Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Social mobilisation and violence at the mining frontier : The case of Honduras
    Middeldorp, Nick ; Morales, Carlos ; Haar, Gemma van der - \ 2016
    The Extractive Industries and Society 3 (2016)4. - ISSN 2214-790X - p. 930 - 938.
    Conflict - Criminalisation of dissent - Honduras - Mining - Social movements
    This paper documents opposition to mining in Honduras, a country at the verge of an attempted ‘mining boom’ since the ratification of a new mining law in April 2013. It analyses how a broad movement – involving NGOs, social movements and local communities – engages in opposition to the extractive industry, declared a national development priority by the Honduran government. The movement emerged in the first decade of the 21st century in response to the establishment of two industrial mines and builds on the negative impact of Goldcorp's open-pit operation in Valle de Siria, where serious health problems have been reported. The anti-mining movement has devised a successful campaign about access to uncontaminated water. By contrast, the extractive industry and the central government have been far less successful in convincing local populations of the potential benefits of mining. As political opportunities for dialogue with the central government have been lacking since the 2009 coup d’état, the anti-mining movement increasingly turned to the local level, motivating communities to declare their municipality free of mining via public referendums. In turn, when legal means to obtain consent from communities for mining projects fail, the ‘extractive frontier’ is frequently extended through criminalisation of dissent and the threat of violence.
    Udder Hygiene Analysis tool
    Borkent, H. ; Bos, I. ; Fleuren, M.M.L. ; Middeldorp, M. - \ 2013
    UHC - 61
    melkkoeien - uiers - rundermastitis - diergezondheid - melkveehouderij - dairy cows - udders - bovine mastitis - animal health - dairy farming
    In this report, the pilot of UHC is described. The main objective of the pilot is to make farmers more aware of how to increase udder health in dairy herds. This goes through changing management aspects related to hygiene. This report firstly provides general information about antibiotics and the processes that influence udder health. Secondly, six subjects are described related to udder health. Thirdly, the tools (checklists and roadmap) are shown and fourthly, advises that are written by UHC are presented. Finally, the evaluation of the farmers is included.
    Reductive dechlorination of B-hexachlorocyclohexane (B-HCH) by a Dehalobacter species in coculture with a Sedimentibacter species
    Doesburg, W.C.J. van; Eekert, M.H.A. van; Middeldorp, P.J.M. ; Balk, M. ; Schraa, G. ; Stams, A.J.M. - \ 2005
    FEMS microbiology ecology 54 (2005)1. - ISSN 0168-6496 - p. 87 - 95.
    gradient gel-electrophoresis - 16s ribosomal-rna - microbial dehalorespiration - gamma-hexachlorocyclohexane - enrichment culture - gen. nov. - degradation - alpha - bacterium - isomers
    An anaerobic coculture was enriched from a hexachlorocyclohexane (HCH) polluted soil. The coculture reductively dechlorinates the ß-HCH isomer to benzene and chlorobenzene in a ratio of 0.5¿2 depending on the amount of ß-HCH degraded. The culture grows with H2 as electron donor and ß-HCH as electron acceptor, indicating that dechlorination is a respiratory process. Phylogenetic analysis indicated that the coculture consists of two bacteria that are both related to gram-positive bacteria with a low G + C content of the DNA. One bacterium was identified as a Dehalobacter sp. This bacterium is responsible for the dechlorination. The other bacterium was isolated and characterized as being a Sedimentibacter sp. This strain is not able to dechlorinate ß-HCH. The Dehalobacter sp. requires the presence of Sedimentibacter for growth and dechlorination, but the function of the latter bacterium is not clear. This is the first report on the metabolic dechlorination of ß-HCH by a defined anaerobic bacterial culture.
