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Effects of age and environment on adaptive immune responses to Mycobacterium avium subsp. paratuberculosis (MAP) vaccination in dairy goats in relation to paratuberculosis control strategies
Koets, Ad ; Ravesloot, Lars ; Ruuls, Robin ; Dinkla, Annemieke ; Eisenberg, Susanne ; Lievaart-Peterson, Karianne - \ 2019
Veterinary Sciences 6 (2019)3. - ISSN 2306-7381
Diagnostics - Immunity - Mycobacterium - Paratuberculosis - Vaccination
Paratuberculosis infection is caused by Mycobacterium avium subsp. paratuberculosis (MAP). In the Netherlands, 75% herd level prevalence of caprine paratuberculosis has been estimated, and vaccination is the principal control strategy applied. Most goat dairy farms with endemic paratuberculosis systematically vaccinate goat kids in the first months of life with a commercially available whole cell MAP vaccine. We hypothesized that the development of adaptive immune responses in goats vaccinated at young age depends on the environment they are raised in, and this has implications for the application of immune diagnostic tests in vaccinated dairy goats. We evaluated the early immune response to vaccination in young goat kids sourced from a MAP unsuspected non-vaccinated herd and raised in a MAP-free environment. Subsequently we compared these with responses observed in birth year and vaccination matched adult goats raised on farms with endemic paratuberculosis. Results indicated that initial adaptive immune responses to vaccination are limited in a MAP-free environment. In addition, adult antibody positive vaccinated goats raised in a MAP endemic environment are less likely to be IS900 PCR-positive as compared to antibody negative herd mates. We conclude that test-and-cull strategies in a vaccinated herd are currently not feasible using available immune diagnostic tests.
Effect of dietary phosphorus deprivation on leukocyte function in transition cows
Eisenberg, S.W.F. ; Ravesloot, L. ; Koets, A.P. ; Grünberg, W. - \ 2019
Journal of Dairy Science 102 (2019)2. - ISSN 0022-0302 - p. 1559 - 1570.
hypophosphatemia - immune function - lymphoproliferation test - phagocytosis assay
Phosphorus depletion and hypophosphatemia have been described to hamper immune function in different species, an effect barely studied in dairy cows commonly developing hypophosphatemia in early lactation. Dietary P deprivation in mid lactating dairy cows was associated with a decline of the number of granulocytes and impaired granulocyte survival, whereas the phagocytic activity remained unaffected. The objective of the study reported here was to determine the effect of P deprivation on the leukocyte function of periparturient dairy cows. Eighteen multiparous and late pregnant dairy cows were randomly assigned to either a treatment group that was offered a markedly P-deficient diet or a control group receiving the same ration with adequate P content. The study consisted of a 2-wk acclimation period that was followed by a P deprivation period extending from 4 wk before to 4 wk after parturition and a P repletion period of 2 wk thereafter. Blood samples for leukocyte counts and leukocyte function analysis were obtained at the end of the acclimation period, after 2 wk of P deprivation, within the first week of lactation, at the end of the P depletion period and after 2 wk of dietary P supplementation. Blood samples for biochemical analysis were obtained weekly. Immune function was assessed by means of a phagocytosis assay and a lymphocyte stimulation test. Dietary P deprivation resulted in pronounced and sustained hypophosphatemia. Time effects were observed on the counts of different leukocyte fractions, the relative number of phagocytic granulocytes, the degree of phagocytosis, and the lymphocyte proliferation. Differences between P-deprived and control cows were only identified for the degree of phagocytosis that was lower in P-deprived cows compared with control cows. The correlation and regression analyses, however, revealed positive associations of the plasma phosphate concentration and the granulocyte count, the relative number of phagocytic granulocytes, and the degree of phagocytosis at the end of the dietary P deprivation when P depletion was most severe. The results of the study reported here indicate a mild negative effect of pronounced and sustained hypophosphatemia on the granulocyte count and the phagocytic activity of granulocytes in transition dairy cows. The clinical relevance of this effect for health and productivity of dairy cows remains to be determined.
