Phagotrophic Protists Modulate Copper Resistance of the Bacterial Community in Soil.

Protist predation is a crucial biotic driver modulating bacterial populations and functional traits. Previous studies using pure cultures have demonstrated that bacteria with copper (Cu) resistance exhibited fitness advantages over Cu-sensitive bacteria under the pressure of protist predation. However, the impact of diverse natural communities of protist grazers on bacterial Cu resistance in natural environments remains unknown. Here, we characterized the communities of phagotrophic protists in long-term Cu-contaminated soils and deciphered their potential ecological impacts on bacterial Cu resistance. Long-term field Cu pollution increased the relative abundances of most of the phagotrophic lineages in Cercozoa and Amoebozoa but reduced the relative abundance of Ciliophora. After accounting for soil properties and Cu pollution, phagotrophs were consistently identified as the most important predictor of the Cu-resistant (CuR) bacterial community. Phagotrophs positively contributed to the abundance of a Cu resistance gene (copA) through influencing the cumulative relative abundance of Cu-resistant and -sensitive ecological clusters. Microcosm experiments further confirmed the promotion effect of protist predation on bacterial Cu resistance. Our results indicate that the selection by protist predation can have a strong impact on the CuR bacterial community, which broadens our understanding of the ecological function of soil phagotrophic protists.

Fifty thousand years of Arctic vegetation and megafaunal diet.

Although it is generally agreed that the Arctic flora is among the youngest and least diverse on Earth, the processes that shaped it are poorly understood. Here we present 50 thousand years (kyr) of Arctic vegetation history, derived from the first large-scale ancient DNA metabarcoding study of circumpolar plant diversity. For this interval we also explore nematode diversity as a proxy for modelling vegetation cover and soil quality, and diets of herbivorous megafaunal mammals, many of which became extinct around 10 kyr bp (before present). For much of the period investigated, Arctic vegetation consisted of dry steppe-tundra dominated by forbs (non-graminoid herbaceous vascular plants). During the Last Glacial Maximum (25-15 kyr bp), diversity declined markedly, although forbs remained dominant. Much changed after 10 kyr bp, with the appearance of moist tundra dominated by woody plants and graminoids. Our analyses indicate that both graminoids and forbs would have featured in megafaunal diets. As such, our findings question the predominance of a Late Quaternary graminoid-dominated Arctic mammoth steppe.

Bioaccumulation of cadmium in soil organisms - With focus on wood ash application.

Harvesting whole-tree biomass for biofuel combustion intensifies removal of nutrients from the ecosystem. This can be partly amended by applying ash from the combustion back to the system and thus recycle the nutrients. However, besides being rich in inorganic nutrients, ash also contains trace amounts of heavy metals. Due to the risk of toxic effects and trophic transfer of heavy metals, especially cadmium, legislation usually restricts the use of ash as a soil amendment. In order to provide researchers and governmental agencies with a tool to assess the risk of cadmium bioaccumulation in specific soil systems after ash application, we review: 1) the properties of ash; 2) the chemical and toxic properties of cadmium; 3) the key factors affecting cadmium bioavailability, cadmium uptake-, storage- and elimination-abilities in soil organisms and the risk of cadmium accumulation and biomagnification in the soil food web; 4) how ash impact on soil can change the risk of cadmium bioaccumulation. We conclude that for assessing the risk of cadmium bioaccumulation for specific sites, it is necessary to consider both the type and composition of ash, the soil conditions and organism composition on the site. On a general basis, we conclude that granulated ashes low in cadmium content, applied to low pH soils with high organic matter content, in systems with low abundances of earthworms, isopods and gastropods, will have a low risk of cadmium accumulation.

A role for copper in protozoan grazing - two billion years selecting for bacterial copper resistance.

