Formalin Extraction Research Articles

Earthworm species in native and planted forests in Brazil.

Over 150 species of earthworms are known from the Brazilian Atlantic rainforest, but many more are expected to live in this megadiverse biome. In the present study, we evaluated earthworm species occurrence and diversity in native and reforested areas in four National Forests in three Brazilian states: Três Barras National Forest (Santa Catarina), Irati and Piraí do Sul National Forests (Paraná) and Capão Bonito National Forest (São Paulo) using formalin and hand sorting methods. A total of 13 species were found, five exotic and eight natives (of which four were new, undescribed species), belonging to six genera and five families (Rhinodrilidae, Glossoscolecidae, Ocnerodrilidae, Benhamiidae, Megascolecidae). In general, higher number of earthworms were collected in Araucaria plantations, except in Capão Bonito where Pinus sp. plantations had higher abundance. Hand sorting also collected more species in all forests, so this should be the preferred sampling method to evaluate earthworm communities. Formalin extraction was efficient only for epi-endogeic earthworms, most of them exotic species. Exotic pine species plantations tend to reduce species richness and abundance, compared with native forests and Araucaria plantations, and substitution of native vegetation for pine plantations should consider potential negative effects on soil animal populations.

Earthworm populations in forestry plantations (Araucaria angustifolia, Pinus elliottii) and Native Atlantic forest in Southern Brazil compared using two sampling methods

Earthworm populations have been little studied in forest systems in South America, and the choice of appropriate sampling methods is an important factor to consider. Hence, the present study evaluated earthworm populations using two methods: handsorting of large soil monoliths (40 × 40 cm × 20 cm depth), and application of formalin (0.5%) to the soil surface. Worms were sampled in fifteen areas at the Embrapa Forestry Research Station, including native Atlantic forest fragments, and native (Araucaria angustifolia) or exotic (Pinus elliottii) tree species plantations. Eight species of earthworms were collected overall, including four native species (Urobenus brasiliensis, Andiorrhinus duseni, Fimoscolex nivae and Glossoscolex embrapaensis) and one exotic (M. schmardae) in low abundance, and three other exotics in higher abundance (Pontoscolex corethrurus, Amynthas gracilis and Amynthas corticis). Total earthworm density ranged from 11.5 to 102 individuals m−2 and biomass from 5.6 to 31.6 g m−2, with higher values being found in P. elliottii plantations. Handsorting was more effective than formalin for sampling earthworms, resulting in higher species richness, overall abundance and biomass (particularly of the endogeic P. corethrurus) in both Pine and Araucaria plantations. Formalin extraction was better for extracting epi-endogeic species (particularly M. schmardae). Eveness and A. corticis densities were higher in native forests, while A. gracilis abundance was higher in Araucaria plantations and P. corethrurus predominated in both plantations, likely due to soil-related differences between the vegetation types. Earthworm population assessments in Brazilian forest systems should prioritize handsorting, while biodiversity inventories may benefit from combining handsorting with formalin extraction.

A rapid extraction method for glycogen from formalin-fixed liver.

Liver glycogen, a highly branched polymer, acts as our blood-glucose buffer. While past structural studies have extracted glycogen from fresh or frozen tissue using a cold-water, sucrose-gradient centrifugation technique, a method for the extraction of glycogen from formalin-fixed liver would allow the analysis of glycogen from human tissues that are routinely collected in pathology laboratories. In this study, both sucrose-gradient and formalin-fixed extraction techniques were carried out on piglet livers, with the yields, purities and size distributions (using size exclusion chromatography) compared. The formalin extraction technique, when combined with a protease treatment, resulted in higher yields (but lower purities) of glycogen with size distributions similar to the sucrose-gradient centrifugation technique. This formalin extraction procedure was also significantly faster, allowing glycogen extraction throughput to increase by an order of magnitude. Both extraction techniques were compatible with mass spectrometry proteomics, with analysis showing the two techniques were highly complementary.

