Perennial Crops Can Compensate for Low Soil Carbon Inputs from Maize in Ley-Arable Systems.

(1) Background: Soil organic carbon (SOC) in agricultural soils plays a crucial role in mitigating global climate change but also, and maybe more importantly, in soil fertility and thus food security. Therefore, the influence of contrasting cropping systems on SOC not only in the topsoil, but also in the subsoil, needs to be understood. (2) Methods: In this study, we analyzed SOC content and δ13C values from a crop rotation experiment for biogas production, established in southern Germany in 2004. We compared two crop rotations, differing in their proportions of maize (0 vs. 50%) and perennial legume-grass leys as main crops (75 vs. 25%). Maize was cultivated with an undersown white clover. Both rotations had an unfertilized variant and a variant that was fertilized with biogas digestate according to the nutrient demand of crops. Sixteen years after the experiment was established, the effects of crop rotation, fertilization, and soil depth on SOC were analyzed. Furthermore, we defined a simple carbon balance model to estimate the dynamics of δ13C in soil. Simulations were compared to topsoil data (0-30 cm) from 2009, 2017, and 2020, and to subsoil data (30-60 cm) from 2020. (3) Results: Crop rotation and soil depth had significant effects, but fertilization had no effect on SOC content and δ13C. SOC significantly differed between the two crop rotations regarding δ13C in both depths but not regarding content. Annual enrichment in C4 (maize) carbon was 290, 34, 353, and 70 kg C ha-1 per maize year in the topsoil and subsoil of the unfertilized and fertilized treatments, respectively. These amounts corresponded to carbon turnover rates of 0.8, 0.3, 0.9, and 0.5% per maize year. Despite there being 50% maize in the rotation, maize carbon only accounted for 20% of the observed carbon sequestration in the topsoil. Even with pre-defined parameter values, the simple carbon model reproduced observed δ13C well. The optimization of model parameters decreased the carbon use efficiency of digestate carbon in the soil, as well as the response of belowground carbon allocation to increased aboveground productivity of maize. (4) Conclusions: Two main findings resulted from this combination of measurement and modelling: (i) the retention of digestate carbon in soil was low and its effect on δ13C was negligible, and (ii) soil carbon inputs from maize only responded slightly to increased above-ground productivity. We conclude that SOC stocks in silage maize rotations can be preserved or enhanced if leys with perennial crops are included that compensate for the comparably low maize carbon inputs.

Effects of Wilting and Dew on the Water Isotope Composition of Detached Grass in Temperate Grassland.

Understanding the water isotopes in feed products derived from grass is fundamental for tracing domestic animal products. Grass silage water was reported to have fewer heavy isotopes than fresh grass, but it is still unknown whether dew formation (either dewfall or dewrise), exchange with soil water, or other processes override the expected enrichment of heavy isotopes due to wilting. The isotopic variations of water (δ2H, δ18O) in fresh grass and cut grass during wilting on soil and on plastic were compared in this study. Drying enriched heavier isotopes, but this was overridden by three processes that finally caused low δ2H and δ18O values: (i) the adsorption of humidity from the surroundings, (ii) the exchange with humidity, and (iii) the depletion of heavy water isotopes close to organic surfaces, called the surface effect, which was the most dominant effect at the end of drying when the water content became low.

Soil organic carbon storage following conversion from cropland to grassland on sites differing in soil drainage and erosion history.

Changing soil use from cropland to grassland influences organic carbon storage in a highly complex way. This includes the root/shoot allocation, the root depth distribution, the incorporation of shoot biomass and lateral organic carbon fluxes, by erosion and removal of harvested carbon, and finally the aeration by tillage. An experiment was designed allowing resampling a number of soils 18 yr after conversion to grassland (either pasture or meadow or set-aside) only 20 cm apart from the original sampling to exclude site variation. Before conversion to grassland the cropland was prone to erosion, with a mean lateral carbon flux during 20 yr prior to conversion of 13 t ha-1. Harvest had removed another 29 t ha-1 of carbon at eroding sites. Colluvial carbon inputs had been up to 18 t ha-1 while harvest had removed 38 t ha-1 at colluvial sites. The carbon fluxes by erosion were negligible during the 18 yr period after conversion. After conversion the carbon losses by harvest also ceased at set-aside grassland and pastures while the net losses on meadows were 45 t ha-1. Conversion to grassland significantly changed depth functions of carbon, stones, bulk density and porosity. Despite the large changes in carbon fluxes, carbon stocks did only change significantly within 18 yr under poorly drained, gleyic soils. Well-aerated soils did not show a significant increase in SOC stocks. This was even true for heavily eroded soils, where conversion from cropland to grassland (without erosion) should foster dynamic replacement of SOC. The widespread drainage of wet grassland soils prior to conversion to cropland thus can cause a large release of carbon, while an influence of tillage by either increasing aeration or erosion could not be detected in this study. Therefore, fostering carbon sequestration by conversion of cropland to grassland requires restoring former draining conditions.

