ehren. andRhodomonas minuta var. nannoplanctonica Skuja, dominated the phytoplankton most of the year. Chlorophytes, euglenophytes, and cyanophytes were observed less frequently. Estuarine and Lake Erie phytoplankton were considered distinct populations; lake taxa were largely confined to the estuary mouth and present only in low biomass. Maxima and minima of estuarine phytoplankton coincided with meteorological and hydrological forcing in the form of rainfall and subsequent storm-water inflows, respectively. Distinct population dynamics between the upper and lower estuary following storm events were attributed to the presence/absence of refugia serving as a source for repopulation by opportunistic taxa, fluctuating light conditions in the water column resulting from influx of particulate matter and resuspension of bottom sediments, and nutrient inputs associated with surface runoff and sub-surface interflow. Additionally, agricultural herbicides introduced by storm-water inflows potentially may affect and/or control the growth and physiological responses of individual taxa.     

In situ bioassessment of dredging and disposal activities in a contaminated ecosystem: Toronto Harbour

The contamination of Toronto Harbour is a very serious problem. The major sources of pollution are the Don River and sewer outflows, as well as industrial, and municipal effluents. The problem is further compounded by perturbations of the toxic sediment caused by dredging, dredge-disposal, navigation, and recreational activities. The impact of contamination and nutrient enrichment was reflected in the size-fractionated primary productivity experiments. Generally, microplankton/netplankton (> 20 µm) productivity was enhanced whereas ultraplankton (< 20 µm) productivity was inhibited. These observations are attributable to interactions between ameliorating nutrients and toxic contaminants as well as to the differential sensitivity of natural phytoplankton size assemblages to the bioavailable chemical regime. In situ environmental techniques applied in Toronto Harbour were effective, sensitive, and rapid, and provided a better understanding of the impact of dredging/disposal activities under natural conditions. These techniques have great potential in the assessment of the ecotoxicology of harbours and other stressed environments.     

Health impacts from cyanobacteria harmful algae blooms: Implications for the North American Great Lakes

Harmful cyanobacterial blooms (cHABs) have significant socioeconomic and ecological costs, which impact drinking water, fisheries, agriculture, tourism, real estate, water quality, food web resilience and habitats, and contribute to anoxia and fish kills. Many of these costs are well described, but in fact are largely unmeasured. Worldwide cHABs can produce toxins (cyanotoxins), which cause acute or chronic health effects in mammals (including humans) and other organisms. There are few attempts to characterize the full health-related effects other than acute incidences, which may go unrecorded. At present these are difficult to access and evaluate and may be ascribed to other causes. Such information is fundamental to measure the full costs of cHABs and inform the need for often-costly management and remediation. This paper synthesizes information on cHABs occurrence, toxicology and health effects, and relates this to past and current conditions in the Great Lakes, a major global resource which supplies 84% of the surface water in North America. This geographic region has seen a significant resurgence of cHABs since the 1980s. In particular we focus on Lake Erie, where increased reporting of cHABs has occurred from the early 1990's. We evaluate available information and case reports of cHAB-related illness and death and show that cHABs occur throughout the basin, with reports of animal illness and death, especially dogs and livestock. Lake Erie has consistently experienced cHABs and cyanotoxins in the last decade with probable cases of human illness, while the other Great Lakes show intermittent cHABs and toxins, but no confirmed reports on illness or toxicity. The dominant toxigenic cyanobacterium is the genus Microcystis known to produce microcystins. The presence of other cyanotoxins (anatoxin-a, paralytic shellfish toxins) implicates other toxigenic cyanobacteria such as Anabaena (Dolichospermum) and Lyngbya.     

