Effects of chlorpyrifos and lead (Pb) on the earthworm Eisenia fetida : a biomarker comparison and a characterisation of recovery

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This thesis is a result of studies conducted at the Department of Biology, University of Oslo, Norway and the Department of Botany and Zoology, Stellenbosch University, South Africa. I feel very lucky that Professors Sophie and Adriaan Reinecke helped me to both live and study in Stellenbosch, and I am deeply grateful. I also want to thank my supervisor Jørgen Stenersen for making this overseas-journey possible in the rst place.

The vast and grand landscapes of South Africa still linger in my mind.

Some of the analyses were carried out at the Norwegian Crop Protection Institute. Despite their busy schedule, the people at the Pesticide Laboratory patiently made space for Heidi and me in the lab, and generously provided us with chemicals, materials and good advice for methods. I am truly grateful to all, and especially to Børge Holen and Kari Stuveseth, for good help. A big thanks also to Gunnar Brunborg and to students and PhDs at the Norwegian Institute for Public Health for providing me with the opportunity to do the scoring of my earthworm cells using their microscope and computer program.

I am especially and hugely grateful to Dr. Heidi Sjursen, who has sacriced of her time to make it possible to carry out the experiments at Ås, has proofread and commented my writings, and been a rich source of advice and encouragement.

Finally, my dear Filip deserves much praise for supporting me throughout the entire process.

Solveig Aamodt

Blindern, 12 August 2005

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List of Figures

1 The nervous system in the anterior part of the earthworm . . . 8

2 The basic structure of organophosphorus pesticides . . . 10

3 The structures of some OP compounds . . . 11

4 Metabolism of chlorpyrifos . . . 16

5 The reaction between chlorpyrifos and a ChE . . . 21

6 An overview of DNA strand break sources . . . 26

7 Molecular interactions behind the Ellman assay . . . 32

8 Sample preparation for the comet assay . . . 38

9 Sample preparation for gas chromatography . . . 42

10 Stability and proportionality between amount of protein and nkatal in three selected enzyme preparates during three minutes in the spectrophotometer 49 11 ChE activity in E. fetida after 6 hours and 1 week of exposure to chlorpyrifos (10 mg/kg) and dierent concentrations of lead . . . 51

12 Olive tail moments and tail extent moments in coelomocytes from control and chlorpyrifos- and lead-exposed E. fetida . . . 52

13 E2 activity in E. fetida after inhibition by dierent concentrations of carbaryl 54 14 Change in total ChE and E2 activity over time during constant chlorpyrifos exposure . . . 56

15 Total ChE activity in control and chlorpyrifos-exposed worms during 12 weeks after an acute exposure . . . 57

16 E1 and E2 activity in E. fetida, controls and chlorpyrifos-exposed worms, during 84 days . . . 60

17 Chlorpyrifos content in E. fetida during 4 weeks after an acute exposure . 61 18 Changes in E. fetida chlorpyrifos content during 1 week after an acute exposure . . . 62

19 Total ChE activity in 10 juvenile and 10 adult worms . . . 63

20 Eects of nutrient availability on ChE activity, worm weight and protein content . . . 64

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21 Schematic diagram of a gas chromatograph . . . 87

22 The role of acetylcholine and AChE in the synapse . . . 89

23 Standard curves for bovine albumin . . . 92

List of Tables

1 Chemical characteristics of chlorpyrifos . . . 15

2 The half-lives and k₊₃ values of some phosphorylated rabbit erythrocyte AChEs . . . 23

3 The composition of the agricultural soil from Ås . . . 29

4 Dierences in sample preparation and ChE measurements . . . 33

5 Eects of Ca²⁺ on total ChE activity . . . 50

6 Eects of soil and chlorpyrifos concentrations on total ChE activity . . . . 55

7 ANOVA results for total ChE and E2 activity at 0, 28, 56 and 84 days after exposure . . . 58

8 Results from the repeated measures ANOVA performed on data from the recovery experiment . . . 59

9 E2 activity (nkatal/mg protein) in E. fetida expressed as a percentage of total ChE activity . . . 59

10 Full data set showing ChE activity in E. fetida exposed to chlorpyrifos and lead . . . 94

11 Data set showing Olive tail moments and tail extent moments in E. fetida exposed to chlorpyrifos and lead . . . 95

12 Full data set showing total ChE and E2 activity in control (unexposed) E. fetida in the recovery experiment . . . 96

13 Full data set showing ChE activity in chlorpyrifos-exposed E. fetida in the recovery experiment . . . 97

14 Full data set showing chlorpyrifos content of chlorpyrifos-exposed E. fetida in the long-term recovery experiment . . . 99

15 Full data set showing chlorpyrifos content of chlorpyrifos-exposed E. fetida in the short-term recovery experiment . . . 99

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1 Summary

Cholinesterase (ChE) activity is a recognised biomarker of organophosphorus (OP) insecticide exposure. It is frequently applied to earthworms in the eld of agriculturally linked ecotoxicology, because of the use of OP insecticides on crops, and the exposure of earthworms due to their inhabiting the soil. The ubiquitously occurring metal lead (Pb) has previously been shown to both inuence and not inuence ChE activity in earthworms, therefore it was of interest to investigate the inuence of lead on chlorpyrifos-induced ChE inhibition in the earthworm Eisenia fetida. Four treatment groups were exposed to chlorpyrifos (10 mg/kg), and three of the groups were also exposed to lead (20, 500 and 1000 mg/kg). Worms were sampled for analyses after 6 hours and after 1 week of exposure. In addition to the ChE activity, the amount of DNA strand breaks in coelomocytes was measured, thereby allowing a comparison of these two biomarkers. After 6 hours, there were no signicant dierences between the ChE activity between any of the treatment groups and the control. The DNA strand breaks could not be assessed at this point due to lack of enough cell samples of good quality. After 1 week, the ChE activity in the worms exposed to chlorpyrifos alone and combined with 20 and 1000 mg/kg lead was signicantly lower than the control (P < 0.05), corresponding to a 35-38 % inhibition. No eect of lead on the chlorpyrifos-induced ChE activity could be stated. DNA strand breaks as expressed by tail extent moments (TEMs) increased signicantly compared to the negative control in the worms exposed to chlorpyrifos and the two highest concentrations of lead (P <

0.01), whereas no signicant dierences in Olive tail moments (OTMs) were detected.

The syringe extraction method was used when sampling coelomocytes from the worms, and based on the results, it is not considered a preferable method when used as described here.

OP insecticides are known to cause an irreversible inhibition of ChE activity, which means that new enzyme must be synthesised for the activity to recover. Recovery of ChE activity after exposure to several OP insecticides has in many animals, including earthworms, been proved a lengthy process, however little is known of the recovery of earthworms following chlorpyrifos exposure. To see whether the slow ChE recovery applies to chlorpyrifos as well, E. fetida specimens were exposed to chlorpyrifos (240 mg/kg) for 48 hours, and subsequently transferred to clean soil. The activity of both types of ChE in E. fetida, E1 and E2, was measured at intervals over a 12 week period, and the appearance and behaviour of the worms were observed. Directly after exposure, the total ChE and E2 activity was inhibited by 63 % and 52 %, respectively, and there was no rise in the activity of the exposed worms compared to the controls during the recovery period. However, after 2-3 weeks, the exposed earthworms could not be distinguished from the control worms by appearance or behaviour. Aspects of applying ChE activity as a biomarker are discussed.

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2 Introduction

The main purpose of this master's degree was to measure eects of the organophosphorus insecticide chlorpyrifos on the earthworm Eisenia fetida. The aim was to assess eects using biomarkers, including the well-established biomarker cholinesterase (ChE) activity.