Behavior of farmers in regard to erosion by water as reflected by their farming practices.

The interplay between natural site conditions and farming raises erosion by water above geological background levels. We examined the hypothesis that farmers take erosion into account in their farming decisions and switch to farming practices with lower erosion risk the higher the site-specific hazard becomes. Erosion since the last tillage was observed from aerial orthorectified photographs for 8100 fields belonging to 1879 farmers distributed across Bavaria (South Germany) and it was modeled by the Universal Soil Loss Equation using highly detailed input data (e.g., digital terrain model with 5×5m2 resolution, rain data with 1×1km2 and 5min resolution, crop and cropping method from annual field-specific data from incentive schemes). Observed and predicted soil loss correlated closely, demonstrating the accuracy of this method. The close correlation also indicted that the farmers could easily observe the degree of recent erosion on their fields, even without modelling. Farmers clearly did not consider erosion in their decisions. When natural risk increased, e.g. due to steeper slopes, they neither grew crops with lower erosion potential, nor reduced field size, nor used contouring. In addition, they did not compensate for the cultivation of crops with higher erosion potential by using conservation techniques like mulch tillage or contouring, or by reducing field size. Only subsidized measures, like mulch tillage or organic farming, were applied but only at the absolute minimum that was necessary to obtain subsidies. However, this did not achieve the reduction in erosion that would be possible if these measures had been fully applied. We conclude that subsidies may be an appropriate method of reducing erosion but the present weak supervision, which assumes that farmers themselves will take erosion into account and that subsidies are only needed to compensate for any disadvantages caused by erosion-reducing measures, is clearly not justified.

Ambient Conditions and Feeding Strategy Influence δ18O of Milk Water in Cows (Bos taurus).

There are increasing concerns by consumers regarding agricultural product traceability and authenticity. Oxygen isotope composition (δ18O) has been used in this context based on the relationship between δ18O of animal products and annual precipitation. However, in dairy products this relationship is affected by the seasonality of δ18O in milk water which in turn depends on the feeding system used. We measured 608 milk samples from 28 farms with various feeding strategies in southern Germany throughout the year, investigating the influences of ambient conditions, drinking water source, and feeding strategies on seasonal variation of δ18O in milk water (δmilk). The mechanistic Munich-Kohn model reflecting these influences predicted the seasonal and farm-specific variation of δmilk well. The relationship between δ18O of precipitation and δmilk varied in different feeding strategies. The interplay of ambient conditions and feeding strategy on δmilk should thus be carefully considered when identifying the origin of milk.

Model explanation of the seasonal variation of δ18O in cow (Bos taurus) hair under temperate conditions.

Oxygen isotopes (δ18O) in animal and human tissues are expected to be good recorders of geographical origin and migration histories. However, seasonal variation of δ18O may diminish the origin information in the tissues. Here the seasonality of δ18O in tail hair was investigated in a domestic suckler cow (Bos taurus) that underwent different ambient conditions, physiological states, keeping and feeding during five years. A detailed mechanistic model was built to explain this variation. The measured δ18O in hair significantly related (p < 0.05) to the δ18O in meteoric water in a regression analysis. Modelling suggested that this relation was only partly derived from the direct influence of feed moisture. Ambient conditions (temperature, moisture) also affected the animal itself (drinking water demand, transcutaneous vapor etc.). The clear temporal variation thus resulted from complex interactions with multiple influences. The twofold influence of ambient conditions via the feed and via the animal itself is advantageous for tracing the geographic origin because δ18O is then less influenced by variations in moisture uptake; however, it is unfavorable for indicating the production system, e.g. to distinguish between milk produced from fresh grass or from silage. The model is versatile but needs testing under a wider range of conditions.

Effects of functional diversity loss on ecosystem functions are influenced by compensation.

