Lime treatment and its effects on the chemistry and biota of hardwater eutrophic lakes

1. The main focus of this study was to investigate the effects of single and multiple moderate doses of lime (slaked lime, Ca(OH)₂, and/or calcite, CaCO₃) on eutrophic hardwater lakes. This information would contribute to strategies to manage phytoplankton and macrophyte biomass in eutrophic lakes.
2. Water chemistry and biota were monitored for up to 7 years after initial lime treatment and results were compared with reference systems.
3. Complementary studies investigated the effect of lime on macrophytes in ponds, irrigation canals and microcosm experiments.
4. When water pH was kept within its natural range (≤ 10), single and multiple lime applications to lakes and ponds controlled macrophyte biomass, without negatively affecting invertebrate communities.
5. Single lime treatments at moderate dosages of lakes and ponds resulted in variable and mostly temporary changes in chlorophyll a (chl a ) and phosphorus (P) concentration. Although sediment P release was reduced in single-dose lakes during the first winter following treatment, reductions appeared temporary.
6. Multiple treatments of lakes and ponds were effective at reducing both chl a and P concentrations over longer periods. Mean winter P release rate was also reduced after initial treatment.
7. In laboratory studies, sediment cores were incubated with eight different treatments to assess P release. Redox-sensitive treatments were no more effective at lowering total P concentration in overlying water than some redox-insensitive treatments. Lime reduced total P concentrations, but was not as effective as treatments with alum.
8. The use of lime in managing macrophyte and phytoplankton biomass in shallow, hardwater lakes and ponds may be preferable over other treatments, because lime is economical and non-toxic as long as pH is kept within a natural range.     

Engineering and biological approaches to the restoration from eutrophication of shallow lakes in which aquatic plant communities are important components

Engineering approaches (nutrient removal, sediment pumping, hypolimnion oxygenation, alum treatments) may be most appropriate to deep lakes where the aim of restoration from eutrophication is simply to reduce the production and crop of one component, the phytoplankton. They do not always give the desired results because the nutrient loading may only be reduced to a limited extent. There are additional problems in shallow lakes where change of state between community dominance (aquatic plants versus plankton) is wanted. Each community has powerful buffering mechanisms and biomanipulation may be essential to switch one state to another even with considerable nutrient reduction. For the phytoplankton-dominated community the buffers include the advantages of early growth, lower diffusion pathways for CO₂, overhead shading, and an absence of large cladoceran grazers. This later is because open-water shallow environments provide no refuges against predation for the large Cladocera which are both the most efficient grazers and the most favoured prey for fish. Restoration of aquatic plants may then require provision of refuges for the grazers. Different sorts of refuge are discussed using case studies of Hoveton Great Broad and Cockshoot Broad in the Norfolk Broadland.     

Limitations of using extracellular alkaline phosphatase activities as a general indicator for describing P deficiency of phytoplankton in Chinese shallow lakes

Extracellular phosphatase can be produced by phytoplankton to utilize organic phosphorus under phosphorus (P) deficiency. However, there is a controversy about its use as an indicator of P deficiency in natural phytoplankton community inferred by such an “induction–repression” mechanism. Size-fractionation of alkaline phosphatase activity (APA), soluble reactive phosphorus (SRP) concentration, algal density, and composition were determined in six Chinese shallow lakes ranking in gradient of trophic status, where a positive relationship between SRP concentration and algal density was observed. Enzyme-labeled fluorescence (ELF) method was used to localize phosphatase on cell membrane of algae. The so-called algal APA that associated with coarser particle (>3.0 μm) accounted for the largest part of total APA. Within a lake with lower SRP concentration, the “induction–repression” mechanism held true. Contrastingly, both algal and total APA were positively related to SRP concentration based on the data across all study lakes with statistical significance, which may be explained firstly by algal composition. The lakes with higher SRP concentration were dominated by diatoms and green algae, while they easily produced extracellular phosphatases as evidenced by ELFA labeling. In parallel, the lakes with lower SRP concentration were dominated by cyanobacteria, while it was never ELFA-positive; secondly, ELFA-positive dots or structures suggested that, in lakes with higher trophic status, attached bacteria or heterotrophic microorganisms could substantially contribute to extracellular phosphatases for hydrolyzing organophosphoric compounds but probably utilizing the organic moiety as an organic carbon source. This process simultaneously produces inorganic P, leading to the co-occurrence of high phosphate concentration and APA. So, the contributor of APA are complex, which may produce extracellular phosphatase species-specific or not exclusively for P nutrient and consequently make it difficult to normalize APA with the exact biomass estimators. Therefore, it is not reasonable to use APA, normalized or not, as a general indicator for describing P deficiency of phytoplankton in shallow lakes especially eutrophic ones.     