    Reductive dechlorination of hexachlorocyclohexane (HCH) isomers in soil under anaerobic conditions
    Middeldorp, P.J.M. ; Doesburg, W.C.J. van; Schraa, G. ; Stams, A.J.M. - \ 2005
    Biodegradation 16 (2005)3. - ISSN 0923-9820 - p. 283 - 290.
    petroleum-contaminated aquifers - beta-hexachlorocyclohexane - alpha-hexachlorocyclohexane - benzene oxidation - degradation - toluene - tetrachloroethene - chlorobenzene - stimulation - bioremediation
    The biological anaerobic reductive dechlorination of -hexachlorocyclohexane under methanogenic conditions was tested in a number of contaminated soil samples from two locations in the Netherlands. Soils from a heavily polluted location showed rapid dechlorination of -hexachlorocyclohexane to benzene and chlorobenzene with lactate as electron donor. Soils from an adjacent slightly polluted location did not show substantial dechlorination of -hexachlorocyclohexane within 4months. A heavily polluted sample was selected to optimise the dechlorination. All tested hexachlorocyclohexane isomers (-, -, -, and -), either added separately or simultaneously, were dechlorinated in this soil sample. The most rapid dechlorination was observed at a temperature of 30°C. Dechlorination of -hexachlorocyclohexane was observed with acetate, propionate, lactate, methanol, H2, yeast extract and landfill leachate as electron donors. In a soil percolation column, packed with a selected heavily polluted soil sample, the presence of 10mM sulphate in the influent led to simultaneous dechlorination of -hexachlorocyclohexane and sulphate reduction. When the column was fed with 10mM nitrate instead of sulphate, dechlorination ceased immediately. After omitting nitrate from the influent, dechlorination activity recovered in about 1month. Also in a separate column, the addition of nitrate from the start of the experiment did not result in dechlorination of -HCH. The significance of these experiments for in situ bioremediation of polluted soils is discussed
    Anaerobic microbial reductive dehalogenation of chlorinated ethenes
    Middeldorp, P.J.M. ; Luijten, M.L.G.C. ; Pas, B.A. van de; Eekert, M.H.A. van; Kengen, S.W.M. ; Schraa, G. ; Stams, A.J.M. - \ 1999
    Bioremediation journal 3 (1999). - ISSN 1088-9868 - p. 3 - 169.
    Bioremediation of contaminated soils: factors influencing the biological process.
    Middeldorp, P.J.M. ; Schraa, G. - \ 1998
    In: Microbial interactions in agriculture and forestry / Subba Roa, N.S., Dommergues, Y.R., - p. 239 - 253.
    Stimulation of reductive dechlorination for in situ bioremediation of a soil contaminated with chlorinated ethenes.
    Middeldorp, P.J.M. ; Aalst, M.A. van; Rijnaarts, H.H.M. ; Stams, A.J.M. ; Kreuk, H.F. de; Schraa, G. ; Bosma, T.N.P. - \ 1998
    Water Science and Technology 37 (1998). - ISSN 0273-1223 - p. 105 - 110.
    Enrichment and properties of a 1,2,4 trichlorobenzene dechlorinating methanogenic microbial consortium.
    Middeldorp, P.J.M. ; Wolf, J. de; Zehnder, A.J.B. ; Schraa, G. - \ 1997
    Applied and Environmental Microbiology 63 (1997). - ISSN 0099-2240 - p. 1225 - 1229.
    Microbial transformation of highly persistent chlorinated pesticides and industrial chemicals
    Middeldorp, P.J.M. - \ 1997
    Agricultural University. Promotor(en): A.J.B. Zehnder; G. Schraa. - S.l. : Middeldorp - ISBN 9789054856429 - 114
    microbiële afbraak - gewasbescherming - pesticidenresiduen - chemische industrie - persistente organische verontreinigende stoffen - microbial degradation - plant protection - pesticide residues - chemical industry - persistent organic pollutants

