Resting spore formation and biochemical composition of the marine planktonic diatom Chaetoceros pseudocurvisetus in culture: ecological significance of decreased nucleotide content and activation of the xanthophyll cycle by resting spore formation

The biochemical composition of vegetative cells and resting spores of Chaetoceros pseudocurvisetus Mangin was compared in cultures under various nutrient and light conditions. The cellular content of major nucleotides such as AMP, ADP, ATP and UTP decreased in the order: vegetative cells, nutrient-starved (vegetative) cells and resting spores, indicating that the general metabolism of resting spores is relatively inactive. ADP-glucose was only abundant in nutrient-starved vegetative cells, suggesting metabolic imbalance in these cells. The chl a–specific fluorescence yield of vegetative cells grown under all culture conditions was low, but very high in resting spores. The ratios of the cellular contents of diadinoxanthin to chl a and of diatoxanthin to chl a were higher in resting spores and nutrient-starved vegetative cells than in nutrient-replete vegetative cells. The diadinoxanthin–diatoxanthin xanthophyll cycle was active in resting spores; the xanthophyll cycle was synchronized with a 14 h light:10 h dark photoperiod. Also, the ratios of cellular content of diadinoxanthin and diatoxanthin to cellular content of chl a in resting spores were relatively high in high irradiance, and decreased gradually in conditions of darkness over long culture periods. Under conditions of strong light and high temperature, most resting spores survived more than 40 d while nutrient-starved vegetative cells died within 33 d. These results suggest that resting spore formation is a strategy for enhancing protection and lowering metabolic rate for survival. These physiological changes accompanying spore formation enable resting spores not only to overwinter but also to “oversummer” in the coastal euphotic layer. Received: 23 March 1999 / Accepted: 11 August 1999     

Survival and recovery of resting spores and resting cells of the marine planktonic diatom Chaetoceros pseudocurvisetus under fluctuating nitrate conditions

Of the two resting life-forms of the planktonic diatom Chaetoceros pseudocurvisetus Mangin formed during periods of nitrate depletion, resting spores survived at least 1 month after spore formation at 24 °C, while resting cells survived only for about 10 d at the same temperature. Under nitrogen limitation, resting cells exhibited higher specific death rates than resting spores at temperatures ranging from 5 to 30 °C. After nitrogen replenishment, resting spores required a certain lag period of about 1 d to initiate vegetative growth at levels of nitrate supply from 0.5 to 20 M, while resting cells initiated vegetative growth almost immediately. Resting spores exhibited an intracellular accumulation of the supplied nitrate during germination and initial vegetative growth. The resting cells, however, exhibited more active vegetative growth, closely coupled with the uptake of the supplied nitrate. The resting spores and resting cells appear to play different roles in the maintenance of populations under nutrient fluctuations depending on the interval length between nutrient fluxes in natural waters. Received: 27 April 1998 / Accepted: 1 March 1999     

Resting spore formation of the marine planktonic diatom Chaetoceros anastomosans induced by high salinity and nitrogen depletion

The marine planktonic diatom Chaetoceros anastomosans, which was isolated from Sagami Bay, was used for a study of resting spore formation mechanisms in batch culture experiments. Vegetative cells could grow at salinities ranging from 20.7 to 45.5‰, and resting spore formation was enhanced significantly in nitrate-depleted, high salinity media (40.0 to 45.5‰). The rate of resting spore formation (1.9 d⁻¹) was comparable to the specific growth rate (1.8 d⁻¹) of vegetative cells in the exponential growth phase in normal salinity medium. The size of resting spores formed under high salinity conditions was smaller than that of spores formed in normal salinity media. Unlike vegetative cells, resting spores seemed to possess some mechanisms to survive over a wider range of salinities by resisting bacterial attacks on their cell walls. Received: 4 August 1996 / Accepted: 27 August 1996     

Life-form population responses of a marine planktonic diatom,Chaetoceros pseudocurvisetus,to oligotrophication in regionally upwelled water