Understanding the impacts of biodiversity loss on ecosystem functioning and services has been a central issue in ecology. Experiments in synthetic communities suggest that biodiversity loss may erode a set of ecosystem functions, but studies in natural communities indicate that the effects of biodiversity loss are usually weak and that multiple functions can be sustained by relatively few species. Yet, the mechanisms by which natural ecosystems are able to maintain multiple functions in the face of diversity loss remain poorly understood. With a long-term and large-scale removal experiment in the Inner Mongolian grassland, here we showed that losses of plant functional groups (PFGs) can reduce multiple ecosystem functions, including biomass production, soil NO3 -N use, net ecosystem carbon exchange, gross ecosystem productivity, and ecosystem respiration, but the magnitudes of these effects depended largely on which PFGs were removed. Removing the two dominant PFGs (perennial rhizomatous grasses and perennial bunchgrasses) simultaneously resulted in dramatic declines in all examined functions, but such declines were circumvented when either dominant PFG was present. We identify the major mechanism for this as a compensation effect by which each dominant PFG can mitigate the losses of others. This study provides evidence that compensation ensuing from PFG losses can mitigate their negative consequence, and thus natural communities may be more resilient to biodiversity loss than currently thought if the remaining PFGs have strong compensation capabilities. On the other hand, ecosystems without well-developed compensatory functional diversity may be much more vulnerable to biodiversity loss.

Fine sediment as environmental stressor affecting freshwater mussel behavior and ecosystem services.

Fine sediment pollution is considered a major stressor for aquatic ecosystems and their biodiversity. In particular, fine sediments have been suggested to play a crucial role in the declines of freshwater mussels which are considered keystone fauna of streams and rivers. Whereas the effects of deposited fine sediments on recruitment failure are well known, effects of suspended fine sediments on adult mussel behavior are less studied. Therefore the aim of this study was to investigate the effects of fine sediment exposure on freshwater mussel behavior and on mussel-dependent ecosystem services. Unio pictorum mussels were used to test three behavioral endpoints: Hall activity, transition frequency and relative water clearance rate. Mussels were exposed to fine sediments of different particle size classes (<45µm, 45-63µm, 63-125µm) and different concentration (0-10gL(-1)) of the smallest particle size class. Hall sensor technology and turbidity measurements were used to detect mussel behavior in presence of suspended sediments. Results revealed that mussels improve clearance of suspended particles out of the water column by 35%, independent of particle size class and concentration. Transition frequency was determined an unsuitable behavioral endpoint for non-soluble substances. Contrary to previous studies, we could demonstrate that fine sediments do not interfere with filtration by mussels and that mussels have a great influence on water purification, providing a valuable ecosystem service.

Establishing mussel behavior as a biomarker in ecotoxicology.

Most freshwater mussel species of the Unionoida are endangered, presenting a conservation issue as they are keystone species providing essential services for aquatic ecosystems. As filter feeders with limited mobility, mussels are highly susceptible to water pollution. Despite their exposure risk, mussels are underrepresented in standard ecotoxicological methods. This study aimed to demonstrate that mussel behavioral response to a chemical stressor is a suitable biomarker for the advancement of ecotoxicology methods that aids mussel conservation. Modern software and Hall sensor technology enabled mussel filtration behavior to be monitored real-time at very high resolution. With this technology, we present our method using Anodonta anatina and record their response to de-icing salt pollution. The experiment involved an environmentally relevant 'pulse-exposure' design simulating three subsequent inflow events. Three sublethal endpoints were investigated, Filtration Activity, Transition Frequency (number of changes from opened to closed, or vice versa) and Avoidance Behavior. The mussels presented a high variation in filtration behavior, behaving asynchronously. At environmentally relevant de-icing salt exposure scenarios, A. anatina behavior patterns were significantly affected. Treated mussels' Filtration Activity decreased during periods of very high and long de-icing salt exposure (p<0.001), however, increased during short de-icing salt exposure. Treated mussels' Transition Frequency increased during periods of very high and long de-icing salt exposure (p<0.001), which mirrored the Avoidance Behavior endpoint observed only by mussels under chemical stress. Characteristics of Avoidance Behavior were tighter shell closures with repeated and irregular shell movements which was significantly different to their undisturbed resting behavior (p<0.001). Additionally, we found that mussels were sensitive to a chemical stressor even when the mussel's valves were closed. Due to the effects of de-icing salt pollution on freshwater mussel behavior, we suggest better management practices for de-icing salt use be implemented. Our experimental method demonstrated that, with the application of current technologies, mussel behavioral response to a chemical stressor can be measured. The tested sublethal endpoints are suitable for mussel ecotoxicology studies. Avoidance Behavior proved to be a potentially suitable endpoint for calculating mussel behavior effect concentration. Therefore we recommend adult mussel behavior as a suitable biomarker for future ecotoxicological research. This method could be applied to other bivalve species and for physical and environmental stressors, such as particulate matter and temperature.

