The metabolism of organic matter in the hyporheic zone of a mountain stream, and its spatial distribution

Community respiration in hyporheic sediments (HCR) was studied in a characteristic riffle-pool-sequence of a mountain stream. HCR activity at the riffle site strongly exceeded that at the corresponding pool site with a mean ratio of 5.3. The vertical distribution of HCR activity was homogeneous in the pool, while there was a distinct maximum in the uppermost layer in the riffle. Similarly, the spatial distribution of certain fractions of particulate organic matter (POM), and their turnover, was largely determined by stream morphology. Mean annual HCR per unit area of stream bed was estimated as 1.71 g O₂ m⁻² d⁻¹. Hence, HCR contributes significantly to total heterotrophic activity in streams, thus enhancing the relative importance of heterotrophic processes in running waters containing hyporheic zones.     

Flow-substrate interactions create and mediate leaf litter resource patches in streams

1. The roles that streambed geometry, channel morphology, and water velocity play in the retention and subsequent breakdown of leaf litter in small streams were examined by conducting a series of field and laboratory experiments. 2. In the first experiment, conditioned red alder (Alnus rubra Bongard) leaves were released individually in three riffles and three pools in a second‐order stream. The transport distance of each leaf was measured. Several channel and streambed variables were measured at each leaf settlement location and compared with a similar number of measurements taken at regular intervals along streambed transects (‘reference locations’). Channel features (such as water depth) and substrate variables (including stone height, stone height‐to‐width ratio, and relative protrusion) were the most important factors in leaf retention. 3. In the second experiment, the role of settlement location and reach type in determining the rate of leaf litter breakdown was examined by placing individual conditioned red alder leaves in exposed and sheltered locations (on the upper and lower edges of the upstream face of streambed stones, respectively) in riffle and pool habitats. After 10 days, percent mass remaining of each leaf was measured. Generally, leaves broke down faster in pools than in riffles. However, the role of exposure in breakdown rate differed between reach types (exposed pool > sheltered pool > sheltered riffle > exposed riffle). 4. In the third experiment, the importance of substrate geometry on leaf litter retention was examined by individually releasing artificial leaves upstream of a series of substrate models of varying shape. Substrates with high‐angle upstream faces (were vertical or close to vertical), and that had high aspect ratios (were tall relative to their width), retained leaves more effectively. 5. These results show that streambed morphology is an important factor in leaf litter retention and breakdown. Interactions between substrate and flow characteristics lead to the creation of detrital resource patchiness, and may partition leaf litter inputs between riffles and pools in streams at baseflow conditions.     

Spatial heterogeneity in water velocity drives leaf litter dynamics in streams

1. Stream hydro-morphology refers to the heterogeneous distribution of hydrologic conditions that occur above a complex benthic surface such as a streambed.
2. We hypothesised that hydro-morphological conditions will influence the retention, re-distribution, and microbial-driven decomposition of leaf litter inputs in stream ecosystems because each process varies with overlying water velocity.
3. We tested this hypothesis using: (1) the spatial distribution of water velocity within a stream reach as a surrogate of stream hydro-morphology; (2) leaf tracer (i.e. Ginkgo biloba L.) additions with serial recovery to examine the relationship between benthic retention and overlying velocity; and (3) measurements of leaf litter decomposition (i.e. Alnus glutinosa [L.] Gaertn.) under different water velocity conditions.
4. Results demonstrate that water velocity exerts a significant influence on the retention and re-distribution of leaf litter inputs within the reach. The observed range of water velocity (from c. 0 to 0.92 cm/s) also strongly influences the range of leaf litter decomposition rates (0.0076–0.0222/day).
5. Our findings illustrate that water velocity influences leaf litter dynamics in streams by controlling leaf litter transport, retention and re-distribution as well as how leaves decompose within recipient stream reaches. Ultimately, the results show that the efficiency of leaf litter inputs in supporting stream ecosystem function is dependent on the hydro-morphological characteristics of the receiving stream ecosystems.