Immunization of young heifers with staphylococcal immune evasion proteins before natural exposure to Staphylococcus aureus induces a humoral immune response in serum and milk
Benedictus, Lindert ; Ravesloot, Lars ; Poppe, Kim ; Daemen, Ineke ; Boerhout, Eveline ; Strijp, Jos Van; Broere, Femke ; Rutten, Victor ; Koets, Ad ; Eisenberg, Susanne - \ 2019
BMC Veterinary Research 15 (2019)1. - ISSN 1746-6148
Cattle - Efb - Experimental immunization - LukM - Mastitis - Milk antibodies - Natural exposure - Non-protective immunity - Staphylococcus aureus
Background: Staphylococcus aureus, a leading cause of mastitis in dairy cattle, causes severe mastitis and/or chronic persistent infections with detrimental effects on the cows' wellbeing, lifespan and milk production. Despite years of research there is no effective vaccine against S. aureus mastitis. Boosting of non-protective pre-existing immunity to S. aureus, induced by natural exposure to S. aureus, by vaccination may interfere with vaccine efficacy. The aim was to assess whether experimental immunization of S. aureus naïve animals results in an immune response that differs from immunity following natural exposure to S. aureus. Results: First, to define the period during which calves are immunologically naïve for S. aureus, Efb, LukM, and whole-cell S. aureus specific serum antibodies were measured in a cohort of newborn calves by ELISA. Rising S. aureus specific antibodies indicated that from week 12 onward calves mounted an immune response to S. aureus due to natural exposure. Next, an experimental immunization trial was set up using 8-week-old heifer calves (n = 16), half of which were immunized with the immune evasion molecules Efb and LukM. Immunization was repeated after one year and before parturition and humoral and cellular immunity specific for Efb and LukM was determined throughout the study. Post-partum, antibody levels against LukM and EfB were significantly higher in serum, colostrum and milk in the experimentally immunized animals compared to animals naturally exposed to S. aureus. LukM specific IL17a responses were also significantly higher in the immunized cows post-partum. Conclusions: Experimental immunization with staphylococcal immune evasion molecules starting before natural exposure resulted in significantly higher antibody levels against Efb and LukM around parturition in serum as well as the site of infection, i.e. in colostrum and milk, compared to natural exposure to S. aureus. This study showed that it is practically feasible to vaccinate S. aureus naïve cattle and that experimental immunization induced a humoral immune response that differed from that after natural exposure only.
Changes in aggregation states of light-harvesting complexes as a mechanism for modulating energy transfer in desert crust cyanobacteria
Bar Eyal, Leeat ; Ranjbar Choubeh, R. ; Cohen, Eyal ; Eisenberg, Ido ; Tamburu, Carmen ; Dorogi, Márta ; Ünnep, Renata ; Appavou, Marie-Sousai ; Revo, Reinat ; Raviv, Uri ; Reich, Ziv ; Garab, Gyozo ; Amerongen, H. van; Paltiel, Yossi ; Keren, Nir - \ 2017
Proceedings of the National Academy of Sciences of the United States of America 114 (2017)35. - ISSN 0027-8424 - p. 9481 - 9486.
In this paper we propose an energy dissipation mechanism that is
completely reliant on changes in the aggregation state of the
phycobilisome light-harvesting antenna components. All photosynthetic
organisms regulate the efficiency of excitation energy transfer
(EET) to fit light energy supply to biochemical demands. Not many do
this to the extent required of desert crust cyanobacteria. Following
predawn dew deposition, they harvest light energy with maximum
efficiency until desiccating in the early morning hours. In the
desiccated state, absorbed energy is completely quenched. Time
and spectrally resolved fluorescence emission measurements of the
desiccated desert crust Leptolyngbya ohadii strain identified (i) reduced
EET between phycobilisome components, (ii) shorter fluorescence
lifetimes, and (iii) red shift in the emission spectra, compared
with the hydrated state. These changes coincide with a loss of the
ordered phycobilisome structure, evident from small-angle neutron
and X-ray scattering and cryo-transmission electron microscopy data.