Contaminant Trends in Freshwater Fish from the Laurentian Great Lakes: A 20-Year Analysis

Although dietary concerns of Laurentian Great Lakes (GL) fish focus on the risk from persistent bioaccumulative toxicant (PBT) contaminants, fish are also an important source of nutrients beneficial to human health such as polyunsaturated fatty acids (e.g., eicosapentanoic acid and docosahexanoic acid). This study presents PBT trend data from the GL tribal fisheries over the past 20 years. PBT contaminants (282 analytes) from fillet portions of lake trout and whitefish were analyzed for trending patterns from 1992 to 2011 and are reported on five of the ATSDR/USEPA Great Lakes biomonitoring legacy contaminants (Hg, ΣDDE, ΣDDT, HCB, mirex, and ΣPCBs), two of the optional biomonitoring PBTs (toxaphene and Σdioxins/furans) and PCB 153 as a specific congener marker. Similar to other recent reports our data indicate that most PBT contaminant concentrations in the GL biota have decreased, which may indicate progress in reducing environmental emissions. Our research confirms that all contaminants demonstrate significant declines except Hg and toxaphene. Both of those remained constant after correcting for known independent factors of age, lipid, and size. These results are potentially encouraging and may provide useful data for the long distance and perhaps global influences of PBTs on the safety of fish consumption.     

Emergence of Viral hemorrhagic septicemia virus in the North American Great Lakes region is associated with low viral genetic diversity

Viral hemorrhagic septicemia virus (VHSV) is a fish rhabdovirus that causes disease in a broad range of marine and freshwater hosts. The known geographic range includes the Northern Atlantic and Pacific Oceans, and recently it has invaded the Great Lakes region of North America. The goal of this work was to characterize genetic diversity of Great Lakes VHSV isolates at the early stage of this viral emergence by comparing a partial glycoprotein (G) gene sequence (669 nt) of 108 isolates collected from 2003 to 2009 from 31 species and at 37 sites. Phylogenetic analysis showed that all isolates fell into sub-lineage IVb within the major VHSV genetic group IV. Among these 108 isolates, genetic diversity was low, with a maximum of 1.05% within the 669 nt region. There were 11 unique sequences, designated vcG001 to vcG011. Two dominant sequence types, vcG001 and vcG002, accounted for 90% (97 of 108) of the isolates. The vcG001 isolates were most widespread. We saw no apparent association of sequence type with host or year of isolation, but we did note a spatial pattern, in which vcG002 isolates were more prevalent in the easternmost sub-regions, including inland New York state and the St. Lawrence Seaway. Different sequence types were found among isolates from single disease outbreaks, and mixtures of types were evident within 2 isolates from individual fish. Overall, the genetic diversity of VHSV in the Great Lakes region was found to be extremely low, consistent with an introduction of a new virus into a geographic region with previously naive host populations.     

Mercury risks versus nutritional benefits of tribal commercial fish harvests in the Upper Laurentian Great Lakes

The Inter-Tribal Fisheries and Assessment Program (ITFAP) of the Chippewa Ottawa Resource Authority (CORA) in Sault Ste. Marie, Michigan, has been monitoring contaminant concentrations in the fillet portions of lake trout (Salvelinus namaycush) and lake whitefish (Coregonus clupeaformis) from the waters of lakes Superior, Huron, and Michigan since 1991. The primary purpose of this article is to present a risk quantification of methylmercury (MeHg) that is adjusted for nutritional benefit, originally presented by Ginsberg and Toal (2009 Ginsberg GL and Toal BF. 2009. Quantitative approach for incorporating methylmercury risks and omega-3 fatty acid benefits in developing species-specific fish consumption advice. Environ Health Perspect 117:267–75[Crossref], [PubMed], [Web of Science ®] , [Google Scholar], 2015) on trends in contaminant concentrations in fillet portions of these commercial fish that we recently reported in Dellinger et al. (2014 Dellinger JA, Moths MD, Dellinger M, et al. 2014. Contaminant trends in freshwater fish from the Great Lakes: A 20 year analysis. Hum Ecol Risk Assess 20:461–78[Taylor &; Francis Online], [Web of Science ®] , [Google Scholar]). Both species of fish caught by tribal fishermen showed clear benefits to cardiovascular health and infant neurodevelopment if consumed at a rate of six ounces per week. However, other popularly consumed fish such as cod, tuna, and tilapia are estimated to have only marginal benefit or net negative effects on cardiovascular health and infant neurodevelopment. This dynamic assessment of benefits and risks further demonstrates the importance of traditionally caught fish in tribal health.     

