Free Cupric Ion Concentrations Research Articles

Distribution and speciation of copper and its relationship with FDOM in the East China Sea

Total dissolved Cu concentrations, concentrations and conditional stability constants of Cu-binding organic ligands, and the intensities of fluorescent dissolved organic matter (FDOM) components in seawater were determined in the East China Sea. Concentrations of organic ligands were determined using CLE-AdCSV with a 5 μM SA detection window. Total dissolved Cu concentrations ranged from 0.59 nM to 4.65 nM and were particularly high in surface waters with low salinity due to influences of the Changjiang Diluted Water. Concentrations of the stronger ligand (L1) were between 0.65 and 6.92 nM and its distribution closely followed that of dissolved Cu. Four FDOM components were detected in surface waters, including two humic-like and two protein-like components. L1 concentrations exhibited similar distribution patterns to the intensities of all four FDOM components. The concentrations of the weaker ligands (L2) ranged from 2.86 nM to 31 nM, with the highest concentrations detected in the surface waters of stations unaffected by the Kuroshio Current. We found the buffering ability of Cu-binding organic ligands in the East China Sea to be sufficient for responding to atmospheric and anthropogenic Cu inputs and keeping the free cupric ion concentrations relatively constant.

Copper requirements of the ammonia‐oxidizing archaeon Nitrosopumilus maritimus SCM1 and implications for nitrification in the marine environment

Ammonia oxidizing archaea (AOA) have recently been recognized as the primary nitrifiers in the marine environment; they thus play an important role in the nitrogen cycle. Available genome sequences of AOA indicate that numerous Cu‐dependent enzymes are essential for both ammonia oxidation and electron transfer, suggesting a particularly high requirement for copper. However, our knowledge of the copper requirements of AOA and their response to copper limitation in the ocean is nonexistent. Here, we examine the copper requirements of the chemolithoautotrophic AOA Candidatus Nitrosopumilus maritimus SCM1 using a combination of the metal chelators ethylenediaminetetraacetic acid and 1,4,8,11‐tetraazacyclotetradecane‐1,4,8,11‐tetraacetic acid and show that ammonia oxidation is limited at free cupric ion concentrations < 10−12.7 mol L−1, which are higher than concentrations frequently reported for many coastal and oceanic regimes. Prolonged exposure of cells to copper starvation for up to 6 d had no effect on the recovery of ammonia oxidation by N. maritimus. In addition, we present evidence that N. maritimus does not produce a copper‐binding ligand (chalkophore) under copper limitation and therefore probably relies mainly on acquisition of copper ions from surrounding media. Copper limitation may be an important constraint on archaeal ammonia oxidation throughout the marine environment.

Metal speciation and toxicity of Tamar Estuary water to larvae of the Pacific oyster, Crassostrea gigas

As part of the PREDICT Tamar Workshop, the toxicity of estuarine waters in the Tamar Estuary (southwest England) was assessed by integration of metal speciation determination with bioassays. High temporal resolution metal speciation analysis was undertaken in situ by deployment of a Voltammetric In situ Profiling (VIP) system. The VIP detects Cd (cadmium), Pb (lead) and Cu (copper) species smaller than 4 nm in size and this fraction is termed ‘dynamic’ and considered biologically available. Cadmium was mainly present in the dynamic form and constituted between 56% and 100% of the total dissolved concentration, which was determined subsequently in the laboratory in filtered discrete samples. In contrast, the dynamic Pb and Cu fractions were less important, with a much larger proportion of these metals associated with organic ligands and/or colloids (45–90% Pb and 46–85% Cu), which probably reduced the toxicological impact of these elements in this system. Static toxicity tests, based on the response of Crassostrea gigas larva exposed to discrete water samples showed a high level of toxicity (up to 100% abnormal development) at two stations in the Tamar, particularly during periods of the tidal cycle when the influence of more pristine coastal water was at its lowest. Competitive ligand-exchange Cu titrations showed that natural organic ligands reduced the free cupric ion concentration to levels that were unlikely to have been the sole cause of the observed toxicity. Nonetheless, it is probable that the combined effect of the metals determined in this work contributed significantly to the bioassay response.