Based on these observations we propose a model where in the hydrated
state the organized rod structure of the phycobilisome supports
directional EET to reaction centers with minimal losses due to
thermal dissipation. In the desiccated state this structure is lost, giving
way to more random aggregates. The resulting EET path will exhibit
increased coupling to the environment and enhanced quenching.
Reisolation of Staphylococcus aureus from bovine milk following experimental inoculation is influenced by fat percentage and specific immunoglobulin G1 titer in milk
Boerhout, E.M. ; Koets, A.P. ; Vernooij, J.C.M. ; Mols-Vorstermans, T.G.T. ; Nuijten, P.J.M. ; Rutten, V.P.M.G. ; Bijlsma, J.J.E. ; Eisenberg, S.W.F. - \ 2016
Journal of Dairy Science 99 (2016)6. - ISSN 0022-0302 - p. 4259 - 4269.
Bovine mastitis - Milk fat percentage - Specific immunoglobulin G titer - Staphylococcus aureus
The associations of management parameters, herd characteristics, and individual cow factors with bovine mastitis have been subject of many studies. The present study aimed to evaluate the association between milk composition parameters, including fat, protein, lactose, urea, and specific immunoglobulin levels, at the time of experimental bacterial inoculation of the mammary gland and subsequent shedding dynamics of Staphylococcus aureus. Sixty-eight cows were experimentally infected with S. aureus and closely monitored for 3 wk. Mixed model analyses were used to determine the influence of management and herd characteristics (farm and experimental group), individual cow factors (days in milk, milk yield, and quarter position), and a challenge-related parameter (inoculation dose) in combination with either the milk components fat, protein, lactose and urea, or the S. aureus-specific antibody isotype titers at the time of bacterial inoculation, on the number of S. aureus reisolated from milk after inoculation. A positive association was observed between the milk fat percentage and the number of S. aureus reisolated from quarter milk, and a negative relationship between the S. aureus-specific IgG1 titer in milk and the number of S. aureus. These findings should be considered in the development of a vaccine against S. aureus-induced bovine mastitis.
Diurnal differences in milk composition and its influence on in vitro growth of Staphylococcus aureus and Escherichia coli in bovine quarter milk
Eisenberg, S.W.F. ; Boerhout, E.M. ; Ravesloot, L. ; Daemen, A.J.J.M. ; Benedictus, L. ; Rutten, V.P.M.G. ; Koets, A.P. - \ 2016
Journal of Dairy Science 99 (2016)7. - ISSN 0022-0302 - p. 5690 - 5700.
Bovine - Escherichia coli - Milk composition - Staphylococcus aureus
In experimental intramammary inoculation studies, it has been observed that mastitis susceptibility is influenced, among others, by cow factors. To identify milk characteristics leading to these differences, quarter milk samples of morning and evening milk were collected and analyzed for their composition (protein, fat, lactose, urea, lactoferrin, lactoperoxidase, and β-lactoglobulin concentrations), somatic cell count, and antibodies against Staphylococcus aureus. Furthermore, in vitro growth of S. aureus and Escherichia coli in fresh quarter milk samples was determined. All measured parameters differed significantly between quarters and also between morning and evening milk with the exception of lactose levels. In addition, quantitative growth of S. aureus and E. coli was significantly different in morning milk compared with evening milk. Mixed model analysis revealed that replication of S. aureus was negatively associated with the presence of fat, S. aureus-specific IgG1 antibodies, contamination of the milk sample and morning milk. Replication of E. coli was negatively associated with fat concentrations, and positively associated with morning milk. The significant difference between morning and evening milk supports the theory that changes in milk composition influence bacterial growth. Although all determined milk components differed significantly between quarters and in time no significant association with bacterial growth could be identified with the exception of fat for both studied species and IgG1 titers for S. aureus. The negative association of fat with bacterial growth was assumed to occur due to activation of lipolysis by milk handling and can most likely be neglected for in vivo relevance. The fact that S. aureus-specific IgG1 titers were negatively associated with S. aureus growth in vitro encourages the ongoing effort to develop a vaccine against S. aureus-induced mastitis.