Earthworm populations in septic system filter fields and potential effects on wastewater renovation

Wastewater renovation in septic-system filter fields can be affected by preferential flow through soil macropores. Anecic earthworm species make deep vertical burrows that may reduce renovation by acting as preferential flow paths that decrease effluent contact with the soil matrix. On the other hand, endogeic earthworms make largely horizontal burrows that may enhance wastewater renovation by distributing the effluent over a larger area. Additionally, the moist, nutrient-rich environment in filter fields may increase earthworm populations by enhancing their survival. Therefore, our objectives were to determine earthworm numbers and biomass with distance from soil treatment trenches, and identify species present to estimate potential effects on wastewater renovation. Five septic systems were investigated. At each site, earthworm populations were measured using formalin extraction at 10 locations along each of three 7-m long transects perpendicular to the trenches. There were an average of 6.4 times more earthworms and 5.4 times more earthworm biomass within 1 m of the trench than in the background (3.5–7.0 m from the trenches) in 13 of the 15 transects. This suggests that earthworms may have a significant effect on the movement of effluent. Because only epigeic and endogeic species were observed, the potential for reduced renovation and groundwater contamination at these sites is likely low. This may not be the case in areas with large numbers of anecic earthworms.

Effect of reduced tillage systems on earthworm communities in a 6-year organic rotation

Chisel plough or no-till practises are clearly attributed to sustainable farming for the support of soil organisms and soil biological functioning. However, in organic farming these techniques are only applied to a very small extent since there is a need for cultivation to control weeds. In organic farming it is important to develop and establish less destructive soil tillage practises to benefit from the support of soil organisms, especially from ecosystem engineers like earthworms. In this study three tillage systems were compared on a silty, loess' derived soil in a 6 crop rotation. The tillage systems established were: (1) “ecomat” shallow (10 cm) inversive ploughing; (2) a ridge culture system; and (3) conventional tillage (inversion tillage) down to 30 cm. In fall 2005, three years after establishment of the field plots earthworm communities were investigated using formalin extraction. In total 6 species were detected. The earthworm abundance was low, ranging from 0 to 84 individuals m −2. Shallow ploughing using the “ecomat” technique was not different than conventional ploughing in its effects on earthworm populations, while the ridge culture system was significantly reduced in earthworm numbers and biomass compared to conventional ploughing. We conclude that both “ecomat” and “ridge culture” do not support earthworms and significantly reduce earthworm densities. The reductions in tillage depth alone are not effective for improving the habitat quality for earthworms in soil.

Indirect gradient analysis at different spatial scales of prorated and non-prorated earthworm abundance and biomass data in temperate agro-ecosystems

We investigated how earthworm communities of agricultural systems vary in abundance, biomass and species composition at different spatial scales. In four farms representative of the main agro-ecological regions of Belgium 14 parcels were sampled using a combined method (hand sorting after formalin extraction). Parcels in both grassland and arable land were studied. In each parcel two or three sample plots (1 m 2) were randomly selected; these were further split up in two, resulting in four or six 0.5 m 2 subplots per parcel. Principal component analysis was applied to earthworm numbers and biomass. Innovative was that we compared the results from data matrices with raw data with prorated matrices, where unidentified juveniles were added to species following certain rules. To test the degree of variability between the different spatial levels, average distances between subplots, plots and parcels based on ordination scores were compared. A general linear model with a nested structure for subplots, plots, and parcels was built to find absolute differences between farms. Earthworm numbers ranged from 10 to 463 individuals m –2, biomass varied between 3 and 186 g m –2 and species numbers ranged from one to seven per parcel. Ordinations separated earthworm communities along a geographical—mainly soil—gradient on the first axis and distinguished between land uses along the second axis. Both in numbers of individuals and in biomass differences in earthworm populations increased from subplot to parcel level. As expected, differences within a pair of subplots were small. Plots within the same parcel tended to be highly similar, but in some cases large dissimilarities were encountered, reflecting a clustered population structure of earthworm communities at the field scale, and, possibly, abiotic gradients. The largest differences were observed at the parcel level. The latter may be attributed to differences in land use. Prorating had a narrowing effect on the data through which differences became less pronounced.