Routing of Fatty Acids from Fresh Grass to Milk Restricts the Validation of Feeding Information Obtained by Measuring (13)C in Milk.

Dairy production systems vary widely in their feeding and livestock-keeping regimens. Both are well-known to affect milk quality and consumer perceptions. Stable isotope analysis has been suggested as an easy-to-apply tool to validate a claimed feeding regimen. Although it is unambiguous that feeding influences the carbon isotope composition (δ(13)C) in milk, it is not clear whether a reported feeding regimen can be verified by measuring δ(13)C in milk without sampling and analyzing the feed. We obtained 671 milk samples from 40 farms distributed over Central Europe to measure δ(13)C and fatty acid composition. Feeding protocols by the farmers in combination with a model based on δ(13)C feed values from the literature were used to predict δ(13)C in feed and subsequently in milk. The model considered dietary contributions of C3 and C4 plants, contribution of concentrates, altitude, seasonal variation in (12/13)CO2, Suess's effect, and diet-milk discrimination. Predicted and measured δ(13)C in milk correlated closely (r(2) = 0.93). Analyzing milk for δ(13)C allowed validation of a reported C4 component with an error of <8% in 95% of all cases. This included the error of the method (measurement and prediction) and the error of the feeding information. However, the error was not random but varied seasonally and correlated with the seasonal variation in long-chain fatty acids. This indicated a bypass of long-chain fatty acids from fresh grass to milk.

Comment on "Rainfall erosivity in Europe" by Panagos et al. (Sci. Total Environ., 511, 801-814, 2015).

Recently a rainfall erosivity map has been published. We show that the values of this map contain considerable bias because (i) the temporal resolution of the rain data was insufficient, which likely underestimates rain erosivity by about 20%, (ii) no attempt had been included to account for the different time periods that were used for different countries, which can modify rain erosivity by more than 50%, (iii) and likely precipitation data had been used instead of rain data and thus rain erosivity is overestimated in areas with significant snowfall. Furthermore, the seasonal distribution of rain erosivity is not provided, which does not allow using the erosivity map for erosion prediction in many cases. Although a rain erosivity map for Europe would be highly desirable, we recommend using the national erosivity maps until these problems have been solved. Such maps are available for many European countries.

Isotope evidence for preferential dispersal of fast-spreading invasive gobies along man-made river bank structures.

Invasive round goby Neogobius melanostomus and bighead goby Ponticola kessleri have successfully colonized freshwater and coastal habitats worldwide. The objective was to use stable isotope analyses to study the foraging and movement of both species at small spatial scales in the Upper Danube River, considering 861 samples from two different years, seasons and sides of the river in an area where limited mixing at a confluence occurs. A difference in δ(13)C of 1 ‰ between gobies from both river sides was observed in both species and reflected the isotope spacing in their dominant benthic prey Dikerogammarus villosus. These results suggest an absence of goby movement across the Danube River which was unexpected, given the fast spread of gobies at invasion fronts. It can be concluded that their dispersal is highly preferential with longitudinal movement likely being facilitated by artificial rip-rap structures along river banks, which provide shelter and food.

(13)C discrimination between diet, faeces, milk and milk components.