A longitudinal study of factors influencing the result of a Mycobacterium avium ssp. paratuberculosis antibody ELISA in milk or dairy cows
Eisenberg, S.W.F. ; Veldman, E. ; Rutten, V.P.M.G. ; Koets, A.P. - \ 2015
Journal of Dairy Science 98 (2015)4. - ISSN 0022-0302 - p. 2345 - 2355.
The influence of milk yield and milk composition on the diagnosis of Mycobacterium avium ssp. paratuberculosis (MAP) by milk ELISA in the context of the total IgG secretion patterns in milk throughout lactation and serum concentrations were investigated. A 2-yr trial was performed in which 1,410 dairy cows were sampled monthly and MAP milk ELISA status and milk yield and composition were determined. Data were analyzed by mixed model analysis. Milk yield was found to significantly influence ELISA results expressed as sample-to-positive (S/P) ratios. For each 5-kg increase in milk, the S/P ratio has to be multiplied by 0.89; therefore, high milk yield can change the MAP milk ELISA outcome of a cow in early infection from positive to negative. Parity influenced ELISA outcome significantly, indicating that cows with a parity >1 are more likely to be identified by milk testing. Also, herd was an important predictor, showing that herd prevalence influences the milk ELISA strongly. Other factors influencing the S/P ratios were protein concentration, somatic cell count, and days in milk. The IgG concentration and mass excreted per day were determined longitudinally in a subset of 41 cows of which samples and data of a complete lactation were available. Again, the IgG concentration in milk was mainly influenced by milk yield. The total IgG mass secreted per day in milk was found to be relatively constant, with a mean of 8.70 ± 5.38 g despite an increasing IgG concentration in serum at the same time. The variation of IgG concentration in milk can be mainly attributed to dilution through changes in milk yield. This supports the assumption that concentrations of MAP-specific antibodies are influenced by changes in milk yield similarly. In conclusion, we confirmed that antibody concentrations, and therefore MAP ELISA outcome, were influenced by milk yield, herd, and parity. To enhance performance, milk ELISA tests should either be performed in early or late lactation, when milk yield is low. From a management perspective, sampling should be done during early lactation before cows are bred again. Based on the slow progressive infection dynamics, only first-parity cows should be preferentially tested at the end of their first lactation to avoid false-negative results.
Immunization routes in cattle impact the levels and neutralizing capacity of antibodies induced against S. aureus immune evasion proteins
Boerhout, Eveline ; Vrieling, Manouk ; Benedictus, Lindert ; Daemen, Ineke ; Ravesloot, Lars ; Rutten, Victor ; Nuijten, Piet ; Strijp, Jos Van; Koets, Ad ; Eisenberg, Susanne - \ 2015
Veterinary Research 46 (2015)1. - ISSN 0928-4249 - 9 p.
Vaccines against S. aureus bovine mastitis are scarce and show limited protection only. All currently available vaccines are applied via the parenteral (usually intramuscular) route. It is unknown, however, whether this route is the most suitable to specifically increase intramammary immunity to combat S. aureus at the site of infection. Hence, in the present study, immunization via mucosal (intranasal; IN), intramuscular (triangle of the neck; IM), intramammary (IMM) and subcutaneous (suspensory ligament; SC) routes were analyzed for their effects on the quantity of the antibody responses in serum and milk as well as the neutralizing capacity of the antibodies within serum. The experimental vaccine comprised the recombinant S. aureus immune evasion proteins extracellular fibrinogen-binding protein (Efb) and the leukotoxin subunit LukM in an oil-in-water adjuvant combined with a hydrogel and alginate. The highest titer increases for both Efb and LukM specific IgG1 and IgG2 antibody levels in serum and milk were observed following SC/SC immunizations. Furthermore, the harmful effects of Efb and leukotoxin LukMF' on host-defense were neutralized by serum antibodies in a route-dependent manner. SC/SC immunization resulted in a significant increase in the neutralizing capacity of serum antibodies towards Efb and LukMF', shown by increased phagocytosis of S. aureus and increased viability of bovine leukocytes. Therefore, a SC immunization route should be considered when aiming to optimize humoral immunity against S. aureus mastitis in cattle.