Population dynamics of ambient and altered earthworm communities in row-crop agroecosystems in the Midwestern U.S.The 7th international symposium on earthworm ecology · Cardiff · Wales · 2002

Summary Earthworms affect agroecosystem processes and few studies have addressed population dynamics when earthworms are intentionally introduced. Handsorting and formalin extraction were used semi-annually from fall 1994 to fall 1997 to measure populations in plots with and without added earthworms under chisel till in a corn-soybean rotation (CT) and a ridge-till system in a corn-soybean-wheat rotation (RT) in Ohio, USA. Earthworm communities were altered by adding ∼76 Lumbricus terrestris (L.) m –2 each spring and fall into plots with no, or very few of these anecic earthworms. Increases in L. terrestris were small (∼7 m –2 ) compared to the number added and their establishment was at the expense of the epigeic earthworm L. rubellus (Hoff.), which declined four and two-fold in CT and RT, respectively. Populations of the endogeic earthworm Octolasion tytaeum (Sav.) were unaffected by the addition of L. terrestris . Sampling 5 years after the additions indicated that L. terrestris persisted only in the RT plots. The decline in L. rubellus populations in plots with added anecic worms was no longer evident. We understand that the increased number and amounts of residues and the minimal level of disturbance found in RT probably increased the extent and quality of earthworm habitat over CT plots, which had fewer crops, less cover, and periodic disturbance. Apparently, population dynamics and competitive relationships among different earthworm functional groups were regulated by agroecosystem type, and their correspondent disturbance regimes and resource distributions.

Population dynamics of ambient and altered earthworm communities in row-crop agroecosystems in the Midwestern U.S.: The 7th international symposium on earthworm ecology · Cardiff · Wales · 2002

Earthworms affect agroecosystem processes and few studies have addressed population dynamics when earthworms are intentionally introduced. Handsorting and formalin extraction were used semi-annually from fall 1994 to fall1997 to measure populations in plots with and without added earthworms under chisel till in a corn-soybean rotation (CT) and a ridge-till system in a corn-soybean-wheat rotation (RT) in Ohio, USA. Earthworm communities were altered by adding ∼76 Lumbricus terrestris (L.) m - 2 each spring and fall into plots with no, or very few of these anecic earthworms. Increases in L. terrestris were small (∼7 m - 2 ) compared to the number added and their establishment was at the expense of the epigeic earthworm L. rubellus (Hoff.), which declined four and two-fold in CT and RT, respectively. Populations of the endogeic earthworm Octolasion tytaeum (Sav.) were unaffected by the addition of L. terrestris. Sampling 5 years after the additions indicated that L. terrestris persisted only in the RT plots. The decline in L. rubellus populations in plots with added anecic worms was no longer evident. We understand that the increased number and amounts of residues and the minimal level of disturbance found in RT probably increased the extent and quality of earthworm habitat over CT plots, which had fewer crops, less cover, and periodic disturbance. Apparently, population dynamics and competitive relationships among different earthworm functional groups were regulated by agroecosystem type, and their correspondent disturbance regimes and resource distributions.

Linking spatio-temporal dynamics of earthworm populations to nutrient cycling in temperate agricultural and forest ecosystemsThe 7th international symposium on earthworm ecology · Cardiff · Wales · 2002

Numerous laboratory and small plot-scale studies have highlighted the importance of earthworm populations in decomposition and nutrient cycling. However, the simple scaling up of such results to explain conditions on a large field scale, across agricultural landscapes under varied management scenarios, or across landscapes made up of different types of ecosystems (agricultural, grassland, forest), is very much constrained by a lack of information about the spatiotemporal distribution of earthworm populations. This study documents the spatiotemporal variation in earthworm populations in temperate ecosystems. A mixed deciduous forest, grass-dominated hayfield and corn agroecosystem were each fitted with a single 50 m × 50 m sampling grid, consisting of twenty-five 10 m × 10 m cells. Earthworms were collected at distinct single georeferenced locations in each of the 25 cells. Locations varied according to the sampling date (May, July or September). The earthworms collected by hand-sorting and formalin extraction, were first separated by species, then counted and weighed. The soil characteristics (temperature and moisture content, extractable nutrients, organic matter, pH) were also assessed for each time and location. Geostatistical and correlation analyses served to generate spatial maps of earthworm populations and to assess which soil factors most closely paralleled the numerical distribution of earthworms. This provided some insight into small-scale heterogeneity in earthworm populations, possibly allowing for the future extrapolation of laboratory and controlled field study results to larger scales, and in turn a more accurate estimation of the role of earthworms in nutrient cycling at the ecosystem and landscape levels.