Laboratory-determined Phosphorus Flux from Lake Sediments as a Measure of Internal Phosphorus Loading

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) loading from lake sediments can account for a substantial portion of the total P load in eutrophic, and some mesotrophic, lakes. Laboratory determination of P release rates from sediment cores is one approach for determining the role of internal P loading and guiding management decisions. Two principal alternatives to experimental determination of sediment P release exist for estimating internal load: in situ measurements of changes in hypolimnetic P over time and P mass balance. The experimental approach using laboratory-based sediment incubations to quantify internal P load is a direct method, making it a valuable tool for lake management and restoration.Laboratory incubations of sediment cores can help determine the relative importance of internal vs. external P loads, as well as be used to answer a variety of lake management and research questions. We illustrate the use of sediment core incubations to assess the effectiveness of an aluminum sulfate (alum) treatment for reducing sediment P release. Other research questions that can be investigated using this approach include the effects of sediment resuspension and bioturbation on P release.The approach also has limitations. Assumptions must be made with respect to: extrapolating results from sediment cores to the entire lake; deciding over what time periods to measure nutrient release; and addressing possible core tube artifacts. A comprehensive dissolved oxygen monitoring strategy to assess temporal and spatial redox status in the lake provides greater confidence in annual P loads estimated from sediment core incubations.     

Effectiveness of Al,Ca, and Fe salts for control of internal phosphorus loading in shallow and deep lakes

Internal P loading can maintain high P concentrations and delay eutrophic lake recovery following abatement of external loading. Sediment P inactivation with Al salts has been shown to provide long-term (5–14 years) control of sediment P release; long-term effectiveness of Fe and Ca salts has not been reported. Al toxicity problems are possible unless pH is maintained in the 6–8 range. Vertical transport of hypolimnetic P is unlikely in small, deep, dimictic lakes (\-ZA₀ > 8), and effectiveness of P inactivation in lowering their mid-summer epilimnetic P has not been demonstrated. To date, P inactivation has been found to be most effective in improving trophic state in shallow, softwater, polymictic lakes where control of sediment P release affects the entire water column. Abatement of external loading, where necessary, is essential for a successful P inactivation treatment.     

Sediments,not plants,offer the preferred refuge for Daphnia against fish predation in Mediterranean shallow lakes: an experimental demonstration

1. Different behavioural responses of planktonic animals to their main predators, fish, have been reported from shallow lakes. In north temperate lakes, large‐bodied zooplankton may seek refuge from predation among macrophytes, whereas in subtropical lakes, avoidance of macrophytes has been observed. The prevalent behaviour probably depends on the characteristics of the fish community, which in Mediterranean lakes is typically dispersed in both the open water zone and in the littoral, as in temperate lakes, and is dominated by small size classes, as in subtropical lakes. 2. We performed ‘habitat choice’ experiments to test the response of Daphnia magna to predation cues at both the horizontal and vertical level by mimicking a ‘shallow littoral’ zone with plants and a ‘deeper pelagic’ zone with sediments. 3. Initial separate response experiments showed that natural plants, artificial plants and predation cues all repelled D. magna in the absence of other stimuli, while sediments alone did not trigger any significant response by D. magna. 4. The habitat choice experiments showed that, in the presence of predation cues and absence of plants, Daphnia moved towards areas with sediment. In the presence of both plants and sediments, Daphnia moved away from the plants towards the sediments under both shallow and deep water treatment conditions. 5. Based on these results, we suggest that Daphnia in Mediterranean shallow lakes avoid submerged macrophytes and instead prefer to hide near the sediment when exposed to predation risk, as also observed in subtropical shallow lakes. This pattern is not likely to change with water level alterations, a common feature of lakes in the region, even if the effectiveness of the refuge may be reduced.     

