There was substantial variability in the ChE activity in both the short- and long-term experiment. In terms of minimum and maximum values, the ChE activity in many of the treatment groups varied four- to vefold (see Appendix F and G). A large variability in earthworm ChE activity has previously been observed by several authors, e.g. Hoel (1999) and Scaps (1997b). Variations in activity of chlorpyrifos-exposed worms are understand-able, and expectunderstand-able, in that the worms may dier in uptake of the compound. However, variations in the ChE activity of control worms are a limitation for this biomarker. It is necessary to have a referance of normal activity when examining potentially aected specimens, and the high control variation of ChE activity necessitates a large control group, especially if the aim is to assess eects of low concentrations of pesticide. Also, it is impossible to suggest whether a randomly sampled earthworm is under the inuence of ChE-inhibiting agents or not, unless the inhibition is drastic. Sancho et al. (1997) oper-ated with 20 % AChE inhibition as the limit that indicates exposure to OP compounds, and refer to additional authors who use this limit for indication of bird, sh or inverte-brate OP insecticide exposure. This limit can denitely not be applied to earthworms, as this would lead to numerous wrong conclusions due to the large variations in normal ChE activity.
Why is the variance in control worms so high? Firstly, a consideration of the method should be done. E2 has been shown sensitive to heat (Stenersen 1980a), however at temperatures higher than used in the current experiments, for which the temperature was xed at 30 ◦C during the spectrophotometric measurements. ChE activity is considered dependant on general state of health, sex and age in many species. For earthworms, age and general state of health may inuence the enzyme activity, and to illuminate these possibilities, the ad hoc-experiments (see section 4.9) were carried out. The number of worms used for these tests were small (n = 10 in all groups), and it was a pseudo-replication experiment. Thus, the results can not be considered representative for the whole population, but they may show the relationships between ChE activity, protein content, weight and cocoon production within the three groups tested. Between the juvenile and adult earthworms, there was no signicant dierence in ChE activity. Further, age is unlikely to be the most inuencing factor to variability in the experiments of this master's degree, since all worms used in the experiments were clitellate and not too old.
There was more variability within the juvenile group, which contained earthworms of
dierent sizes, which may suggest that ChE activity diers between maturation stages.
ChE activity is very often measured per amount of total protein in the sample, and it is plausible that a worm's protein content depends on the nutrient availability in its surroundings. This value is the denominator in the equation expressing the ChE activity, therefore a lower protein content would yield a higher value for the ChE activity4. In the ad hoc experiment, there were no signicant dierences between the protein contents of the groups after two weeks of dierent feeding schemes. There were admittedly no values for comparison with protein contents at the start of the experiment, but the worms were all taken from the same batch culture where they had equal nutrient availabilities and therefore expectedly the same points of departure. There were also no signicant dierences between the ChE activity of the three groups, so the only parameter for which they diered was weight gain. During the work with this master's degree, it has been speculated upon expressing the enzyme activity per g of worm rather than per mg og protein. However, the results of the ad hoc experiment suggest that amount of protein may in fact be a more stable denominator in the activity equation than weight, since weight gain probably is more sensitive to nutrient availability, and thus may cause more variation if there are slight dierences between the amounts of food ingested by worms in an experiment. An issue to consider regarding expressing ChE activity per mg of protein, however, is that the amount of total protein and amount of ChE in the tissue may not be proportional. It would perhaps be better to search for another protein by which to express the ChE activity, e.g. a protein more specic for the nervous system.
4The sensitivity of the protein quantitation method can also be discussed, however this is beyond the scope of this thesis.
6 Conclusion
After 1 week of exposure to a chlorpyrifos concentration of 10 mg/kg, there was a sig-nicant decrease in the ChE activity of E. fetida. No eects of lead on ChE activity were observed. Similarly, although based on a scant dataset, no eects of chlorpyrifos (10 mg/kg) alone on DNA strand breaks were observed, but chlorpyrifos (10 mg/kg) and lead concentrations of 500 and 1000 mg/kg produced signicantly higher levels of DNA strand breaks, as expressed by TEMs, compared to the control. The syringe extraction method was not successful, in that the impurities of the obtained cell suspension made several of the comet slides impossible to score, and was thus not shown to be a good alternative to the established, but more time-consuming extrusion uid method.