     

Colonization of leaf patches at topographically different locations by insect shredders in a small mountain stream

We examined physical constraints on the colonization of leaf patches by shredder individuals by comparing the colonizations of artificially standardized leaf patches placed at different locations within a stream reach (i.e., riffles, middles and edges of pools). Stonefly taxa (Nemoura, Protonemura) colonized riffle patches 2–10 times more often than pool (middle, edge) patches, whereas caddisfly taxa (two species of Lepidostoma, Nothopsyche) almost exclusively colonized pool patches. Colonization also differed between the middle and edge patches in pools for most taxa; it was 2–5 times greater in edge patches for Nemoura and in middle patches for Lepidostoma. The abilities of species to cope with low oxygen circulation and high shear stress appear to determine differences in colonization between riffle and pool patches, whereas species-specific dispersion behavior (e.g., return time from drift) may differentiate colonization between middle and edge patches in pools. Our results suggest that changes in leaf distribution within a reach can affect the suitability of stream reaches in terms of food acquisition for shredder individuals.     

Impact of highway construction on leaf processing in aquatic habitats of eastern Tennessee

Rates of leaf litter processing at eight sites were used in conjunction with other methods to evaluate the impact of highway construction on aquatic habitats. Monthly processing of white oak leaves from four different mesh size bags at unimpacted reference sites indicated that the three larger mesh sizes were useful for comparing sites, as they did not restrict invertebrate colonization and provided similar rates of processing within sites. Small mesh size (0.12 mm) prevented leaf shredding macroinvertebrates from colonizing bags, and caused significantly slower leaf processing in a riffle.Leaf processing was fastest in a reference riffle above an area of highway construction but was significantly slower in the reference pool due to low current velocity and the absence of shredders. Leaf processing in a riffle below the highway was slower than the reference riffle, and the number of shredders was reduced. Removal of streamside vegetation during highway construction caused increased stream temperatures and reduced the amount of natural leaf accumulations, thereby reducing shredder habitat. At other sites highway construction caused less of an impact than preexisting environmental influences. Leaf processing in an riffle of a lower pH stream was significantly slower than in the reference riffle. Shredders were absent from the low pH stream, and rates of leaf processing in the acid impacted riffle and pool were similar to those of the reference pool. Comparisons of the low pH stream with the reference stream indicated that physical processes were less important than biological processes of leaf decay. Leaf processing in ponds was similar to that of stream pools. Processing was faster in a small versus large pond, and a high density of invertebrates was associated with leaf bags in the small pond after one year.     

Spatial distribution and population dynamics of a benthic rotifer, Embata laticeps (Murray) (Rotifera, Bdelloidea) in the bed sediments of a gravel brook

• 1 The spatial and temporal distributions of the most abundant bdelloid rotifer, Embata laticeps (Murray), inhabiting the sediment surface and the hyporheic interstitial of a gravel stream were investigated between October 1991 and October 1992. Three temporal peaks of population density occurred during the year at the sediment surface differing in their riming from the density peaks detected within the bed sediments.
• 2 The depth distributions of E. laticeps in pool and riffle areas differed significantly. Higher abundances were found between 10 and 20 cm depth in the pool and between 20 and 30 cm in the riffle area. In these two sites a significant effect of surface discharge (estimated 1 week before sampling) on rotifer densities was found. Discharge greater than 0.424 m³s^?1 significantly reduced the densities in the riffle, while only at values greater than 1.660m³s^?1 were abundances influenced in the pool area.
• 3 The percentages of egg-bearing females were higher within the bed sediment than in the surface layer, and in the pool area than in the riffle. Thus, overall mean values of population increase were higher in the pool habitat (x?= 0.015 ± SE 0.029 day^?1) than in the riffle and at the sediment surface (x?= 0.004 ± 0.025 and 0.005 ± 0.026 day^?1, respectively). In addition, population growth fluctuated more strongly at the sediment surface than within the hyporheic intersiritial. Comparison of the observed values of population increase with a randomization test, where observed densities were randomly permuted, revealed no significant differences between those observed and values from a random model. Thus, a clear trend in population growth of E. laticeps over time could not be demonstrated in this gravel stream.

     

Do pools impede drift dispersal by stream insects?