State of the art in the functioning of shallow Mediterranean lakes: workshop conclusions

Studies on shallow lakes from the north temperate zone show that they alternate between clear and turbid water states in response to control factors. However, the ecology of semi-arid to arid shallow Mediterranean lakes is less explored. Hydrological effects (e.g. water level fluctuations, water residence time) on major ions and nutrient dynamics and processes, and ecology of submerged macrophytes appear to have a crucial role for food webs in shallow Mediterranean lakes. Nutrient control may be of greater priority in eutrophicated warm shallow lakes than in similar lakes at higher latitudes. This will be relevant for the implementation of the European Water Framework Directive, and conservation and management of these ecosystems. Strong trophic cascading effects of fish resulting from dominance of omnivorous and benthivorous fish species, whose diversity is usually high, together with frequent spawning and absence of efficient piscivores, seem to be the reason for the lack of large-bodied grazers that could control phytoplankton. However, such effects may vary within the region depending on fish distribution and community. These factors need elaboration in order to allow shallow lake ecologists and managers to develop better restoration strategies for eutrophicated shallow Mediterranean lakes. Consequently, modifications for the implementation of the European Water Framework Directive for determining ecological status in shallow Mediterranean lakes appear to be necessary. Furthermore, the implications of climate warming may be even more challenging than in high latitude lakes since shallow lakes in the Mediterranean region are among the most sensitive to extreme climate changes. There is an urgent need for data on the ecology of shallow lakes in the region. An appeal is made for international cooperation, development of large-scale research and information exchange to facilitate this and a web-based discussion list has been implemented.     

Phosphorus transformations in the epilimnia of small humic forest lakes

Research on epilimnetic phosphorus transformations in lakes had led to the establishment of a generalised picture which is promoted in reviews and textbooks. However, it would be wrong to believe that this established view can be applied to all lakes. Certainly the transformations of phosphorus in the summer epilimnia of small, coloured, humic forest lakes in southern Finland show features which deviate markedly from the generally accepted paradigm. (1) The plankton appears to be limited simultaneously by P and N rather than being strongly P limited. (2) Particulate P is typically a rather small fraction of the total P. (3) Molybdate reactive P may be a large part of the total P, although much of this MRP is not free orthophosphate, especially in highly humic lakes. (4) Turnover times for PO₄-P are normally long (50–1000 minutes); turnover times appear to be longer in highly humic lakes. (5) Added 32 PO₄ passes rapidly to macrozooplankton, apparently with rapid turnover of zooplankton phosphorus. (6) Sedimentation of phosphorus from the typically shallow epilimnion appears high, but may be partially compensated by biological retrieval of phosphorus from the phosphorus-rich hypolimnion.     

Seasonal dynamics of macrophytes and phytoplankton in shallow lakes: a eutrophication‐driven pathway from plants to plankton?

1. Seasonal relationships between macrophyte and phytoplankton populations may alter considerably as lakes undergo eutrophication. Understanding of these changes may be key to the interpretation of ecological processes operating over longer (decadal‐centennial) timescales. 2. We explore the seasonal dynamics of macrophytes (measured twice in June and August) and phytoplankton (measured monthly May–September) populations in 39 shallow lakes (29 in the U.K. and 10 in Denmark) covering broad gradients for nutrients and plant abundance. 3. Three site groups were identified based on macrophyte seasonality; 16 lakes where macrophyte abundance was perennially low and the water generally turbid (‘turbid lakes’); 7 where macrophyte abundance was high in June but low in August (‘crashing’ lakes); and 12 where macrophyte abundance was high in both June and August (‘stable’ lakes). The seasonal behaviour of the crashing and turbid lakes was extremely similar with a consistent increase in nutrient concentrations and chlorophyll‐a over May–September. By contrast in the stable lakes, seasonal changes were dampened with chlorophyll‐a consistently low (<10–15 μg L^?1) over the entire summer. The crashing lakes were dominated by one or a combination of Potamogeton pusillus, Potamogeton pectinatus and Zannichellia palustris, whereas Ceratophyllum demersum and Chara spp. were more abundant in the stable lakes. 4. A long‐term loss of macrophyte species diversity has occurred in many shallow lakes affected by eutrophication. One common pathway is from a species‐rich plant community with charophytes to a species‐poor community dominated by P. pusillus, P. pectinatus and Z. palustris. Such compositional changes may often be accompanied by a substantial reduction in the seasonal duration of plant dominance and a greater tendency for incursions by phytoplankton. We hypothesise a slow‐enacting (10–100 s years) feedback loop in nutrient‐enriched shallow lakes whereby increases in algal abundance are associated with losses of macrophyte species and hence different plant seasonal strategies. In turn such changes may favour increased phytoplankton production thus placing further pressure on remaining macrophytes. This study blurs the distinction between so‐called turbid phytoplankton‐dominated and clear plant‐dominated shallow lakes and suggests that plant loss from them may be a gradual process.     