Three months were spent at the Department of Botany and Zoology at Stellenbosch University, South Africa. Given this opportunity, another goal was to get a perspective on pesticide use in developing countries. The rest of the work was carried out at the Program for Toxicology and Ecophysiology, Institute of Biology at the University of Oslo, Norway.

The experimental goals can be summarised as follows:

• Short-term experiment: Compare combined eects of chlorpyrifos and metal exposure on ChE activity and DNA damage.

• Long-term experiment: Investigate the recovery of the activity of both ChEs of E.

fetida after an acute exposure to chlorpyrifos.

In the short-term experiment, assessing combined eects of contaminants was of interest since organisms frequently are exposed to a mixture of chemicals in the eld. This is particularly the case in agricultural elds, and in this experiment, the focus was more specically directed towards vineyards and orchards in the Stellenbosch region. Here, pesticides, e.g. chlorpyrifos, are sprayed and metals are deposited as a result of general pollution and motorised vehicle use on the elds.

The long-term experiment was meant to extend the growing knowledge about the inhibition of ChEs by organophosphorus insecticides. It was especially of interest to investigate the recovery of both types of ChEs of E. fetida. The importance of intact levels of ChEs in animals in general is unclear, and in this context, it was of interest to investigate how E. fetida would manage with a strongly depressed ChE activity, and at which rate the levels would be restored to normal.

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2.1 Biomarkers

Toxicants can induce various changes in an organism, e.g. upregulation of proteins, acti- vation or inhibition of enzymes, alteration of membranes, reduction of sperm cell number or DNA damage. These types of change can be used as biomarkers. The term biomarker is being used extensively and in many elds of science. In a broad toxicological denition, a biomarker can include any measurement reecting an interaction between a biological system and an environmental agent (WHO 2000), although it has become more common to use the term more restrictively about a biochemical or cellular sublethal change resulting from individual exposure to xenobiotics (Hyne & Maher 2003).

A highly important aspect of biomarkers on molecular or cellular level, e.g. protein induction or DNA damage, is that these changes can be detected before eects occur on the individual, population and ecosystem levels (Depledge et al. 1993). Thus, molecular and cellular biomarkers are sensitive, and thereby allow preventive actions to be taken at an early stage (McCarthy & Shugart 1990).

Earthworms and biomarkers In ecotoxicology, the use of biomarkers has become widespread. Much has happened since the initial work of Stenersen (1979) of applying well-established biomarkers to earthworms. Spurgeon et al. (2003) categorises earthworm biomarkers as follows:

1. Cellular biomarkers: lysosomal membrane stability (neutral red retention time, reviewed by Svendsen et al. 2004), immune responses (Goven et al. 1993), DNA strand breaks as measured by the single cell gel electrophoresis (SCGE) assay, also known as the comet assay (Verschaeve & Gilles 1995) and sperm number and morphology (Cikutovic et al. 1993).

2. Protein biomarkers: activity of detoxication enzymes and proteins, including cy- tochrome P450 (CYP) enzymes (Achazi et al. 1998), glutathione transferase (GST) (Hans et al. 1993) and antioxidising enzymes (e.g. catalases and peroxidases) (Saint- Denis et al. 1998), and induction of metallothioneins (Kille et al. 1999).

3. Biomarkers depicting the eect of exposure rather than detoxication mechanisms, e.g. induction of heat shock proteins (Marino et al. 1999), mitochondrial proteins and lysosomal membrane-linked proteins (Stürzenbaum 1997).

4. Comprehensive metabolic proling: the use of magnetic resonance allows a full overview over many related metabolic changes (Gibb et al. 1997).

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ChE activity has been used as an ecotoxicological biomarker for a long time, i.e. it is well-established, and can be measured in virtually any sample containing nervous tissue.

The comet assay, however, is new, but promising, in the eld of ecotoxicology. Although originally developed for medical toxicological testing, it has more recently been applied to cells from several invertebrate species, including various aquatic organisms (reviewed in Mitchelmore & Chipman 1998) and earthworms (reviewed in Reinecke & Reinecke 2003b), in order to assess eects of environmental pollutants on organisms. DNA damage in plant cells has also been investigated using this method (Koppen & Verschaeve 1996).

In many experiments, a battery of biomarkers is used to give an overview of multiple modes of action or eects of the toxicant. This is useful for looking at the relevance of the dierent biomarkers in relation to each other. Using biomarkers from the molecular together with the cellular level, is a good approach to assessing the relevance of the molecular responses on a larger scale.

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2.2 Earthworms in ecotoxicological testing

In the eld of pesticide ecotoxicology, earthworms are widely used test organisms. Earth- worms inhabit many types of soils, including agricultural soils in which pesticides are deposited, and are benecial for the soil qualities. Earthworms are non-target organisms, i.e. the pesticides are not aimed at them. There are several reasons why earthworms are being used so extensively in pesticide ecotoxicology. The more practical reasons are that they are easily accessible, handled and identied (Hønsi 1999), whereas the more ecological, and agricultural, reasons have risen from the understanding and recognition of earthworms as soil benefactors and important parts of the food chain.

The rst standardised earthworm acute toxicity test was established by OECD in 1984.

According to Spurgeon et al. (2003), the test acted as a catalyst for the use of earthworms as model organisms in terrestrial ecotoxicology. Enchytraeids, soil micro-organisms (for assessment of nitrogen and carbon transformation) and earthworms are today (2005) the only soil-dwellers, aside from plants, connected to standardised OECD toxicity tests.

2.2.1 Taxonomy and geographical distribution

Earthworms belong to the class Oligochaeta under the phylum Annelida. The oligochaete species include both aquatic and terrestrial worm species, of which the latter are called earthworms (see Lawrence (2000)). Examples of other annelid species are leeches (class Hirudinea) and bristle worms (class Polychaeta). The class names Oligo- and Polychaeta refer to the amount of bristles, or setae, on the body wall of the worms, who are few- and many-bristled, respectively.

There are several theories on the phylogenetic age of the oligochaetes. While Bather (1920) thought that early oligochaetes could be found in England during the Silurian period (about 400 million years ago) Stephenson (1930) claimed that the class arose during the Cretaceous period (about 100 million years ago), after the dicotyledons had appeared. Benham (1922) hypothesised that they showed up prior to the dicotyledons, but that they thrived and prospered to a larger extent in the presence of these plants.

The lumbricids (Lumbricidae) are probably the earthworm family the most known to humans (Edwards & Lofty 1977). The lumbricids can inhabit various types of soils, and have been transported around the world as a result of human activities. Thus, the family has representatives on many continents, in several climatic zones (Edwards & Lofty 1977).

In Norway, there are at least 18 species of earthworms (Sims & Gerard 1999), all of which are lumbricids.

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2.2.2 Ecology

Earthworms are an important part of the food chain, between predators, plants, decom- posers and rotting organic material. Their signicance in the food chain was rst realised after a robin poisoning case on the grounds of Michigan State University in the 1950s, where DDT was sprayed on trees to avoid Dutch elm disease. Dr. Roy Barker published an article in 1958, explaining how the insecticide had reached the robins via the earthworms. Rachel Carson thoroughly describes the solving of the jigsaw puzzle of the doomed robins in her book Silent Spring from 1962:

Dr. Barker's work, published in 1958, traced the intricate cycle of events by which the robins' fate is linked to the elm trees by way of the earthworms.

The trees are sprayed in the spring (usually at the rate of 2 to 5 pounds of DDT per 50-foot tree, which may be the equivalent of as much as 23 pounds per acre where elms are numerous) and often again in July, at about half this concentration. Powerful sprayers direct a stream of poison to all parts of the tallest trees, killing directly not only the target organism, the bark beetle, but other insects, including pollinating species and predatory spiders and beetles.