Dam Mycobacterium avium subspecies parartuberculosis (MAP) infection status does not predetermine calves for future shedding when raised in a contaminated environment : a cohort study
Eisenberg, S.W.F. ; Rutten, Victor P.M.G. ; Koets, A.P. - \ 2015
Veterinary Research 46 (2015). - ISSN 0928-4249 - 8 p.
Uptake of Mycobacterium avium subsp. paratuberculosis (MAP) by calves in the first days of life from colostrum, milk and faeces is regarded an important moment of transmission. The objective of this study was to quantify the
association between the MAP status of dams as determined by the presence of MAP DNA and antibody in colostrum and that of DNA in faeces and the environment with subsequent MAP shedding of their daughters. A cohort of 117
dam-daughter pairs giving birth/being born on eight commercial dairy farms with endemic paratuberculosis was followed where colostrum, faecal and environmental samples (dust) were analysed for the presence of MAP using an
IS900 real-time PCR. Antibodies in colostrum were measured by ELISA. Analysis of dust samples showed that on all farms environmental MAP exposure occurred continuously. In significantly more colostrum samples (48%) MAP DNA
was detected compared to faecal samples (37%). MAP specific antibodies were present in 34% of the colostrum samples. In total MAP DNA was present in faecal samples of 41% of the daughters at least once during the sampling
period. The association between faecal shedding in the offspring and the dam MAP status defined by MAP PCR on colostrum, MAP PCR on faeces or ELISA on colostrum was determined by an exact cox regression analysis for discrete
data. The model indicated that the hazard for faecal shedding in daughters born to MAP positive dams was not significantly different compared to daughters born to MAP negative dams. When born to a dam with DNA positive
faeces the HR was 1.05 (CI 0.6; 1.8) and with DNA positive colostrum the HR was 1.17 (CI 0.6; 2.3). When dam status was defined by a combination of both PCR outcomes (faeces and colostrum) and the ELISA outcome the HR was
1.26 (CI 0.9; 1.9). Therefore, this study indicates that neither the presence of MAP DNA in colostrum, MAP DNA in faeces nor the presence of MAP antibodies in colostrum of the dam significantly influences the hazard of MAP
shedding in their subsequent daughters up to the age of two years when raised in a contaminated environment.
Characterization of histatin 5 with respect to amphipathicity, hydrophobicity, and effects on cell and mitochondrial membrane integrity excludes a candidacidal mechanism of pore formation
Helmerhorst, E.J. ; Hof, W. van 't; Breeuwer, P. ; Veerman, E.C.I. ; Abee, T. ; Troxler, R.F. ; Nieuw Amerongen, A.V. ; Oppenheim, F.G. - \ 2001
Journal of Biological Chemistry 276 (2001). - ISSN 0021-9258 - p. 5643 - 5649.