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Importance of transient storage zones for ammonium and phosphate retention in a sandy-bottom Mediterranean stream

1. The ability of hyporheic sediments to exchange water and retain ammonium and phosphate in the Riera Major stream ,North-East Spain, under different discharge conditions was measured by conducting short-term nutrient and chloride additions. 2. The mean exchange coefficients from free-flowing water to the storage zone (k₁) and vice versa (k₂) were 0.82 × 10–4 s^??1 and 7 × 10^??3 s^??1, respectively. The ratio of storage zone cross-sectional area to stream cross-sectional area (A_S/A) averaged 2.8 × 10–2 and was negatively correlated with discharge (r = –0.85, d.f. = 13, P < 0.001). 3. The percentage of hyporheic zone water which came from surface water varied as a function of discharge and hyporheic depth, ranging between 33% and 95% at 25 cm depth, and between 78% and 100% at 10 cm depth. 4. The nutrient retention efficiency in the hyporheic zone at 10 cm depth measured as uptake length (S_wh) was less than 3.3 cm for ammonium and 37 cm for phosphate. Higher nutrient retentions were measured in the sediments at 10 cm depth than at 25 cm, indicating that near-surface sediments were involved more actively in phosphate retention than the deeper hyporheic sediments. The lack of ammonium at any depth of the hyporheic zone showed that ammonium was very rapidly taken up in the surfacial sediments.     

Essai d'interprétation des cinétiques d'échange isotopique entre C18O2 et eau d'une feuille: Expériences à l'obscurité

Summary The interpretation of photosynthesis experiments using ¹⁸O as a tracer becomes very difficult on account of the exchange rate between oxygen atoms of carbon dioxide and water. The mathematical analysis of the kinetics of isotope exchange between CO₂ highly enriched with ¹⁸O and the water of an aerial leaf kept in darkness allows the determination of two velocity coefficients, k _e and k. k _e, related to the gaseous CO₂ diffusion, makes it possible to evaluate the CO₂ flow between the gaseous phase and the leaf, as well as the diffusion resistance of the boundary layer and the leaf stomata. k, related to the CO₂ hydration, gives the in situ enzymic activity of carbonic anhydrase. Measurements carried out on a great number of leaves show that k _e and k vary in the same direction.In addition, a simple relation exists between the isotopic composition of the gaseous phase and the corresponding values of the C¹⁸O₂ inside the leaf. Thus it is possible to determine the conditions for a leaf that may be favourable for light experiments designed to ascertain the origin of photosynthetic oxygen by use of ¹⁸O.     

Reduced nitrogen allocation to expanding leaf blades suppresses ribulose-1,5-bisphosphate carboxylase/oxygenase synthesis and leads to photosynthetic acclimation to elevated CO<Subscript>2</Subscript> in rice

Net photosynthetic rate (P _N) measured at elevated CO₂ concentration (C _e), ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and nitrogen (N) content in rice leaves decreased significantly after exposure to long term C _e. The reduction in P _N, Rubisco, and leaf N at C _e was similar for the last fully expanded leaf blade (LFELB) and expanding leaf blade (ELB). Spatial leaf N content in the ELB was highest in the zone of cell division, sharply declined as cell expansion progressed and gradually increased with cell maturation. Maximum reduction in spatial leaf N and Rubisco content was found at C _e only within cell expansion and maturation zones. The spatial leaf N content correlated well with the amount of Rubisco synthesized during leaf expansion, suggesting that N deposition into the expanding leaf blade may be the key for Rubisco synthesis and possibly photosynthetic acclimation to C _e.     

Habitat use of Atlantic salmon Salmo salar parr in a dynamic winter environment: the influence of anchor‐ice dams

The effect of anchor‐ice dams on the physical habitat and behavioural responses of Atlantic salmon Salmo salar parr in a small, steep stream was investigated. Anchor‐ice dams formed periodically, leading to a dynamic winter environment as the study reach alternated between riffle and walk dominated habitat. Parr demonstrated large individual variation in habitat use, utilizing most of the wetted stream width, and were generally unaffected by diel changes in the mesohabitat composition. Furthermore, parr displayed high site fidelity in areas with low embedded substrata, and demonstrated few large movements between the three mesohabitat classes present: shallow riffle, walk and pool. Findings from this study question the importance of hydraulic variables such as water depth, flow velocity and dynamic ice formation as single habitat features for juvenile stream salmonids during winter and emphasize the importance of access to substratum cover.     