The Great Oxidation Event resulted in integration of soft metals in a wide range of biochemical processes including, in our opinion, killing of bacteria by protozoa. Compared to pressure from anthropologic copper contamination, little is known on impacts of protozoan predation on maintenance of copper resistance determinants in bacteria. To evaluate the role of copper and other soft metals in predatory mechanisms of protozoa, we examined survival of bacteria mutated in different transition metal efflux or uptake systems in the social amoeba Dictyostelium discoideum. Our data demonstrated a strong correlation between the presence of copper/zinc efflux as well as iron/manganese uptake, and bacterial survival in amoebae. The growth of protozoa, in turn, was dependent on bacterial copper sensitivity. The phagocytosis of bacteria induced upregulation of Dictyostelium genes encoding the copper uptake transporter p80 and a triad of Cu(I)-translocating PIB -type ATPases. Accumulated Cu(I) in Dictyostelium was monitored using a copper biosensor bacterial strain. Altogether, our data demonstrate that Cu(I) is ultimately involved in protozoan predation of bacteria, supporting our hypothesis that protozoan grazing selected for the presence of copper resistance determinants for about two billion years.

A brief history of metal recruitment in protozoan predation.

Metals and metalloids are used as weapons for predatory feeding by unicellular eukaryotes on prokaryotes. This review emphasizes the role of metal(loid) bioavailability over the course of Earth's history, coupled with eukaryogenesis and the evolution of the mitochondrion to trace the emergence and use of the metal(loid) prey-killing phagosome as a feeding strategy. Members of the genera Acanthamoeba and Dictyostelium use metals such as zinc (Zn) and copper (Cu), and possibly metalloids, to kill their bacterial prey after phagocytosis. We provide a potential timeline on when these capacities first evolved and how they correlate with perceived changes in metal(loid) bioavailability through Earth's history. The origin of phagotrophic eukaryotes must have postdated the Great Oxidation Event (GOE) in agreement with redox-dependent modification of metal(loid) bioavailability for phagotrophic poisoning. However, this predatory mechanism is predicted to have evolved much later - closer to the origin of the multicellular metazoans and the evolutionary development of the immune systems.

Microeukaryotic plankton evolutionary constraints in a subtropical river explained by environment and bacteria along differing taxonomic resolutions.

Microeukaryotic plankton communities are keystone components for keeping aquatic primary productivity. Currently, variations in microeukaryotic plankton diversity have often been explained by local ecological factors but not by evolutionary constraints. We used amplicon sequencing of 100 water samples across five years to investigate the ecological preferences of the microeukaryotic plankton community in a subtropical riverine ecosystem. We found that microeukaryotic plankton diversity was less associated with bacterial abundance (16S rRNA gene copy number) than bacterial diversity. Further, environmental effects exhibited a larger influence on microeukaryotic plankton community composition than bacterial community composition, especially at fine taxonomic levels. The evolutionary constraints of microeukaryotic plankton community increased with decreasing taxonomic resolution (from 97% to 91% similarity levels), but not significant change from 85% to 70% similarity levels. However, compared with the bacterial community, the evolutionary constraints were shown to be more affected by environmental variables. This study illustrated possible controlling environmental and bacterial drivers of microeukaryotic diversity and community assembly in a subtropical river, thereby indirectly reflecting on the quality status of the water environment by providing new clues on the microeukaryotic community assembly.

Application of wood ash leads to strong vertical gradients in soil pH changing prokaryotic community structure in forest top soil.

Wood ash is alkaline and contains base-cations. Application of wood ash to forests therefore counteracts soil acidification and recycle nutrients removed during harvest. Wood ash application to soil leads to strong vertical gradients in physicochemical parameters. Consequently, we designed an experimental system where small-scale vertical changes in soil properties and prokaryotic community structure could be followed after wood ash application. A mixed fly and bottom ash was applied in dosages of 3 and 9 t ha-1 to the surface of soil mesocosms, simulating a typical coniferous podzol. Soil pH, exchangeable cations and 16S prokaryotic community was subsequently assessed at small depth intervals to 5 cm depth at regular intervals for one year. Wood ash significantly changed the prokaryotic community in the top of the soil column. Also, the largest increases in pH and concentrations of exchangeable cations was found here. The relative abundance of prokaryotic groups directionally changed, suggesting that wood ash favors copiotrophic prokaryotes at the expense of oligotrophic and acidophilic taxa. The effect of wood ash were negligible both in terms of pH- and biological changes in lower soil layers. Consequently, by micro-vertical profiling we showed that wood ash causes a steep gradient of abiotic factors driving biotic changes but only in the top-most soil layers.

Is wood ash amendment a suitable mitigation strategy for N2O emissions from soil?