Histatin 5 is a 24-residue peptide from human saliva with antifungal properties. We recently demonstrated that histatin 5 translocates across the yeast membrane and targets to the mitochondria, suggesting an unusual antifungal mechanism (Helmerhorst, E. J., Breeuwer, P., van`t Hof, W., Walgreen-Weterings, E., Oomen, L. C. J. M., Veerman, E. C. I., Nieuw Amerongen, A. V., and Abee, T. (1999) J. Biol. Chem. 274, 7286-7291). The present study used specifically designed synthetic analogs of histatin 5 to elucidate the role of peptide amphipathicity, hydrophobicity, and the propensity to adopt -helical structures in relation to membrane permeabilization and fungicidal activity. Studies included circular dichroism measurements, evaluation of the effects on the cytoplasmic transmembrane potential and on the respiration of isolated mitochondria, and analysis of the peptide hydrophobicity/amphipathicity relationship (Eisenberg, D. (1984) Annu. Rev. Biochem. 53, 595-623). The 14-residue synthetic peptides used were dh-5, comprising the functional domain of histatin 5, and dhvar1 and dhvar4, both designed to maximize amphipathic characteristics. The results obtained show that the amphipathic analogs exhibited a high fungicidal activity, a high propensity to form an -helix, dissipated the cytoplasmic transmembrane potential, and uncoupled the respiration of isolated mitochondria, similar to the pore-forming peptide PGLa (Peptide with N-terminal Glycine and C-terminal Leucine-amide). In contrast, histatin 5 and dh-5 showed fewer or none of these features. The difference in these functional characteristics between histatin 5 and dh-5 on the one hand and dhvar1, dhvar4, and PGLa on the other hand correlated well with their predicted affinity for membranes based on hydrophobicity/amphipathicity analysis. These data indicate that the salivary protein histatin 5 exerts its antifungal function through a mechanism other than pore formation
Electrochemical metal speciation in natural and model polyelectrolyte systems
Hoop, M.A.G.T. van den - \ 1994
Agricultural University. Promotor(en): J. Lyklema, co-promotor(en): H.P. van Leeuwen. - S.l. : Van den Hoop - ISBN 9789054852179 - 131
metalen - elektrolyten - chemische speciatie - elektrochemie - dubbelzouten - metals - electrolytes - chemical speciation - electrochemistry - double salts
The purpose of the research described in this thesis was to examine the applicability of electro-analytical techniques in obtaining information on the speciation of metals, i.e. their distribution over different physico-chemical forms, in aquatic systems containing charged macromolecules. In chapter 1 a general introduction is given to (i) metal speciation in aquatic systems, (ii) (bio)polyelectrolytes and their counterion distributions and (iii) electrochemical methods emphasizing their apllication to metal speciation.
Chapter 2 deals with the conductometric: measurement of counterion association with macromolecules. First, we have surveyed theoretical developments concerning ion association for purely electrostatic interaction and as reflected in the conductivities of polyelectrolyte solutions. It will be shown that for the salt free case, the distribution of monovalent counterions can be obtained from plots of the molar conductivity of the polyelectrolyte solution versus the molar conductivity of the monovalent counterion, so-called Eisenberg plots. Experimental results for various alkali polymethacrylate concentrations show that the fraction of conductometrically- free monovalent counterions is in close agreement with theoretical predictions, which are based on a two-state appoach. Furthermore, for linear polyelectrolytes a recently proposed model for the case of counterion condensation in systems with ionic mixtures is presented. Finally, the treatment of conductometric data for polyelectrolyte solutions with either one type of counterion or mixtures of two types of counterions in terms of free and bound fractions is discussed.
In chapter 3 we describe a voltammetric methodology for the analysis of labile homogeneous heavy metal-ligand complexes in terms of a stability K . The method takes into account the difference between the diffusion coefficients of the free and bound metal. Since the relationship between voltammetric current and mass transport properties under stripping voltammetric conditions is not yet well esthablished, we propose a relationship between the experimentally obtained current and the mean diffusion coefficient of the metal-complex system. A sensitivity analysis of this expression for different parameters, such as the stability and the ratio of the diffusion coefficients of the bound and free metal is performed.
Natural complexing agents are often heterogeneous with regard to their affinity to metal ions. Therefore, we will discuss the evaluation of the heterogeneity of these complexes from voltammetric data for various metal-to-ligand ratios. For the case of a large excess of ligand over the metal atom concentration, the stability of the metal-complex system may be obtained independently from the potential shift. For this an equation is given similar to the classical one derived by DeFord and Hume. Finally, we present an experimental procedure based on adding ligand to the solution of the metal and measuring its voltammetric characteristics. The procedure takes into account (i) possible adsorption of metal ions to elements of the equipement and (ii) measuring all protolysis of the polyacids involved.