It was shown that chlorpyrifos causes irreversible ChE inhibition in the earthworm E.
fetida, in that neither the E1 nor the E2 activity recovered during 12 weeks after an expo-sure to chlorpyrifos (240 mg/kg) for 48 hours, despite the rapid breakdown of chlorpyrifos in the worms. There was no increase in the activity of the exposed worms compared to the control during the recovery period, i.e. there was no rise in ChE activity what-soever following the exposure. However, the worms' general motility and behaviour was indistinguishable from that of the control worms after 2-4 weeks of recovery.
Further research is suggested:
• Experiments conducted along the same lines as the recovery experiment described in this thesis, only with juvenile in stead of adult worms, in order to see whether de novo synthesis of enzyme occurs in juvenile earthworms following irreversible ChE inhibition.
• Finding eects of repeated exposure to OP insecticides and how this aects worms that already have depressed levels from previous exposures. If this is conducted with concentrations likely to be found in the eld, it will be a highly relevant experiment.
• Further investigation of possible side-eects of depressed ChE activity, by assessing reproductory parameters in earthworms with depressed ChE levels but in a seem-ingly good shape.
References
Abbasi, S. A. & Soni, R. (1983). Stress-induced enhancement of reproduction in earth-worm (Octochaetus pattoni) exposed to chromium(VI) and mercury(II) - implica-tions in environmental management, International Journal of Environmental Studies 22: 4347.
Abu-Qare, A. W. & Abou-Donia, M. B. (2001). Simultaneous determination of chlorpyri-fos, permethrin, and their metabolites in rat plasma and urine by high-performance liquid chromatography, Journal of Analytical Toxicology 25: 275279.
ACGIH (1986). American Conference of Governmental Industrial Hygienists, Inc.: Doc-umentation of the Threshold Limit Values and Biological Exposure Indices, 5. edn.
Achazi, R. K., Flenner, C., Livingstone, D. R., Peters, L. D., Schaub, K. & Scherwe, E.
(1998). Cytochrome P450 and dependent activities in unexposed and PAH-exposed terrestrial annelids, Comparative Biochemistry and Physiology B: Biochemistry and Molecular Biology 121: 339350.
Adler, M. & Filbert, M. G. (1990). Role of butyrylcholinesterase in canine tracheal smooth muscle function, FEBS Letters 267: 107110.
Al Addan, F. et al. (1991). Relations entre peuplements lombriciens et propriétés des sols méditerranéens, in G. K. Veereesh et al. (eds), Advances in management and conservation of soil fauna, Vedams Books International, New Delhi, India.
Alberts, B., Johnson, A., Lewis, J., Ra, M., Roberts, K. & Walter, P. (2002). Molecular Biology of the Cell, 4 edn, Garland Science, New York.
Aldridge, W. N. (1993). The esterases: perspectives and problems, Chemo-Biological Interactions 87: 513.
Aldridge, W. N. & Johnson, M. K. (1971). Side eects of organophosphorus compounds:
delayed neurotoxicity, Bulletin of WHO 44: 259263.
Aldridge, W. N. & Reiner, E. (1972). Enzyme Inhibitors as Substrates: Interactions of Esterases with Esters of Organophosphorus and Carbamic acids, Vol. XVI, North-Holland Publishing Company, Amsterdam.
Arpagaus, M., Richier, P., Berge, J. B. & Toutant, J. P. (1992). Acetylcholinesterases of the nematode Steinerma carpocapsae. Characterization of two types of amphiphilic forms diering in their mode of membrane association, European Journal of Bio-chemistry 207: 11011108.
Barker, R. (1958). Notes on some ecological eects of DDT sprayed on elms, Journal of Wildlife Management 22: 269274.
Bather, E. A. (1920). Pantoscolex latus, a new worm from Lower Ludlow, Annual Maga-zine of Natural History 9: 5.
Bengtsson, G., Gunnarsson, T. & Rundgren, S. (1986). Eects of metal pollution on the earthworm Dendrobaena rubida (Sav.) in acidied soils, Water Air and Soil Pollution 28: 361383.
Benham, W. B. (1922). Oligochaeta of Macquarie Island. Australian Antarctic Expedition, Scientic Reports, Zoology and Botany 6.
Benke, G. M. & Murphy, S. D. (1974). Anticholinesterase action of methyl parathion, parathion and azinphosmethyl in mice and sh: Onset and recovery of inhibition, Bulletin of Environmental Contamination and Toxicology 12: 117122.
Black, M. C., Ferrell, J. R., Honning, R. C. & Martin, L. K. (1996). DNA strand breakage in freshwater mussels (Anodonta grandis) exposed to lead in the laboratory and eld, Environmental Toxicology and Chemistry 15: 802808.