The poison forms a tenacious lm over the leaves and bark. Rains do not wash it away. In the autumn the leaves fall to the ground, accumulate in sodden layers, and begin the slow process of becoming one with the soil. In this they are aided by the earthworms, who feed in the leaf litter, for elm leaves are among their favorite foods. In feeding on the leaves the worms also swallow the insecticide, accumulating and concentrating it in their bodies. [...] In the spring the robins return to provide another link in the cycle. As few as 11 large earthworms can transfer a lethal dose of DDT to a robin. And 11 worms form a small part of a day's rations to a bird that eats 10 to 12 earthworms in as many minutes.

The soil quality is aected by earthworms, as they eat and grind soil containing plant residues and rotting organic matter, rendering this more accessible for other soil dwelling organisms. Thus, they mediate several steps of the nitrogen cycle. Some of the worms also dig deep tunnels that increase the air and water supply to lower soil layers (Al Addan et al. 1991, Bouché 1992). The tunnel diggers are mainly the anecic worms, i.e. the largest and darkest earthworms, which feed on the surface at night and digest during the day. When feeding, an anecic worm frequently keeps the posterior part of its body in the tunnel, while the anterior part searches for food around the tunnel opening. In the deep tunnels, there are gradients of temperature and moisture, in which the anecics can

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position themselves according to their preferred conditions. There are two other ecological groups of earthworms: epigeic and endogeic worms (Bouché 1992). The epigeics, which include E. fetida, are homochromic, which means that their body pigmentation assimilates the colours of their environment, and they inhabit the top layer of soils rich in organic matter. Because of this, E. fetida is used by many vermicomposters. Endogeic worms, on the other hand, inhabit lower soil layers. They have no pigmentation of the body wall, and their physiology is closely related to the soil conditions. Low temperatures and dry soils cause the worms to hibernate. Endogeics are a diverse group, highly adaptive and capable of inhabiting many soil layers and types (Bouché 1992).

Epigeics are easier to culture than anecics and endogeics (Bouché 1992). This fact, in addition to the established OECD guideline tests where E. fetida is the recommended species, renders E. fetida a frequently used test species. The species' suitability as a representative of all earthworms is however being debated, as it seems to be less susceptible to several compounds, including pesticides, than other species (Reinecke 1992, Edwards

& Coulson 1992). A major advantage of using E. fetida as a test species, however, is the built up knowledge about and experience with the species. Because of the frequent use of E. fetida, using this species renders good possibilities of comparing data with other experiments done around the world.

2.2.3 A brief summary of earthworm physiology

Respiration In earthworms, all respiration occurs over the skin. The surface of the earthworm serves the same task that human alveoles do: It is an aqueous layer on a surface in which the gases dissolve before they can be transported over to the blood (Krüger 1952). The rate of respiration rises with the temperature. Earthworms lack the ability to keep their body temperature constant, therefore the range of respiration rates is wide within a single species. In average, tropical species respire faster than temperate species (Johnson 1942).

Digestion Earthworms derive nutrition from ingesting either soil or leaves, or a mixture of these. Generally, endogeic worms have stronger preferences for soil than anecic worms, who feed primarily on leaf litter (Neilson & Boag 2003). Leaf litter is often colonised by fungi, and earthworm species have been shown to selectively eat litter colonised by certain fungus species (Cooke & Luxton 1980). As the nutrients pass into the mouth and further into the gut, they are exposed to amylase secreted from the pharyngeal gland, grinding movements of the strong, muscular body wall of the gizzard, and various proteases secreted from the intestine. What is not absorbed from the intestine is excreted through the anus

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Figure 1: The nervous system in the anterior part of the earthworm. Illustration strongly inspired by Hess (1925).

(Edwards & Lofty 1977).

Excretion through nephridia Nephridia serve as primitive kidneys, excreting waste materials from the coelomic uid¹ out through nephridiopores in the worm's body wall.

The coelomic uid consists of several cell types: basophils, acidophils, neutrophils, gran- ulocytes and chloragogen cells (Stein et al. 1977). Chloragogen cells are important in removing waste materials by means of phagocytocis and depositing them in the coelomic uid for excretion. The excretion uid consists of waste materials, urea and ammonia.

Not all the nitrogen compounds are excreted through the nephridiopores, as some are excreted as mucus through other pores in the body wall. These secretions function as lining for the worm's soil burrows (Edwards & Lofty 1977). Some metals have been shown to accumulate in the nephridia (Prinsloo et al. 1999).

Circulation Earthworms have a dorsal and a ventral blood vessel, and vertical vessels connecting these. In addition, many branchings see to that the blood can be delivered to and collected from all parts of the body. In the anterior segments, the vertical vessels are wide and pulsating, functioning as hearts (Edwards & Lofty 1977).

The nervous system The nervous system of earthworms consists of the lateral and the medial system. The medial is the ventral nerve chord, while the lateral are the segmental

1Many earthworm species excrete coelomic uid through dorsal pores in the body wall when irritated.

The coelomic uid of E. fetida has a strong, characteristical odour, which has given the species its name.

Lat. foetor =stench (see Sinclair 2000). The species has sometimes, although incorrectly, been called E.

foetida.

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nerves running from the ventral nerve chord, forming a local neural net in each segment of the worm. In the front lies a cluster of neuronal bodies, forming the cerebral ganglion, the equivalence of a brain (Fig. 1) (Edwards & Lofty 1977). In addition, they have an enteric nervous system: a net of neurons and ganglia within the gut wall. The axons are myelinated (Toman & Sabelli 1968). The synapses of invertebrates are simpler than those of vertebrates, but essentially they function in the same way.

Reproduction and life cycle Earthworms are hermaphrodites. Two worms copulate by positioning themselves close to each other, the ventral side of their clitellar regions adjoined. Each worm secretes seminal uid which fertilises the ova of the opposing worm.

During the whole process, mucus is excreted from the clitellar region. After the copulation, each worm has a belt of mucus around its clitellum, containing the fertilised eggs. The mucus partly hardens, and the worm moves backwards, drawing the mucus belt over its head. The belt of partly hardened mucus closes up in the edges and becomes a cocoon (Edwards & Lofty 1977).

The life span of earthworms vary according to species and climate (Edwards & Lofty 1977).

In protected labs and given favourable conditions, E. fetida has been reported to live for four and a half years, but the worms are unlikely to survive for such a long time in the eld.

Cocoons take several weeks to hatch, varying between species and dependent on climatic conditions. In a study conducted by Tripathi and Bhardwaj (2004), E. fetida produced an average of nine cocoons per month, and the fertilised eggs developed into adults in four months. Poor conditions delay this developmental process (Edwards & Lofty 1977, Viljoen et al. 1992). The amount of cocoons produced may be strongly inuenced by the nutrient availability and temperature. Engelstad (1991) found that the cocoon production of E. fetida declined with an increasing number of individuals in the same container (2-16 individuals in 280 mL boxes) and was higher at 25^◦C than at 15^◦C. It is also possible that some kinds of pollutants induce an increase in cocoon production (Satchell 1967, Abbasi

& Soni 1983). However, this is unclear, as many papers showing the opposite have been published (Ma 1984, Bengtsson et al. 1986, Brown et al. 2004, Spurgeon et al. 2004).