Life-form population responses of a centric planktonic diatom,Chaetoceros pseudocurvisetus Mangin, were investigated in summer 1986 and 1988 in the Izu Islands, Japan, in regionally upwelled water where nutrient concentration changed from favorable to unfavorable conditions for active growth and reproduction (oligotrophication). Two types of life form were observed: vegetative cells of healthy and unhealthy looking conditions and resting spores. The observed life-form responses were experimentally evaluated along with a depletion of limiting nutrients. The algal population ceased vegetative growth and initiated resting spore formation with a disappearance of limiting nitrate, and this life-form response seemed to be triggered by the decrease of cellular nitrogen content below a certain level. Since a large amount of silicon was required for the resting spore formation, a part of vegetative cells were unable to form resting spores and formed unhealthy looking vegetative cells under insufficient concentrations of silicic acid. Percentage shares of the resting spores in the population were linearly related to the amounts of available silicic acid. Vegetative cells which did not form resting spores showed greater mortality than resting spores by attack of bacteria and protozoa; however, vegetative cells could respond quickly to possible nutrient replenishment.     

Uptake and release of phosphorus by phytoplankton in the Chesapeake Bay estuary,USA

The phytoplankton uptake and release rates for inorganic phosphate, dissolved organic phosphate and polyphosphate were estimated during 5 cruises on the Chesapeake Bay over a 9-month period. Phosphorus in all pools turned over in several minutes to 100 h, and each soluble pool appeared to contain fractions which were metabolically useful to the phytoplankton. Maximal uptake rates (V _m ) for orthophosphate ranged from 0.02 to 2.95 μg-at P (1.h)^-1 with half saturation constants (K _s ) between 0.09 and 1.72 μg-at P l^-1. At low soluble reactive phosphorus concentrations, the uptake rate of trace ³²P orthophosphate was initially rapid, but declined after 15 to 60 min incubation. The data suggest that the initial uptake phase was dominated by exchange of ³²PO₄ ^≡ for ³¹PO₄ ^≡ in the membrane transport systems whereas the subsequent phase represented the net incorporation of orthophosphate into phytoplankton cells.     

Aspects of the biology of resting spores of Thalassiosira nordenskioeldii and Detonula confervacea

Large numbers of resting spores of Thalassiosira nordenskioeldii Cleve and Detonula confervacea (Cleve) Gran were produced when these species were grown at low levels of ammonia and nitrate. The production of resting spores by T. nordenskioeldii was inversely related to temperature. At 0° and 5°C between 68 and 96% of the total cells were resting spores, while at 15°C resting spores were not produced. Resting spores of both species would not survive 7 days in the dark at 20°C. At 0°, 5°, 10° and 15°C, the length of time that the resting spores of both T. nordenskioeldii and D. confervacea remained viable was inversely related to temperature. At 0°C, T. nordenskioeldii remained viable for 576 days. The data suggest that the production of resting spores by these two species does not aid them in the survival of unfavorably high temperatures such as are found in temperate estuaries during the summer. Rather, they appear to be an adaptation for the survival of long periods of darkness in polar regions.     

Phosphorus metabolism of oceanic dinoflagellates: phosphate uptake,chemical composition and growth of Pyrocystis noctiluca

The phosphorus metabolism of Pyrocystis noctiluca Murray (Schuett) 1886 has characteristics which may enhance its potential for success in orthophosphate impoverished waters. The steady-state phosphate uptake rates were equal in the light and dark, and were directly proportional to both the phosphorus cell quota and the cell division rate. In contrast, nutrient-saturated uptake rates were multiphasic, faster in the light than the dark, 2 to 4 orders of magnitude greater than steady-state rates, and were inversely proportional to both the phosphorus cell quota and the cell division rate. These uptake characteristics suggest that P. noctiluca may take up phosphate coincidently at their typically low ambient concentrations as well as to exploit episodic nutrient events in nature. Cell division rates were a hyperbolic function of the ambient orthophosphate concentration. The shortest doubling time was 8.7 d, the phosphate concentration at half the maximum division rate was 0.15 M and the threshold, concentration for cell division was ca 0.05 M PO ₄ ^3- . Division rates of P. noctiluca in the ocean are much faster than predicted from the measured ambient orthophosphate concentrations. Since this dinoflagellate has high naturally occurring alkaline phosphatase activities, and can utilize organic-P compounds, we suggest that organic-P can be as important as orthophosphate in supporting the observed division rates of P. noctiluca in the sea.     