Stable isotope analysis is a fundamental tool in food origin and authenticity testing. Its use in livestock production requires knowledge of isotope discrimination between product and diet. Here, we report (13)C discrimination ((13)Δ) for milk, milk components (fat, casein and lactose) and faeces in eight lactating dairy cows, which grazed pasture or were fed fresh pasture herbage in the stall. Cows were supplemented with grain maize at 1.72 kg d(-1) (dry matter). Feed components were collected daily, and faeces, milk fat, casein, lactose and whole milk 4 times per week during an 8-week-long sampling period. Carbon isotope composition (δ(13)C) of each sample was analysed. δ(13)C was lowest in milk fat (-29.8‰) and highest in casein (-26.4‰). Compared to the diet, whole milk was depleted in (13)C ((13)Δ = 0.4‰) due to a strong (13)C-depletion of fat ((13)Δ = 2.2‰), which was not fully compensated by the (13)C-enrichment of casein ((13)Δ = -1.1‰) and lactose ((13)Δ = -0.7‰). Faeces were also depleted in (13)C ((13)Δ =1.7‰). Influences of feeding environment (stall vs. pasture) and herbage quality were minor (<0.4‰). A review of literature data shows large variation between studies. We consider that the present results are superior, as they are based on a much larger data set regarding the number of cows and milkings (total n = 256) with greater detail in analyses of diet and milk products. Also, the study covered both stall- and pasture-feeding scenarios in realistic settings with long periods of equilibration. This is the first comprehensive analysis of (13)C discrimination between diet and all main milk components (as well as faeces). Thus, the results will improve the use of stable isotope analyses in regard to authenticity testing and proof of origin.

Transamination governs nitrogen isotope heterogeneity of amino acids in rats.

The nitrogen isotope composition (δ¹5N) of different amino acids carries different dietary information. We hypothesized that transamination and de novo synthesis create three groups that largely explain their dietary information. Rats were fed with ¹5N-labeled amino acids. The redistribution of the dietary ¹5N labels among the muscular amino acids was analyzed. Subsequently, the labeling was changed and the nitrogen isotope turnover was analyzed. The amino acids had a common nitrogen half-life of ∼20 d, but differed in δ¹5N. Nontransaminating and essential amino acids largely conserved the δ¹5N of the source and, hence, trace the origin in heterogeneous diets. Nonessential and nontransaminating amino acids showed a nitrogen isotope composition between their dietary composition and that of their de novo synthesis pool, likely indicating their fraction of de novo synthesis. The bulk of amino acids, which are transaminating, derived their N from a common N pool and hence their δ¹5N was similar.

Oxygen and hydrogen isotope composition of silage water.

Silage is an important dietary water source that influences the oxygen and hydrogen isotopic composition of domestic herbivores and their products. Silage sampled fresh from the silo had (18)O- and (2)H-depleted tissue water when compared with fresh pasture grass sampled around midday during the silage-making seasons. During exposure in the feed bunk, silage water became increasingly enriched in (18)O and (2)H. When δ(18)O was plotted against δ(2)H, the slope of the regression was less during daytime than during night-time. Exposure to (18)O- and (2)H-enriched or -depleted water vapor inside sealed glass containers led to strong changes in the isotope composition of silage water. The results resembled predictions from the Craig-Gordon isotope model of evaporation. The atmospheric conditions during exposure (relative humidity, exposure time, and isotopic composition of the air vapor) in the feed bunk thus strongly affect the isotopic composition of silage water ingested by domestic herbivores.

Forward modeling of fluctuating dietary 13C signals to validate 13C turnover models of milk and milk components from a diet-switch experiment.

Isotopic variation of food stuffs propagates through trophic systems. But, this variation is dampened in each trophic step, due to buffering effects of metabolic and storage pools. Thus, understanding of isotopic variation in trophic systems requires knowledge of isotopic turnover. In animals, turnover is usually quantified in diet-switch experiments in controlled conditions. Such experiments usually involve changes in diet chemical composition, which may affect turnover. Furthermore, it is uncertain if diet-switch based turnover models are applicable under conditions with randomly fluctuating dietary input signals. Here, we investigate if turnover information derived from diet-switch experiments with dairy cows can predict the isotopic composition of metabolic products (milk, milk components and feces) under natural fluctuations of dietary isotope and chemical composition. First, a diet-switch from a C3-grass/maize diet to a pure C3-grass diet was used to quantify carbon turnover in whole milk, lactose, casein, milk fat and feces. Data were analyzed with a compartmental mixed effects model, which allowed for multiple pools and intra-population variability, and included a delay between feed ingestion and first tracer appearance in outputs. The delay for milk components and whole milk was ~12 h, and that of feces ~20 h. The half-life (t½) for carbon in the feces was 9 h, while lactose, casein and milk fat had a t½ of 10, 18 and 19 h. The (13)C kinetics of whole milk revealed two pools, a fast pool with a t½ of 10 h (likely representing lactose), and a slower pool with a t½ of 21 h (likely including casein and milk fat). The diet-switch based turnover information provided a precise prediction (RMSE ~0.2 ‰) of the natural (13)C fluctuations in outputs during a 30 days-long period when cows ingested a pure C3 grass with naturally fluctuating isotope composition.