Seasonal survey of copper-complexing ligands and thiol compounds in a heavily utilized, urban estuary: Elizabeth River, Virginia

Concentrations of thiol compounds, copper-complexing ligands, and total dissolved copper were followed over the course of 1 year (October 2002 until September 2003) in the Elizabeth River, Virginia to evaluate seasonality. Copper-complexing ligand concentrations were determined by competitive ligand equilibration-adsorptive cathodic stripping voltammetry (CLE/ACSV). Thiol detection was carried out by high performance liquid chromatography (HPLC) and calibration using a suite of nine thiol compounds (cysteine, glutathione, mercaptoacetic acid, 2-mercaptoethanesulfonic acid, 2-mercaptoethanol, 2-mercaptopropionic acid, 3-mercaptopropionic acid, mercaptosuccinic acid, and monothioglycerol). Total dissolved copper concentrations reached a January low of 13.1 nM to a June high of 24.7 nM and were found to vary seasonally with higher concentrations occurring from June to September. With a low of 26 nM during April to a high of 56 nM in October, copper-complexing ligand (average log K′ CuL of 12.0 ± 0.2) concentrations displayed a similar seasonal pattern to that of total dissolved copper. Free cupric ion concentrations remained below 1.5 pM for a majority of the year except during March, April, and December when values reached pM levels greater than 1.5. Six of the nine thiol compounds surveyed were detected in the Elizabeth River samples and ranged in concentration from below detectable concentrations (< 5 nM) to individual highs ranging from 25.3 to168.5 nM. The thiol compound concentrations displayed a clear seasonality fluctuating at below detection limits during November to February then increasing with increasing surface water temperatures from March to July. CLE/ACSV was used to assess whether or not the suite of thiol compounds detected by HPLC could contribute to the copper-complexing ligand pool. Conditional stability constants for each one of six thiol standards (average log K′ CuL ∼ 12.1 ± 0.5) were found to be statistically equivalent to the naturally occurring copper-complexing ligands (average log K′ CuL ∼ 12.0 ± 0.2). This suggests that these thiol compounds could act as copper-complexing ligands in natural samples and could contribute to the copper-complexing ligand pool detected by CLE/ACSV. This study involving seasonality of copper-complexing ligands and thiols in an industrialized, urban estuary underscored several points that have to be substantiated in future research efforts including copper-complexing ligands sources and the role that thiol compounds as well as other unidentified organic compounds play in the copper-complexing ligand pool.

Modeling chemical speciation of copper in River Yamuna at Delhi, India

Chemical modeling of copper in the river Yamuna was attempted by an equilibrium constant method, using a computer program COCSOC developed for the purpose. Concentrations of metals (Cu, Ca and Mg), ligands (organics, carbonates and bicarbonates) and stability constants of the probable complexes were the inputs for the modified Newton-Raphson iteration. The model adequately predicted concentrations of various copper species, even without taking into consideration solid phases, other competing metals and ligands. The free cupric ion concentrations estimated were in the range of 10-17 to 10-16M, undetectable by ion selective electrodes in the water from the river. The non-convergence of equilibrium computations for certain data sets at some sampling sites indicates the prevailing non-equilibrium conditions in the river due to large-scale release of ligands and metal ions through several drains.

An evaluation of sodium loss and gill metal binding properties in rainbow trout and yellow perch to explain species differences in copper tolerance.

The main objective of the study was to use a species comparison approach in order to understand sensitivity and tolerance differences to copper. We hypothesized that species differences in toxicity would be reflected by differences in copper binding to high-affinity sites on the gill. Specifically, the strength of copper binding (affinity, logK) and maximum number of binding sites (saturation, Bmax) for copper at the gill surface would vary among different species of fish. Two species that are different in their copper sensitivity are the rainbow trout (Oncorhynchus mykiss) and yellow perch (Perca flavescens). We explicitly compared acute toxicity (median lethal concentrations via 96-h LC50s) and whole-body Na+ loss in both organisms in two distinct water chemistries (i.e., hard and soft water). For both species, the copper binding sites at the gill surface were characterized for their affinity and saturability. The binding properties of the gill were quite similar between the two species in each water chemistry. Based on estimations of the free cupric ion concentration, the affinity, or logK, was 8.4 for both species in soft water, whereas in hard water, the affinity was higher (approximately 9.7). The Bmax value in soft water was 1.88 nmol/g for rainbow trout and yellow perch, while in hard water, saturation occurred at 3.63 nmol/g for rainbow trout and 9.01 nmol/g for yellow perch. More importantly, the amount of copper bound to the gills at 50% mortality (i.e., lethal accumulation; the LA50) was different between the two species (yellow perch LA50s were nine times higher than those of rainbow trout in soft water and hard water), indicating that the copper binding to the yellow perch gill must not have been 'biologically reactive.' According to 96-h LC50s, yellow perch were less sensitive to copper than were rainbow trout; however, the difference between the two species was similar in hard water (1.05 vs 4.16 microM) and soft water (approximately 0.10 vs 0.44 microM). Perch were more tolerant because they lost less sodium upon exposure to copper; yet this mechanism of tolerance was not reflected by the amount of copper at the gill surface. The influence of water chemistry on the binding properties of the gill demonstrates the dynamic nature of the gill in maintaining ionoregulatory homeostasis, a key issue in the future development of the chronic biotic ligand model.