Wood ash, the by-product of biomass combustion to energy, can return important nutrients back to the soil and counteract acidification. However, the application of wood ash may affect the emission of greenhouse gases. Here, the effect of wood ash application on nitrous oxide (N2O) emissions from different soil environments were investigated in a 40 days incubation experiment comprising ten different soil types amended with five different wood ash concentrations (0, 3, 9, 20, and 54 t ash ha-1). The emitted N2O was measured continuously, and initial soil properties without ash application (carbon (C), nitrogen (N), ammonium (NH4+), nitrate (NO3-), and pH) and resulting soil properties (pH, NH4+, and NO3-) were measured prior and after the incubation period, respectively. The Random Forests (RF) model was used to identify which factors (initial and resulting soil properties, vegetation, management, wood ash doze, and respiration rate) were the most important to predict the development of emitted N2O after ash application. Wood ash either increased, decreased, or had no effect on the amount of emitted N2O depending on soil type and ash dose. The RF model identified the final resulting pH as the most important factor for the prediction of emitted N2O. The results suggest that wood ash can mitigate N2O emissions from soil, however, this effect depends on soil type where a mitigating effect of wood ash application was observed mainly in low pH soils with high soil organic matter whereas an increase in N2O emissions was observed in mineral soils that had previously received N fertilization. This study emphasises the importance of pH manipulation in regards to N2O emissions from soil.

Seven-year dynamics of testate amoeba communities driven more by stochastic than deterministic processes in two subtropical reservoirs.

Testate amoebae are widely distributed in natural ecosystems and play an important role in the material cycle and energy flow. However, community assembly of testate amoebae is not well understood, especially with regard to the relative importance of the stochastic and deterministic processes over time. In this study, we used Illumina high-throughput sequencing to explore the community assembly of testate amoebae from surface waters in two reservoirs of subtropical China over a seven-year period. Majority of testate amoebae belonged to the rare taxa because their relative abundances were typically lower than 0.01% of the total eukaryotic plankton community. The testate amoeba community dynamics exhibited a stronger interannual than seasonal variation in both reservoirs. Further, species richness, rather than species turnover, accounted for the majority of community variation. Environmental variables explained less than 20% of the variation in community composition of testate amoebae, and the community assembly appeared to be strongly driven by stochastic processes. Based on the Sloan neutral community model, it was found that neutral processes explained more than 65% of community variation. More importantly, the Stegen null model analysis showed that the stochastic processes (e.g., ecological drift) explained a significantly higher percentage of community assembly than deterministic processes over seven years, although deterministic processes were more influential in certain years. Our results provide new perspectives for understanding the ecological patterns, processes and mechanisms of testate amoeba communities in freshwater ecosystems at temporal scale, and have important implications for monitoring plankton diversity and protecting drinking-water resources.

Effect of ash application on the decomposer food web and N mineralization in a Norway spruce plantation.

In the face of global climate change there is an increasing demand for biofuel, which exerts pressure on production and thus management of biofuel plantations. The intensification of whole-tree harvest from biofuel plantations increases export of nutrients. Returning ash from biofuel combustion to the forest plantations can amend the soil nutrient status and thus facilitate sustainable forest management. However, ash affects the forest floor decomposer food web, potentially changing organic matter turnover, carbon sequestration and nitrogen availability. Our aim was to examine the response of decomposer organisms, food web structure and nitrogen mineralization function after ash application. In a coniferous forest plantation amended with 0, 3, 4.5 or 6 t ash ha-1, we sampled in several depths of the forest floor for key organisms of the decomposer food web (fungal biomass, 0-12 cm; bacteria, protozoa, nematodes and enchytraeids, 0-3 cm and 3-6 cm; microarthropods and earthworms, 0-5 cm), 2, 14 and 26 months after ash application. We used structural equation modelling (SEM) to detangle the direct and indirect effects of ash application on organisms in the decomposer food web and on nitrogen availability. We found that ash increased the abundance of bacteria and protozoa, as well as the inorganic nitrogen pool at 0-3 cm depth, whereas the effect of ash was negligible at 3-6 cm depth. Earthworm abundance increased, whereas enchytraeid abundance decreased 2 years after ash application. The structural equation modelling showed that ash application stimulated the bacterial feeding pathway and increased nitrogen mineralization. Contrary, ash had a negative effect on fungal biomass at the first sampling, however, this effect subdued over time. Our results suggest that as the soil decomposer food web is resilient to ash application, this is a viable option for sustainable management of biofuel plantations.

