NMR investigation of the behavior of an organothiophosphate pesticide, chlorpyrifos, sorbed on soil components

Phosphorus-31 nuclear magnetic resonance (31P NMR) was used to follow the decomposition of chlorpyrifos (an organothiophosphate pesticide) adsorbed on soil, humic acid, partially hydrated kaolin clay, and partially hydrated montmorillonite clay at high concentration (typically 2-10 wt %). Solid-state 31P NMR (using magic-angle spinning and cross polarization or direct polarization) and liquid-solution 31P NMR of DMSO and acetone extracts indicate that chlorpyrifos is initially physisorbed, appearing by solid-state 31P NMR to exhibit significant motion on the molecular level, which results in almost liquidlike solid-state spectra. Over periods ranging from hours to years, the signals due to unreacted chlorpyrifos sorbed on the clays diminish and are replaced by new 31P NMR peaks resulting from hydrolysis, isomerization, mineralization, and oxidation reactions. The 31P NMR signal characteristics indicate that these decomposition products are much more tightly bound to the clay than is chlorpyrifos. Solid-state 13C and 27AI NMR spectra were less useful for following the decomposition of chlorpyrifos than those obtained by 31P NMR. Solid-state 31P NMR results indicate that a chlorpyrifos loading level of 10% by weight, used in some of the samples to facilitate 31P NMR detection of less-than-dominant decomposition products, exceeds the adsorption capacity of the soil, humic acid, and kaolinite tested, but not Ca2+ -exchanged montmorillonite. This pattern is consistent with intercalation into the montmorillonite, but only surface adsorption on kaolinite.     

NMR investigation of the behavior of an organothiophosphate pesticide, methyl parathion, sorbed on clays

The decomposition of methyl parathion (an organothiophosphate pesticide) sorbed on partially hydrated kaolin and montmorillonite clays (in Ca2+, Cu2+, Zn2+, and Al3+ forms) at high concentration (typically 1-10 wt %) has been examined by nuclear magnetic resonance (NMR), using solid-state 31P NMR (based on magic-angle spinning and cross polarization or direct polarization) and liquid-state 31P NMR of DMSO and acetone extracts. The results indicate that methyl parathion is initially physisorbed, appearing by solid-state 31P NMR to exhibit substantial molecular-level motion. The signals due to unreacted methyl parathion diminish and are replaced by new 31P NMR peaks resulting from hydrolysis, isomerization, and oxidation reactions over periods ranging from hours to years. 31P NMR characteristics indicate that these decomposition products are much more tightly bound to the clay than is methyl parathion. Methyl parathion decomposition is most effectively catalyzed by partially hydrated Cu(II)- and Al-montmorillonites (but with different product distributions); Ca-montmorillonite and kaolin were least effective.     

P NMR spectroscopic investigations of caseins treated with microbial transglutaminase

Caseins - the main constituents of bovine milk proteins - self-assemble into large supramolecular aggregates, so-called casein micelles. The enhancement of the stability of casein micelles is advantageous with respect to technological milk processing. A promising approach to accomplish this goal is the cross-linking of caseins using microbial transglutaminase (mTG). The present paper describes the combined use of liquid- and solid-state ³¹P NMR spectroscopy as well as dynamic light scattering in order to characterize the influence of an mTG treatment upon the structure of micelles in ultrahigh temperature (UHT)-treated skim milk at a molecular level. Liquid-state ³¹P NMR spectroscopy was applied to characterize milk, milk serum and casein dispersions. A narrow SerP signal in the liquid-state ³¹P NMR spectra of UHT-treated milk is shown to be due to casein molecules in the milk serum whereas the casein molecules bound in the micelles give rise to broad signals. Most of the caseins contribute to a 3 kHz broad background signal which can be visualized in the spectrum of suspensions of re-dispersed micellar material derived from UHT-treated milk. Treatment with mTG results in a cross-linking of caseins, which could be followed by liquid-state ³¹P NMR spectroscopy. Especially, the cross-linking of β-casein was demonstrated by quantitative liquid-state ³¹P NMR experiments. Furthermore, the stability of cross-linked micellar aggregates against EDTA could be investigated by liquid-state ³¹P HR NMR in combination with dynamic light scattering (DLS). Solid-state ³¹P NMR was used to show that the motional state of the κ-caseins located at the outer surface of the micelles derived from UHT-treated milk is not significantly changed by the applied mTG treatment.     