Ecosystem-level effects of re-oligotrophication and N:P imbalances in rivers and estuaries on a global scale

Trends and ecological consequences of phosphorus (P) decline and increasing nitrogen (N) to phosphorus (N:P) ratios in rivers and estuaries are reviewed and discussed. Results suggest that re-oligotrophication is a dominant trend in rivers and estuaries of high-income countries in the last two–three decades, while in low-income countries widespread eutrophication occurs. The decline in P is well documented in hundreds of rivers of United States and the European Union, but the biotic response of rivers and estuaries besides phytoplankton decline such as trends in phytoplankton composition, changes in primary production, ecosystem shifts, cascading effects, changes in ecosystem metabolism, etc., have not been sufficiently monitored and investigated, neither the effects of N:P imbalance. N:P imbalance has significant ecological effects that need to be further investigated. There is a growing number of cases in which phytoplankton biomass have been shown to decrease due to re-oligotrophication, but the potential regime shift from phytoplankton to macrophyte dominance described in shallow lakes has been documented only in a few rivers and estuaries yet. The main reasons why regime shifts are rarely described in rivers and estuaries are, from one hand the scarcity of data on macrophyte cover trends, and from the other hand physical factors such as peak flows or high turbidity that could prevent a general spread of submerged macrophytes as observed in shallow lakes. Moreover, re-oligotrophication effects on rivers may be different compared to lakes (e.g., lower dominance of macrophytes) or estuaries (e.g., limitation of primary production by N instead of P) or may be dependent on river/estuary type. We conclude that river and estuary re-oligotrophication effects are complex, diverse and still little known, and in some cases are equivalent to those described in shallow lakes, but the regime shift is more likely to occur in mid to high-order rivers and shallow estuaries.     

Iron:phosphorus ratio in surface sediment as an indicator of phosphate release from aerobic sediments in shallow lakes

Analysis of Danish lakes showed that both mean winter and mean summer concentrations of lake water total phosphorus in the trophogenic zone correlated negatively with the total iron to total phosphorus ratio (Fe:P) in surface sediments. No correlation was found between the water total phosphorus concentration and either the sediment phosphorus concentration alone or with sediment calcium concentration. The increase in total phosphorus from winter to summer, which is partly a function of net internal P-loading, was lowest in lakes with high Fe:P ratios in the surface sediment.A study of aerobic sediments from fifteen lakes, selected as representative of Danish lakes with respect to the sediment Fe and phosphorus content, showed that the release of soluble reactive phosphorus was negatively correlated with the surface sediment Fe:P ratio. Analysis of phosphate adsorption properties of surface sediment from 12 lakes revealed that the capability of aerobic sediments to buffer phosphate concentration correlated with the Fe:P ratio while the maximum adsorption capacity correlated with total iron. Thus, the Fe:P ratio may provide a measure of free sorption sites for orthophosphate ions on iron hydroxyoxide surfaces.The results indicate that provided the Fe:P ratio is above 15 (by weight) it may be possible to control internal P-loading by keeping the surface sediment oxidized. Since the Fe:P ratio is easy to measure, it may be a useful tool in the management of shallow lakes.     

Maximum growing depth of macrophytes in Loch Leven,Scotland, United Kingdom,in relation to historical changes in estimated phosphorus loading

Eutrophication is common in shallow lakes in lowland areas. In their natural state, most shallow lakes would have clear water and a thriving aquatic plant community. However, eutrophication often causes turbid water, high algal productivity, and low species diversity and abundance of submerged macrophytes. A key indicator of the ecological state of lake ecosystems is the maximum growing depth (MGD) of aquatic plants. However, few studies have yet quantified the relationship between changes in external phosphorus (P) input to a lake and associated variation in MGD. This study examines the relationship between these variables in Loch Leven, a shallow, eutrophic loch in Scotland, UK. A baseline MGD value from 1905 and a series of more recent MGD values collected between 1972 and 2006 are compared with estimated P loads over a period of eutrophication and recovery. The results suggest a close relationship between changes in MGD of macrophytes and changes in the external P load to the loch. Variation in MGD reflected the ‘light history’ that submerged macrophytes had been exposed to over the 5-year period prior to sampling, rather than responding to short term, within year, variations in water clarity. This suggests that changes in macrophyte MGD may be a good indicator of overall, long term, changes in water quality that occur during the eutrophication and restoration of shallow lakes.     