Total RNA sequencing reveals multilevel microbial community changes and functional responses to wood ash application in agricultural and forest soil.

Recycling of wood ash from energy production may counteract soil acidification and return essential nutrients to soils. However, wood ash amendment affects soil physicochemical parameters that control composition and functional expression of the soil microbial community. Here, we applied total RNA sequencing to simultaneously assess the impact of wood ash amendment on the active soil microbial communities and the expression of functional genes from all microbial taxa. Wood ash significantly affected the taxonomic (rRNA) as well as functional (mRNA) profiles of both agricultural and forest soil. Increase in pH, electrical conductivity, dissolved organic carbon and phosphate were the most important physicochemical drivers for the observed changes. Wood ash amendment increased the relative abundance of the copiotrophic groups Chitinonophagaceae (Bacteroidetes) and Rhizobiales (Alphaproteobacteria) and resulted in higher expression of genes involved in metabolism and cell growth. Finally, total RNA sequencing allowed us to show that some groups of bacterial feeding protozoa increased concomitantly to the enhanced bacterial growth, which shows their pivotal role in the regulation of bacterial abundance in soil.

Wood ash effects on growth and cadmium uptake in Deschampsia flexuosa (Wavy hair-grass).

Wood ash recycling to forests is beneficial because it regains nutrients and prevents acidification, but wood ash application is restricted due to its cadmium (Cd) content. We question if Cd in wood ash represents a problem, since decreases in Cd bioavailability due to ash-induced pH changes may counteract increased total Cd concentration. We studied effects of wood ash (0, 3, 9 and 30 t ha-1) and lime (pH increase equivalent to the wood ash treatments) on growth and Cd uptake in Deschampsia flexuosa. After four months, we measured plant biomass and Cd accumulation, and extracted Cd from the soil using three different methods; HNO3 (total), EDTA (chelator-based) and NH4NO3 (salt-based). Wood ash and lime strongly stimulated plant growth. Cd concentration in the plant tissue decreased with wood ash and lime addition, and correlated positively with the NH4NO3 extractable fraction of Cd in the soil. In contrast, HNO3 and EDTA extracted more Cd with increased wood ash application. We conclude that wood ash amendment increases soil pH, total Cd concentration, nutrient levels and stimulates plant growth. However, it does not increase Cd accumulation in D. flexuosa, as pH-driven decreases in Cd bioavailability leads to reduced plant Cd uptake. Finally, soil bioavailable Cd is best determined using NH4NO3-extraction.

Metabolic Inactivity and Re-awakening of a Nitrate Reduction Dependent Iron(II)-Oxidizing Bacterium Bacillus ferrooxidans.

Microorganisms capable of anaerobic nitrate-dependent Fe(II) (ferrous iron) oxidation (ANDFO) contribute significantly to iron and nitrogen cycling in various environments. However, lab efforts in continuous cultivation of ANDFO strains suffer from loss of activity when ferrous iron is used as sole electron donor. Here, we used a novel strain of nitrate-dependent Fe(II)-oxidizing bacterium Bacillus ferroxidians as a model and focused on the physiological activity of cells during ANDFO. It was shown that B. ferrooxidans entered a metabolically inactive state during ANDFO. B. ferrooxidans exhibited nitrate reduction coupled with Fe(II) oxidation, and the activity gradually declined and was hardly detected after 48-h incubation. Propidium monoazide (PMA) assisted 16S rRNA gene real-time PCR suggested that a large number of B. ferrooxidans cells were alive during incubation. However, 2H(D)-isotope based Raman analysis indicated that the cells were metabolically inactive after 120-h of ANDFO. These inactive cells re-awakened in R2A medium and were capable of growth and reproduction, which was consistent with results in Raman analysis. Scanning electron microscopy (SEM) observation and x-ray diffraction (XRD) revealed the formation of Fe minerals in close proximity of cells in the Fe(II)-oxidizing medium after Fe(II) oxidation. Overall, our results demonstrated that continued ANDFO can induce a metabolically inactive state in B. ferrooxidans, which was responsible for the loss of activity during ANDFO. This study provides an insight into the ANDFO process and its contribution to iron and nitrogen cycling in the environments.

A common soil flagellate (Cercomonas sp.) grows slowly when feeding on the bacterium Rhodococcus fascians in isolation, but does not discriminate against it in a mixed culture with Sphingopyxis witflariensis.