Booth, L., Heppelthwaite, V. & Eason, C. T. (1998). Cholinesterase and glutathione S-transferase in the earthworms Aporrectodea caliginosa as biomarkers of organophos-phate exposure, Proceedings of the 51st New Zealand Plant Protection Conference, pp. 138142.
Booth, L. & O'Halloran, K. (2001). A comparison of biomarker responses in the earth-worm Aporrectodea caliginosa to the organophosphorus insecticides diazinon and chlorpyrifos, Environmental Toxicology and Chemistry 20: 24942502.
Booth, L., Palasz, F., Darling, C., Lanno, R. & Wickstrom, M. (2003). The eect of lead-contaminated soil from canadian prairie skeet ranges on the neutral red retention assay and fecundity in the earthworm Eisenia fetida, Environmental Toxicology and Chemistry 22: 24462453.
Bouché, M. B. (1992). Earthworm species and ecotoxicological studies, in P. e. a. Greig-Smith (ed.), Exotoxicology of earthworms, Intercept Ltd., United Kingdom.
Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Analytical Bio-chemistry 72: 248254.
Brown, P. J., Long, S. M., Spurgeon, D. J., Svendsen, C. & Hankard, P. K. (2004).
Toxicological and biochemical responses of the earthworm Lumbricus rubellus to pyrene, a non-carcinogenic polycyclic aromatic hydrocarbon, Chemosphere 57: 1675 1681.
Carson, R. (1987). Silent Spring, twenty-fth anniversary edition edn, Houghton Miin Company, Boston, chapter 8. And No Birds Sing, pp. 103127.
Chatonnet, A. & Lockridge, O. (1989). Comparison of butyrylcholinesterase and acetyl-cholinesterase, Biochemical Journal 260: 625634.
Cikutovic, M. A., Fitzpatrick, L. C., Venables, B. J. & Goven, A. J. (1993). Sperm count in earthworms (Lumbricus terrestris) as a biomarker for environmental toxicology:
eects of cadmium and chlordane, Environmental Pollution 81: 123125.
Cooke, A. & Luxton, M. (1980). Eects of microbes on food selection by Lumbricus terrestris L., Revue d'Ecologie et Biologie du Sol 17: 365370.
Coppage, D. L. & Matthews, E. (1974). Short-term eects of organophosphate pesticides on cholinesterases of estuarine shes and pink shrimp, Environmental Contamination and Toxicology 11: 483488.
Cousin, X., Strähle, U. & Chatonnet, A. (2005). Are there non-catalytic functions of acetylcholinesterases? Lessons from mutant animal models, Progress in neurobiology 27: 189200.
Danadevi, K., Rozati, R., Banu, B. S., Rao, P. H. & Grover, P. (2003). DNA damage in workers exposed to lead using comet assay, Toxicology 187: 183193.
Dell'Omo, G., Turk, A. & Shore, R. F. (1999). Secondary poisoning in the common shrew (Sorex araneus) fed earthworms exposed to an organophosphate pesticide, Environmental Contamination and Toxicology 18: 237240.
Dembélé, K., Haubruge, E. & Gaspar, C. (1999). Recovery of acetylcholinesterase activ-ity in the common carp (Cyprinus carpio L.) after inhibition by organophosphate and carbamate compounds, Bulletin of Environmental Contamination and Toxicol-ogy 62: 731742.
Dep (1996). South African Water Guidelines, Vol. 1: Domestic Water Use (1st edn.).
Depledge, M. K., Aramat-Mendes, J. J., Daniel, B., Halbrook, R. S., Kloepper-Sams, P., Moore, M. N. & Peakall, D. B. (1993). The conceptual basis of the biomarker approach, in D. Peakall & L. Shugart (eds), Biomarker Research and Application in the Assessment of Environmental Health.
Dishburger, H. J., McKellar, R. L., Pennington, J. Y. & Rice, J. R. (1977). Determination of residues of chlorpyrifos, its oxygen analogue, and 3,5,6-trichloro-2-pyridinol in tis-sues of cattle fed chlorpyrifos, Journal of Agricultural Food and Chemistry 25: 1325 1329.
Donaldson, D., Kiely, T. & Grube, A. (2004). 2000-2001 Pesticide Market Estimates, U.S. Environmental Protection Agency.
Dow AgroSciences LLC (copyright c1998-2005). Chlorpyrifos europe frequently asked questions, http://www.dowagro.com/chlorp/eu/faq/index.htm#faq4.
