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Final Report: Evaluating the Effects of Pesticide Mixtures to Aquatic Organisms: Mechanisms of Synergistic Toxicity
EPA Grant Number: R827589E04Title: Evaluating the Effects of Pesticide Mixtures to Aquatic Organisms: Mechanisms of Synergistic Toxicity
Investigators: Lydy, Michael , Hendry, Bill , Siegfried, Blair , Zhu, Kun Yan
Institution: Wichita State University , Kansas State University , University of Nebraska at Lincoln
EPA Project Officer: Winner, Darrell
Project Period: June 1, 1999 through March 30, 2003
Project Amount: $107,011
RFA: EPSCoR (Experimental Program to Stimulate Competitive Research) (1998)
Research Category: EPSCoR (The Experimental Program to Stimulate Competitive Research)
Description:
Objective:The overall objective of this research project was to conduct a multicomponent investigation to examine mechanisms to explain the synergism between atrazine and various organophosphorus (OP) insecticides noted previously for Chironomus tentans. The specific objectives of this research project were to: (1) determine biotransformation rates for C. tentans to selected OPs by determining the ratio of parent compounds to metabolites in the body residues of midges exposed to either OP insecticides alone or animals treated with atrazine and OP insecticides; (2) examine the effects that a known inducer and inhibitor of the P450 system may have on OP toxicity; (3) determine whether atrazine induces detoxification enzymes involved with OP insecticide metabolism in C. tentans and, if so, quantify these induction levels; and (4) examine the effects of atrazine exposure on the inhibition of acetylcholinesterase (AChE) by OP insecticides.
Summary/Accomplishments (Outputs/Outcomes):Acute toxicity of selected OP insecticides (chlorpyrifos, methyl parathion, diazinon, and malathion) was determined for individual OPs and binary combinations of the OPs with atrazine to the midge larvae, C. tentans. Atrazine individually was not acutely toxic even at high concentrations (10,000 µg/L); however, the presence of atrazine at much lower concentrations (40-200 µg/L) increased the toxicity of chlorpyrifos, methyl parathion, and diazinon. Atrazine did not increase the toxicity of malathion. Possible mechanisms for the synergistic toxicity found between atrazine and chlorpyrifos were investigated, including increased uptake rate and increased biotransformation into a more toxic metabolite. Although the uptake rate was increased by more than 40 percent, the resulting increase in toxicity would be minimal as compared to the 400 percent decrease estimated to occur in EC50 values for the same atrazine exposure (200 µg/L). Body residue analysis of midges exposed in vivo to atrazine and chlorpyrifos mixtures for 96 hours indicated that a larger amount of metabolites was generated in atrazine treatments as compared to controls. Additionally, in vitro assays of microsomal proteins obtained from treated and control midges indicated that an increase in toxic metabolite (chlorpyrifos-O-analog) was generated in atrazine-treated midges. Therefore, the increase in toxicity is thought to be attributed to an increase in biotransformation rates of the OPs, resulting in more O-analog within the organism.
This study also examined the joint toxicity of atrazine and three OP insecticides
(chlorpyrifos, methyl parathion, and diazinon) exposed to Hyalella azteca and
Musca domestica. A factorial design was used to evaluate the toxicity of binary
mixtures, in which the LC1/LD1, LC5/LD5, LC15/LD15, and LC50/LD50 of each OP
was combined with atrazine concentrations of 0, 10, 40, 80, and 200 µg/L
for H. azteca and 0, 200, and 2,000 ng/mg for M. domestica. Atrazine concentrations
(
40 µg/L), in combination with each OP, caused a significant increase
in toxicity to H. azteca compared to the OPs dosed individually. AChE activity
also was examined for the individual OPs with and without atrazine treatment.
Atrazine, in combination with each OP, resulted in a significant decrease in
AChE activity compared to the OPs dosed individually. In addition, H. azteca that were pretreated with atrazine ( 40 µg/L) were much more sensitive
to the OP insecticides compared to H. azteca that were not pretreated with
atrazine before being tested. Topical exposure to atrazine concentrations did
not significantly increase OP toxicity to M. domestica. The results of this
study indicate the potential for increased toxicity in organisms exposed to
environmental mixtures.