Direct speciation of phosphorus in alum-amended poultry litter: solid-state 31P NMR investigation

Amending poultry litter (PL) with aluminum sulfate (alum) has proven to be effective in reducing water-soluble phosphorus (P) in the litter and in runoff from fields that have received PL applications; it has therefore been suggested as a best management practice. Although its effectiveness has been demonstrated on a macroscopic scale in the field, little is known about P speciation in either alum-amended or unamended litter. This knowledge is important forthe evaluation of the long-term stability and bioavailability of P, which is a necessary prerequisite forthe assessment of the sustainability of intensive poultry operations. Both solid-state MAS and CP-MAS 31P NMR as well as 31P[27Al]-TRAPDOR were used to investigate P speciation in alum-amended and unamended PL. The results indicate the presence of a complex mixture of organic and inorganic orthophosphate phases. A calcium phosphate phase, probably a surface precipitate on calcium carbonate, could be identified in both unamended and alum-amended PL, as well as physically bound HPO4(2-). Phosphate associated with Al was found in the alum-amended PL, most probably a mixture of a poorly ordered wavellite and phosphate surface complexes on aluminum hydroxide that had been formed by the hydrolysis of alum. However, a complex mixture of organic and inorganic phosphate species could not be resolved. Phosphate associated with Al comprised on average 40 +/- 14% of the total P in alum-amended PL, whereas calcium phosphate phases comprised on average 7 +/- 4% in the alum-amended PL and 14 +/- 5% in the unamended PL.     

Adsorption of organophosphates into microporous and mesoporous NaX zeolites and subsequent chemistry

Due to the neurotoxicity of organophosphate (OP) pesticides and nerve agents synthesized as military or terror agents, their safe destruction and disposal is of considerable current importance. A representative OP, trimethyl phosphate (TMP), was adsorbed onto NaX zeolite, two mesoporous modifications, and a low-silica X zeolite. The nucleophilic chemical reactions of TMP with the zeolites were investigated by solid-state 13C and 31P nuclear magnetic resonance (NMR) and the solvent extracts by 1H, 13C, and 31P NMR. Nucleophilic substitution and subsequent hydrolysis reaction schemes are proposed. All of the zeolites have similar TMP decomposition yields, supporting the hypothesis that slow or incomplete diffusion of TMP in the microporous zeolite regions limits TMP decomposition.     

Accurate quantification of aromaticity and nonprotonated aromatic carbon fraction in natural organic matter by 13C solid-state nuclear magnetic resonance

An improved approach for accurately determining the aromatic carbon fraction (fa) and nonprotonated aromatic carbon fraction (faN) in natural organic matter by solid-state 13C NMR is described. Quantitative peak areas are obtained from direct polarization 13C nuclear magnetic resonance (NMR) under high-speed magic angle spinning (MAS). The problem of overlap between aromatic and alkyl carbon resonances around 90-120 ppm in 13C NMR spectra is solved by a 13C chemical shift anisotropy (CSA) filter technique. After correction for residual spinning sidebands, an accurate value of the aromaticity fa is obtained. To obtain a quantitative faN fraction, dipolar dephasing was adapted for high-speed MAS 13C NMR; the separation of the signals of nonprotonated alkyl and aromatic carbons was achieved by CSA filtering plus dipolar dephasing. The method is demonstrated on a peat humic acid, yielding fa = 45 +/- 2% and faN = (0.64 +/- 0.07) x 45%.     

Solid-State 31P NMR, a Relevant Method to Evaluate the Distribution of Phosphates in Semi-hard Cheeses

The potentiality of solid-state ³¹P nuclear magnetic resonance (NMR) to determine in a non-destructive way the different states of phosphates in cheeses was examined. Sixteen semi-hard cheeses of various compositions were studied, and three fractions of phosphates (P) were distinguished according to their mobility: (1) mobile soluble P (ca. 10 % of total P), (2) mobile insoluble P (70 %) and (3) immobile insoluble P (20 %). In accordance with chemical composition and buffering capacities of the cheeses, these fractions could represent respectively (1) soluble inorganic P, (2) inorganic colloidal calcium P and phosphorylated serine residues (Pser) involved in a loose structure and (3) Pser involved in a tight environment. This method was shown to be of sufficient accuracy to evidence the effect of ripening on the different P fractions. It was thus demonstrated that solid-state NMR is an appropriate method to observe the distribution of phosphates in cheese matrix and their evolution during cheese-making.     