The characteristic features of applying the two electrochemical techniques conductometry and voltammetry to the study of ion binding by polyelectrolytes are discussed and compared in chapter 4. Analysis of data on K +/Zn(II)/polyacrylate and K +/Zn(II)/polymethacrylate systems illustrates a certain complementary of the two methodologies. Conductometry primarily measures the Zn(II)/K +exchange ratio. Voltammetry measures the Zn(II)/polyion binding strength; its dependence on the (excess) K +concentration also yields information on the Zn(II)/K +exchange ratio. The different results seem to be fairly coherent.
Experimental conductometric and voltammetric speciation data of metal-synthetic polyacid systems are presented and discussed in chapter 5. The competitive binding of monovalent and divalent counterions has been studied by the conductometric procedure described in chapter 2 for aqueous solutions of alkali metal polymethacrylates in the presence of Ca(NO 3 ) 2 and Mg(NO 3 ) 2 . The experimentally obtained fractions of conductometrically free counterions are compared with theoretical values computed according to a new thermodynamic model described in the same chapter. For the systems studied, the fractions of free monovalent and divalent counterions can be fairly well described by the theory. In fact, the results support the assumption that under the present conditions the conductometrically obtained distribution parameters f 1 and f 2 approximate the equilibrium fractions of free monovalent and divalent counterions. The experimentally obtained M +/M 2+exchange ratios agree well with the theoretical ones. Similar experiments have been performed for the Zn(II)/polyacrylate and Zn(II)/polymethacrylate systems. It seems that, compared to Ca 2+and Mg 2+ions, the Zn(II)-ions are bound more strongly. This could be due to some specific binding of Zn(II)-ions. Since the theoretical model does not incorporate this mechanisme, the experimental results do not agree well with the theoretical ones.
Furthermore, chapter 5 collects the results of a systematic study of the stripping voltammetric behaviour of Zn(II)- and Cd(II)-ions in polyacrylate and polymethacrylate solutions. All metal- ligand complexes involved apprear to be voltammetrically labile over the whole range of metal-to- ligand ratios under the various experimental conditions employed. Hence, the voltammetric data could be analyzed in terms of a stability K according to the methodology presented in chapter 3. The first set of experiments is concerned with the influence of the molar mass of the polyacrylate anion on the stability. Analysis of the data in terms of a mean diffusion coefficient, which decreases with increasing molecular mass, yields a consistent picture with molar mass- independent complex stabilities. The speciation of Zn(II) in such a polyelectrolyte system varies with the concentration of carboxylate groups, but it is invariant with the polyionic molar mass. Secondly, the competition between monovalent (K +) and divalent (Zn(II) and Cd(II)) counterions has been investigated by varying the concentration of electroinactive supporting electrolyte. The results show that the stability of the heavy metal/polymethacrylate complex decreases with increasing KNO 3 concentration. This effect is largely due to the reduction of the electrostatic component of the metal/polyanion interaction, which is generally the case for polyelectrolytes with high charge densities. For the Zn(II)/polymethacrylate system, a comparison with conductometric data representing the competitive behaviour of monovalent and divalent counterions has been made in chapter 4. The influence of the polyelectrolyte charge density of the polymethacrylic acid on the stability K has been studied by varying the degree of neutralization of the polyanion. For the Zn(II)/PMA complexes, the stability increases approximately linearly with increasing degree of neutralization, i.e. with increasing polyionic charge density. This is in accordance with the general polyelectrolytic feature that counterion binding is stronger with higher polyionic charge density. Finally, for later comparison with natural complexing agents, the chemical homogeneity of the macromolecules involved has been verified by varying the total metal ion concentration for a given polyelectrolyte concentration. The results indeed confirm that the Zn(II)/polymethacrylate and Cd(II)/polymethacrylate complexes have a homogeneous energy distribution. This is in line with expectation, since these macromolecules consist of only one repeating chemical binding site, i.e. the carboxylate group.