Heavy metal contamination of sediments in the Upper Connecting Channels of the Great Lakes

In 1985, sampling at 250 stations throughout the St. Marys, St. Clair, and Detroit rivers and Lake St. Clair — the connecting channels of the upper Great Lakes — revealed widespread metal contamination of the sediments. Concentrations of cadmium, chromium, copper, lead, mercury, nickel, and zinc each exceeded U.S. Environmental Protection Agency sediment pollution guidelines at one or more stations throughout the study area. Sediments were polluted more frequently by copper, nickel, zinc, and lead than by cadmium, chromium, or mercury. Sediments with the highest concentrations of metals were found (in descending order) in the Detroit River, the St. Marys River, the St. Clair River, and Lake St. Clair. Although metal contamination of sediments was most common and sediment concentrations of metals were generally highest near industrial areas, substantial contamination of sediments by metals was present in sediment deposition areas up to 60 km from any known source of pollution.Contribution 735 of the National Fisheries Research Center-Great Lakes, U.S. Fish and Wildlife Service, 1451 Green Road, Ann Arbor, MI 48105.     

Effects of modified nutrient concentrations and ratios on the structure and function of the native phytoplankton community in the Neuse River Estuary, North Carolina, USA

A variety of analyses were used to assess the structure (community composition) and function (assimilation number, nitrogen fixation) of phytoplankton in the Neuse River Estuary (NRE), NC under ambient and modified nutrient concentrations. Dilution bioassays were employed to reduce the concentration of nitrogen (N) or both N and phosphorus (P) and thus compare varied DIN:DIP ratios. Experimental manipulations created conditions that may result from mandated N load reductions to the estuary. We hypothesized that unilateral reduction of N loading to the NRE would increase the activity, abundance and diversity of N₂ fixing cyanobacteria. Changes in phytoplankton primary productivity, N₂ fixation (nitrogenase activity), genetic potential for N₂ fixation (presence of nifH), phytoplankton taxonomic composition (diagnostic photopigment concentration) and abundances of N₂ fixing cyanobacteria (microscopy) were determined. Decreasing ambient DIN:DIP ratios in NRE samples resulted in increased rates of N₂ fixation when seed populations were present and environmental conditions were amenable. Decreasing the DIN:DIP ratio did not lead to an increase in the abundance or diversity of N₂ fixing cyanobacteria. Because N₂ fixing cyanobacteria were only actively fixing nitrogen during periods of low riverine N discharge (summer and early autumn), lowering nutrient ratios may not have a major impact on the NRE. However, the maximum potential amount of N from N₂ fixation was calculated using rates from this study and was found to be approximately 3% of total riverine loading of N to the NRE. Because N₂ fixation occurs farther downstream and later in the year than riverine N loading to the NRE, there is potential for N₂ fixation to modify N dynamics. Analyses of the phytoplankton community as a whole in these relatively short term experiments indicated that reduced DIN:DIP may not have a major impact on their structure and function.     