一株产高活性多酚氧化酶菌株筛选鉴定及其酶学性质

采用变色圈法从土壤样品中分离筛选产多酚氧化酶的菌株,对其进行形态学观察、分子生物学鉴定,并对其所产多酚氧化酶的酶学性质进行研究。结果表明,筛选得到1株产多酚氧化酶活性较高的菌株,命名为ZL-2,其被鉴定为竹黄菌属（Shiraia sp.）。菌株ZL-2发酵8 d产多酚氧化酶酶活最高,达到521 U/mL。菌株ZL-2产多酚氧化酶最适底物为邻苯二酚,其最适pH值为6、最适温度为30 ℃;酶活在温度20～40 ℃及pH 3～6范围内稳定。金属离子Cu2+、Mg2+、Mn2+、Fe3+、Zn2+对多酚氧化酶都有激活作用,其中Cu2+激活作用最强;Ca2+、Al3+对酶活有一定抑制作用;L-抗坏血酸和亚硫酸氢钠对该酶抑制作用较强。     

好食脉孢霉发酵产类胡萝卜素的鉴定、抗氧化性及稳定性研究

利用液相色谱-质谱联用技术对好食脉孢霉(Neurospora sitophila)固态发酵生产的类胡萝卜素进行初步鉴定,研究了类胡萝卜素粗提物的抗氧化活性和稳定性。N.sitophila来源的类胡萝卜素的主要成分之一为β-胡萝卜素;当质量浓度为6μg/mL时,类胡萝卜素的DPPH自由基清除率显著高于α-生育酚、丁基羟基茴香醚(butyl hydroxylanisole,BHA)和β-胡萝卜素(P <0.05),羟自由基清除率显著高于α-生育酚、BHA、特丁基对苯二酚(tert-butyl hydroquinone,TBHQ)和β-胡萝卜素(P <0.05),总还原力显著高于α-生育酚和β-胡萝卜素(P <0.05);类胡萝卜素在避光、低温及不含K⁺、Na⁺、Mg²⁺、Ca²⁺、Ba²⁺、Cu²⁺、Fe³⁺的条件下较稳定。研究结果表明好食脉孢霉类胡萝卜素具有较强的抗氧化活性,具有开发为天然抗氧化剂的巨大潜力。     

One-Dimensional Nuclear Magnetic Resonance Studies of Starch and Starch Products

The use of one-dimensional Nuclear Magnetic Resonance (NMR) spectroscopy as applied to structural studies of starch, starch products and glycogen is reviewed. Proton, carbon-13 and phosphorus-31 NMR spectra of a range of starches and starch products are described. These spectra, particularly carbon-13 NMR spectra, permit one to obtain structural information regarding these molecules. From the proton NMR spectra, which can be obtained with greater sensitivity, it is possible to determine the DP (degree of polymerization, the average number of glucose units per molecule) and the degree of branching (the proportion of glucose units which are (1→6) linked) without recourse to chemical or enzymatic methods. Moreover, the anomer distribution of the reducing sugars can be determined by ¹H or ¹³C NMR. Finally, some examples of the application of NMR to follow the time course of enzymatic digestion and sugar anomerization are discussed, as is the solid-state NMR of starch and glycogen. The phosphorus-31 NMR spectra provide information about the nature of the phosphate groups in potato starch.     

不同金属离子对多酚氧化酶的影响

采用匀浆法从茄子中提取多酚氧化酶(PPO),以绿茶粉为底物,研究Cu2+、Mn2+、Mg2+、Ca2+、Na+、Al3+和Fe3+7种金属离子对PPO活性的影响。结果表明:Cu2+、Mn2+和Mg2+3种金属离子对PPO的活性有不同程度的促进作用,而Ca2+、Na+、Al3+和Fe3+对PPO的活性有抑制作用。其中,Cu2+浓度为3mmol/L时对PPO有激活作用,此时茶黄素总量达到7.45mg;Fe3+浓度为5mmol/L对PPO有显著抑制作用,此时茶黄素总量降低至0.26mg。     

Solid substrate production of Epicoccum nigrum conidia for biological control of brown rot on stone fruits