Short-term biogeochemical influence of a diatom bloom on the nutrient and trace metal concentrations in South San Francisco Bay microcosm experiments

Two laboratory microcosm experiments were conducted to mimic an annual spring diatom bloom in South San Francisco Bay by isolating the phytoplankton community from the benthic grazing pressure to induce a phytoplankton bloom. The purpose of these experiments was to isolate the impact of a spring diatom bloom on the nutrient and trace metal geochemical cycling. Microcosms were created in 2.5 L incubation bottles and subjected to one of 4 treatments (control, copper [Cu] addition, manganese [Mn] addition, and both Cu and Mn addition) to investigate the toxicity of Cu on the resident plankton and the potential antagonistic effects of Mn on reducing Cu toxicity. Dissolved macronutrient (nitrate + nitrite, phosphate, and silicate), and dissolved and particulate trace metal (Cu, Ni, Mn) concentrations were monitored in the grow-out incubations on a daily basis. Chlorophylla concentrations were also monitored over the course of the experiment and used to calculate diatom-specific growth rates. In the experiments containing ambient South San Francisco Bay surface waters, average specific growth rates were on the order of 1.1 d−1. The induced diatom blooms resulted in significant removal of macronutrients from the microcosms over the course of the experiments. Our research supports previous suggestions that dissolved Ni and Cu concentrations in South San Francisco Bay have a very low biological availability as a result of organic chelation. Ni(EDTA)2− has been found to be the dominant dissolved Ni species by other researchers and Cu speciation analyses from this study and others indicate that > 99% of the dissolved Cu in South San Francisco Bay is strongly chelated as CuL1. The free cupric ion concentration was on the order of 10−12 M. Marked removal of dissolved Mn was observed in the control treatments, well exceeding expected dissolved Mn removal by diatom uptake. Additions of 375 nM Cu resulted in the complete titration of the chelating ligand (L1) concentrations. The elevated [Cu2+] (≈10−8MM) appeared to have a toxic effect on the diatom community observed in the significant decreases in their specific growth rates (μ=0.4 d−1). The suppression of dissolved Mn removal from solution was also observed in treatments spiked with high levels of dissolved Cu, providing support that Mn precipitation was due to biologically mediated oxidation not phytoplankton assimilation. The observed geochemical behavior in the concurrent Cu and Mn addition treatments provide evidence in support of Mn alleviation of Cu toxicity. The biological role in the ambient short-term biogeochemical cycling of Cu and Ni in South San Francisco Bay appears to be minimal due to the inert character of the organic ligand-metal complexes. A significant portion of the annual macronutrient and Mn cycling occurs as a result of spring diatom blooms in South San Francisco Bay.