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2.3 Organophosphorus insecticides

Organophosphorus insecticides are designed to kill crop-detrimental insects by inhibiting enzymes in their nervous system (see section 2.4). These pesticides are esters constructed after the same, basic structure (Fig. 2). The entire group of organophosphorus pesticides is sometimes referred to as organophosphates, however, the compounds can be divided into several groups: organophosphates, -phosphonates, -phosphoroamidates and -phosphinates (Fig. 3). Which group a compound belongs to, depends on its structure:

Organophosphates have two P-O-R-bonds, organophosphonates have one P-O-R- and one P-R-bond, organophosphoroamidates have at least one P-N-R-bond, and organophosphi- nates have two P-R-bonds (WHO 1986). Within these groups, there are thione as well as oxon compounds, the rst having a P=S- instead of a P=O-bond. These compounds are named organophosphorothioates, -phosphonothioates, -phosphoroamidothioates and -phosphinothioates, respectively (WHO 1986). The thione compounds are less toxic than their oxon analogs, but they are bioactivated to oxons when ingested or absorbed by animals, resulting in an increase of toxicity (see Ecobichon 2001). In this thesis, I will use the term organophosphorus insecticides (OP insecticides) when dealing with the group as a whole.

Although most of the OP compounds are synthesised by humans, a few naturally occuring OP substances exist. These substances have the same mechanism of action as the synthesised OP insecticides. Anatoxin-a(S) produced by the cyanobacterium Anabaena lemmermannii (see Hallegrae et al. 2003) and the toxin from the soil bacterium Strep- tomyces antibioticus (see Stenersen 2004) are two examples.

Figure 2: The basic structure of organophosphorus pesticides, after Schrader (1951). The R- groups are alkyl groups, and the acyl group is the leaving group when binding to an enzyme occurs.

The acyl group of strong inhibitors is highly electrophilic, and thereby draws electrons from the phosphorus atom. Compounds originally containing a thione group, are bioactivated to the oxon analog in the organism (see Stenersen 2004).

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Figure 3: The structures of some a) organophosphates, b) -phosphonates, c) -phosphoroamidates and d) -phosphinates. Chlorpyrifos, trichlorfon, bromophos, leptophos and mipafox are insecticides. Sarin is a warfare agent and phenyl-di-pentyl-phosphinate (PDPP) is a compound used for theoretical research on inhibitory mechanisms.

2.3.1 The history of OP insecticides

In 1854, Philip de Clermont presented the synthesis of tetraethyl-pyrophosphate (TEPP) to the French Academy of Sciences. This inspired, among others, the German Dr. Willy Lange, who began researching uorophosphates and -sulfates. In 1932 he and his gradu- ate student Gerda von Krueger prepared and investigated esters of monouorophosphoric acid. During this work, Dr. Lange and von Krueger probably became the rst to investigate the physiological eects of organophosphorus esters on humans. In her thesis from 1933, von Krueger wrote (based on translation from German by Stenersen 1988):

The strong eect of monouorophosphoric acid alkyl ester on the human organism is interesting. The vapours of the compounds smell pleasant and aromatic. However, just a few minutes after breathing there is a strong pres-

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sure against the larynx, connected to a shortage of breath. Then a light unconsciousness and visual problems with painful hypersensitivity of the eyes against light appears. First after several hours, these symptoms disappear.

Gerhard Schrader at Farbenfabriken Bayer AG was given the task of developing new insecticides, since many of the compounds in use (mainly organochlorines and metals) had to be imported and were expensive. Schrader began looking into the possible use of organophosphorus esters. In 1937, he and his colleague Hans Kükenthal investigated the properties of esters of phosphoric acid, which were used as conservatives in plastics. They discovered that some of them were toxic, and more importantly, they discovered how the structure was related to the toxicity. This lead to Schrader's basic structure of a neu- rotoxic organophosphorus ester (Fig. 2). His ndings were reported to Heereswaenamt (the German Army Weapons Oce), as was mandatory by law, and unfortunately, this information made it possible for the Nazis to develop warfare agents like tabun, sarin and soman (Pngsten 2003).

During and after the war, the research on OP insecticides continued. The rst ones in use include TEPP, ethion, parathion and schradan (WHO 1986, Stenersen 1988). These new compounds became an alternative to the previously dominating organochlorine pesticides, although not without complications for the applicants. When parathion replaced DDT in the 1950's, a number of poisonings occurred due to the ignorance about the insecticides' mode of action (Ecobichon 2001). Thus, the farmers' knowledge of the new products' toxicity towards humans was achieved on a somewhat empirical basis.

2.3.2 Pesticide use in developing countries

Even though many OP insecticides marketed today are more selective and less toxic towards mammals, many of the older ones are still used in developing countries. A major problem is the widespread lack of knowledge about safe handling of the pesticides:

99 % of the over 200.000 annual acute pesticide poisoning deaths happen in developing countries (Jeyaratnam 1990). Wrong or unwary use of OP insecticides cause the majority of the acute poisonings (WHO & UNEP 1990, Mbakaya et al. 1994, Kishi et al. 1995 and Ngowi et al. 2001). According to a report from the United Nations Food and Agriculture Organisation (FAO), many farmers believe in high volumes and high doses when applying pesticides. Farmers in Brazil have been reported to spray with amounts of which less than 10 % would have been sucient. Much of the equipment also needs upgrading. An FAO study showed that approximately 58 % of the manual spray equipment in Indonesia was leaking. In the Philippines, the majority of the farmers never recieve formal training in using the application equipment.

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The easy access to and poor regulation of OP insecticides is possibly the reason that these pesticides are common suicidal remedies in some developing countries (Niwaz &

Faridi 1999). WHO numbers actually indicate that approximately 1 million accidental poisonings and 2 million suicide attempts connected to various pesticides occur annually (Joshi et al. 2005).

A poor regulation also leads to a poor surveillance. In South Africa, the pesticide legis- lation is complexly organised, fragmented across 14 Acts and 7 government departments (Rother & London 1998). Here, as well as in many other countries, it is not mandatory by law for farmers to report how much pesticide they are using. In India, there are na- tional standards for application of pesticides, but many food producers do not overhold the standards. According to FAO, the levels of pesticide residues found on Indian foods are high compared to the world average.

Chronic poisonongs as a result of long-term exposure to low doses, can be a problem to both farmers and other citizens. In South Africa, the Department of Water Aairs and Forestry (1996) has set drinking water guidelines for several metals and some organic contaminants, but for only one pesticide, i.e. atrazine (London et al. 2005). This is a contrast to international organisations, e.g. WHO, which has set drinking water limits for virtually all existing pesticides. It is a problem in South Africa, in that pesticides in low concentrations are widespread in surface- and groundwater (see London et al. 2005).

Toxicological expertise and competence in laboratory analysis is growing, but considering the enormous social dierences in South Africa, introducing a more sensible pesticide policy countrywide is a big challenge.

In developed countries, the overall most used pesticides are herbicides (Donaldson et al.

2001). The OP insecticides are gradually facing a phase-out. This phase-out is not seen in developing countries.

2.3.3 Chlorpyrifos

Introduction According to Dow AgroSciences, chlorpyrifos (IUPAC name: O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate) was discovered in 1962 by The Dow Chem- ical Company, and the rst report on the compound was written by Kenaga et al. (1965).

Chlorpyrifos is used against a number of pests in both agricultural and urban areas, on grains, cotton, wheat, alfalfa, sugarcanes, nuts, fruit trees and vegetables. It is also used for prevention against damage caused by termites, cockroaches, eas and ants in or around buildings. Chlorpyrifos is registered for use in 98 nations, and like other OP insecticides, it is especially common in developing countries. Today (2005), about 350 registered chlorpyrifos products are marketed (Dow AgroSciences LLC).