Dietary protein content affects isotopic carbon and nitrogen turnover.

RATIONALE: Isotopic turnover quantifies the metabolic renewal process of elements in organs and excreta. Knowledge of the isotopic turnover of animal organs and excreta is necessary for diet reconstruction via stable isotope analysis, as used in animal ecology, palaeontology and food authentication. Effects of dietary protein content on the isotopic carbon and nitrogen turnover (i.e. delay, representing the time between ingestion and start of renewal, and half-life) are unknown for most mammalian organs and excreta. METHODS: To examine the effect of dietary protein content on turnover (delay and turnover rate), we fed 18 rats either a diet at protein maintenance or above protein maintenance, and quantified their isotopic carbon and nitrogen turnover in ten organs and excreta. These included the excreta faeces and urine, the visceral organs blood plasma, liver, kidney, lung and spleen, the cerebral tissue brain, and the muscular tissues heart and muscle. For data analysis, we used piecewise linear/non-linear exponential modelling that allows quantifying delay and turnover rate simultaneously. RESULTS: Delays were ~0.5 days for carbon and nitrogen turnover and were not affected by dietary protein content. Half-lives during the following reaction progress were in the range of 1 to 45 days, increasing from excreta to visceral organs to muscular and cerebral organs. Rats fed the higher protein amount had 30% shorter nitrogen half-lives, and 20% shorter carbon half-lives. CONCLUSIONS: The renewal times of organs and excreta depend on the dietary protein content. Hence, isotopic diet reconstruction is confronted with variation in half-lives within the same organ or excrement, altering the time window through which information can be perceived.

Sheep wool δ13C reveals no effect of grazing on the C3/C4 ratio of vegetation in the inner Mongolia-Mongolia border region grasslands.

We tested whether the abundance of C(4) vegetation in grasslands of the Mongolian plateau is influenced by grazing conditions. The analysis exploited the politically originated contrast that exists between Mongolia (low stocking rate, transhumant system) and the district of Inner Mongolia, China (high stocking rate, sedentary system). We estimated the proportion of C(4) carbon (P(C4)) in grazed vegetation from the relative carbon isotope ratio (δ(13)C) of sheep wool sampled from 298 annual shearings originating from 1996 to 2007. Annual stocking rates varying over time and between the districts of both countries were taken from regional statistics. The P(C4) pattern within the 0.7 million km(2) sampling area was geostatistically analyzed and related to stocking rates and temperature gradients. For similar climatic conditions, P(C4) was the same in both countries. Further, a unique relationship was found between P(C4) and July temperature on both sides of the border, which explained 71% of the pattern. Stocking rate and grazing system had no significant influences on present-day C(3)/C(4) abundance ratio. This finding suggests that recent changes in the C(3)/C(4) ratio of these grasslands are mainly a consequence of regional warming, not overgrazing.

Drivers and spatio-temporal extent of hyporheic patch variation: implications for sampling.

The hyporheic zone in stream ecosystems is a heterogeneous key habitat for species across many taxa. Consequently, it attracts high attention among freshwater scientists, but generally applicable guidelines on sampling strategies are lacking. Thus, the objective of this study was to develop and validate such sampling guidelines. Applying geostatistical analysis, we quantified the spatio-temporal variability of parameters, which characterize the physico-chemical substratum conditions in the hyporheic zone. We investigated eight stream reaches in six small streams that are typical for the majority of temperate areas. Data was collected on two occasions in six stream reaches (development data), and once in two additional reaches, after one year (validation data). In this study, the term spatial variability refers to patch contrast (patch to patch variance) and patch size (spatial extent of a patch). Patch contrast of hyporheic parameters (specific conductance, pH and dissolved oxygen) increased with macrophyte cover (r(2)=0.95, p<0.001), while patch size of hyporheic parameters decreased from 6 to 2 m with increasing sinuosity of the stream course (r(2)=0.91, p<0.001), irrespective of the time of year. Since the spatial variability of hyporheic parameters varied between stream reaches, our results suggest that sampling design should be adapted to suit specific stream reaches. The distance between sampling sites should be inversely related to the sinuosity, while the number of samples should be related to macrophyte cover.