An in vitro approach for modelling branchial copper binding in rainbow trout

The main objective of this study was to characterize the individual effects of water chemistry (Ca 2+, Na +, dissolved organic matter (DOM), pH, alkalinity) on the rapid binding of copper to the gill surface of rainbow trout using an in vitro gill binding assay. In this assay, individual gill arches were exposed for 5 min to 64Cu labelled copper solutions ranging from 0.02 to 0.16 μM in water chemistries reflecting the full range of fresh water values for the Great Lakes. The gills displayed saturable Cu binding within this Cu range but gill–Cu binding was completely unaffected over the full range of calcium, sodium and alkalinity concentrations used. Only low pH (pH 4.0) and commercial DOM (Aldrich humic acid at ⩾3 mgC/l) altered copper binding to rainbow trout gills in vitro. These findings were consistent with the results of geochemical modelling of our water chemistry (using mineql+, Version 4.5) which showed that H + and DOM affected the free cupric ion concentration. However, DOM (up to 80 mgC/l) was only able to reduce Cu on the gills by 50%. We hypothesize that in the range of 0.02–0.16 μM Cu there are two high affinity Cu binding sites on the gills, one having a substantially higher affinity for copper than DOM. The absence of a calcium effect on gill copper binding was in accord with in vivo evidence that calcium primarily acts to alter the physiology of the gill binding sites through acclimatory processes, rather than through competitive interactions. It was a surprise that water chemistry parameters influence rapid gill–metal binding in a manner different to their influence on acute toxicity and different from the effects on long-term binding reported in other studies. Currently, the biotic ligand model uses the rapid increase of gill copper (believed to reflect binding to the physiologically active receptor sites) to model gill binding characteristics. The distinction between rapid surface binding and metal uptake obviously plays an important role in determining the toxic effects of copper, especially when regulators need to predict the modifying effects of water chemistry.

Copper toxicity and cyanobacteria ecology in the Sargasso Sea

The closely related cyanobacteria Synechococcus and Prochlorococcus have different distributions in stratified water columns in the northern Sargasso Sea. The abundance of Synechococcus is relatively uniform with depth, but Prochlorococcus cell numbers are low within shallow mixed layers and high in and below the thermocline. Because free cupric ion (free Cu2+) concentrations are high (up to 6 pM) in shallow mixed layers and lower in deeper water, there is an inverse relationship between Prochlorococcus densities and the free Cu2+ concentration. We explored the possibility of a causal underpinning for this relationship by examining the relative copper sensitivities of Prochlorococcus and Synechococcus in cultures and field populations. Prochlorococcus isolates from both the highߚ and lowߚlight adapted ecotypes were inhibited at free Cu2+ concentrations that had no effect on Synechococcus. However, the highߚlight adapted strains were more copper resistant than their lowߚlight adapted counterparts. When copper was added to Prochlorococcus from environments where the in situ free Cu2+ was low (in deeply mixed water columns and below the mixed layer in stratified conditions), net growth rates were substantially reduced and cells arrested in the G1 and early S phases of the cell cycle. Prochlorococcus in shallow mixed layers were less sensitive to copper and were probably members of the copperߚresistant highߚlight adapted ecotype. Synechococcus were relatively copper resistant across a range of environments in the Sargasso Sea. These observations are consistent with our hypothesis that copper plays a role in cyanobacteria ecology in the Sargasso Sea.

Intercomparison of voltammetric techniques to determine the chemical speciation of dissolved copper in a coastal seawater sample

The chemical speciation of dissolved copper in a coastal seawater sample was determined using a variety of voltammetric methods by three different research groups. This intercomparison study was performed on a sample from near the mouth of Narragansett Bay, Rhode Island, chosen to be representative of coastal seawater from the East Coast of North America. Three research groups used distinct methods of competitive ligand equilibration/adsorptive cathodic stripping voltammetry (CLE-ACSV) with different competing ligands (salicylaldoxime, benzoylacetone and 8-hydroxyquinoline), two of which employed a range of added ligand concentrations. Two groups used anodic stripping voltammetry (ASV) with a thin mercury film (TMF) rotating glassy carbon disk electrode (RGCDE). Combining these various approaches, resulted in analytical competition strengths ( α Cu(AL) 2 ) ranging from 10 2.7 to 10 6.5 for the CLE-ACSV approaches and 10 1.3 ( α Cu) for the ASV methods. As the value of the analytical competition strength for the added competing ligand increased, the value determined for the natural Cu-binding ligand concentration decreased, while its conditional stability constant, K CuL i ,Cu 2+ cond, increased. This type of behavior is consistent with the natural Cu-binding ligands consisting of a continuum of Cu-binding ligands with increasingly smaller concentrations of increasingly stronger ligands. Results from the different approaches carried out at similar analytical competition strengths compared very favorably. All of the approaches determined that dissolved copper in this sample was overwhelmingly chelated with strong Cu-binding ligands (>99.97%). Thus, although the total dissolved copper concentration was 12.7 nM (10 −7.9 M), the Cu-binding ligands lowered the free cupric ion concentration, [Cu 2+], to ∼10 −13.0 M, a concentration that is non-toxic to even the most sensitive marine microorganisms.