Persistent internal phosphorus loading during summer in shallow eutrophic lakes

Nutrient availability, in particular of phosphorus (P), is a key factor for the structure and functioning of shallow lakes, and not least the sediment plays an important role by acting as both a nutrient source and sink. We used 21 years of monthly mass balance and lake water data from six shallow (mean depth = 1.2–2.7 m) and fast flushed (mean hydraulic retention time = 0.6–2.6 months) eutrophic Danish lakes (mean summer P concentrations ranging from 0.09 to 0.61 mg/l) to investigate long-term trends in yearly and seasonal patterns of P retention. To one of the lakes, the external P input was reduced by 70% in the early 1990s, whereas none of the other lakes have experienced major changes in external P loading for more than 20 years. All lakes showed a distinct seasonal pattern with high P concentrations and typically negative P retention during summer (up to ?300% of the external loading from May to August). During winter, P retention was overall positive (up to 50% of the external loading from December to April). Internal P loading from the sediment delayed lake recovery by approximately 10 years in the lake with the most recently reduced external loading, but in all the lakes net release of P from the sediment occurred during summer. P release in the six lakes has not abated during the past decade, indicating that the sediment of eutrophic and turbid shallow lakes remains a net source of P during summer. The seasonal variations in P retention became more pronounced with increasing P levels, and retention decreased with increasing temperature, but increased if clear water conditions were established.     

Resuspension behaviour of aluminium treated lake sediments: effects of ageing and pH

Lake restoration with aluminium (Al) has been widely used in shallow lakes, but it is unknown how ageing of the Al floc affect resuspension behaviour, sediment stability and entrainment of Al and phosphorus (P). High primary production in shallow lakes can lead to high pH in the water column with a potential release of Al and P during resuspension events. A laboratory resuspension experiment at defined hydrodynamic conditions using a calibrated erosion chamber was performed with intact sediment cores (with intact vs. disturbed biofilm on the surface) from previously Al-treated shallow Lake Möllen, NE Germany. Newly applied Al reduced surface sediment stability, but ageing led to the same stability as untreated sediment within 2 months with an intact biofilm and within 4 months with a disturbed biofilm. Dissolved Al increased markedly at resuspension after 2 days and 2 weeks of ageing and with elevated pH (9–11) in the water. The Al floc were redissolved and dissolved Al increased with elevated pH and shear velocity. Dissolved P concentration was constantly low due to excess binding capacity of the Al floc. In conclusion, Al application to shallow lakes prone to resuspension and with a high production must be done in periods with less resuspension risk to allow 2–4 months time for floc stabilization. Otherwise, possible resuspension and high(er) pH may lead to elevated concentrations of dissolved Al in the water column.     

Alternate Stable States and the Shape of the Lake Trophic Distribution

Ecological theory applied to small and shallow lakes suggests that water clarity and primary productivity may be bimodal, reflecting alternate stable states. This hypothesis was tested using remote sensing data. We used estimated Secchi disk transparency derived from reflected light measured by Landsat satellite photosensors. Lake trophic state indices (TSI) were estimated from the transparency estimates. Because alternate stable state theory is typically applied to productive lakes, we predicted that planktonic primary productivity would be especially bimodal in the small and shallow eutrophic lakes of southern Wisconsin. We found that overall, trophic state for over 8000 lakes in Wisconsin was multimodal (at least bimodal). Frequency distributions for lake size categories appeared to be distinctly bimodal. The largest and smallest lakes for all of Wisconsin had significantly bimodal curves, and southern (more productive) lakes had a more distinct multimodal curve than northern lakes. These results support the basic predictions of alternate stable state theory. The significantly trimodal distribution for southern lakes suggests that they may exist in more than just two alternate stable states.     