Cell-cell recognition system in gorgonians: description of the basic mechanism

A comparative investigation of the chemical composition of Thalassiosira antarctica var. antarctica vegetative and resting stages revealed C:N and C:chl a ratios to be lower in vegetative cells. These trends primarily reflect vegetative levels of C/cell below, and N/cell and chl a/cell levels above those of spores. There was a change in chemical composition with the initial formation of resting spores, and spores continued to modify their composition while maintained in a cyclic light/dark regime for about one and one half weeks. Most notable was a net increase in carbon and chlorophyll a per cell. Spores then subjected to darkness for over one week appeared to retain most of the carbon and chlorophyll a previously synthesized. These findings support the idea that resting spores enhance the survival capabilities of a species under adverse conditions.     

“Seed bank” of coastal planktonic diatoms in bottom sediments of Hiroshima Bay, Seto Inland Sea, Japan

Abundance and temporal distribution of viable (able to germinate) resting stage cells of planktonic diatoms in bottom sediments have been investigated almost monthly during 1989 to 1992 in Hiroshima Bay, western part of Seto Inland Sea, Japan. The abundance of viable resting stages in bottom sediments was enumerated with the extinction dilution method (most probable number method, MPN). In bottom sediments of Hiroshima Bay, dominantly distributed species and/ or genera of the diatom resting stages were Skeletonema costatum, Chaetoceros spp. and Thalassiosira spp. Viable resting stages of these diatoms were densely distributed on the orders of 10³ to 10⁶ (MPN g⁻¹ wet sediments), and persisted in bottom sediments throughout the investigation period. Conversely, vegetative cells of these diatoms fluctuated remarkably in the water column and disappeared sporadically. Survival of the resting stages in a collected sediment sample was also determined with the MPN method, at different storage temperatures (5, 10, 15, 20, 25 °C). The survival test demonstrated that the diatom resting stages could survive in the dark for several months or years in sediments. Resting stages survived longer at the lower storage temperature, and the order of longevity was consistent within three diatoms (Chaetoceros spp. > Thalassiosira spp. > S. costatum) at each storage temperature. The present study suggests that these diatom resting stages in the coastal bottom sediments could serve as a “seed bank”, analogous to those of terrestrial plants. The seed bank would ensure the survival of diatoms within highly fluctuating coastal environments, while it would also be the source of sporadic and autochthonous diatom blooms in coastal waters. Received: 29 November 1996 / Accepted: 16 December 1996     

The role of diatom resting stages in the onset of the spring bloom in the East China Sea

The abundance and species composition of diatoms were investigated along the PN line from the Okinawa Islands to the inner continental shelf in the East China Sea in the early spring of 1996. Viable diatom resting stages in sediments on the shelf were also enumerated by the extinction dilution method (most probable number method). Clear differentiation in the water masses was observed, with less saline, cold water (shelf water) on the shelf region, and warm, saline water (Kuroshio water) prevalent off the shelf and on the shelf edge. In the Kuroshio water, the abundance of diatoms was generally low but species diversity of diatoms was relatively high. In contrast, the spring bloom of diatoms was clearly observed in the shelf water where the water column was weakly stratified. The bloom was dominated by Chaetoceros debilis, contributing occasionally >80% of the diatom community. Resting stages of this species were also the most abundant taxon in the sediments on the shelf. Resuspension of the sediment during winter mixing of the water column should have enabled the resting stages to germinate at the surface. Subsequent vegetative growth after germination led to the formation of an early spring bloom of C. debilis when the water column was stratified and light availability had increased. Intermittent resuspension of sediment on the shelf, driven by strong winds and tidal currents, probably provides opportunities for diatoms with a resting stage to exploit favorable conditions for their germination and subsequent vegetative growth. It is further suggested that complex hydrographic conditions in the East China Sea result in a dynamic bloom with contributions by both autochthonous and allochthonous species.Communicated by T. Ikeda, Hakodate     

Effect of inorganic phosphorus on the growth rate of diatoms

The effect is studied of different inorganic phosphorus concentrations on changes in growth rates of 7 diatom species isolated from the plankton of the Black Sea. All species examined increase their cell-division rate with increasing phosphorus concentration in the medium. The phosphorus concentrations have been determined above which division rate is not limited by phosphorus content in the medium. The non-limiting concentration amounts to V/V _m =0.9. In the species studied, non-limiting concentrations ranged from 1 to 30 g P/1. The highest values were obtained for relatively large-sized species. The ratio of cell surface to volume tended to be inversely related to the growth-rate-limiting concentration. Minimal values of phosphorus content in cells have been calculated to range from 0.6 to 4.0·10^–17 g.at–P/^–3. Based on a comparison of phosphate levels in the Black Sea with experimentally-derived rate-limiting concentrations, it is concluded that phosphorus does not limit the reproduction rate of phytoplankton in the more productive regions of the Black Sea or in the lower strata of the euphotic zone.     