Comparison of [Cu2+] Measurements in Lake Water Determined by Ligand Exchange and Cathodic Stripping Voltammetry and by Ion-Selective Electrode

While there has been a proliferation of techniques for measuring free metal ion concentrations and complexation in natural waters, there has been little direct intercomparison among methods. Here we report the results of an intercomparison among different methods used to determine free cupric ion concentration ([Cu2+]) and organic complexation in lake water samples. The techniques used were potentiometric measurement with a cupric ion-selective electrode (Cu-ISE) and ligand exchange methods involving the addition of three well-characterized ligands with dif ferent binding affinities for copper (catechol, 8-hydroxyquinoline, and tropolone) followed by measurement of the Cu chelates of these ligands by adsorptive differential pulse cathodic stripping voltammetry (DPCSV). Good agreement was found among relationships between [Cu2+] and the total concentration of natural copper species ([Cunat]) determined via titration with the three ligand exchange/DPCSV methods. Relationships between [Cu2+] and [Cunat] measured by these methods also agreed with those determined with a cupric ion electrode within the overlapping range of detection windows for each method ([Cunat] = 40−100 nM). At lower Cu concentrations, the Cu electrode gave er roneously high [Cu2+] readings, apparently due to a failure of the electrode membrane to equilibrate with the sample. The agreement among the different methods provides support for the validity of each individual method of measuring [Cu2+] and natural Cu complexation. By combining Cu titration data measured by DPCSV and Cu-ISE, we obtained a more complete description of the Cu-complexing characteristics of water samples from two Swiss lakes than could be obtained with any single method. Equilibrium modeling of this composite data indicated the presence of at least three ligand classes. These ranged in concentration from 38 ± 19 nM for the strongest binding ligand class, which had conditional binding constants in the range of 1015 at pH 7.8−8.0, to 8 ± 2 μM for the weakest class of ligands, which had a conditional constant of 108.6 M-1.

Free cupric ion concentrations and Cu complexation in selected Swiss lakes and rivers

[Cu2+] and Cu complexation parameters in some selected freshwater systems in Switzerland were determined by the technique of ligand-exchange and DPCSV. Results from the water columns of some eutrophic and oligotrophic lakes are presented and compared to small acid lakes. Cu is strongly complexed by organic ligands which with very high stability constants at low concentrations are probably biologically produced, as indicated by the seasonal variations in the eutrophic lakes and by the relationship between Cu complexation and algal activity in the eutrophic (pCu=15−16), oligotrophic (pCu=13−14) and acidic (pCu=9−10) lakes. The extent of Cu complexation in river waters was generally lower than in the eutrophic lakes, at similar DOC levels. No obvious correlation between Cu complexation and DOC was observed, indicating that Cu complexing ligands are specific organic compounds.

Regulation of copper concentration in the oceanic nutricline by phytoplankton uptake and regeneration cycles

Similar sigmoidal relationships were observed between cellular Cu: C ratios and free cupric ion concentration for the neritic alga Thalassiosira pseudonana and two oceanic species (Thalassiosira oceanica and Emiliania huxleyi) grown in trace metal ion buffered media. Only 5–9‐fold variations in cell Cu: C were observed for these species over the [Cu2+] range 3 × 10−15 to 3 × 10−12 M, with increasing cell copper vs. [Cu2+] slopes above and below this range. At the mean [Cu2+] for the euphotic zone of the North Pacific (10−13.2 M), cell Cu: C ratios for the three species were 4.4, 4.4, and 3.8 µmol mol−1, similar to values for plankton taken from North Pacific waters. These values also match the mean Cu: C ratio of 4.1 µmol mol−1 determined from slopes of linear relationships between Cu and PO4 in the nutricline of the central North Pacific and the “Redfield” C : PO4 ratio in plankton of 106 : 1. This agreement provides strong evidence that copper concentrations in remote oceanic nutriclines are regulated by phytoplankton uptake and regeneration processes.

