The effect of organophosphate and carbamate pesticides on the Australian Freshwater crayfish Cherax destructor

Pham, B 2018, The effect of organophosphate and carbamate pesticides on the Australian Freshwater crayfish Cherax destructor, Doctor of Philosophy (PhD), Science, RMIT University.


Document type: Thesis
Collection: Theses

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Title The effect of organophosphate and carbamate pesticides on the Australian Freshwater crayfish Cherax destructor
Author(s) Pham, B
Year 2018
Abstract Australian freshwater environments and the aquatic organisms that live within them, including the yabby, Cherax destructor Clark, 1936, may be threatened by the presence of anthropogenic pollutants, such as insecticides. The effect of such stressors may be magnified by climate change. The application of insecticides to control crop pests close to aquatic environments increases the likelihood that the said insecticides will be transported to the water and cause nonlethal effects to aquatic species. Organophosphate (OP) and carbamate (CB) chemicals are the most widely used insecticides in Australia and throughout the world. These insecticides have been detected in aquatic ecosystems and have been the subject of many reports and studies, though none of them concentrating specifically on the effects of these insecticides and their mixtures on C. destructor. In addition, there are no studies currently available on the nonlethal effects of these insecticides on this species under conditions of thermal stress, predicted as a possible effect of climate change. The current study was initiated to determine the nonlethal effects of these insecticides, alone and as mixtures, on the Australian native freshwater crayfish species C. destructor, using two OP and one CB insecticides, and to investigate the effect of temperature as an additional stressor with the insecticides.

Chlorpyrifos (CPF), malathion (MAL) and methomyl (METH) were chosen for evaluation in the present study as they are insecticides of major importance in the control of crop pests. The residues of these chemicals have been detected in the natural environment and have been reported to be toxic to aquatic organisms. The test species, the yabby (C. destructor), is a native decapod crustacean (crayfish) that is widely distributed throughout Australia. It has been proposed as a bio-monitor of trace metal contamination, and has a number of characteristics that make it suitable as a bio-indicator for pesticide contamination.

Juveniles of C. destructor were exposed to CPF, MAL and METH for 96 hours at different environmentally relevant concentrations (0.5, 2 and 5 µg.L-1). The toxicity of these insecticides to C. destructor was assessed by determining the activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in the nervous system, the activity of glutathione S-transferase (GST) in the hepatoponcreas, and the activity of Na+/K+ ATPase in their gills. Recovery of these enzyme activities were also investigated after transferring exposed C. destructor to clean water for 14 days. Yabbies exposed to all three insecticides at 2 and 5 µg.L-1 exhibited significant AChE, BChE, GST and Na+/K+ ATPase inhibition, although the activities of these enzymes recovered significantly after 14 days. These findings demonstrate that these enzyme inhibitions are sensitive biomarkers for CPF, MAL and METH exposure in C. destructor.

In an attempt to replicate field conditions where insecticides are commonly detected in aquatic ecosystems and predominantly occur as mixtures of varying complexity, C. destructor were exposed to different binary and ternary mixtures of CPF, MAL and METH insecticides. The concentration addition approach (CA) was used to estimate mixture toxicity based on the concentration causing 25% enzyme inhibition (EC25). When estimated using the CA model, the observed inhibition of AChE activity caused by binary mixtures of CPF plus MAL was both greater than additive and less than additive depending on the relative ratios of these chemicals in the mixtures. The observed inhibition of AChE activity in the CPF plus METH and MAL plus METH, was greater than additive, less than additive and synergistic, again depending on the relative ratios of these chemicals in the mixture. In ternary mixtures, all combinations of CPF, MAL and METH conformed to less than additive and antagonistic. The effect of mixtures of these three insecticides on C. destructor has not previously been assessed, and the data suggests that individual chemical risk assessments are likely to incorrectly estimate the effect of these insecticides on C. destructor in the aquatic environment where combinations of such chemicals occur.

Aquatic species are exposed to multiple stressors in their environment, in which the toxicity of pesticides and water temperature are some of the most important factors to be considered within aquatic systems. Therefore, in the present study, insecticide exposures under thermal stress were also applied simultaneously on juvenile C. destructor. Combined effects were tested with 2 µg.L-1 CPF, MAL, METH and their mixtures at 20°C and 25°C. Interaction between the two factors was found for all AChE, BChE, GST and Na+/K+ATPase activities. CPF, MAL, METH and mixture of MAL-METH produced AChE inhibition at 25°C higher than that of 20°C. CPF, MAL, and mixture of MAL-METH, CPF-MAL-METH produced BChE inhibition at 25°C, higher than that of 20°C. CPF, MAL, and mixture of CPF-METH, CPF-MAL- METH produced Na+/K+ATPase inhibition at 25°C, higher than that of 20°C. CPF, MAL, and mixture of CPF-METH, MAL-METH, CPF-MAL- METH produced GST inhibition at 25°C, higher than that of 20°C. These findings highlight how environmental stress can increase the relative toxicity of OP and CB pesticides. These findings also clearly demonstrate the importance of pollution reduction strategies to enhance ongoing yabbies conservation and recovery efforts. These findings can also be used to inform pollution reduction strategies in an effort to enhance ongoing conservation and recovery efforts directed toward yabbies, but also potentially for a range of aquatic organisms.

The gas chromatography mass spectrometry (GC-MS) based metabolomics approach is a technique that can be used to investigate the metabolic profiles of an organism; therefore, in the present study, we also used this approach to quantify metabolites identified in the yabby muscle exposed to CPF, MAL, METH insecticides and their mixtures. These insecticides disrupted amino acids, organic acids, fatty acids and sugar metabolism in the yabby muscle. Fifteen biomarkers of effect in yabby muscle tissue were identified by GC-MS, in which alanine, glycine and sucrose were reliable biomarkers for exposure to CPF, methionine, glycine, and sucrose were the unique biomarkers for exposure to MAL, and alanine and sucrose were two reliable biomarkers for exposure to METH. Proline and stigmasterol were the unique biomarkers for exposure to mixtures of CPF-METH. Tocopherol and campesterol were the unique biomarkers for exposure to mixtures of MAL-METH. These findings suggest that the metabolomics analysis is more sensitive than regular clinical observation and pathological examination for detecting the toxicity of used insecticides, even at low levels. These results also identified unique biomarkers in the metabolome of yabbies for exposure to to CPF, MAL and METH exposure, which may provide new insights into the mechanism of their toxicity.

This study presents novel information on the response to chemicals and thermal stress in C. destructor, an Australian native species. It identified a strong and prolonged effect of insecticide exposure and thermal stress on the AChE, BChE, GST and Na+/K+ATPase activities in C. destructor, and provides evidence for the use of these biomarkers during CPF, MAL, METH exposure and thermal stress. These findings in the present study indicate that C. destructor is sensitive to CPF, MAL, METH insecticides and their mixtures. Combined effects of insecticides and thermal stress are generally considered to be worse than individual stress. This study also demonstrates the potential of the metabolomics approach in investigating nonlethal effect of insecticides on non-target species in aquatic ecosystems.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Science
Subjects Environmental Monitoring
Environmental Management
Environmental Impact Assessment
Keyword(s) Cherax destructor
Chlorpyrifos
Malathion
Methomyl
Acetylcholinesterase
Butyrylcholinesterase
Glutathione S-transferase
Na+/K+ATPase
Metabolomics
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Created: Wed, 28 Nov 2018, 10:22:34 EST by Anna Koh
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