The influence of anthropogenic shoreline changes on the littoral abundance of fish species in German lowland lakes varying in depth as determined by boosted regression trees

Residential development on lake shores is regularly associated with the conversion of natural littoral habitats to riprap, sheet piles, beaches, parks, or marinas. The subsequent loss of littoral vegetation induces a decline of structural diversity and impacts littoral fish communities. These impacts may be shaped by lake morphology. Using boosted regression trees (BRT) to relate fish abundance data from 57 north-east German lowland lakes to various factors characterizing trophic state, lake morphology, and shoreline development, we investigated the response of 11 fish species to shoreline development. The analyses revealed that mean depth followed by trophic level and shoreline length (SL) contributed most in explaining littoral fish abundance. BRT models built for deep and shallow lakes separately confirmed that primarily trophic level and SL influenced fish abundance but that littoral vegetation was relatively more important in deep compared to shallow lakes, indicating that the effects of shoreline development may be more pronounced in deep lakes where the littoral makes up a smaller proportion of the lake area as compared to shallow lakes. The BRT further demonstrated species-specific responses to shoreline degradation, indicating that the reliability of ecological quality assessments of lakes can be improved by applying separate metrics for individual species.     

A Comparison of Predictive Phosphorus Load-Concentration Models for Lakes

Lake models that predict phosphorus (P) concentrations from P-loading have provided important knowledge enabling successful restoration of many eutrophic lakes during the last decades. However, the first-generation (static) models were rather imprecise and some nutrient abatement programs have therefore produced disappointingly modest results. This study compares 12 first-generation models with three newer ones. These newer models are dynamic (time-dependent), and general in the sense that they work without any further calibration for lakes from a wide limnological domain. However, static models are more accessible to non-specialists. Predictions of P concentrations were compared with empirical long-term data from a multi-lake survey, as well as to data from transient conditions in six lakes. Dynamic models were found to predict P concentrations with much higher certainty than static models. One general dynamic model, LakeMab, works for both deep and shallow lakes and can, in contrast to static models, predict P fluxes and particulate and dissolved P, both in surface waters and deep waters. PCLake, another general dynamic model, has advantages that resemble those of LakeMab, except that it needs three or four more input variables and is only valid for shallow lakes.     

Chemical and Microbiological Processes as Regulators of the Exchange of Substances between Sediments and Water in Shallow Eutrophic Lakes

It is generally believed that excessive P release from lake sediments, i. e. internal P load, is only a problem in deep lakes with stagnant anaerobic bottom waters. However, substantial amounts of P can also be released from sediments in shallow, well-mixed lakes. The dynamics and magnitude of P release from sediments in these types of lakes are affected primarily by physical factors, such as seasonal variations in water temperature and year-to-year differences in water renewal. These factors, in turn, induce chemical and microbiological processes which regulate the exchange of substances between sediments and water. The fractional distribution of sedimentary P and the chelating capacity of the water are also important factors that can provide insights to the processes involved and their quantitative impact on the P status in shallow, eutrophic lakes.     

Phosphorus release from sediments in hardwater eutrophic lakes: the effects of redox-sensitive and -insensitive chemical treatments

1. Phosphorus (P) release from bottom sediments is an important source of nutrient enrichment in many lakes in sedimentary basins, such as those in western Canada. On the Boreal Plain, sediment P release is particularly strong during periods of seasonal anoxia.
2. In this study, the effects of reduction–oxidation (redox)-sensitive and -insensitive chemicals on P release were examined in sediment cores collected from three eutrophic lakes.
3. Contrary to expectations, redox-sensitive treatments were no more effective at lowering total phosphorus (TP) in sediment cores than some redox-insensitive treatments. Redox-sensitive treatments with FeCl₃ and FeCl₃ + O₂ reduced TP to 8 and 6%, respectively, of reference concentrations, whereas redox-insensitive treatments with alum or lime + alum reduced TP to 14% of reference concentrations. Lime and O₂ treatments reduced TP concentrations to 35 and 52% of reference concentrations, respectively.
4. The fraction of P that adsorbed and co-precipitated with iron and aluminium in the sediment cores was low (non-apatite phosphorus fractions < 5%), suggesting that P release was controlled by apatite solubility and bacterial metabolism.     

Nuclear stains with soluble metachrome metal mordant dye lakes. The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues.

Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe2+ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO4 sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control. Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe2+ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results. Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes. The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes. Benzil at pH 13, which prevents the beta-naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.