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In USA in 2000, about 11 million pounds of chlorpyrifos were applied by the agricultural sector. About 5 million pounds were used by industrial, commercial and government agencies, and about 3 million pounds were sold in the home and garden market. In 2001, malathion was the most used OP insecticide (as determined by total weight of active ingredient) in USA, whereas Chlorpyrifos ranked second, followed by diazinon (Donaldson et al. 2004). Chlorpyrifos is produced in Germany, Luxembourg and Switzerland and applied in many European countries. In Sweden, four registered chlorpyrifos compounds have approvals lasting to 2008, mostly for household use (Esbjörnsson 2002). In Norway, chlorpyrifos is not registered for use. It is manufactured and used in South Africa, although it is dicult to nd exact numbers on the amounts applied.

The most common trade names are Dursban (formulations used for household products) and Lorsban (formulations used for agricultural products). Various types of formulations exist: baits, dusts, granules, wettable powders, owables, impregnated plastics and pressurised liquids.

Dow AgroSciences, which market many pesticides, write on their internet pages:

No other pest control product has been researched more thoroughly.

In 1988, the Environmental Protection Agency (EPA) in USA started a scrutinising in- vestigation of all pesticides introduced to the market prior to 1985. This process lasted for nearly ten years. Chlorpyrifos made it through the investigations, and is still (2005) approved as an agricultural product. There were, however, made recommendations for better safety equipment and longer intervals between application, as well as strong restrictions on household use (Klassen 2002). There was made an agreement to stop the sale of chlorpyrifos household products from December 31, 2001. The household restrictions were partly based on the Food Quality Protection Act, which aims to review products that may adversely aect children. Chlorpyrifos is shown to have adverse eects on the development of rat foetus brains (e.g. Garcia et al. 2005).

Resistance Several insect species have developed a resistance towards chlorpyrifos.

These include populations of the German cockroach Blattella germanica (Rust & Reierson 1991, Pai et al. 2005), mosquito strains in Alabama and Florida (Liu et al. 2004) and the fruit y Drosophila melanogaster in Egypt (Ringo et al. 1995).

Chemical properties The information on the chemical properties and acute toxicity of chlorpyrifos (Fig. 3) is taken from the Pesticide Manual, 12^th edition (2000).

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Table 1: Chemical caracteristics of chlorpyrifos ( O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate)

Molecular weight 350,6

Molecular formula C9H11Cl3NO3PS

Form Colourless crystals with mild mercaptan

odour

Melting point 42-43,5^◦C

Vapour pressure (25 ^◦C) 2,7 mPa

KOW log P = 4,7

Solubility in water (25 ^◦C) 1,4 mg/L

Solubility in organic solvents benzene: 7900, acetone: 6500, chloroform:

(25 ^◦C) 6300, carbon disulde: 5900, diethyl ether:

5100, xylene: 5000, iso-octanol: 790 and methanol: 460 g/kg

Chemical characteristics of the compound are shown in Table 1.

Acute toxicity: Moderately toxic (EPA and WHO class II²) to humans. Details on human poisoning are given in section 2.4.1. Mammals: Oral LD50for rats=135-163 mg/kg, for guinea pigs=504 mg/kg and for rabbits=1000-2000 mg/kg. Percutaneous LD50for rats

> 2000 mg/kg and for rabbits > 5000 mg/kg. Birds: Oral LD50 for chickens = 32-102 mg/kg and for house sparrows = 122 mg/kg. Fish: 96-hour LC50 for rainbow trout = 0,007-0,051 mg/L and for fathead minnow=0,12-0,54 mg/L. Invertebrates: Oral LD50

for honey bees =360 ng/bee and 14-day LC50 for E. fetida = 210 mg/kg soil.

Chronic toxicity: Delayed symptoms are reported for humans (see section 2.4.2). In dogs given chlorpyrifos (3 mg/kg/day) for two years, there was an increase in liver weight in addition to inhibition of ChE. In rats and mice given chlorpyrifos for 104 weeks, there was ChE inhibition only (EPA 1989). No long-term health eects occured in two-year feeding studies with rats and dogs (ACGIH 1986, Gallo & Lawryk 1991). Fathead minnows exposed to 0,002 mg/L for 200 days produced progeny with decreased survival and growth.

For mammals, there are few indices of reproductional toxicity. The available research suggests that the insecticide is neither mutagenic nor carcinogenic.

Fate in soil: In the soil, chlorpyrifos is degraded by UV light, chemical hydrolysis and micro-organisms. The half-life in soil usually lies between 60 and 120 days, but can range from two weeks to over a year, depending on the soil and climate conditions (Howard 1991). The persistence declines with increasing soil pH and in anaerobic conditions. Contamination of ground water happens to a low extent, because of chlorpyrifos'

2EPA and WHO have arranged products in classes I-IV according to their toxicity. I is the most and IV the least toxic. I: DANGER-POISON, II: WARNING and III-IV: CAUTION.

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Figure 4: An overview of various metabolic pathways for chlorpyrifos. In animals, several of these reactions are mediated by the CYP enzyme system. TCP is the dominating metabolite both in animals and plants, and is the main degradation product in soil.

immobility in the soil due to strong adsorption to soil particles (Racke 1992).

Metabolism In mammals, chlorpyrifos is rapidly absorbed from the intestine, and to a small extent over the skin. Earthworms take up the compound directly over the skin as well as from their gut. Plants do not absorb chlorpyrifos from the soil, but through plant tissues above ground (see Tomlin 2000).

Like many OP insecticides, chlorpyrifos undergoes both biotransformation and detoxication mediated by CYP enzymes. Chlorpyrifos is a phosphorothioate, and must therefore be bioactivated to chlorpyrifos-oxon in order to be toxic. Hydrolysis of chlorpyrifos- oxon (Fig. 4) results in the formation of 3,5,6-trichloro-2-pyridinol (TCP), which can be metabolised to 3,5,6-trichloro-2-methoxypyridine (TMP) or conjugated and excreted. An alternative route is hydrolysis of one of the P-O-R-bonds, resulting in desethyl-chlorpyrifos which is further metabolised to TCP (see Kennedy 1991 or Ecobichon 2001).

In mammals, TCP is rapidly excreted in the urine. It is the major metabolite in plants, soil and animals, and is not reported to be toxic. Chlorpyrifos can accumulate in fat tissue

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to some extent, but the half-life in animals is short (see Tomlin 2000). Few studies have been done on the metabolism in chlorpyrifos in earthworms, however one was conducted by Lydy and Linck (2003), where they exposed E. fetida individuals to ¹⁴C-chlorpyrifos (1 ng/cm²), using a standard lter paper test (OECD 1984), both alone and together with two concentrations of atrazine, for 96 hours. Then they investigated the presence of chlorpyrifos, chlorpyrifos-oxon and TCP in prepared worm samples. Of the chlorpyrifos and derivates left in the worms treated with chlorpyrifos alone, approximately 60 % was the parent compound, 7-8 % was the oxon analog and 7 % was TCP. In the worms treated with the highest atrazine concentration (1,0 µg/cm²), the distribution was dierent: approximately 40 % was parent compound whereas 20 % was TCP. However, the percentage of the oxon analog was the same. Lydy and Linck suggested the reason to be that the oxon analogs were bound to the ChE-receptors and thereby immobilised.

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2.4 Cholinesterases

The cholinesterase (ChE) enzymes are widely distributed throughout the animal kingdom (Scaps et al. 1997). They are part of a group of enzymes called B-esterases, which are esterases that are inhibited by OP compounds (Aldridge 1993). ChEs are serine esterases, meaning that a serine residue in the active site of the enzyme interacts with the substrate or inhibitor.