Flagellates are very important predators on bacteria in soil. Because of their high growth rates, flagellate populations respond rapidly to changes in bacterial numbers. Previous results indicate that actinobacteria are generally less suitable than proteobacteria as food for flagellates. In this study, we investigated the growth of the flagellate Cercomonas sp. (ATCC 50334) on each of the two bacteria Sphingopyxis witflariensis (Alphaproteobacteria) and Rhodococcus fascians (actinobacteria) separately and in combination. The growth rate of the flagellate was lower and the lag phase was longer when fed with R. fascians than when fed with S. witflariensis. This supports our initial hypothesis that the actinobacterium is less suitable as food than the alphaproteobacterium. However, after longer periods of growth the peak abundance of flagellates was higher on R. fascians, indicating that the food quality of bacterial prey depends on the time perspective of the flagellate-bacterial interaction. There was no evidence that the flagellates selected against the actinobacterium when feeding in mixed cultures of the two bacteria. Experiments where flagellates were fed with washed bacterial cells or with bacteria growing with different substrate concentrations suggested that the low food quality of R. fascians is related both to the intrinsic cell properties and to the extracellular metabolites.

Toxicity of cadmium and zinc to small soil protists.

Small heterotrophic protists (flagellates and naked amoebae) are very abundant in soil and play a key role in maintaining soil services. Hence, knowledge on how xenobiotics affect these organisms is essential in ecosystem management. Cadmium (Cd) is an increasing environmental issue as both industrial deposition and recycling of heavy metal rich waste products have led to Cd enrichment of soils. Evaluation of toxicity of Cd to micro-organisms is often performed using a solution of pure Cd (e.g. CdCl) in liquid culture. This approach may be highly misleading as interactions between Cd and other substances, e.g. various ions or inherent soil components often strongly modify Cd toxicity. Hence, we compared the toxic effect of Cd to small heterotrophic protists in soil microcosms and liquid culture. We also evaluated how zinc (Zn) affects Cd toxicity, as Zn usually accompanies Cd in a ratio of c. 100:1, and is known to impede Cd toxicity. In the soil microcosms, we also monitored the primary food source of the protists, i.e. culturable bacteria, and used soil respiration as a proxy of soil functioning. Finally, we examined to what extent Cd actually sorbs to soil. We found 1) that c. 103 times more Cd was required to obtain the same effect in the soil microcosms compared to the liquid culture, 2) that soil sorption explains why Cd, even though highly toxic in aqueous solutions, has very limited effect when applied to soil, and 3) (very surprisingly) that in our experimental systems Zn was as toxic as Cd. Our study suggests that Cd toxicity to soil protists will be small because most Cd in soil will be sorbed to the soil matrix and because the Zn:Cd ratio of 100:1 in most substances, incl. pollutants, will mean that lethal Zn effects will occur before Cd reaches toxic levels.

Dynamics and determinants of amoeba community, occurrence and abundance in subtropical reservoirs and rivers.

Free-living amoebae are widespread in freshwater ecosystems. Although many studies have investigated changes in their communities across space, the temporal variability and the drivers of community changes across different habitat types are poorly understood. A total of 108 surface water samples were collected on a seasonal basis from four reservoirs and two rivers in Xiamen city, subtropical China. We used high throughput sequencing and qPCR methods to explore the occurrence and abundance of free-living amoebae. In total, 335 amoeba OTUs were detected, and only 32 OTUs were shared by reservoir and river habitats. The reservoirs and rivers harbored unique amoebae communities and exhibited distinct seasonal patterns in community composition. High abundance of the 18S rRNA gene of Acanthamoeba was observed in spring and summer, whereas the abundance was low in autumn and winter. In addition, the abundance of Hartmannella was significantly higher when isolated from reservoirs in summer/autumn and from river in spring/summer. Moreover, the temporal patterns of amoebae communities were significantly associated with water temperature, indicating that temperature is an important variable controlling the ecological dynamics of amoebae populations. However, our comparative analysis indicated that both environmental selection, and neutral processes, significantly contributed to amoeba community assembly. The genera detected here include pathogenic species and species that can act as vectors for microbial pathogens, which can cause human infections.

Crosslinks rather than strand breaks determine access to ancient DNA sequences from frozen sediments.