Epilithic bacterial and algal colonization in a stream run, riffle, and pool: a test of biomass covariation

Epilithic bacterial and algal biomass were compared among a run, riffle, and pool along an open-canopy section of a third-order, temperate stream. Epilithic biofilms were sampled after 3, 7, 14, 21, 28, and 35 days colonization on unglazed ceramic tiles that were attached to plastic trays (n = 3) placed across each of the three habitats (i.e., run, riffle, pool). The diverse habitats and sampling regime were selected to provide a range in algal biomass so that potential covariation between epilithic bacterial and algal biomass could be assessed. There were significant differences among habitats and among trays within each habitat for both chlorophyll a and AFDM. Chlorophyll a and AFDM increased in the run and pool throughout the colonization period. In the riffle, chlorophyll a and AFDM increased rapidly early in colonization, then decreased. Epilithic bacterial biomass increased rapidly with no significant differences among the three habitats throughout colonization. Further, bacterial biomass did not correlate with either chlorophyll a or AFDM in any of the three habitats or on any of the sampling days. These results suggest that epilithic algal and bacterial biomass may be regulated by independent controls in some stream environments.     

Nitrogen retention in the riparian zone of catchments underlain by discontinuous permafrost

1. Riparian zones function as important ecotones that reduce nitrate concentration in groundwater and inputs into streams. In the boreal forest of interior Alaska, permafrost confines subsurface flow through the riparian zone to shallow organic horizons, where plant uptake of nitrate and denitrification are typically high. 2. In this study, riparian zone nitrogen retention was examined in a high permafrost catchment (approximately 53% of land area underlain by permafrost) and a low permafrost catchment (approximately 3%). To estimate the contribution of the riparian zone to catchment nitrogen retention, we analysed groundwater chemistry using an end‐member mixing model. 3. Stream nitrate concentration was over twofold greater in the low permafrost catchment than the high permafrost catchment. Riparian groundwater was not significantly different between catchments, averaging 13 μm overall. Nitrogen retention, measured using the end‐member mixing model, averaged 0.75 and 0.22 mmol N m^?2 day^?1 in low and high permafrost catchments, respectively, over the summer. The retention rate of nitrogen in the riparian zone was 10–15% of the export in stream flow. 4. Our results indicate that the riparian zone functions as an important sink for groundwater nitrate and dissolved organic carbon (DOC). However, differences in stream nitrate and DOC concentrations between catchments cannot be explained by solute inputs from riparian groundwater to the stream and differences between streams are probably attributable to deeper groundwater inputs or flows from springs that bypass the riparian zone.     

Characterizing nitrogen dynamics, retention and transport in a tropical rainforest stream using an in situ15N addition

1. This study was part of the Lotic Intersite Nitrogen eXperiment (LINX); a series of identical ¹⁵NH₄ tracer additions to streams throughout North America. ¹⁵NH₄Cl was added at tracer levels to a Puerto Rican stream for 42 days. Throughout the addition, and for several weeks afterwards, samples were collected to determine the uptake, retention and transformation pathways of nitrogen in the stream. 2. Ammonium uptake was very rapid. Nitrification was immediate, and was a very significant transformation pathway, accounting for over 50% of total NH₄ uptake. The large fraction of NH₄ uptake accounted for by nitrification (a process that provides energy to the microbes involved) suggests that energy limitation of net primary production, rather than N limitation, drives N dynamics in this stream. 3. There was a slightly increased ¹⁵N label in dissolved organic nitrogen (DON) the day after the ¹⁵NH₄ addition was stopped. This DO¹⁵N was < 0.02% of DON concentration in the stream water at the time, suggesting that nearly all of the DON found in‐stream is allochthonous, or that in‐stream DON production is very slow. 4. Leptophlebiidae and Atya appear to be selectively feeding or selectively assimilating a very highly labelled fraction of the epilithon, as the label found in the consumers became much higher than the label found in the food source. 5. A large spate (>20‐fold increase in discharge) surprisingly removed only 37% of in‐stream fine benthic organic matter (FBOM), leaves and epilithon. The fraction that was washed out travelled downstream a long distance (>220 m) or was washed onto the stream banks. 6. While uptake of ¹⁵NH₄ was very rapid, retention was low. Quebrada Bisley retained only 17.9% of the added ¹⁵N after 42 days of ¹⁵N addition. Most of this was in FBOM and epilithon. Turnover rates for these pools were about 3 weeks. The short turnover times of the primary retention pools suggest that long‐term retention (>1 month) is minimal, and is probably the result of N incorporation into shrimp biomass, which accounted for < 1% of the added ¹⁵N.     