    Organic pollutants can be transformed, both in unsaturated and saturated areas of the soil, by means of biologically mediated reactions. The potential of soil microorganisms to clean up polluted soils is enormous. However, soil systems are highly heterogeneous with respect to the spatial distribution of substrates, nutrients and microorganisms, and also with respect to various phases and interfaces (e.g. water, air, minerals, organic matter). To come to the development of appropriate soil bioremediation techniques, comprehensive knowledge is required about the biological and physico- chemical processes and parameters that determine the rate, the nature and the degree of degradation of organic compounds within a certain time scale in soil. The most important processes and parameters that influence the behavior of pollutants in soil are shown in Figure 7.1.

    Several aspects, which may arise when assessing the application of microorganisms in soil remediation. techniques, have been studied in this thesis.

    A major part of this thesis concerns the environmental conditions, that are essential for biodegradation. The biodegradability of a pollutant depends not only on the molecular structure of this pollutant, but also on the occurrence of the appropriate enviromnental conditions. The biodegradation of many enviromnental pollutants has been studied primarily under aerobic conditions, since these conditions are prevailing in polluted locations and easy to maintain. However, the anaerobic degradation of pollutants forms a relatively new potential of interesting reactions, which may be used for bioremediation purposes.

    For instance, β-hexachlorocyclohexane (β-HCH), a waste product from the production of the pesticide lindane, is considered extremely recalcitrant under aerobic conditions.

    This may be due to the spatial arrangement of the chlorine atoms in this HCH isomer. Disappearance of β-HCH under anaerobic conditions has been reported, but product identifications and mass balances were lacking in these studies. In Chapter 2, the biodegradation of β-HCH under anaerobic conditions is described. It is shown that β-HCH can be dechlorinated under methanogenic conditions. The observed dechlorination products of β-HCH, benzene and chlorobenzene, have individually been found for the anaerobic biodegradation of γ-HCH, but they were never reported to be formed simultaneously from any HCH isomer by microorganisms.

    The dechlorination products of β-HCH, benzene and chlorobenzene, accumulate under the conditions studied. The formation of these products, which are potentially toxic, introduces a new problem since chlorobenzene is recalcitrant under methanogenic conditions and anaerobic benzene degradation has been observed in only a few cases. However, many studies have shown that these compounds are degradable in the presence of molecular oxygen. Therefore a bioremediation process in which subsequently anaerobic and aerobic conditions are created would lead to a total mineralization of β-HCH.

    Such a sequential change from anaerobic to aerobic conditions also applies to the decontamination of soils polluted with polychlorinated benzenes (CBs) and biphenyls (PCBs). These compounds, when substituted with five or more chlorine atoms, generally persist under aerobic conditions. However, anaerobic reductive dechlorination of these compounds has abundantly been shown. The products of this dechlorination process are important for several reasons. From a toxicological point of view, because ortho -chlorinated PCBs are considered 10-100 times less toxic than non ortho -chlorinated ones (coplanar PCBs), but also from a biotechnological point of view, since the dechlorination products are the substrates in an eventually following aerobic treatment. Microbial reductive dechlorination of PCBs is mainly limited to meta - and para -dechlorination, and therefore reduces the toxicity of the PCBs. Dechlorination of CBs and PCBs also increases the water solubility and therefore the mobility of these compounds in the soil. This may be advantageous in the case of an ex situ bioremediation. However, since this process has been shown to occur in undisturbed polluted sediment and will also occur in biological soil treatments in situ , one should prevent the pollutant to move away from the polluted site.

    The dechlorination of CBs and PCBs is carried out by different groups of microorganisms, originating from different locations. Chapter 3 describes the dechlorination of these compounds in a methanogenic consortium, enriched from a mixture of Polluted sediments on 1,2,4-trichlorobenzene (TCB). CBs appear to be extensively dechlorinated by this consortium, with 1,2- and 1,3-dichlorobenzene (DCB) as well as chlorobenzene as the final products. This is in contrast with the performance of previously reported enrichments, which have been obtained mainly using hexa- and pentachlorobenzene as substrates for dechlorination. Such cultures show a lesser extent of dechlorination and produce 1,3,5-TCB or 1,3-TCB as final products, which are problematic compounds for an eventually following aerobic mineralization. Chapter 3 also shows that, whereas mono- through pentachlorobenzenes are dechlorinated within 2 days, hexachlorobenzene (HCB) and several PCBs are dechlorinated after a lag phase of about two weeks. Dechlorination of the latter compounds occurs via a different pathway, in which only chlorines with two adjacent chlorines are removed. Chapter 3 also shows that dechlorination of highly chlorinated benzenes can be directed to the formation of chlorobenzene as the only product by the addition of 2-bromoethanesulfonic acid (BrES), a methanogenic inhibitor. These observations show that different dechlorinating activities are present in the consortium. They are probably carried out by different physiological groups of microorganisms. In contrast, Chapter 4 describes a different methanogenic consortium which has the capacity to dechlorinate both CBs and PCBs with the same specificity toward chlorine substution pattern of these compounds. This and the fact that similar lag phases for the dechlorination of CBs and PCBs were observed, shows that the dechlorination of these compounds may be carried out by the same type of microorganisms.