Diagenesis was studied in DNA obtained from Siberian permafrost (permanently frozen soil) ranging from 10,000 to 400,000 years in age. Despite optimal preservation conditions, we found the sedimentary DNA to be severely modified by interstrand crosslinks; single- and double-stranded breaks; and freely exposed sugar, phosphate, and hydroxyl groups. Intriguingly, interstrand crosslinks were found to accumulate approximately 100 times faster than single-stranded breaks, suggesting that crosslinking rather than depurination is the primary limiting factor for ancient DNA amplification under frozen conditions. The results question the reliability of the commonly used models relying on depurination kinetics for predicting the long-term survival of DNA under permafrost conditions and suggest that new strategies for repair of ancient DNA must be considered if the yield of amplifiable DNA from permafrost sediments is to be significantly increased. Using the obtained rate constant for interstrand crosslinks the maximal survival time of amplifiable 120-bp fragments of bacterial 16S ribosomal DNA was estimated to be approximately 400,000 years. Additionally, a clear relationship was found between DNA damage and sample age, contradicting previously raised concerns about the possible leaching of free DNA molecules between permafrost layers.

Wood Ash Induced pH Changes Strongly Affect Soil Bacterial Numbers and Community Composition.

Recirculation of wood ash from energy production to forest soil improves the sustainability of this energy production form as recycled wood ash contains nutrients that otherwise would be lost at harvest. In addition, wood-ash is beneficial to many soils due to its inherent acid-neutralizing capabilities. However, wood ash has several ecosystem-perturbing effects like increased soil pH and pore water electrical conductivity both known to strongly impact soil bacterial numbers and community composition. Studies investigating soil bacterial community responses to wood ash application remain sparse and the available results are ambiguous and remain at a general taxonomic level. Here we investigate the response of bacterial communities in a spruce forest soil to wood ash addition corresponding to 0, 5, 22, and 167 t wood ash ha-1. We used culture-based enumerations of general bacteria, Pseudomonas and sporeforming bacteria combined with 16S rRNA gene amplicon sequencing to valuate soil bacterial responses to wood ash application. Results showed that wood ash addition strongly increased soil pH and electrical conductivity. Soil pH increased from acidic through neutral at 22 t ha-1 to alkaline at 167 t ha-1. Bacterial numbers significantly increased up to a wood ash dose of 22 t ha-1 followed by significant decrease at 167 t ha-1 wood ash. The soil bacterial community composition changed after wood ash application with copiotrophic bacteria responding positively up to a wood ash dose of 22 t ha-1 while the adverse effect was seen for oligotrophic bacteria. Marked changes in bacterial community composition occurred at a wood ash dose of 167 t ha-1 with a single alkaliphilic genus dominating. Additionally, spore-formers became abundant at an ash dose of 167 t ha-1 whereas this was not the case at lower ash doses. Lastly, bacterial richness and diversity strongly decreased with increasing amount of wood ash applied. All of the observed bacterial responses can be directly explained by the wood ash induced changes in pH, electrical conductivity and the addition of wood ash inherent nutrients.

Wood ash application increases pH but does not harm the soil mesofauna.

Application of bioash from biofuel combustion to soil supports nutrient recycling, but may have unwanted and detrimental ecotoxicological side-effects, as the ash is a complex mixture of compounds that could affect soil invertebrates directly or through changes in their food or habitat conditions. To examine this, we performed laboratory toxicity studies of the effects of wood-ash added to an agricultural soil and the organic horizon of a coniferous plantation soil with the detrivore soil collembolans Folsomia candida and Onychiurus yodai, the gamasid predaceous mite Hypoaspis aculeifer, and the enchytraeid worm Enchytraeus crypticus. We used ash concentrations spanning 0-75 g kg-1 soil. As ash increases pH we compared bioash effects with effects of calcium hydroxide, Ca(OH)2, the main liming component of ash. Only high ash concentrations above 15 g kg-1 agricultural soil or 17 t ha-1 had significant effects on the collembolans. The wood ash neither affected H. aculeifer nor E. crypticus. The estimated osmolalities of Ca(OH)2 and the wood ash were similar at the LC50 concentration level. We conclude that short-term chronic effects of wood ash differ among different soil types, and osmotic stress is the likely cause of effects while high pH and heavy metals is of minor importance.