Environmental factors influencing the regional distribution and local density of a small benthic fish: the stoneloach (<Emphasis Type="Italic">Barbatula barbatula</Emphasis>)

We investigated the relationships between different environmental variables and the spatial distribution patterns of the stoneloach (Barbatula barbatula) at the stream system, the stream site, and the mesohabitat (riffle/pool) scales in south-western France. Stoneloach occurred at 240 sites (out of 554 sampling sites), chiefly close to the source, in areas at low elevation and with weak slopes. Population density at a site was primarily influenced by physical conditions. Stream width was positively related to the probability of presence of stoneloach within the stream system, but negatively related to local density. These results indicate that stoneloaches can occur in a wide range of streams, but they are less abundant in wide rivers, probably because of lower habitat heterogeneity. Slope was negatively correlated to both fish presence at the regional scale and local density, suggesting that stoneloach’s swimming performance were weak under greater erosive forces. These results suggested that the distribution of populations and the density of stoneloach were governed by the suitability of physical habitat. Multi-scale studies of factors influencing a species’distribution allow to integrate patterns observed at different scales, and enhance our understanding of interactions between animals and their environment. The use of few pertinent variables in successful final models could reduce the effort and cost of data collection for water management applications.     

Two lowland stream riffles – linkages between physical habitats and macroinvertebrates across multiple spatial scales

The physical structure of two riffles in a lowland Danish stream was studied and its importance for the composition and density of the macroinvertebrate communities was evaluated. The two riffles were visually assessed to be very similar, but measurements revealed that they differed in overall hydraulic conditions, stability, substratum composition and consolidation. Differences affected abundance of both burrowing and surface dwelling macroinvertebrates. The unstable unconsolidated riffle had higher total macroinvertebrate abundance (4137 m⁻² vs. 1698 m⁻²), diptera abundance (2329 m⁻² vs. 386 m⁻²) and total estimated species richness (31.7 vs. 28.8) as well as lower evenness (0.77 vs. 0.83) than the compact riffle. Among samples within the unconsolidated riffle, variations in macroinvertebrate communities were related to differences in mean substratum particle size. Here a linear log–log relationship existed between macroinvertebrate abundance, the abundance of EPT taxa and the median particle size (r ² _total = 0.46, p = 0.002; r ² _EPT = 0.73, p < 0.001). No similar relationships were evident on the consolidated riffle. Moreover, macroinvertebrate communities on the unconsolidated riffle were dominated by species with a high colonising potential. Despite being assessed to the same morphological unit, physical variation between riffles was surprisingly high as the riffles differed substantially with respect to consolidation, substratum heterogeneity and overall hydraulic structure. Macroinvertebrate community structure and composition also differed between riffles despite being drawn from the same species pool. The findings address the question if we use the correct methods and parameters when assessing the macroinvertebrate communities at the scale of the morphological unit.     

Urban catchment hydrology overwhelms reach scale effects of riparian vegetation on organic matter dynamics

1. Urbanisation severely affects stream hydrology, biotic integrity and water quality, but relatively little is known about effects on organic matter dynamics. Coarse particulate organic matter (CPOM) is a source of energy and nutrients in aquatic systems, and its availability has implications for ecosystem productivity and aquatic communities. In undisturbed environments, allochthonous inputs from riparian zones provide critical energy subsidies, but the extent to which this occurs in urbanised streams is poorly understood. 2. We investigated CPOM inputs, standing stocks, retention rates and retention mechanisms in urban and peri‐urban streams in Melbourne, Australia. Six streams were chosen along a gradient of catchment urbanisation, with the presence of reach scale riparian canopy cover as a second factor. CPOM retention was assessed at baseflow via replicate releases of marked Eucalyptus leaves where the retention distance and mechanism were recorded. CPOM and small wood (>1 cm diameter) storage were measured via cores and direct counts, respectively, while lateral and horizontal CPOM inputs were assessed using riparian litter traps. Stream discharge, velocity, depth and width were also measured. 3. CPOM inputs were not correlated with urbanisation, but were significantly higher in ‘closed’ canopy reaches. Urbanisation and riparian cover altered CPOM retention mechanisms, but not retention distances. Urban streams showed greater retention by rocks; while in less urban streams, retention by small wood was considerably higher. CPOM and small wood storage were significantly lower in more urban streams, but we found only a weak effect of riparian cover. 4. These findings suggest that while riparian vegetation increases CPOM inputs and has modest/weak effects on storage, catchment scale urbanisation decreases organic matter availability. Using an organic matter budget approach, it appears likely that the increased frequency and magnitude of high flows associated with catchment urbanisation exerts an overriding influence on organic matter availability. 5. We conclude that to maintain both organic matter inputs and storage, the restoration and protection of streams in urban or rapidly urbanising environments relies on the management of both riparian vegetation and catchment hydrology.     