ChEs are classied according to their substrate specicities, and named after the substrate they hydrolyse the fastest. Based on studies of vertebrate ChEs, the enzymes are classied into two major types: acetylcholinesterase (AChE, IUB number 3.1.1.7), which most importantly hydrolyses the neurotransmitter acetylcholine in synapses, and butyrylcholinesterase (BChE, 3.1.1.8, also known as pseudocholinesterase, non-specic cholinesterase or serum cholinesterase), which has a broader substrate specicity. Its function remains unclear (see Kennedy 1991 or Stenersen 2004). A third, but less common type is propionylcholinesterase (PrChE).

All OP insecticides exert their toxicity mainly through inhibiting AChEs. For an overview of the role of acetylcholine and AChE in the synapse, see Appendix B.

2.4.1 Localisation and molecular forms

In mammalian nervous tissue, AChEs are located in 1) the parasympathetic autonomic nervous system, 2) the neuromuscular junctions and 3) the central nervous system (Johnson 1993). The inhibition can therefore yield symptoms resulting from stimulation of these areas, including 1) increased secretions, bronchoconstriction, miosis, gastrointestinal cramps, diarrhea, urination, bradycardia, 2) tachycardia, hypertension, muscle fasciculation, tremors, muscle weakness, accid paralysis and 3) restlessness, emotional liability, ataxia, lethargy, mental confusion, loss of memory, generalised weakness, convulsions, cyanosis and coma (see Ecobichon 2001). There are also AChEs in erythrocytes, of which the functions remain unclear. However, erythrocyte AChE activity is a commonly applied biomarker, because it can easily be measured in blood samples of pesticide workers or other suspect- edly exposed individuals (reviewed by Nigg & Knaak 2000).

11 forms of BChE have been identied in humans, mainly located in the blood plasma.

Their exact functions are unknown (Chatonnet & Lockridge 1989), but they may be involved in hydrolysis of drugs such as cocaine (Mattes et al. 1996) or possibly play some coregulatory role in the hydrolysis of acetylcholine (Adler & Filbert 1990). Most animals, including earthworms, have at least two enzymes corresponding to human AChEs and

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BChEs. In earthworms, both of these enzyme types are highly aected by OP insecticides (Stenersen 1980b).

Cholinergic systems in earthworms are involved in muscle activity in the body wall, blood vessels and gut, and they are present in some central synapses (Laverack 1963). Falugi and Davoli (1993) investigated the ChE activity of dierent parts of embryonic and adult E. fetida individuals, using dierent substrates. They found the total ChE activity to be higher in the developing central nervous system (CNS) of embryos than in the developed adult CNS, where the test for ChE activity was scarcely positive. ChE activity was also found in blastomeres of embryos and, showing higher activity than in the CNS, in the body wall of adults. AChE was the dominating form in the CNS. Pilot studies conducted by Dell'Omo et al. (1999) showed that the highest ChE activity in E. fetida and E.

veneta could be found within the rst ve segments. The cerebral ganglion and the sensitive prostomium possibly accounts for much of this activity, although this contrasts the ndings of Falugi and Davoli.

ChEs are thought to play a critical part in the development of the nervous system in many animals, including earthworms (as reviewed by a.o. Layer 1991). ChEs are present prior to the dierentiation of nerve and muscle tissues in both vertebrates and invertebrates.

Both vertebrate and invertebrate AChEs are shown to include several dierent molecular forms. In the nematode Steinerma carpocapsae, two major AChEs are involved in acetylcholine hydrolysis, of which one is more sensitive to eserine than the other (Arpa- gaus et al. 1992). Two AChEs, one being more sensitive to eserine and DFP than the other, have also been identied in the medicinal leech Hirudo medicinalis (Talesa et al.

1995). The earthworm Dendrobaena veneta (equals E. veneta) is shown to have three distinct AChEs with dierent sensitivities to the inhibitors edrophonium, procainamide and tetra(monoisopropyl)-pyrophosphortetramide (Talesa et al. 1996).

E. fetida has at least two ChEs, labelled E1 (possibly E1a and E1b) and E2 (Stenersen 1980a, Stenersen & Brekke 1992). Stenersen (1980a) suggested that E1 shares properties with AChEs in that it is highly sensitive towards selective AChE inhibitors. However, it resembles propionylcholinesterases (PrChEs) more in that it shows a higher substrate specicity towards propionylcholine than acetylcholine. This property nevertheless renders E1 more similar to AChEs than BChEs, since the rst have a very high specicity for propionylcholine compared to butyrylcholine. E2 was found to hydrolyse butyrylcholine the fastest, thereby showing a closer resemblance to BChEs. Further, E2 was stabilised by calcium ions whereas E1 was not aected by their presence. Also, the carbamate insecticide carbaryl inhibits E1 whereas it has no eect on E2. The only other species for which this is reported, is the close relative E. andrei. Thus, E. fetida and E. andrei

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tolerate high doses of carbaryl compared to other earthworm species.

Within many species, there are large variations in the ChE activity. Enzyme activity may depend on general state of health, sex and age (see Kennedy 1992). Hoel (1999) observed considerable inter-individual dierences in both unexposed and parathion-exposed E. veneta, in that within some of the control groups, the lowest-activity-individuals had approximately 50 % of the activity of the highest-activity-individuals. Scaps et al. (1997b) measured eects of cadmium and lead on ChE activity and three metabolic pathway enzymes (malate dehydrogenase, phosphoglucomutase and glutamate oxalate transferase) in E. fetida, and found the variability in ChE activity to be large compared to the other enzymes.

There is some disagreement concerning the degree of ChE inhibition necessary to cause death, but the agreement is that is must be high. For sh, the range is thought to be between 70-80 % inhibition (Coppage & Matthews 1974), for starlings, over 74 % (Grue

& Shipley 1984). Eels are shown to survive up to 64 % reduction of AChE activity over a 96-hour period (Sancho et al. 1997), and earthworms have survived over 90 % inhibition (Stenersen 1973).

2.4.2 The reaction between enzyme and inhibitor

The current model of AChE's active site depicts a 20 Å deep gorge, penetrating halfway into the structure of the enzyme (Silver 1974, Koellner et al. 2000). The gorge is narrow, and it is believed that acetylcholine and inhibitors can enter due to large-amplitude oscil- lations. OP insecticides bind to a specic serine hydroxyl group in the active site of the enzyme, to produce O,O-dialkylphosphoserine (see Kennedy 1991). The serine residue is, together with a glutamate and a histidine residue also situated in the active site, essential for the hydrolytic activity of AChE. OP insecticides are competitive inhibitors, i.e. the dialkylphosphorus group occupies the active site, preventing other substrates from enter- ing. A peripheral site which probably interacts with OP insecticides and thereby alters the potency of inhibition at the active site, is also described (Kousba et al. 2004).

Carbamate insecticides, e.g. carbaryl, also inhibit AChE by binding to the serine residue in the active site. However, OP compounds and carbamates dier in their abilities to stay bound to the enzyme, i.e. decarbamylation of AChE happens more rapidly than dephos- phorylation. Therefore, OP compounds and carbamates are referred to as irreversible and reversible inhibitors, respectively (Ecobichon 2001).

Aging Two things can happen after the OP insecticide has bound to AChE (Fig. 5), the enzyme can be dephosphorylated and thereby return to its normal, active form, or the

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Figure 5: Inhibition (a), reactivation (b) or aging (c) of a chlorpyrifos-oxon-inhibited (phosphorylated) ChE.