Large-scale terrestrial gastropod community composition patterns in the Great Lakes region of North America

Abstract: Previous ordination studies of land snail community composition have been limited to four or fewer habitat types from sites separated by no more than 300 km. To investigate the nature of large-scale patterns, North American land snail assemblages at 421 sites, representing 26 habitat types and covering a 1400 × 800 km area, were ordinated using global, nonmetric multi-dimensional scaling (NMDS). These data were then subjected to model-based cluster analysis and kmeans clustering to identify the main compositional groups and most important environmental covariables. Six primary compositional groups were identified. Three of these largely represent upland forest and rock outcrop sites, while the remaining largely represent either lowland forest, lowland grassland or upland grassland habitats. The geographical location and moisture level of sites also influences community composition. A strong compositional difference exists between sites having duff vs. turf soil surface layers. Only 8% of sites were improperly classified when soil surface architecture was used as the sole predictor variable. Fully 43% of taxa exhibited significant preferences towards one of these surface types, while only 15% of relatively common (10 + occurrence) taxa showed no preferences. Twelve groups of closely related taxa within the same genus had members that favoured different surface types, indicating that differential selection pressures have existed over evolutionary time scales. While turf faunas appeared unaffected by anthropogenic disturbance, duff faunas were strongly impacted, suggesting that their conservation will require protection of soil surface architecture.     

Hot summer temperatures may stop movement of Papilio canadensis butterflies and genetic introgression south of the hybrid zone in the North American Great Lakes Region

The reasons that the northern tiger swallowtail butterfly Papilio canadensis does not move south of the Great Lakes hybrid zone, where it meets P. glaucus , may be largely due to natural temperature-induced stress on diapausing pupae. Temperatures of 36°C for only four days killed all P. canadensis (from northern Michigan) and most of the Papilio troilus , a species that lives south of the hybrid zone (sympatrically with P. glaucus ). In contrast, interspecific hybrids ( P. glaucus mother× P. canadensis father) had significant adult eclosion or pupal survival at both 30° and 36°C. All surviving hybrid pupae were only females (the heterogametic sex, which are known to express the prolonged diapause due to Haldane effects).
The southern species, P. troilus , had almost all (81%) normal (non-deformed) adults eclose at 30°C, whereas only 20% of the P. canadensis females and ca 30% of the P. canadensis males eclosed to produce non-deformed adults. Unlike the case with P. troilus and P. canadensis , no hybrid females eclosed at 30°C (only males did). Unlike P. troilus and P. canadensis pupae, hybrid (female) pupae remain viable, some of which have already successfully emerged after the chamber experiments.
A follow-up study using P. glaucus , P. canadensis (from Vermont), and their hybrids with more normal lower thermal regimes included (27°, 30°, 33°, and 36°C) again showed higher hybrid survival as uneclosed (living) pupae at 36°C. In addition, P. glaucus and P. canadensis showed high mortality and wing deformity of eclosing adults at 36°C, suggesting that geographic source of the P. canadensis may reflect differential tolerances of the extreme 36°C temperature.     

Ecosystem integrity in the Great Lakes Basin: an historical sketch of ideas and actions

The concepts of ecosystem and integrity effectively entered the binational political arena in the Great Lakes Basin in the early 1970's. They were brought together explicitly in the statement of the purpose of the 1978 Great Lakes Water Quality Agreement. The 1987 Protocol to that Agreement has helped to specify the practical meaning of ecosystem integrity of the Great Lakes Basin. The proceedings of a binational workshop in 1988, titled An Ecosystem Approach to the Integrity of the Great Lakes Basin in Turbulent Times, helped to clarify the conceptual meaning. An Ecosystem Charter for the Great Lakes-St. Lawrence River Basin was proposed in 1989 to help achieve more thorough implementation of the commitment to ecosystem integrity. The evolutionary emergence of this political concept and related practice is described in the paper.List of abbreviations used in the text CUSIS Canada-U.S. Inter-University Seminar Series - LAA Environmental Lakes Area - EPA Environmental Protection Agency - GLBC Great Lakes Basin Commission - GLC Great Lakes Commission - GLER Great Lakes Ecosystem Rehabilitation - GLFC Great Lakes Fishery Commission - GLSAB Great Lakes Science Advisory Board - GLU Great Lakes United - GLWQA Great Lakes Water Quality Agreement - HASP Heritage Area Security Plan - IBP International Biological Program - IJC International Joint Commission - IZAP Inundation Zone Adaptive Plan - LAMP Lake-wide Management Plan - LLRS Lake Levels Reference Study - MAB Man and the Biosphere program - NEPA National Environmental Policy Act - PLUARG Pollution from Land Use Activities Reference Group - RAP Remedial Action Plan - SCOL Salmonid Communities in Oligotrophic Lakes - SGLFMP Strategic Great Lakes Fishery Management Plan - SPOF Strategic Plan for Ontario Fisheries - UNESCO United Nations Educational, Scientific and Cultural Organization Aquatic Ecosystem Health and Management Society. Symposium at the University of Waterloo, July 23–25, 1990.     