Lability of organic carbon in lakes of different trophic status

1. We used first‐order kinetic parameters of biological oxygen demand (BOD), the constant of aerobic decomposition (k) and the asymptotic value of BOD (BOD_ult), to characterise the lability of organic carbon pools in six lakes of different trophic state: L. Naroch, L. Miastro and L. Batorino (Belarus), L. Kinneret (Israel), L. Ladoga (Russia) and L. Mendota (U.S.A.). The relative contributions of labile and refractory organic carbon fractions to the pool of total organic carbon (TOC) in these lakes were quantified. We also determined the amounts of labile organic carbon within the dissolved and particulate TOC pools in the three Belarus lakes. 2. Mean annual chlorophyll concentrations (used as a proxy for lake trophic state) ranged from 2.3 to 50.6 μg L⁻¹, labile organic carbon (OC_L = 0.3BOD_ult) from 0.75 to 2.95 mg C L⁻¹ and k from 0.044 to 0.14 day⁻¹. 3. Our data showed that there were greater concentrations of OC_L but lower k values in more productive lakes. 4. In all cases, the DOC fraction dominated the TOC pool. OC_L was a minor component of the TOC pool averaging about 20%, irrespective of lake trophic state. 5. In all the lakes, most (c. 85%) of the DOC pool was refractory, corresponding with published data based on measurements of bacterial production and DOC depletion. In contrast, a larger fraction (27–55%) of the particulate organic carbon (POC) pool was labile. The relative amount of POC in the TOC pool tended to increase with increasing lake productivity. 6. Long‐term BOD incubations can be valuable in quantifying the rates of breakdown of the combined particulate and dissolved organic carbon pools and in characterising the relative proportions of the labile and recalcitrant fractions of these pools. If verified from a larger number of lakes our results could have important general implications.     

Kinetics and mechanisms of dissolved organic carbon retention in a headwater stream

Freeze-dried aqueous extracts of autumn-shed maple leaves, birch leaves, and spruce needles were added to a third-order reach of Bear Brook, New Hampshire at concentrations similar to those predicted to occur during peak leaf fall. Leachate from each species was rapidly removed from solution. With initial concentrations of added leachate of approximately 5 mgl^–1, dissolved organics (DOC) uptake ranged from 73 to 130 mg m^–2 h^–1 for the first five hours of travel downstream from the point of addition. There was no preferential removal of DOC of low molecular weight, or of monomeric carbohydrates relative to phenolics or unidentified DOC.Stream sediments and organic debris rapidly removed DOC from solution in laboratory experiments. No significant flocculation or microbial assimilation of sugar maple leachate occurred in stream water alone. Stream sediments showed small increases in respiration with addition of leaf leachate, but no increase in respiration occurred upon addition of leachate to organic debris. Abiotic adsorption due to the high concentrations of exchangeable iron and aluminium in stream sediments may be responsible for much of the rapid removal of leaf leachate observed in field experiments. Abiotic processes appear to retain DOC within the stream, thereby allowing subsequent metabolism of dissolved organic carbon by stream microflora.     

New retention mechanism of mononucleotides with buffer concentrations in ion-suppressing RP-HPLC

HPLC separation of ionic samples tends to be more complicated and difficult to understand than that of non-ionic compounds. On the other hand, band spacing is much more easily manipulated for ionic than for neutral samples. Ion-suppressing RP-HPLC method was used with organic modifier and aqueous buffer solution. In this work, five mononucleotides of cytidine-5-monophosphate (5′-CMP) disodium salt, uridine-5-monophosphate disodium salt (5′-UMP), guanosine-5-monophosphate disodium salt (5′-GMP), inosine-5-monophosphate disodium salt (5′-IMP), and adenosine-5-monophosphate disodium salt (5′-AMP) were examined. Acetic acid and sodium phosphate were used as buffers, and methanol as an organic modifier. A new relationship between the retention factor and the buffer concentration at a fixed modifier content (5% of methanol) could be expressed by fol|lowing:k=(k ₋₁+k ₀ (K _B/K _S)C _B ^a)/(1+(K _B/K _S)C _B ^a), whereC _B was the concentration of buffer. Using this relationship, the calculated values closely matched the experimental data. The derived relationship showed that as the buffer concentration increased, the retention factor approached a certain value, and this was buffer dependent.