Production of conidia of Epicoccum nigrum, a biocontrol agent of the fungal pathogen Monilinia laxa, was tested in liquid- and solid-state fermentation. Liquid fermentation was conducted in 250 ml Erlenmeyer flasks containing 50 ml of a mineral medium (containing per litre: 20 g lactose, 10 g NO3K, 1 g K2HPO4, 0.5 g MgSO4.7H2O, and 1 ml of a minor-element solution), inoculated with 2 x 10(5) E. nigrum conidia ml(-1), and incubated at 20-25 degrees C and 150 rpm for 7 days. Solid-state fermentation was carried out in specially designed plastic bags (600 cm3) (VALMIC) containing either 50 g of peat/vermiculite (1:1, w/w), or 50 g of peat/vermiculite/lentil meal (1:1:1, w/w/w) with 40% (v/w) initial moisture content. Substrate was inoculated with a conidial suspension of E. nigrum to give 10(5) conidia g(-1) substrate, and bags were incubated at 20-25 degrees C for 7 days in darkness. The amount of conidia of E. nigrum obtained in solid-state fermentation with substrate based on peat/vermiculite/lentil meal was 10-fold higher than with substrate based on peat/vermiculite or in liquid fermentation. Conidial production under these conditions was maintained in the range of 10(8) conidia g(-1) substrate from 10 to 150 days after inoculation. Germinability of these conidia was >90%. Addition of other nutrients than lentil meal to peat/vermiculite did not enhance production of conidia. Presence of peat in the substrate was necessary for good conidia production, but change in the kind of peat or vermiculite did not improve conidial production. Conidial production was similar when the substrate was inoculated with 10(5), 10(6) or 10(7) conidia g(-1) dry substrate. Incubation of bags in light conditions did not enhance conidial production. Fresh conidia produced in this solid-state fermentation system reduced the incidence and lesion diameter induced by M. laxa on peaches.     

Emissions of chromium, copper, arsenic, and PCDDs/Fs from open burning of CCA-treated wood

Aged and weathered chromated copper arsenate (CCA) treated wood was burned in an open burn research facility to characterize the air emissions and residual ash. The objectives were to simulate, to the extent possible, the combustion of such waste wood as might occur in an open field or someone's backyard; to characterize the composition and particle size distribution (PSD) of the emitted fly ash; to determine the partitioning of arsenic, chromium, and copper between the fly ash and residual ash; and to examine the speciation of the CCA elements. This work reports preliminary air emission concentrations and estimated emission factors for total particulate matter, arsenic (As), chromium (Cr), copper (Cu), and polychlorinated dibenzodioxins/dibenzofurans (PCDD/F) totals and toxic equivalents (TEQs). The partitioning of As, Cr, and Cu between the emitted fly ash and residual ash is examined and thermochemical predictions from the literature are used to explain the observed behavior. Results indicate a unimodal fly ash PSD between 0.1 and 1.0 microm diameter. In addition to a large carbonaceous component, between 11 and 14% of the As present in the burned CCA treated wood was emitted with the air emissions, with the remainder present in the residual ash. In contrast, less than 1% of both the Cr and Cu present in the wood was emitted with the air emissions. PCDD/F levels were unremarkable, averaging 1.7 ng TEQ/kg of treated wood burned, a value typical for wood combustion. Scanning electron microscopy (SEM) was unable to resolve inorganic particles consisting of Cu, Cr, or As in the wood samples, but X-ray absorption fine structure (XAFS) spectroscopy confirmed that the oxidation states of the CCA elements in the wood were Cu2+, Cr3+, and As5+. SEM examination of the fly ash samples revealed some inorganic microcrystals within the mostly carbonaceous fly ash, while XAFS spectroscopy of the same samples showed that the oxidation states after combustion were mixed Cu+ and Cu2+, Cr3+, and mixed As3+ and As5+. Estimates of the ratios of the mixed oxidation states based on the XAFS spectra were As3+/(total As) = 0.8-0.9 and Cu+/(total Cu) = 0.65-0.7. The Cu and Cr present in the fly ash were determined to coexist predominantly in the two oxide phases CuCrO2 and CuCr2O4. These results indicate that the open burning of CCA-treated wood can lead to significant air emissions of the more toxic trivalent form of As in particle sizes that are most respirable.     

Colloidal calcium phosphates in casein micelles studied by slow-speed-spinning 31P magic angle spinning solid-state nuclear magnetic resonance.

The composition of bovine casein micelles was analyzed by 31P magic angle spinning solid-state nuclear magnetic resonance spectroscopy. By looking at isotropic and anisotropic 31P chemical shift parameters, resonance line shapes, the combination of single-pulse and 1H to 31P cross-polarization spectra, and comparison with spectra for various model compounds combined with multiple-component simulation and iterative fitting procedures, we were able to identify and quantify a variety of inorganic and organic phosphates in the micelles. These include phosphates from mobile and immobile inorganic hydroxyapatite-type phosphates as well as phosphates from kappa-casein and the Ca2+-binding phosphoserines from alphas1-, alphas2-, and beta-casein. This information is discussed in relation to previous knowledge and various models for the colloid formation.     