Free cupric ion concentration and Cu(II) speciation in a eutrophic lake

The free cupric ion concentrations, [Cu2+], in the water column ofeutrophic Lake Greifen were evaluated by means of ligand exchange with catechol and cathodic stripping voltametry of the Cu-catechol complexes. Total dissolved Cu, [Cu],, was in the range 0.5-2.8 x 10 -8 M, while [Cu2-I] ranged from lo-l6 to lo-l4 M at different times and depths. These values of [CU~-~ ] are much lower than literature values for the open sea. Equilibrium models of titration data indicate the presence of very strong ligands with conditional stability constants of 1013~y-10t4~9 and corresponding concentrations of 40-90 nM. Measured [Cu2+] was especially low during the algal bloom in spring and was higher in winter. p[Cu] and ratios of [Cu], to [Cu2 t ] thus exhibit a seasonal pattern similar to that of algal productivity, suggesting that the Cu-complexing ligands are produced by algae.

The use of chemiluminescence and ligand competition with EDTA to measure copper concentration and speciation in seawater

A batch chemiluminescence method for measurement of copper in seawater was developed from an existing flow injection technique. The method involved the reaction of copper in the sample with 1,10-phenanthroline, and subsequent release of photons from the oxidation of copper(II)-phenan-throline chelates by H 2O 2 at alkaline pH. The method was extremely sensitive with a detection limit in the range of 0.05–for 200 μl samples. Because of its sensitivity, it could be used to measure copper directly in seawater without a preconcentration step. To measure total copper, samples had to be acidified to pH 2 and stored for weeks to months (and in some cases also photo-oxidized by UV light) to release copper from kinetically inert species. Total copper concentrations measured by the chemiluminescence method in natural seawater agreed well with those obtained by more traditional methods. The chemiluminescence method was sensitive to copper speciation due to the slow dissociation of many bound copper species. This property was utilized to develop a ligand competition technique for measurement of free cupric ion concentrations. The method involved additions of EDTA and copper to seawater and was based on the lack of detectability of CuEDTA chelates by the chemiluminescence method. Free cupric ion concentrations measured by this method ranged from 10 −12.9 M for a relatively clean estuarine sample containing 5 nM total dissolved copper to 10 −10.9 M for a sample from a polluted harbor which contained 38 nM copper. Cupric ion concentrations measured by the new method were in good agreement with those measured in the same samples by a previously published method: EDTA competition combined with copper adsorption on to C 18 Sep—Pak cartridges. Copper was found to be heavily (>99%) complexed by natural organic ligands in both shelf and estuarine water samples and the concentrations of these ligands generally increased with dissolved copper concentration and decreasing sample salinity. These results agree with those of several previous studies.

Toxicity of trace metals to Acartia tonsa in the Elizabeth River and southern Chesapeake Bay

Dissolved zinc and copper and free cupric ions were present at high concentrations in water from the Elizabeth River estuary (a polluted tributary of the southern Chesapeake Bay) when compared to values in nearby Hampton Roads and lower Chesapeake Bay. Zinc concentrations at three stations in the Elizabeth River ranged from 87 to 1550 nM compared to values of 3·1 to 16 nM at four stations in the southern Chesapeake Bay. Likewise, free cupric ion concentration ranged from 10 −11·6 to 10 −10·1 M at the Elizabeth River stations, but was appreciably lower (10 −12·3 to 10 −12·6 M) in samples from Hampton Roads and the lower bay. In bioassays conducted with the copepod Acartia tonsa, the survival of naupliar larvae was much lower in Elizabeth River samples, containing high levels of copper and zinc, than in samples from the Chesapeake Bay or Newport River estuary which contained much lower levels of these metals. Based on previous results in trace metal ion buffered media, measured free cupric ion concentrations and estimated free zinc ion concentrations appear to have been high enough in the Elizabeth River samples to account for at least some of the observed decrease in larval survival. Furthermore, the addition of chelators, EDTA and NTA, that complex and detoxify copper and zinc (as well as cadmium, nickel and lead) significantly increased larval survival in the Elizabeth River samples. These results strongly support the hypothesis that elevated levels of copper and zinc (and possibly other toxic trace metals) occur at sufficiently high concentrations in Elizabeth River water to adversely affect Acartia tonsa and other sensitive estuarine organisms.

Potentiometric flow injection determination of total water hardness in tap water by using cupric ion buffer.