Eastman, A. & Barry, M. A. (1992). The origins of DNA breaks: a consequence of DNA damage, DNA repair, or apoptosis?, Cancer Investigation 10(3): 229240.
Ecobichon, D. J. (2001). in C. D. Klaassen (ed.), Casarett & Doull's Toxicology: The Basic Science of Poisons, 6. edn, McGraw-Hill, Medical Publishing Division, chapter 22.
Toxic Eects of Pesticides, p. 1236.
Edwards, C. A. & Lofty, J. R. (1977). Biology of earthworms, 2. edn, Chapman and Hall.
Edwards, P. J. & Coulson, J. M. (1992). Choice of earthworm species for laboratory tests, in P. e. a. Greig-Smith (ed.), Exotoxicology of earthworms, Intercept Ltd., United Kingdom, pp. 3643.
Ellman, G. L., Courtney, K. D., Andres, V. J. & Featherstone, R. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity, Biochemical Phar-macology 7: 8895.
Engelstad, F. (1991). Characteristics of Eisenia species (Oligochaeta, Lumbricidae) rele-vant for application in ecotoxicology and waste management, Department of Biology, University of Oslo and Centre for Soil and Environmental Research, JORDFORSK, Ås.
EPA (1989). Registration Standard (Second Round Review) for the Reregistration of Pesti-cide Products containing Chlorpyrifos. US Environmental Protection Agency, Wash-ington, DC.
Esbjörnsson, C. (2002). Klorpyrifos, Naturvårdsverket, Sweden.
Eyambe, G. S., Goven, A. J., Fitzpatrick, L. C., Venables, B. J. & Cooper, E. L. (1991). A noninvasive technique for sequential collection of earthworm (Lumbricus terrestris) leukocytes during subchronic immunotoxicity studies, Laboratory Animals 25: 6167.
Falugi, C. & Davoli, C. (1993). Localization of putative biochemical messengers during Eisenia foetida (Annelida, Oligochaeta) development, Tissue and Cell 25: 311323.
Gallo, M. A. & Lawryk, N. J. (1991). Organic phosphorus pesticides, in W. Hayes &
E. Laws (eds), Handbook of Pesticide Toxicology, Academic Press, New York.
Garcia, S. J., Seidler, F. J. & Slotkin, T. A. (2005). Developmental neurotoxicity of chlor-pyrifos: targeting glial cells, Environmental Toxicology and Pharmacology 19: 455 461.
Gibb, J. O. T., Svendsen, C., Weeks, J. M. & Nicholson, J. K. (1997). H-1 NMR spec-troscopic investigations of tissue metabolite biomarker response to Cu(II) exposure in terrestrial invertebrates: identication of free histidine as a novel biomarker of exposure to copper in earthworms, Biomarkers 2: 295302.
Goven, A. J., Eyambe, G. S., Fitzpatrick, L. C., Venables, B. J. & Cooper, E. L. (1993).
Cellular biomarkers for measuring toxicity of xenobiotics: Eects of polychlorinated biphenyls on earthworm Lumbricus terrestris coelomocytes, Environmental Toxicol-ogy and Chemistry 12: 863870.
Grue, C. E. & Shipley, B. K. (1984). Sensitivity of nestling and adult starlings to dicro-tophos, an organophosphate pesticide, Environmental Research 35: 454465.
Hallegrae, G. M., Anderson, D. M. & Cembella, A. D. (eds) (2003). Manual on Harmful Marine Microalgae, UNESCO Publishing.
Hans, R. K., Khan, M. A., Farooq, M. & Beg, M. U. (1993). Glutathione S transferase activity in an earthworm (Pheretima posthuma) exposed to 3 insecticides, Soil Biology and Biochemistry 25: 509511.
Hess, W. N. (1925). Nervous system of the earthworm, Journal of Morphology 40: 235 260.
Heywood, R., James, R. W., Street, A. E., Rudd, C. J. & Barry, P. S. I. (1978). In vitro and in vivo study of the ability of the tetraethyl lead to inhibit cholinesterase activity, Toxicology Letters 2: 1116.
Hoel, L. (1999). Bruk av enzymer i meitemark som biomarkører for forurensning av tungmetaller og organofosfater, Master's thesis, University of Oslo.
Howard, P. E. (ed.) (1991). Handbook of Environmental Fate and Exposure Data for Organic Chemicals, Vol3: Pesticides, Lewis Publishers, Chelsea.