Linking spatio-temporal dynamics of earthworm populations to nutrient cycling in temperate agricultural and forest ecosystems: The 7th international symposium on earthworm ecology · Cardiff · Wales · 2002

Numerous laboratory and small plot-scale studies have highlighted the importance of earthworm populations in decomposition and nutrient cycling. However, the simple scaling up of such results to explain conditions on a large field scale, across agricultural landscapes under varied management scenarios, or across landscapes made up of different types of ecosystems (agricultural, grassland, forest), is very much constrained by a lack of information about the spatiotemporal distribution of earthworm populations. This study documents the spatiotemporal variation in earthworm populations in temperate ecosystems. A mixed deciduous forest, grass-dominated hayfield and corn agroecosystem were each fitted with a single 50 m × 50 m sampling grid, consisting of twenty-five 10 m × 10 m cells. Earthworms were collected at distinct single georeferenced locations in each of the 25 cells. Locations varied according to the sampling date (May, July or September). The earthworms collected by hand-sorting and formalin extraction, were first separated by species, then counted and weighed. The soil characteristics (temperature and moisture content, extractable nutrients, organic matter, pH) were also assessed for each time and location. Geostatistical and correlation analyses served to generate spatial maps of earthworm populations and to assess which soil factors most closely paralleled the numerical distribution of earthworms. This provided some insight into small-scale heterogeneity in earthworm populations, possibly allowing for the future extrapolation of laboratory and controlled field study results to larger scales, and in turn a more accurate estimation of the role of earthworms in nutrient cycling at the ecosystem and landscape levels.

Earthworm communities in a wet agricultural landscape of the Seine Valley (Upper Normandy, France)The 7th international symposium on earthworm ecology · Cardiff · Wales · 2002

This study was carried out to describe the driving factors responsible for earthworm community structure in a wet landscape of the Seine Valley (Upper Normandy, France). Fifteen plots were selected along a 1300 m transect, representing the main agroecosystems (i.e. wooded systems, orchards, grasslands, crops) and soil types (i.e. Fluviosol, Reductisol, Histosol and Colluviosol) in the area. Five samples were taken in each plot using a combination of formalin extraction and hand sorting. Earthworms were identified to species level, counted and weighed. Communities responded markedly to agricultural intensification. The highest mean biomass, density and diversity were observed in the medium intensity systems (up to 119.7 g fresh weight m –2 , 309.3 ind m –2 , 7.2 sp m –2 in orchards, respectively). Soil type only had significant effects on community composition and diversity. A principal component analysis was performed with broad descriptive parameters of communities. The first two axes accounted for 50.8% of the total inertia. It separated wooded systems (endogeics and epigeics dominant, low total density, biomass and diversity) from orchards and grasslands (endogeics and/or anecics dominant, high diversity and density) and crops (low diversity, biomass and density). The ordination of plots according to soil type segregated low-lying soils (endogeics and epigeics dominant, low diversity, high density) from better-drained soils (anecics dominant, high density and diversity). Agricultural activities and natural soil heterogeneity are environmental filters that shape community composition and structure at the landscape scale. In the present study, soil type mainly determined earthworm community composition at the scale of soil type patches, while agricultural practices had a stronger impact on community structure at the scale of the agricultural plot.

Earthworm communities in a wet agricultural landscape of the Seine Valley (Upper Normandy, France): The 7th international symposium on earthworm ecology · Cardiff · Wales · 2002

This study was carried out to describe the driving factors responsible for earthworm community structure in a wet landscape of the Seine Valley (Upper Normandy, France). Fifteen plots were selected along a 1300 m transect, representing the main agroecosystems (i.e. wooded systems, orchards, grasslands, crops) and soil types (i.e. FLUVIOSOL, REDUCTISOL, HISTOSOL and COLLUVIOSOL) in the area. Five samples were taken in each plot using a combination of formalin extraction and hand sorting. Earthworms were identified to species level, counted and weighed. Communities responded markedly to agricultural intensification. The highest mean biomass, density and diversity were observed in the medium intensity systems (up to 119.7 g fresh weight m - 2 , 309.3 ind m - 2 , 7.2 sp m - 2 in orchards, respectively). Soil type only had significant effects on community composition and diversity. A principal component analysis was performed with broad descriptive parameters of communities. The first two axes accounted for 50.8 % of the total inertia. It separated wooded systems (endogeics and epigeics dominant, low total density, biomass and diversity) from orchards and grasslands (endogeics and/or anecics dominant, high diversity and density) and crops (low diversity, biomass and density). The ordination of plots according to soil type segregated low-lying soils (endogeics and epigeics dominant, low diversity, high density) from better-drained soils (anecics dominant, high density and diversity). Agricultural activities and natural soil heterogeneity are environmental filters that shape community composition and structure at the landscape scale. In the present study, soil type mainly determined earthworm community composition at the scale of soil type patches, while agricultural practices had a stronger impact on community structure at the scale of the agricultural plot.