Linking cesium and strontium uptake to kaolinite weathering in simulated tank waste leachate

Weathering behavior of kaolinite was studied in batch systems under geochemical conditions characteristic of tank waste released to the vadose zone at the Hanford Site, WA (0.05 M Al(T), 2 M Na+, 1 M N03-, pH approximately 14, Cs+ and Sr2+ present as co-contaminants). Time series experiments were conducted from 0 to 369 d, with initial Cs+ and Sr2+ concentrations ranging from 10(-5) to 10(-3) M. Dissolution of kaolinite increased soluble Si and Al to maximum levels at 7 d (Cs and Sr concentrations of 10(-5) and 10(-4) M) or 33 d (Cs and Sr concentrations of 10(-3) M). Subsequent precipitation of Si and Al was coupled to the formation of oxalate-extractable solids that incorporated Cs and Sr. Strontium sorption was nearly complete within 24 h for initial Sr concentrations (Sr0) < or = 10(-4) whereas Cs uptake increased over the full year of the experiment for all initial Cs concentrations. Spectroscopic analyses revealed neoformed solids including the zeolite Na-Al silicate (Al-chabazite), and feldspathoids sodium aluminum nitrate silicate (NO3-sodalite), and sodium aluminum nitrate silicate hydrate (NO3-cancrinite), which can incorporate Cs. Single-pulse 27Al solid-state nuclear magnetic resonance (NMR) spectroscopyyielded first-order rate constants (k)for mineral transformation that decreased from 3.5 x 10(-3) to 2 x 10(-3) d(-1) as Cs and Sr concentrations were increased from 10(-5) to 10(-3) M. Discrete strontium silicate solids were also observed. The incongruent dissolution of kaolinite promoted the sequestration of contaminants into increasingly recalcitrant solid phases over the 1-yr time period.     

Removal of E. coli from water using surface-modified activated carbon filter media and its performance over an extended use

Modification of activated carbon (AC) by aluminum hydroxychloride (AHC), and diatomaceous earth by zinc hydroxide changed the zeta potentials of these filter media from negative to positive. The modification method is amenable to room temperature, and eliminates the essential requirement of strong base treatment for making metal hydroxide coated filter media. Solid-state MAS 27Al NMR spectra suggested the presence of Al13-mer in the AHC-treated AC. AHC-modified AC samples were further treated with silver halide, and two antibacterial compounds to prevent microbial growth on filter media. In situ precipitation of silver bromide on AC resulted in formation of nanosized AgBr crystals. Bacteria removal performances of the modified media were tested in columns. For the first time, we demonstrated that only 30 g of either AHC-treated AC (60 x 200 mesh) or nano AgBr supported AC could provide >6 log E. coli removal over approximately 1000 L when the input water had a bacterial load of 10(7) CFU/mL. The filter media were robust enough to perform even when water was passed at superficial velocities 3-10 times the typical velocity (6 cm/min) of water treatment processes. Metal leaching from the modified media was found to be less than the USEPA specified Maximum Contaminant Level.     

High-resolution characterization of organic phosphorus in soil extracts using 2D 1H-31P NMR correlation spectroscopy

Organic phosphorus (P) compounds represent a major component of soil P in many soils and are key sources of P for microbes and plants. Solution NMR (nuclear magnetic resonance spectroscopy) is a powerful technique for characterizing organic P species. However, (31)P NMR spectra are often complicated by overlapping peaks, which hampers identification and quantification of the numerous P species present in soils. Overlap is often exacerbated by the presence of paramagnetic metal ions, even if they are in complexes with EDTA following NaOH/EDTA extraction. By removing paramagnetic impurities using a new precipitation protocol, we achieved a dramatic improvement in spectral resolution. Furthermore, the obtained reduction in line widths enabled the use of multidimensional NMR methods to resolve overlapping (31)P signals. Using the new protocol on samples from two boreal humus soils with different Fe contents, 2D (1)H-(31)P correlation spectra allowed unambiguous identification of a large number of P species based on their (31)P and (1)H chemical shifts and their characteristic coupling patterns, which would not have been possible using previous protocols. This approach can be used to identify organic P species in samples from both terrestrial and aquatic environments increasing our understanding of organic P biogeochemistry.     