Hunt, L. M., Gilbert, B. N. & Schlinke, J. C. (1969). Rapid gas chromatographic method for analysis of 0,0 -diethyl-0 -3,5,6-trichloro-2-pyridyl phosphorothioate (Dursban) in turkey and chicken, Journal of Agricultural Food and Chemistry 17: 11681170.
Hurlbert, S. H. (1984). Pseudoreplication and the design of ecological eld experiments, Ecological Monographs 54: 187211.
Hyne, R. V. & Maher, W. A. (2003). Invertebrate biomarkers: links to toxicosis that predict population decline, Ecotoxicology and Environmental Safety 54: 366374.
Hønsi, T. G. (1999). Bruk av meitemark som bioindikator for forurensninger i det ter-restriske miljø, Biologisk Institutt, MCB, Universitetet i Oslo.
Jeyaratnam, J. (1990). Acute pesticide poisoning: a major global health problem, World Health Statistics Quarterly 43: 139144.
Johnson, M. K. (1993). The target for initiation of delayed neurotoxicity by organophos-phorus esters: biochemical studies and toxicological applications, in C. Travis (ed.), Use of Biomarkers in assessing Health and Environmental Impacts of Chemical Pol-lutants, Plenum Press, New York.
Johnson, M. L. (1942). The respiratory function of the haemoglobin of the earthworm, Journal of Experimental Biology 18: 266277.
Joshi, S., Biswas, B. & Malla, G. (2005). Management of organophosphorus poisoning, Update in Anaesthesia pp. 12.
Kallander, D. B., Fisher, S. W. & Lydy, M. J. (1997). Recovery following pulsed exposure to organophosphorus and carbamate insecticides in the midge, Chironomus riparius, Archives of Environmental Contamination and Toxicology 33: 2933.
Kenaga, E. E., Whitney, W. K., Hardy, J. L. & Doty, A. E. (1965). Laboratory tests with Dursban insecticide, Journal of Economic Entomology 58: 10431650.
Kennedy, S. W. (1991). The mechanism of organophosphate inhibition of cholinesterase proposal for a new approach to measuring inhibition, in P. Mineau (ed.), Cholinesterase-inhibiting insecticides their impact on wildlife and the environment, Elsevier Science Publishers B.V., Amsterdam, the Netherlands, pp. 7487.
Kille, P., Stürzenbaum, S. R., Galay, M., Winters, C. & Morgan, A. J. (1999). Molecular diagnosis of pollution impact in earthworms towards integrated biomonitoring, Pedobiologia 43: 602607.
Kishi, M. H. N., Djajadisastra, M., Satterlee, L. N., Strowman, S. & Dilts, R. (1995).
Relationship of pesticide spraying to signs and symptoms in Indonesian farmers, Scandinavian Journal of Workers' Environmental Health 21: 124133.
Klassen, P. (2002). OP insecticide phaseouts: silence means consent, California Farmer Magazine .
Koellner, G., Kryger, G., Millard, C. B., Silman, I., Sussman, J. L. & Steiner, T.
(2000). Active-site gorge and buried water molecules in crystal structures of acetyl-cholinesterase from Torpedo californica, Journal of Molecular Biology 296: 713735.
Koppen, G. & Verschaeve, L. (1996). The alkaline comet test on plant cells: a new genotoxicity test for DNA strand breaks in Vicia faba root cells, Mutation Research 360: 193200.
Kousba, A. A., Sultatos, L. G., Poet, T. S. & Timchalk, C. (2004). Comparison of chlorpyrifos-oxon and paraoxon acetylcholinesterase inhibition dynamics: potential role of a peripheral binding site, Toxicological Sciences 80: 239248.
Krüger, F. (1952). Über die Beziehung des Sauerstoverbauchs zum Gewicht bei Eisenia foetida, Zeitung der vergleichende Physiologie 34: 15.
Labrot, F., Ribera, D., Saint-Denis, M. & Narbonne, J. F. (1996). In vitro and in vivo studies of potential biomarkers of lead and uranium contamination: Lipid per-oxidation, acetylcholinesterase and glutathione peroxidase activities in three non-mammalian species, Biomarkers 1: 2128.
Laverack, M. S. (1963). The Physiology of Earthworms, The MacMillan Company, New York.
Lintern, M. C., Wetherell, J. R. & Smith, M. E. (1998). Dierential recovery of acetyl-cholinesterase in guinea pig muscle and brain regions after soman treatment, Human
& Experimental Toxicology 17: 157162.