O,O-dialkylphosphoserine can be dealkylated to an O-alkylphosphoserine with a negative charge. This complicates the reactivation reaction, and the enzyme is said to be aged (see Aldridge 1993). The reason why aging complicates the reactivation is not clear, but it may be due to the change in charge. If the enzyme is inhibited by OP compounds and no aging has occurred, some nucleophiles, e.g. oximes, can accelerate the reactivation. Carbamates, however, can react with the oxime to produce a more potent inhibitor. The nucleophiles have no eect if AChE is aged. In a study on postnatal exposure to chlorpyrifos on developing rats (Richardson & Chambers 2005), reactivation by the oxime TMB-4 was incomplete at certain stages after the exposure. This indicates that chlorpyrifos has the ability to cause aging of AChE. Aldridge and Johnson (1971) thought that the ability to cause aging increases with the size and structure of the R-groups.

For the AChE activity to recover naturally (see section 2.4.3) in cases of irreversible binding or enzyme aging, synthesis of new enzyme is required (Mikalsen et al. 1982).

Enzyme kinetics The reaction between enzyme and inhibitor can be said to encompass three steps:

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EH+AB ←→^k⁺¹

k−1

EHAB (1)

EHAB −→^k⁺² EA+BH (2)

EA+H2O −→^k⁺³ EH+AOH (3)

Terms used: E = enzyme, AB = substrate/inhibitor, EHAB = Michaelis complex, EA = acylated (phosphorylated or carbamated) enzyme, BH and AOH = Break down products of substrate/inhibitor.

From these steps, the following equations can be derived:

[EH][AB]

[EHAB] = k−1

k₊₁ =K_d (4)

d[AE]

dt = k₊₂[EHAB]−k₊₃[AE] (5)

k_i = k₊₂

K_d (6)

The ratio between K_d and k₊₂ equals k_i (6), the bimolecular inhibition constant (see Stenersen 2004). k_i quanties the dissolution of the Michaelis complex on ground of the rate of formation of the same complex, and is often used to describe the potency of an AChE inhibitor. A high k_i, i.e. a high potency, requires a high k₊₂ and a low K_d, meaning that both the formation of the Michaelis complex and its dissolution, followed by inhibition of the enzyme, must happen rapidly.

k₊₃ (3, 5) is related to the half-life of EA (2) by the equation t¹

2 = _k^ln2

+3. Thus, the more stable EA is, the lower is k₊₃ for the reaction. For acetylcholine, EA is very unstable, giving a highk₊₃. For carbamates, the interval between inhibition and hydrolysis can last several days, and for OP insecticides, several months (Matsumura 1975). Thus, k₊₃ is lower for OP compounds than for carbamates. For some OP agents, e.g. DFP, the toxicity is mainly determined by the low k+3 value. DFP is not a very potent AChE inhibitor, but once inhibited, the enzyme will not be reactivated (k+3 ≈ 0) (see Stenersen 2004).

Aldridge and Reiner (1972) found that the half-life of inhibited rabbit erythrocyte AChEs increased with the size of the R-groups. Inhibition by chlorpyrifos renders a diethylphosphoserine in the active site of the enzyme. According to Aldridge and Reiner's ndings, this would give chlorpyrifos-inhibited rabbit erythrocyte AChE a half-life of 500 minutes and a k₊₃ of 1,4 × 10⁻³ min⁻¹ (Table 2).

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Table 2: The half-lives and k₊₃ values of some phosphorylated AChEs. The enzyme source is rabbit erythrocytes. The inhibiting group refers to the group that binds to the serine in the active site of the enzyme. Data of Aldridge and Reiner (1972).

Inhibiting group Insecticide examples Half-life k₊₃ (min⁻¹) Dimethylphosphoserine Trichlorfon, fenitrothion 80 min. 8,7 × 10⁻³

Diethylphosphoserine Chlorpyrifos, bromophos 500 min. 1,4 × 10⁻³

Dipropylphosphoserine DFP ∞ ≈ 0

Neuropathy target esterase (NTE) is another enzyme that can be inhibited and aged by OP insecticides. NTE, or homologs, is present in various organisms, including yeast, insects, nematodes and mammals (Lush et al. 1998). Aging of NTE results in organophos- phate induced delayed neuropathy (OPIDN), which is caused by a degeneration and de- myelinisation of peripheral nerves, in humans characterised by weakness and tremors in the limbs, especially the legs. Several OP insecticides can cause the OPIDN syndrome, among those reported are omethoate, trichloronate, trichlorfon, parathion, methami- dophos, fenthion and chlorpyrifos (see Ecobichon 2001).

2.4.3 Recovery after ChE inhibition

Recovery of enzyme activity can happen either by hydrolysis of the enzyme-inhibitor bond, i.e. regeneration of free enzyme, or de novo synthesis of enzyme. When the half-life of EA and the dose of inhibitor is high, recovery happens mainly by de novo synthesis (Benke & Murphy 1974).

The recommended application frequencies of OP insecticides vary according to the persistence of the insecticide. In order to assess the impacts of the application frequencies on the non-target organisms, it is of importance to have data on their ability to recover after exposure. Panda and Sahu (2004) exposed the tropical earthworm Drawida willsi to single and double recommended agricultural doses of malathion and carbofuran, and measured the AChE activity for a period of 105 days. They found that during the malathion treatment, the activity recovered fully after 45 days, while during the carbofuran treatment, the recovery time was 75 days. They suggested the interval between application of malathion and carbofuran, single or double dose, to be at least 90 and 105 days, respectively.

Mikalsen et al. (1982) conducted a study on the recovery of earthworm, chicken, guinea pig, mouse and rat ChE activities after acute soman and DFP exposure (LD50 doses). For all the animals except the earthworm (E. fetida), the recovery (new synthesis of enzyme) was well established ve days after the exposure. The whole body ChE activity of E.

fetida remained low for at least 15 days.

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A prolonged ChE depression in earthworms was also found by Dell'Omo et al. (1999).

They exposed E. fetida and E. veneta to dimethoate-contaminated soil (150-200 mg/kg) for 48 hours, and measured the ChE activity on day 1, 5, 10 and 40 after the exposure.

On day 1, the activity was approximately 10 % of the control activity, and on day 40, the activity had only increased to 35 % of the control activity.

Pradhan and Mishra (1998) exposed the tropical earthworms Drawida calebi and Oc- tochaetona surensis to a carbaryl concentration of 9 mg/kg soil for 24 hours, then they transferred the worms to clean soil to assess the enzyme recovery. After the exposure period, the mean AChE activity was 37,45 % and 17,89 % lower than the control activity for D. calebi and O. surensis, respectively. The recovery time for the AChE activity in clean soil was 6 days for D. calebi and 9 days for O. surensis.

Sancho et al. (1997) exposed the European eel Anguilla anguilla to fenitrothion in a continuous ow-through system for 4 days. The concentrations were 0,02 and 0,04 µg/L, resulting in a 44 % reduction of brain AChE activity for the lowest, and 64 % for the highest concentration. The brain AChE activity did not recover during the one week recovery period. According to Morgan et al. (1990), the recovery time depends on the concentration of pesticide. They found that brain AChE activity in Salmo salar exposed to low fenitrothion concentrations (0,004 µg/L) recovered in one week, while concentrations above 0,08 µg/L resulted in a recovery time of 6 weeks or more. Dembélé et al.

(1999) exposed the common carp Cyprinus carpio to the OP insecticide chlorfenvinphos (0,24 ng/L) and the carbamate carbofuran (3 ng/L) in a static system. The recovery time of brain AChE activity was 15 days and less than one day, respectively.

Lintern et al. (1998) administered a single dose of soman (27 µg/kg body weight, approximately the LD50) to guinea pigs, and observed the AChE activity in dierent parts of the brain for one week. The activity in the striatum, cortex and medulla-pons regions had recovered after one week, but the activity in the cerebellum, hippocampus and midbrain had not.