    Chapter 5 applies to the environmental conditions which are necessary for a fast dechlorination of PCBs. Anaerobic reductive dechlorination of PCBs is a slow process, in which lag periods of weeks to months are commonly observed before dechlorination starts. Also the maintenance and subcultivation of PCB dechlorinating microbial consortia are extremely difficult. The optimal enviromnental conditions for the dechlorination process are not known. This is partly due to our lack of knowledge about the physiology of microbial reductive dehalogenation in general and about dechlorination of PCBs in particular. In Chapter 5, the lag phase of the reductive dechlorination of 2,3,4,5-tetrachlorobiphenyl (TeCBP), performed by the consortium described in Chapter 4, was reduced by means of different types of additions to the medium. Sterile Rhine sand appears to shorten the lag phase and to be essential for the maintenance of the consortium. A similar positive effect of sand was also observed in other dechlorinating consortia (Chapter 2 and 3) and for the degradation of toluene in a manganese reducing consortium.

    The mechanism of the reduction of the lag phase by Rhine sand is not known. The Rhine sand that we added to our medium, may contain additional trace metals, necessary for dechlorination. Although the organic carbon content in the Rhine sand we used is very low (<0.05% w/w), another explanation may be that the organic matter served as a supply of growth factors, like vitamins, for the dechlorinating bacteria. Sorption of 2,3,4,5-TeCBP to the sand might also improve the contact between the PCBs and the dechlorinating cells, if these cells tend to settle out or grow on the sand surface (adhesion).

    The addition of anaerobic granular sludge (AGS), either autoclaved or not, also reduced the lag phase before dechlorination of 2,3,4,5-TeCBP drastically. The decrease of the lag phase, achieved by the addition of AGS, may be caused by factors, originating from microorganisms in the AGS. However, the addition of the redox mediator vitamin B 12 , Which is abundantly present in AGS, had little effect on the lag phase. Reduction of the lag phase for PCB dechlorination was also observed by Abramowicz et al. who added fluid thioglycolate medium with beef extract (FTMBE) to polluted Hudson River sediments. In fact, the use of undefined additions such as AGS and FTMBE illustrates how little is known about the mechanism of reductive dechlorination of PCBs. It is of practical importance that a relatively cheap and easily available material such as AGS also reduces the lag phase for dechlorination in a methanogenic enrichment, which is capable of dechlorinating the complex PCB mixture Aroclor 1260 (Chapter 5). It may be worth trying to introduce AGS into methanogenic river sediments polluted with PCBs, to stimulate in situ dechlorination.

    Another aspect one should consider when taking biodegradation into practice is the availability of the pollutant for biodegradation. This is illustrated in Chapter 6, which deals with the biodegradation of pentachlorophenol (PCP) in different soil types by inoculated Rhodococcus chlorophenolicus. After an initially high mineralization rate of the PCP in the inoculated soils, this rate decreased during time and levelled off, leaving behind a residual amount of PCP. Such a non- degraded residue has been observed in many soil biodegradation studies and is referred to as a fraction that is not available for biotransformation, i.e. not bioavailable. Limited bioavailability is considered as one of the most important problems impeding the development of successful bioremediation techniques.

    The rate at which microorganisms can degrade pollutants during bioremediation depends on the intrinsic metabolic activity of the cell, and on the transfer of the pollutant to the cell. Thus, bioavailability of a pollutant is determined by the rate of mass transfer, relative to the activity of the microbial cells. As bacteria generally degrade pollutants to concentrations which are much lower than the residual concentrations observed in soils, it appears that mass transfer is the limiting factor for further degradation. This was confirmed by Bosma et al. who quantified bioavailability by modelling the effect of both mass transfer and the intrinsic activity of the microbial cells. Bioavailability of a pollutant is controlled by a number of physico-chemical processes, such as sorption, diffusion and dissolution. Especially in old polluted sites, part of the pollutants appears to be inaccessible to degrading bacteria. This was shown for a soil contaminated with chlorophenols for over 40 years, in which bioremediation through composting resulted in a residual concentration of about 40 mg/kg. No further degradation was observed when the soil was inoculated with PCP mineralizing bacteria. However, 14C-labelled PCP which was freshly added to this soil led immediately to a complete mineralization of the added PCP. Similar results were reported for two soils contaminated with polycyclic aromatic hydrocarbons and for the reductive dechlorination of hexachlorobenzene in sediment. Rijnaarts et al. showed that biodegradation rates of α-hexachlorocyclohexane in soil could be increased by breaking up the soil particles.