Chapter 6 deals with the pretreatment and characterization of humic material. The pretreatment procedure is used to purify the humic material in such a way that (i) the molecules are soluble under the experimental conditions employed in chapter 7, (ii) the amount of impurities is minimized and (iii) the resulting humic material is transferred into the acid form. Furthermore, a fractionation method based on the solubility of the humic substances is described. The humic material is characterized in terms of (i) the amount of chargeable groups by means of conductometric titration and (ii) molar mass distribution by flow field-flow-fractionation. It will be shown that although the fractionation by varying pH results in samples with different molar masses, the separation is far from ideal.
As was done with the synthetic polyacids, experiments have been performed for natural occuring polyelectrolytes. Conductometric and voltammetric results forvarious metal humic acid systems are presented in chapter 7. Solutions of humic acids were conductometrically titrated with potassium, sodium, lithium, calcium and barium hydroxide solutions. The results have been analyzed in terms of fractions of free and bound metal. The conductance properties of humic acids are basically different from those of a linear polyelectrolyte such as polymethacrylate. A marked difference was observed between the shapes of the curves for alkali metal hydroxides and those for alkaline earth metal hydroxides. It appears that monovalent cations are hardly bound by the humate polyion, whereas divalent counterions show a strong interaction. The latter feature may be fruitfully utilized in quantitative analysis.
The association of the heavy metals zinc(II) and cadmium(II) with humic acid samples has been furthere studied by differential pulse anodic stripping voltammetry (i) for various concentrations of supporting electrolyte (KNO 3 and Ca(NO 3 ) 2 ), (ii) for different degrees of neutralization of the humate polyion, (iii) for different metal-to-ligand ratios and (iv) for different fractions of the humic acid. Under the experimental conditions employed, all heavy metal/humate complexes have been found to be voltammetrically labile over the whole range of metal-to-ligand ratios. Hence, the stability (K) of the complex could be computed taking into account the difference between the diffusion coefficients of the free and bound metal. The dependence of K on the concentration of 1-1 electrolyte (KNO 3 ) is of comparable extent for various metal-humate complexes, but significantly smaller than in the case of the highly charged linear polyelectrolyte polymethacrylic acid. For the humic acid systems, it has been concluded that the relatively weak dependency of K on the salt concentration mainly reflects screening effects. The influence of the concentration of 2-1 electrolyte (Ca(NO 3 ) 2 ) on the stability of the heavy metal/humate complex is more pronounced than for the corresponding case of 1-1 electrolyte. By taking into account the association of calcium with the humate polyion, the stability of the heavy metal/humate complex was found to be more or less constant over the range of Ca(NO 3 ) 2 concentrations studied and comparable to the stability of the corresponding complex in the absence of calcium.
The stability of the heavy metal/humate complex has been found to increase with increasing degree of neutralization, i.e. with increasing charge density of the humate polyion. It seemed that the increase of K is less pronounced for higher values of α n . This observation could not be interpreted from an electrostatic point of view, and is in fact a further indication that the binding of heavy metals with the humate polyion is mainly governed by the chemical characteristics of the humic acid. The chemical heterogeneity of the humic acids was investigated by varying the metal-to-ligand ratio for different total concentrations of the heavy metals but in a range of comparable ligand concentrations. The results show that the stability K of the heavy metal/humate complex decreases with increasing total metal ion concentration, reflecting a certain chemical heterogeneity of the humic acid. For various heavy metal/fractionated humate complexes, the stability K was found to be comparable to the K value for the corresponding unfractionated humic acid system. This means that the distribution of functional groups is more or less the same for different molar masses of the humic acid.
For the present metal/humate complexes, the general conclusion is that the distribution of counterions over the free and bound states is mainly governed by the chemical heterogeneity of the humate polyion.