Early murine immune responses from endotracheal exposures to biotechnology-related Bacillus strains

An immunology-based in vivo screening regime was used to assess the potential pathogenicity of biotechnology-related microbes. Strains of Bacillus cereus (Bc), Bacillus subtilis (Bs), Bacillus thuringiensis (Bt), and Bt commercial products (CPs) were tested. Balb/c mice were endotracheally instilled with purified spores, diluted CP, or vegetative cells (VC) (live or dead). Exposed mice were evaluated for changes in behavioral and physical symptoms, bacterial clearance, pulmonary granulocytes, and pulmonary and circulatory pyrogenic cytokines (interleukins (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α), as well as acute phase biomarkers (fibrinogen and serum amyloid A). Except for some differences in clearance rates, no marked effects were observed in mice exposed to any spore at 10⁶ or 10⁷ colony forming units (cfu). In contrast, live Bc or Bt VCs (10⁵ or 10⁶?cfu) produced shock-like symptoms (lethargy, hunched appearance, ruffled fur, and respiratory distress), and 11–200-fold elevations in pyrogenic cytokines at 2-h post-exposure. In the study, 4-h effects included increased lethargy, ocular discharge, and 1.5–4-fold rise in circulatory acute phase markers, but no indications of recovery. Bs VC did not produce any changes in symptoms or biomarkers. After 2 or 4?h of exposure to dead VC, increases of only plasma IL-1β and TNF-α (4.6- and 12.4-fold, respectively) were observed. These findings demonstrate that purified spores produced no marked effects in mice compared to that of metabolically active bacteria. This early screening regime was successful in distinguishing the pathogenicity of the different Bacillus species, and might be useful for assessing the relative hazard potential of other biotechnology-related candidate strains.     

Waste-water assay with continuous algal cultures: The effect of mercuric acetate on the growth of some marine dinoflagellates

The effect of mercuric acetate was studied in culture experiments with the dinoflagellates Scrippsiella faeroense (Paulsen) Balech et Soares, Prorocentrum micans Ehrenberg and Gymnodinium splendens Lebour. Impairment of growth rates, in vivo chlorophyll fluorescence, maximum cell densities and morphological changes served as criteria for assessing sublethal influences. Tests were made using the batch- and continuous-culture techniques. Addition of Hg at concentrations of 0.001 mg.1^-1 and higher resulted in reduction of relative growth rates. In a few cases populations recovered from the initial decline and showed new growth. Cell counts corresponded very closely to in vivo chlorophyll fluorescence measurements. Morphological variations were observed in S. faeroense, which responded (even in sublethal concentrations) by bursting it's thecae, releasing naked motile cells and forming vegetative resting stages. The problems of optimal algal-bioassay methods are discussed also, in the light of results obtained by other authors.     

有机磷细菌的分离鉴定及其对MC-RR的响应(The Isolation and Identification of Organic Phosphorus Bacteria and Its Response to MC-RR)

从云南滇池水样中分离出具有解磷能力的有机磷细菌P-2,并利用现代分子生物学技术进行了初步鉴定.用0.01、5mg·L-1微囊藻毒素(MC-RR)处理有机磷细菌P-2,研究了MC-RR对其生长、细胞内酸碱磷酸酶活性(ACP和AKP)以及培养液中可溶性磷酸盐含量的影响.结果表明,高浓度MC-RR能显著抑制有机磷细菌的生长,延缓其细胞增殖,抑制细胞内酸碱磷酸酶活性以及培养液中可溶性磷酸盐含量的升高,因而可能改变或减缓生态系统中磷循环的进程,这表明微囊藻毒素在一定程度上可能调节水体细菌功能群落.     