    Such observations indicate a reduced bioavailability due to a so-called "ageing" of the pollutant in the soil. This may be caused by different processes. The first process is a slow diffusion of the pollutant into the deeper and smaller pores of the soil particles and absorption into organic matter as visualized in Figure 7.2. The pollutant can only reach the degrading microorganisms - which are not small enough to penetrate into the deeper pores - by means of diffusion.

    A second process is the occurrence of the pollution as solid particles or as non-aqueous phase liquids (NAPLs) with surrounding semi-rigid films, from which mass transfer into the water phase is limited. Here, bioavailability is controlled by the actual dissolution rate. The third process is bound residue formation and involves the incorporation of pollutants into humic compounds via oxidative coupling reactions. This process especially applies to naphtolic compounds, chlorinated phenols, benzoic acids and anilines, due to their similarity to natural organic compounds. Oxidative coupling can be catalyzed by inorganic materials, such as sesquioxides, clay minerals, oxides and oxohydroxides of iron, silica, and allophane, but also by peroxidases and phenol monooxygenases. Bound residue formation alters the original chemical and biological activity of the pollutants, which therefore become less available, less toxic and less mobile in the soil system. Chapter 6 shows that PCP, freshly added to a sandy and a peaty soil, can be partly recovered as extractable organic halogen (EOX) after 4 months of incubation. However, a chloride mass balance reveals that in the peaty soil, not all measured EOX can be ascribed to PCP, its transformation product pentachloroanisole, or any other possible low molecular weight organic chlorinated compound (MW < 512). This indicates that bound residue formation may have taken place in a time period of 4 months.

    The use of the oxidative enzymes has been proposed as a method to immobilize organic pollutants into the humic material of the soil by stimulating bound residue formation. However, uncontrollable release and distribution of the pollutant cannot be guaranteed. Macromolecular components of dissolved organic matter may form colloids which serve as carriers facilitating the transport of contaminants that bind to the organic matter. Furthermore, fungal oxidative enzymes have also been shown to catalyze unwanted reactions, like the formation of highly toxic chlorinated dibenzodioxins from coupling reactions with chlorinated phenols as substrate.

    It can be concluded that bioavailability is more a physico-chemical problem rather than a microbiological problem. Solutions have therefore to be sought in physico-chemical techniques to increase the transport of the pollutants to the degrading bacteria. These techniques may comprise pulverizing soil particles, increasing the water content of the soil (slurry systems), heating the soil, and using detergents to increase solubility and/or desorption.

    In this thesis, the ever increasing capability of microorganisms to degrade chlorinated pesticides and industrial chemicals is demonstrated, provided that the appropriate enviromnental conditions are created. Despite this enormous potential, the limited bioavailability of pollutants in old contaminated sites and strict soil quality standards, largely hamper the application of microorganisms in soil remediation techniques. The mentioned techniques to increase the availability of pollutants have economical or enviromnental drawbacks. Alternative remediation techniques like incineration or solvent-extraction yields soils without any biological activity, which' destiny is mainly to be used as raw material for building purposes.

    On the other hand, one should ask what the consequences can be of the slow release of the residual amount of a pollutant from a soil that has been biologically treated. (Eco)toxicological assessment of such soil may serve the development of guidelines for the urgency to remove residual pollutants from such soils. Such an approach would stimulate the use of in situ bioremediation techniques, which eventually prevents polluted soil from being excavated. Of course, a continuous monitoring of the soil quality and an appropriate groundwater management of run-off water are essential.