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2.5 DNA damage

DNA can be damaged in several ways (Fig. 6). Ionising radiation (e.g. γ radiation or X-rays), UV radiation, carcinogens and alkylating agents can cause breaks in the DNA strand, directly or via reactive oxygen species. DNA strand breaks also occur as part of the DNA repair, recombination, replication and transcription systems, with or without the inuence of exogenous contaminants (Eastman & Barry 1992, Mitchelmore & Chipman 1998).

Endogenous metabolism, e.g. the Fenton reaction, can result in the formation of reactive oxygen species (Mitchelmore & Chipman 1998, Reinecke & Reinecke 2004). These compounds, e.g. hydrogen peroxide, react readily with the DNA strand and can produce both single and double strand breaks (see Alberts et al. 2002). Ionising radiation can be another origin of reactive oxygen species, although it can also produce single or double strand breaks directly. DNA modications, e.g. depurinations, deaminations, hydrolytic attacs, methylation or the formation of covalent bonds between adjacent nitrogen bases, can be a result of ionising or UV radiation, alkylating agents, base instability or reactive oxygen species (Eastman & Barry 1992). Modied DNA can be repaired, and during this process, transient strand breaks occur. See Appendix C for details on the DNA repair system and strand breaks occuring as part of the repair process.

During meiosis, chromosomes exchange segments of DNA when they are paired and aligned in a DNA synapsis complex. Both single and double strand breaks are essential for these recombination processes. During replication, two types of enzymes cleave DNA: topoisomerase I and topoisomerase II. The rst produces single and the latter double strand breaks, both necessary for the functioning of the replication fork (see Alberts et al. 2002).

Alkylating agents represent the highest danger for the DNA molecule (see Alberts et al.

2002). These agents are electrophilic molecules who add alkyl groups to the nitrogen bases. The result is bulky structures on the strands. Alkylating agents come from a range of sources, and some are bioactivated inside the cell. The ultimate carcinogens of benzo[a]pyrene and aatoxin B1 are alkylating agents that arise as a result of CYP enzyme activities (Ecobichon 2001).

2.5.1 Lead (Pb)

During my stay in South Africa, lead was detected in concentrations of approximately 20 mg/kg in vineyard soils in the Stellenbosch area. Lead is emitted from a.o. battery manufacturers, smelters and, where tetraethyl lead still is used in fuel, from the transport

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Figure 6: An overview of DNA strand break sources. Inspired by Eastman & Barry (1992). The dotted line separates DNA cycle events from other, exogenous or endogenous, agents.

sectors. Lead emitted from combustion processes is likely bound in a lead salt compound.

Larger particles fall to the ground, smaller ones remain in the atmosphere and may fall to the ground with rain. In the soil, lead can eect soil-dwelling organisms, including earthworms.

Many metals are found to be genotoxic, e.g. nickel, chromium, cadmium and lead (Saint- Denis et al. 2001, Ecobichon 2001, Reinecke & Reinecke 2003b). Lead is found to induce the formation of reactive oxygen species and to inhibit specic enzymes possibly related to the stability of the DNA molecule (Saint-Denis et al. 2001), and has in humans been shown to induce DNA strand breaks as measured by the comet assay (Danadevi et al.

2003). There are done several studies on the eect of lead on earthworms (e.g. Booth et al. 2003, Reinecke & Reinecke 2003), showing that it can have adverse eects on various biomarkers, including lysosomal stability (as measured by the neutral red retention time assay). Also, a small number of studies (Heywood et al. 1978, Olson & Christensen 1980) have shown that heavy metals can inhibit both AChE and catalase (CAT) activities in sh.

Labrot et al. (1996) and Saint-Denis et al. (2001) have found lead to cause a signicant decrease of the AChE activity of E. andrei. However, Scaps et al. (1997b) found no eect of lead on E. fetida ChE activity, and therefore concluded that ChE activity is inappropriate as a biomarker of heavy metal pollution for this species. Little is known

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about the ability of lead to inuence ChE inhibition mediated by OP insecticides.

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3 Materials and methods

3.1 List of chemicals

Toxicants:

• Chlorpyrifos: 1) Dursban 480 EC insecticide (EF-1551), solution containing 44.53

% chlorpyrifos, 1-5 % sodium dodecyl benzene sulphonate and for the rest aromatic hydrocarbons, obtained from Dow AgroSciences, and 2) Chlorpirifos, commercial product, solution containing 480 g/L chlorpyrifos, obtained from Efekto

• Lead nitrate Pb(NO3)2 obtained from Saarchem For measuring enzyme activity and protein content:

• Tris-(hydroxymethyl)-aminomethane obtained from Angus Buers & Biochemicals and Saarchem

• Acetyltiocholine iodide, minimum 98 % (TLC), powder, 5,5'-ditiobis (2-nitrobenzoic acid) (DTNB), Ellman's reagent, and hydrated calcium chloride (CaCl2 · 2H2O) obtained from Sigma-Aldrich and Saarchem

• Bradford Reagent, bovine albumin, 97-99 % and carbaryl obtained from Sigma- Aldrich

• Acetone, >99.5 %, obtained from Merck Eurolab AS For the single cell gel electrophoresis assay:

• Triton X-100, sodium chloride (NaCl), normal melting point agarose (NMPA), low melting point agarose (LMPA), phosphate buered saline (PBS) and Trypan Blue all obtained from Saarchem

• Tris-(hydroxymethyl)-aminomethane, ethylene-di-tetra-amide (EDTA) and Di- methyl-sulphone-amide (DMSO) obtained from Saarchem and Sigma-Aldrich

• SYBR Gold nucleic acid gel stain, 10.000x concentration in DMSO, obtained from Molecular Probes

For gas chromatographic analyses:

• Standard solutions of ditalimfos and chlorpyrifos obtained from Dr. Ehrenstorfer

• Petrol ether, iso-octane, acetone and toluene obtained from SDS

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3.2 Earthworms

The species Eisenia fetida was used in all the experiments. Healthy worms, i.e. active and not too dark in colour, were chosen from the batch cultures. Dark colour indicates old age, thus old worms can easily be distinguished and avoided. The batch cultures are maintained at room temperature and are regularly fed horse dung. For all but one experiment (see section 3.5.9), clitellate individuals were used. 24 hours prior to the start of the experiments, the worms were placed on moist lter paper to empty their guts.

3.3 Soil

3.3.1 Soil characteristics

The soil used for the most of the experiments was collected at an agricultural site in Ås, Norway. This soil is previously characterised, and the composition is given in Table 3.

Table 3: The composition of the agricultural soil from Ås.

Component Particle diameter (mm) Percentage of total weight

Sand 63-2000 76,1

Silt 2-63 14,6

Clay < 2 9,3

The total organic carbon content of the soil is 1,6 % and the pH is approximately 6.2.

The moisture content was kept at 25 % (v/w) during all experiments, with one exception (a preliminary experiment see section 3.5.5).

For one of the preliminary experiments (see section 3.5.5), commercial plant soil bought at the garden centre Oasen Hageland was used. This soil consists of 95 % peat moss and 5 % sand, and holds a pH of 5.5-6.5.

The earthworms were fed horse dung. All the amounts of horse dung mentioned in this thesis refer to the wet weight of the dung. The dung was dried prior to use, and resoaked directly before added to the soil. Dry dung was scooped into a container and saturated with water, after which the wanted amount was weighed out.

For one of the experiments (see section 3.5.3), OECD soil, i.e. soil prepared according to OECD guidelines (OECD 1984), was prepared and used. This soil consists of 70 % sand (1/3 rough and 2/3 ne sand), 10 % sieved peat moss and 20 % clay (dry weight percentages). After preparing the dry components, distilled water was added, giving a