The nearshore shunt and the decline of the phytoplankton spring bloom in the Laurentian Great Lakes: insights from a three-dimensional lake model

Dreissenid mussels have been hypothesized to cause selective decreases of phytoplankton in nearshore areas (nearshore shunt hypothesis) as well as the near-complete loss of the offshore phytoplankton spring bloom in some Laurentian Great Lakes. To evaluate whether mussels can reasonably be expected to mediate such changes, we extended the three-dimensional hydrodynamic-ecological model (ELCOM-CAEDYM) to include mussels as a state variable and applied it to Lake Erie (USA-Canada). Mussel-mediated decreases in mean phytoplankton biomass were highly sensitive to the assigned mussel population size in each basin. In the relatively deep east basin, mussels were predicted to decrease phytoplankton in both nearshore and offshore zones, even during periods of thermal stratification but especially during the spring phytoplankton maximum. Spatially, impacts were associated with mussel distributions but could be strong even in areas without high mussel biomass, consistent with advection from areas of higher mussel biomass. The results supported the nearshore shunt hypothesis that mussel impacts on phytoplankton should be greater in nearshore than offshore waters and also supported suggestions about the emerging importance of deep water offshore mussels. The results of this study provide an important insight into ecological role of mussels in lowering plankton productivity in some world’s largest lakes.     

Beta diversity and latitude in North American mammals: testing the hypothesis of covariation

Several hypotheses attempt to explain the latitudinal gradient of species diversity, but some basic aspects of the pattern remain insufficiently explored, including the effect of scales and the role of beta diversity. To explore such components of the latitudinal gradient, we tested the hypothesis of covariation, which states that the gradient of species diversity should show the same pattern regardless of the scale of analysis. The hypothesis implies that there should be no gradients of beta diversity, of regional range size within regions, and of the slope of the species-area curve. For the fauna of North American mammals, we found contrasting results for bats and non-volant species. We could reject the hypothesis of covariation for non-volant mammals, for which the number of species increases towards lower latitudes, but at different rates depending on the scale. Also, for this group, beta diversity is higher at lower latitudes, the regional range size within regions is smaller at lower latitudes, and z, the slope of the species-area relationship is higher at lower latitudes. Contrarily bats did not show significant deviations from the predictions of the hypothesis of covariation: at two different scales, species richness shows similar trends of increase at lower latitudes, and no gradient can be demonstrated for beta diversity, for regional range size, or for the slopes of the species-area curve. Our results show that the higher diversity of non-volant mammals in tropical areas of North America is a consequence of the increase in beta diversity and not of higher diversity at smaller scales. In contrast, the diversity of bats at both scales is higher at lower latitudes. These contrasting patterns suggest different causes for the latitudinal gradient of species diversity in the two groups that are ultimately determined by differences in the patterns of geographic distribution of the species.     

First occurrence of the North American white sucker Catostomus commersoni in Great Britain

The first specimen of white sucker Catostomus commersoni encountered in Great Britain is reported: a female of 320 mm s.l. , 710 g wet body weight, estimated age of 3 +, and ripe with eggs (≃21% of the body weight). Isoelectric focusing of a tissue sampSe was also undertaken.