Impact of raking and bioturbation-mediated ecological manipulation on sediment–water phosphorus diagenesis: a mesocosm study supported with radioactive signature

The study examined the impact of raking and fish bioturbation on modulating phosphorus (P) concentrations in the water and sediment under different trophic conditions. An outdoor experiment was set to monitor physicochemical and microbiological parameters of water and sediment influencing P diagenesis. A pilot study with radioactive ³²P was also performed under the agency of raking and bacteria (Bacillus sp.). Raking was more effective in release of P under unfertilized conditions by significantly enhancing orthophosphate (35%) and soluble reactive phosphate (31.8%) over respective controls. Bioturbation increased total and available P in sediments significantly as compared to control. The rates of increase were higher in the unfertilized conditions (17.6–28.4% for total P and 12.2 to 23.2% for available P) than the fertilized ones (6.5–12.4% for total P and 9.1 to 15% for available P). The combined effects of raking and bioturbation on orthophosphate and soluble reactive phosphate were also stronger under unfertilized state (54.5 and 81.8%) than fertilized ones (50 and 70%). The tracer signature showed that coupled action of introduced bacteria and repeated raking resulted in 59.2, 23 and 16% higher counts of radioactive P than the treatments receiving raking once, repeated raking and bacteria inoculation, respectively. Raking alone or in sync with bioturbation exerted pronounced impact on P diagenesis through induction of coupled mineralization and nutrient release. It has significant implication for performing regular raking of fish-farm sediments and manipulation of bottom-grazing fish to regulate mineralization of organic matter and release of obnoxious gases from the system. Further, they synergistically can enhance the buffering capacity against organic overload and help to maintain aquatic ecosystem health.

     

Relationship between phytoplankton cell size and the rate of orthophosphate uptake: in situ observations of an estuarine population

Orthophosphate uptake by a natural estuarine phytoplankton population was estimated using two methods: (1) ³²P uptake experiments in which filters of different pore sizes were used to separate plankton size-fractions; (2) ³³P autoradiography of phytoplankton cells. Results of the first method showed that plankton cells larger than 5 m were responsible for 2% of the total orthophosphate uptake rate. 98% of the total uptake rate occurred in plankton composed mostly of bacteria, which passed the 5 m screen and were retained by the 0.45 m pore-size filter. There was no orthophosphate absorption by particulates in a biologically inhibited control containing iodoacetic acid. Orthophosphate uptake rates of individual phytoplankton species were obtained using ³³P autoradiography. The sum of these individual rates was very close to the estimated rate of uptake by particulates larger than 5 m in the ³²P labelling experiment. Generally, smaller cells were found to have a faster uptake rate per m³ biomass than larger cells. Although the nannoplankton constituted only about 21% of the total algal biomass, the rate of phosphate uptake by the nannoplankton was 75% of the total phytoplankton uptake rate. Results of the plankton autoradiography showed that the phosphate uptake rate per unit biomass is a power function of the surface: volume ratio of a cell; the relationship is expressed by the equation Y=2x10^-11 X ^1.7, where Y is gP m^-3 h^-1 and X is the surface: volume ratio. These results lend support to the hypothesis that smaller cells have a competitive advantage by having faster nutrient uptake rates.     

Characterization of alkaline phosphatase and organic phosphorous utilization in the oceanic dinoflagellate Pyrocystis noctiluca

Phosphate depleted Pyrocystis noctiluca (Murray) Schuett 1895 has at least one phosphomonoesterase (EC 3:1:3:1) which is triphasic between 0.1 and 222 mol P. The enzyme has a broad temperature range with maximum activity at 50 °C and a Q₁₀ of 1.4 to 1.5. A break in the Arrhenius plot at 35 °C implies the enzyme is membrane-bound. Cytological staining of whole cells and cell fractionation studies (showing 26 times higher specific activity in the particulate compared with the cytoplasmic fraction) suggest the enzyme is plasmalemma-bound. The enzyme has an absolute metal requirement which would be satisfied by Mg⁺⁺ but not Mn⁺⁺, Zn⁺⁺, Fe⁺⁺, or Co⁺⁺ at seawater concentrations. Alkaline phosphatase is a stable enzyme whose activity is not altered by inhibitors of protein synthesis. Orthophosphate inhibition of enzyme activity was largely eliminated in the presence of these inhibitors. Apparently, a protein induced by PO₄ ^3-, rather than PO₄ ^3- itself, inhibits alkaline phosphatase. Cell-free alkaline phosphatase can hydrolyze a variety of phosphate esters and linear polymers of inorganic phosphorus as well as disolved organic phosphorus from tropical oceanic waters. These same hydrolysable organic and inorganic phosphorus compounds support the axenic culture growth of P. noctiluca, suggesting that naturally occurring hydrolysable organic phosphorus compounds may also support the growth of this alga.     