Binding of pyrene to hydrophobic fractions of dissolved organic matter: effect of polyvalent metal complexation

The effects of polyvalent metal cations on pyrene binding to hydrophobic acid and neutral fractions (HoA and HoN, respectively) of dissolved organic matter (DOM) were elucidated. The DOM was isolated from sewage sludge; pyrene binding was estimated from fluorescence measurements. Isotherms of pyrene binding to both fractions were nonlinear. Pyrene binding was higher for HoN due to the combined effect of greater hydrophobicity, aromaticity and the large molecular size of this fraction relative to HoA. The complexation of HoA with Cu2+, Al3+, and Fe3+ increased the binding of pyrene only when the HoA was equilibrated with polyvalent cations before pyrene was added. The maximal increase in pyrene binding to HoA was 56%, 64%, and 118% when pre-equilibrated with Cu2+, Fe3+, and Al3+, respectively. Pyrene binding to HoN was not affected by the presence of metal cations. HoA complexation with metal cations increased the apparent molecular size of this fraction. We suggest that the presence of metal cations induces the formation of pseudomicelles, which are more efficient in binding pyrene than the low-molecular-weight components. Our results demonstrate that HoA and HoN components can significantly affect the transport of organic contaminants in soils irrigated with treated wastewater or amended with sewage sludge.     

光合细菌类胡萝卜素的稳定性研究

本实验研究了金属离子、NaOH、光和H2O2 对光合细菌类胡萝卜素稳定性的影响。结果表明：K+、Na+、Mg2+、Al3+、Ca2+ 以及低浓度NaOH(≤ 0.01mol/L)等对光合细菌类胡萝卜素的稳定性几乎没有影响,而Fe3+、Cu2+、高浓度NaOH(≥ 0.5mol/L)以及光照等会使类胡萝卜素的稳定性显著降低(p ＜ 0.05)。不同来源的光对类胡萝卜素的破坏程度依次为：自然光＞白炽灯＞日光灯＞紫外灯。此外,光合细菌类胡萝卜素具有一定的耐氧化性,当过氧化氢存在时,色素发生轻微降解。     

Toward a biotic ligand model for freshwater green algae: surface-bound and internal copper are better predictors of toxicity than free Cu2+-ion activity when pH is varied

The freshwater green microalgae Chlorella sp. and Pseudokirchneriella subcapitata (P. subcapitata) were chronically (48 and 72 h, respectively) exposed to copper at various pH levels, i.e., pH 6-7.5 and pH 5.9-8.5, respectively. Concentrations resulting in 50% inhibition of exponential growth rate (EC50) were determined as dissolved Cu, estimated chemical activity of the free Cu2+ ion (as pCu = - log{Cu2+ activity as molarity}), and as external (surface-bound) Cu and internal Cu in the algal cells. With increasing pH, EC50dissolved decreased from 30 to 1.1 microg of Cu L(-1) for Chlorella sp. and from 46 to 18 microg of Cu L(-1) for P. subcapitata. The pH effect on copper toxicity was even more obvious when expressed as Cu2+ activity. The EC50pCu increased on average 1.4 pCu unit per pH unit for Chlorella sp. and 1.1 pCu unit per pH unit for P. subcapitata, thus indicating a marked increase of Cu2+ toxicity at higher pH (more than 1 order of magnitude per pH unit). In contrast, it was found that EC50 values expressed as surface bound or external copper (EC50external) and as internal copper (EC50internal) did not vary substantially when pH was increased. External Cu was operationally defined as the Cu fraction removable from the algal cell by short-term contact with ethylenediaminetetraacetic acid; internal copper was defined as the nonremovable fraction. For Chlorella sp. the EC50external varied between 5 and 10 fg of Cu/ cell (factor of 2 difference) and the EC50internal between 25 and 40 fg of Cu/cell (factor of 1.6 difference). For P. subcapitata the EC50external varied between 10 and 28 fg of Cu/cell (factor of 2.8 difference) and the EC50internal between 42 and 71 fg of Cu/cell (factor of 1.7 difference). Because the observed variation in EC50external and EC50internal is much less than the variation in EC50Cu2+, it is concluded that both external and internal copper are better predictors of copper toxicity than Cu2+ when pH is varied. From the perspective of toxicity modeling, this observation is the first step toward considering the use of the cell surface as the algal biotic ligand for Cu in a similar way as fish gills fulfill this role in the biotic ligand model for predicting metal toxicity to fish species.