    Mass transfer limitation of biotransformation: quantifying bioavailability.
    Bosma, T.N.P. ; Middeldorp, P.J.M. ; Schraa, G. ; Zehnder, A.J.B. - \ 1997
    Environmental Science and Technology 31 (1997)1. - ISSN 0013-936X - p. 248 - 252.
    Biotransformation is controlled by the biochemical activity of microorganisms and the mass transfer of a chemical to the microorganisms. A generic mathematical concept for bioavailability is presented taking both factors into account. The combined effect of mass transfer of a substance to the cell and the intrinsic activity of the cell using the substance as primary substrate, is quantified in a bioavailability number (Bn). The concept can easily be extended to secondary substrates. The approach has been applied to explain the observed kinetics of the biotransformation of organic compounds in soil slurries and in percolation columns. The model allowed us to predict threshold concentrations below which no biotransformation is possible. Depending on the environmental system and the chemical involved, predicted threshold concentrations span a range of 11 orders of magnitude from nanograms to grams per liter and match with published experimental data. Mass transferand not the intrinsic microbial activityis in most cases the critical factor in bioremediation.
    Complete anaërobe biologische in situ sanering van met gechloreerde ethenen verontreinigde grond.
    Middeldorp, P.J.M. ; Schraa, G. ; Bosma, T.N.P. ; Aalst, M.A. van; Kreuk, J.F. de - \ 1997
    In: Bodem Breed '97 Nat. Symp. Bodemonderzoek Lunteren. Programmabureau Bodemonderzoek, Wageningen - p. 55 - 55.
    Degradation of ß-HCH in soils by methanogenic consortia.
    Eekert, H.A. van; Middeldorp, P.J.M. ; Ras, N.J.P. van; Schraa, G. ; Stams, A.J.M. ; Field, J.A. - \ 1996
    In: Samenvattingen Nationaal Symposium Bodemonderzoek, Lunteren / Lexmond, M.J., Wageningen : PGB - p. 161 - 161.
    Anaerobic reductive dechlorination of PCBs and chlorinated benzenes.
    Middeldorp, P. ; Wolf, J. de; Schraa, G. - \ 1996
    In: Abstract Symp. on Biodegradation of organic pollutants, Mallorca, Spain - p. 174 - 174.
    Bio transformation of _-, ß-, _-, and _-hexachlorocyclohexane under methanogenic conditions.
    Middeldorp, P.J.M. ; Jaspers, M. ; Zehnder, A.J.B. ; Schraa, G. - \ 1996
    Environmental Science and Technology 30 (1996). - ISSN 0013-936X - p. 2345 - 2349.
    The influence of solid phases and alternative reducing agents on the reductive dechlorination of pentachlorobenzene.
    Wolf, J. de; Middeldorp, P.J.M. ; Schraa, G. - \ 1995
    BioSpektrum (1995)spec issue. - ISSN 0947-0867 - p. 3 - 3.
    Dechlorination and rate optimisation of the anaerobic reductive dechlorination of polychlorinated biphenyls.
    Middeldorp, P.J.M. ; Wolf, J. de; Schraa, G. ; Zehnder, A.J.B. - \ 1995
    Mededelingen van de Faculteit Landbouwwetenschappen, Rijksuniversiteit Gent 60 (1995)4b. - p. 2535 - 2538.
    Biotransformation of ß-hexachlorocyclohexane to monochlorobenzene under methanogenic culture conditions.
    Middeldorp, P.J.M. ; Jaspers, M. ; Schraa, G. ; Zehnder, A.J.B. - \ 1995
    BioSpektrum (1995)spec issue. - ISSN 0947-0867 - p. 3 - 3.
    Biotransformation of ß-hexachlorocyclohexane to monochlorobenzene under methanogenic culture conditions.
    Middeldorp, P.J.M. ; Jaspers, M. ; Kaper, T. ; Schraa, G. ; Zehnder, A.J.B. - \ 1995
    In: Abstracts Int. Meeting on Anaerobic processes for Bioenergy and Environment, Copenhagen (1995) section 28: 1
    The fate of organic pollutants in soils and sediments and the development of biological soil remediation techniques.
    Middeldorp, P.J.M. ; Schraa, G. - \ 1994
    Unknown Publisher - 43 p.
    Biotransformation of ß-hexachlorocyclohexane to monochlorobenzene under methanogenic culture conditions.
    Middeldorp, P.J.M. ; Jaspers, M. ; Kaper, T. ; Schraa, G. ; Zehnder, A.J.B. - \ 1994
    In: Abstract 3rd Int. HCH & Halogenated Pesticides Forum, Bilbao, Spain (1994)
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