A rapid and simple flow injection method for the determination of total water hardness using a cupric ion buffer and a flow-through type cupric ion selective electrode detector is proposed. The flow injection system was constructed with three stream channels. A sample solution of calcium or magnesium ion (200μl) was injected into a carrier (water) stream and was merged with a stream of 0.5 M sodium nitrate solution to maintain a constant ionic strength in the sample. This mixed stream was merged with the stream of cupric ion buffer solution consisting of 5×10-3 M cupric nitrate and 1×10-2 M EDTA adjusted to pH 9.4. The calcium or magnesium ion formed a complex with EDTA. In the reaction coil (0.5 mm i.d.×5.4 m) decreasing the concentration of EDTA in the buffer and as a result liberating the cupric ion from the EDTA-cupric ion complex. The change in the concentration of the free cupric ion was monitored with the cupric ion selective electrode and the concentration of the calcium or magnesium ion could be determined from the peak height of the electrode response. In order to make the sensitivity to the calcium ion equal to that to the magnesium ion, the pH of the cupric ion buffer was adjusted to pH 910 for determination of total water hardness. In this pH region, the amounts of EDTA reacted with the calcium and magnesium ions are almost the same and thus the change in the free cupric ion concentration is almost the same. A small amount of triethylenetetramine was added to the cupric ion buffer to suppress the effect of the chloride or bromide ion coexisting in the sample. The present method was successfully applied to determination of total water hardness in tap water with a sampling of 30 h-1 and a relative standard deviation of 0.7%. The results obtained by the proposed method were in good accordance with those obtained by the conventional chelate titration method.

Measurement of free cupric ion concentration in seawater by a ligand competition technique involving copper sorption onto C18 SEP‐PAK cartridges1

Knowledge of ion speciation and free ion concentrations is important in predicting the biological availability and geochemical behavior of trace metals. Here we describe a new technique for measuring free cupric ion concentration in seawater based on sorption of copper onto SEP‐PAK C18 cartridges and internal free cupric ion calibration. Calibration was achieved by adding cupric ion buffers, consisting of copper and excess concentrations of synthetic chelator, ethylenediaminetetraacetic acid (EDTA). EDTA competes with natural organic ligands for copper complexation, but its copper chelate is not retained by the C18 cartridges. This latter property allows EDTA to be used for free cupric ion calibration.Titrations in seawater utilizing the new technique revealed that copper was highly complexed. Free cupric ion concentrations in three samples of seawater from lower Narragansett Bay, Rhode Island, and one from off the coast of Peru were measured to be 10−12.3, 10−12.0, 10 12.1, and 10−11.5 M at total dissolved copper concentrations of 26.7, 16.5, 19.4, and 3.7 nM. From these values and from computed and experimental values for inorganic complexation, we compute that only 0.03–2% of the dissolved copper can be accounted for as inorganic species (Cu2+, CuCO3, CuOH +, etc.). The remainder, 99.97–98%, appears to be organically complexed. These results are in general agreement with several other recent studies, using several techniques, of copper complexation in seawater.

A field comparison of two methods for the determination of copper complexation: Bacterial bioassay and fixed-potential amperometry

Complexation of copper added to seawater was determined by bacterial bioassay and fixedpotential amperometry. Consistent results were obtained by these two fundamentally different methods. The results of this study support the validity of both techniques and the field applicability of fixed-potential amperometry. The intercomparison studies were performed on samples collected at the N.Y.C. sewage sludge dumpsite and in relatively unpolluted coastal waters. In this limited study, the calculated free cupric ion concentrations at ambient total copper concentrations were similar at both sites.

RELATIONSHIPS BETWEEN FREE CUPRIC ION CONCENTRATIONS IN SEA WATER AND COPPER METABOLISM AND GROWTH IN CRAB LARVAE.

Crab larvae (Rhithropanopeus harrisiiere exposed to a range of free cupric ion concentrations, [Cu2+], regulated in sea water by a metal chelate buffer system. We found a biphasic relationship between intracellular copper distribution and [Cu2+] in sea water. At [Cu2+] within the ambient range (10-12.4 to 10-10.6 M), cytosolic copper was associated with both metallothionein (MT) and high molecular weight (HMW) ligands, and was independent of external [Cu2+]. At higher [Cu2+], copper was also associated with very low molecular weight (VLMW) ligands, and accumulated in this ligand pool and the MT pool as external [Cu2+] increased. In marked contrast, copper in the HMW ligand pool did not correlate with [Cu2+] in sea water over the entire range of exposures. Reductions in larval growth occurred at greater than estimated ambient [Cu2+] and correlated with copper accumulation in the MT and VLMW pools.