Appraisal of the electrical octet method for estimating earthworm populations in arable land

SummaryQuantitative methods are needed for the assessment of the size and composition of earthworm communities. A poorly documented electrical sampling method, Thielemann's octet method, was compared with two long‐established methods, formalin extraction and soil hand sorting, in conventional and direct‐drilled wheat cropping systems at two sites with medium to heavy textured soils in Ireland. Under all agronomic conditions tested, the electrical method extracted significantly higher earthworm numbers than formalin, but earthworm biomasses were not significantly different. When used routinely over two years during periods of high earthworm activity, the electrical method yielded community estimates that were comparable in both size and species composition to those obtained by soil hand sorting (25 cm depth), except in recently ploughed land. However,Murchieona minuscule, a minute endogeic species, was underestimated by electrical extraction. It is concluded that the electrical octet method can be a reliable and useful alternative to other dynamic methods for estimating earthworm populations, especially in situations where minimum soil disturbance is desirable.

Earthworm contribution to infiltration and surface runoff after 15 years of different soil management

The contribution of earthworm burrows to infiltration and surface runoff was investigated in a 15-year-old tillage experiment. The tillage treatments were autumn mouldboard ploughing, autumn stubble cultivation and spring stubble cultivation, each replicated four times. At the time of the study, all treatments had been left untilled following the harvest. Earthworms were sampled by combined formalin extraction and hand-sorting. Undisturbed soil samples taken from formalin-sampled areas were subjected to simulated rainfall treatments in the laboratory, and percolating water and surface runoff were collected. After the rainfall treatments the saturated hydraulic conductivity ( K SAT) of each sample was measured, and a dye was applied to study the preferential pathways of soil water. The earthworm fauna consisted of the dominant Aporrectodea caliginosa and clearly less abundant Lumbricus terrestris and L. rubellus. The only discernible difference between the tillage treatments was that L. terrestris was present only in unploughed soils, although in very low numbers. During the rains, there were no significant differences between the treatments in volumes of percolated water, but there was significantly less runoff from spring stubble-cultivated soil than from autumn-tilled soils. K SAT was also significantly higher in the spring stubble-cultivated soil. K SAT was positively related to the volume of percolation, and negatively to the volume of surface runoff collected during the second rain. Total areas of stained earthworm burrows at the depths of 20 and 30 cm were correlated positively with K SAT.

Relative abundance and seasonal activity of earthworms (Lumbricidae and Megascolecidae) as determined by hand-sorting and formalin extraction in forest soils on the southern Appalachian Piedmont

The relative efficiency of different sampling methods for earthworms may vary with site characteristics, season and earthworm species. We conducted a study in which earthworms were collected by hand-sorting and by formalin extraction in a successional, mixed hardwood-pine forest. The efficiency of each collecting technique was assessed in terms of seasonal trends, soil physical properties and species collected. Earthworm community age structure and seasonal temperature and moisture trends were the factors most integral to the efficiency of each technique. Results indicate that for the part of the year in which small juvenile earthworms were most abundant (November-May), hand-sorting was far superior to formalin extration. However, for the remainder of the year, the efficiency of the two techniques did not vary significantly. The formalin extraction method was more effective than hand-sorting for the collection of Lumbricus terrestris L., but not for any other species. Soil physical properties such as pH and texture, while important in distributional patterns of earthworms, did not have a significant effect on the efficiency of the collection techniques.