Effects of the Interactions Between Selenium and Phosphorus on the Growth and Selenium Accumulation in Rice (Oryza Sativa)

The solution culture, paddy soil culture and the simulation experiments in the laboratory were conducted to clarify the interactions between selenium and phosphorus, and its effects on the growth and selenium accumulation in rice. Results revealed that a suitable supply of selenium could promote rice growth and excessive selenium could injure rice plant, causing lower biomass, especially in the roots. The supply of selenite could enhance the selenium contents of rice shoots and roots in solution culture and in soil culture. The selenium concentrations in roots were much higher than those in shoots supplied with the same rates of selenium and phosphorus. The interaction between selenium and phosphorus was evident. When the phosphorus supply increased to meet the needs of plant growth, phosphorus could promote absorption and accumulation of selenium in the shoots. If the phosphorus supply was excessive, phosphorus could inhibit the accumulation of selenium in the shoots at the lower selenite level (2 mol l^–1), but could not at the higher selenite level (10 mol l^–1). With the supply of phosphate increased, the selenium concentrations in the roots decreased significantly at both selenite levels. The presence of phosphate could decrease Se sorption on the soil surface and increase the selenium concentration in the soil solution. The concentrations of selenium in shoots and roots supplied with 0.08 g kg^–1 phosphorus were lower than those with no phosphorus supplied. With the increase of phosphorus added to 0.4 g kg^–1, the selenium concentration in shoots and roots increased. The effect of phosphorus on the concentration was statistically significant at all three selenium levels.     

锌在不同磷源条件下对铜绿微囊藻生长与产毒的影响

针对磷和微量元素对藻类生长的共同作用,研究不同磷源条件下锌对藻细胞生长与产毒的影响。实验选用铜绿微囊藻为藻种,分别以无机磷磷酸氢二钾(K_2HPO_4)、小分子有机磷甘油磷酸钠(NaGly)和大分子有机磷卵磷脂(LEC)为磷源,研究不同锌(Zn~(2+))含量对藻细胞的藻密度、碱性磷酸酶活性(alkaline phosphatase activity,APA)以及胞内藻毒素(MC-LR)的影响。研究发现:以NaGly为磷源时微量元素锌对藻细胞生长的促进效果显著,而以K_2HPO_4或LEC为磷源时,锌含量的变化对藻细胞生长无显著影响。APA不仅与磷源有关而且与锌含量相关,以LEC为磷源时的APA显著高于以K_2HPO_4或NaGly为磷源时的APA,且锌含量越低APA越低,以K_2HPO_4为磷源时锌含量越低APA越高,而锌对以NaGly为磷源时的APA几乎没有影响。磷源与微量元素锌对藻细胞的产毒均产生影响,NaGly有利于藻毒素的产生; LEC不利于藻细胞的产毒,但锌含量越低藻细胞的产毒量越多。综上所述,磷源与微量元素锌共同作用对藻细胞的生长与产毒产生影响,小分子有机磷NaGly与锌的效果显著。     

Peak assignments for phosphorus-31 nuclear magnetic resonance spectroscopy in pH range 5–13 and their application in environmental samples

Phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy has become popular for the characterization of P species in environmental samples. However, these are commonly made alkaline (pH?>?13) to facilitate sample comparison and ease peak identification, but this may cause hydrolysis of some compounds. This study examined the chemical shift of known P compounds and supplemented this with published data to determine the viability of examining samples at their native pH, thereby minimizing sample disturbance. A ³¹P NMR pH titration of known P compounds resulted in chemical shifts ranging from about ?22 to 8 ppm in the pH range 5–13. Categorization and calculation of chemical shifts for over 100 naturally occurring compounds indicated that good distinction between orthophosphate diesters, orthophosphate monoesters, nucleotides, phosphonates, and phosphagens was best at ≥pH 7, but unlikely below this pH. Analysis of several water extracts of soil and dung, overland flow samples, and lake water indicated a wide variety of well-defined peaks that were assigned to orthophosphate, orthophosphate monoesters, orthophosphate diesters, pyrophosphate, polyphosphate, or phosphonates. Changing the sample pH to >13 caused many species (such as phosphonates, orthophosphate diesters, and polyphosphates) to decrease either by hydrolysis or precipitation. Hence, it is recommended that samples be analysed at their native pH but, if poorly resolved, should have their pH raised to ≥7.