Liu, H., Cupp, E. W., Guo, A. G. & Liu, N. N. (2004). Insecticide resistance in Alabama and Florida mosquito strains of Aedes albopictus, Journal of Medical Entomology 41: 956952.
London, L., Dalvie, M. A., Nowicki, A. & Cairncross, E. (2005). Approaches for regulating water in South Africa for the presence of pesticides, Water SA 31: 5360.
Lush, M. J., Li, Y., Read, D. J., Willis, A. C. & Glynn, P. (1998). Neuropathy target esterase and a homologous Drosophila neurodegeneration-associated mutant protein
contain a novel domain conserved from bacteria to man, Biochemical Journal 332: 1 4.
Lydy, M. J. & Linck, S. L. (2003). Assessing the impact of triazine herbicides on organophosphate insecticide toxicity to the earthworm Eisenia fetida, Archives of Environmental Contamination and Toxicology 45: 343349.
Ma, W.-c. (1984). Sublethal toxic eects of copper on growth, reproduction and litter breakdown activity in the earthworm Lumbricus rubellus, with observations on the inuence of temperature and soil pH, Environmental Pollution Series A, Ecological and Biological 33: 207219.
Marino, F., Winters, C. & Morgan, A. J. (1999). Heat shock proteins (hsp60, hsp70, hsp90) expression in earthworms exposed to metal stressors in the eld and labora-tory, Pedobiologia 43: 615624.
Matsumura, F. (1975). Toxicology of Insecticides, Plenum Press, New York.
Mattes, C., Bradley, R., Slaughter, E. & Browne, S. (1996). Cocaine and bu-tyrylcholinesterase (BChE): determination of enzymatic parameters, Life Sciences 58: L257L261.
Mbakaya, C., Ohayo-Mitoko, G. J., Ngowi, V. A., Mbabazi, R., Simwa, J. M., Maeda, D. N., Stephens, J. & Hakuza, H. (1994). The status of pesticide usage in East Africa, African Journal of Health Sciences 1: 3741.
McCarthy, J. F. & Shugart, L. R. (1990). Biological markers of environmental nation, in J. McCarthy & L. Shugart (eds), Biomarkers of environmental contami-nation, Lewis, Boca Raton, pp. 314.
McKellar, R. L., Wetters, J. H. & Dishburger, H. J. (1972). Unpublished report, Dow Chemical U.S.A, Midland, MI.
Mikalsen, A., Andersen, R. A., Barstad, J. A. B. & Lilleheil, G. (1982). Recovery of activity and molecular forms of cholinesterase in dierent animal species follow-ing irreversible cholinesterase inhibition, Comparative Biochemistry and Physiology 71C: 2735.
Mineau, P. (ed.) (1991). Cholinesterase-inhibiting Insecticides Their Impact on Wildlife and the Environment, Elsevier Science Publishers B.V., Amsterdam, the Netherlands.
Mitchelmore, C. L. & Chipman, J. K. (1998). DNA strand breakage in aquatic organisms and the potential value of the comet assay in environmental monitoring, Mutation Research 399: 135147.
Morgan, M. J., Fancey, L. L. & Kiceniuk, J. W. (1990). Response and recovery of brain acetylcholinesterase activity in atlantic salmon Salmo salar exposed to fenitrothion, Canadian Journal of Fish Aquatic Sciences 47: 16521654.
Neilson, R. & Boag, B. (2003). Feeding preferences of some earthworm species common to upland pastures in Scotland, Pedobiologia 47: 18.
Ngowi, A., Maeda, D. N., Wesseling, C., Partanen, T. J., Sange, M. P. & Mbuse, G. (2001).
Pesticide-handling practices in agriculture in Tanzania: observational data from 27 coee and cotton farms, International Journal of Occupational and Environmental Health 7: 326332.
Nigg, H. N. & Knaak, J. B. (2000). Blood cholinesterases as human biomarkers of organophosphorus pesticide exposure, Reviews in Contamination and Toxicology 163: 29111.
Niwaz, A. & Faridi, M. A. (1999). Organophosphate insecticide poisoning (case report), Anaesth Pain Intensive Care 3: 3436.
OECD (1984). Guidelines for the testing of chemicals, No. 207 Earthworm acute toxicity test, Organisation for Economic Cooperation and Development, Paris, France.
OECD (1984). Guidelines for the testing of chemicals, No. 207 Earthworm acute toxicity test, Organisation for Economic Cooperation and Development, Paris, France.