Population dynamics of the earthworm Amynthas alexandri (Megascolecidae: Annelida) in cultivated soils of the Kumaun Himalayas

The population dynamics of the earthworm Amynthas alexandri was observed over 2 years in cultivated soils at Chanfi and Khurpatal near Nainital. Both hand sorting and formalin extraction were used to sample earthworms from the top 20 cm of soil. The density and biomass of earthworms fluctuated substantially during the year. Maximum density (58.4 m −2) and biomass (19.6 g m −2) were recorded in the rainy period and minimum during the dry period. Within the top 20 cm, 51–69% of the total numbers of earthworms were collected from the 0–10 cm soil layer and the proportion of biomass in the same layer was 51–83%. Most (80–90%) of the worms collected were clitellate. The C: N ratio of the soil decreased with increasing depth in both localities.

Detoxification of jack beans (Canavalia ensiformis): I.—Extrusion and canavanine elimination

AbstractIn order to remove canavanine from jack bean (Canavalia ensiformis) the authors conducted extrusion and extraction experiments. In the extrusion experiments, using jack bean without testa and conditioned to 240 g kg−1 moisture content, different temperatures of the compression section of the extruder and screw rates were tried. Under these conditions the parameters measured were expansion, final moisture, haemagglutinins, urease activity, available lysine and soluble nitrogen index. The screw rate did not affect the extrusion of jack bean. The best temperature was 110°C. The extraction experiments were carried out with extruded jack bean (EJB) at 110°C and 70 rpm using one or two acid or formalin extractions. EJB amount, times and pH were evaluated with a surface response model in one acid extraction experiment. The best results were observed with 100 g kg−1 of EJB, at pH 55 for 2 h (1AEJB), which reduced the canavanine content to 8.5 g kg−1. In the same conditions using a second acid extraction (2AEJB) we obtained a product with 4.9 g kg−1 of canavanine. With the formalin extraction at pH 5.5 and 7.5, and a molar ratio of canavanine: formalin 1 : 1 and 1 : 2, we obtained a product with 8.2 g kg−1 of canavanine.

Species composition and seasonal variation of earthworms and their effect on soil properties in southern Ontario, Canada

Four never cultivated, well drained forest sites representing the range of soil textures in the Lake Erie Basin of southern Ontario (approx. 42°N, 80°W) were characterized monthly during the growing season as to their earthworm populations, using the formalin extraction technique. Earthworm densities and biomasses ranged from 5 to 114 m −2, and 0.5 to 11 g dry wt m −2 respectively. There were a total of six lumbricid species at the four sites, but the anecic species, Lumbricus terrestris (L.) was the dominant contributor to faunal biomass at three of the sites. Earthworms at the fourth site were two small endogeous species that had the lowest abundance and biomass for earthworms of the four sites. The influence of faunal activity on soil properties was measured by the 137Cs (originating as fallout from atmospheric nuclear tests) distribution within 1 cm thick layers to a depth of 15 cm. Organic matter and 137Cs distribution with depth were functions of both earthworm abundance and species. Sites with anecic earthworms had 137Cs distributed throughout the surface horizons, whereas the site with endogeous species had higher 137Cs levels remaining at or near the soil surface.

Impact of landspread sewage sludge and earthworm introduction on established earthworms and soil structure

Sewage sludge was applied to twelve 4-m2 plots in two forest (mixed hardwood, Norway spruce plantation) site and one old field site. The earthworm Eisenia fetida was introduced to half the control and half the treated plots. Earthworm populations were sampled by formalin extraction and hand-sorting five times in the year following treatment. One year after treatment, soil samples were wet-sieved and water-stable aggregate size-class arrays were determined.

Experimental Manipulation of Soil Invertebrate Populations for Trophic Studies

The experimental application of pesticides is developed for manipulation of populations of soil microarthropods, earthworms, and flora to provide controlled populations of soil invertebrates for studies of their role in litter and soil turnover. The technique employs a soil fumigrant (dichloropropene,D—D), an insecticide (aldrin), and an earthworm extractant (formalin) for elimination of various biotic groups. D—D fumigation effectively eliminated earthworms, and reduced microarthropod populations by 98.3%. With periodic application of aldrin to fumigated soil, recolonization by microarthropods was held to 6.5% of control densities after 8 months. Formalin extraction of earthworms remained 80% efficient 6 months after presents the methods of treatment, subsequent population changes, and rates of recolonization of soil by specific invertebrate groups.