A standard Organisation for Economic Cooperation and Development (OECD) filter paper test was used to assess the acute toxicity of chlorpyrifos atrazine, cyanazine, and simazine to the earthworm, Eisenia foetida. Acute toxicity of chlorpyrifos also was determined in conjunction with the three-triazine herbicides. Surprisingly, atrazine and cyanazine caused lethality to E. foetida at concentrations lower than that for chlorpyrifos. Atrazine and cyanazine also significantly increased the toxicity of chlorpyrifos. On the other hand, simazine caused no toxicity to the worms and did not affect chlorpyrifos toxicity in binary mixture experiments. Possible mechanisms for the greater-than-additive toxicity for the binary combinations of atrazine and cyanazine with chlorpyrifos were investigated, including changes in uptake and biotransformation rates of chlorpyrifos in the presence of the triazine herbicides. Uptake of chlorpyrifos decreased slightly when atrazine was present in the system, eliminating increased uptake as a possible explanation for the increased toxicity. Body residue analysis of worms indicated increased metabolite formation, suggesting that the greater than additive response was attributed to increased biotransformation to more toxic oxon metabolites. Finally, the OECD filter paper test appears to be a good screening tool for assessing the effects of pesticide mixtures to E. foetida.
We also evaluated the toxicities of two triazine herbicides (atrazine and cyanazine) and an OP insecticide (chlorpyrifos) individually and with each herbicide in binary combination with chlorpyrifos using fourth instar larvae of the aquatic midge, C. tentans. Chlorpyrifos at 0.25 µg/L resulted in an effect in less than 10 percent of midges in 48-hour acute toxicity bioassays. Neither atrazine nor cyanazine alone at relatively high concentrations (up to 1,000 µg/L) caused significant acute toxicity to C. tentans. Atrazine and cyanazine, however, caused significant synergistic effects on the toxicity of chlorpyrifos when midges were exposed to mixtures of atrazine or cyanazine (10, 100, 1,000 µg/L) with chlorpyrifos (0.25 µg/L). At fixed atrazine and cyanazine concentrations (200 µg/L), toxicity of chlorpyrifos was enhanced by 1.8-fold and 2.2-fold, respectively, at the EC50 levels. Although atrazine and cyanazine are not effective inhibitors of AChE in vitro, the synergism of the two triazine herbicides with chlorpyrifos was associated with increased in vivo inhibition of AChE in midges. We observed a positive correlation between the degree of inhibition of AChE and the concentration of atrazine or cyanazine in the presence of a fixed concentration of chlorpyrifos. It is possible that these herbicides may affect cytochrome P450 enzymes to confer synergistic effects on the toxicity of chlorpyrifos.
We also evaluated toxicities of two chloroacetanilide herbicides (alachlor and metolachlor) and chlorpyrifos individually and with each herbicide in binary mixture with chlorpyrifos. Alachlor alone at concentrations up to 1,000 µg/L did not exhibit significant toxicity, whereas metolachlor at the same concentration resulted in an effect in 58 percent of midges in a 72-hour acute toxicity bioassay. Both alachlor and metolachlor at 1,000 µg/L showed significant synergistic effects on the toxicity of chlorpyrifos (0.25 µg/L). At a fixed concentration of 200 µg/L, alachlor did not show a significant effect on the toxicity of chlorpyrifos, whereas metolachlor enhanced the toxicity of chlorpyrifos by 1.5 fold. Neither alachlor nor metolachlor significantly enhanced the inhibition of AChE by chlorpyrifos. Therefore, the mechanism of the observed synergistic effects of alachlor and metolachlor on the toxicity of chlorpyrifos might be different from that of the triazine herbicides, in which the synergisms were associated with increased inhibition of AChE in the midges. Our results suggest that the coexistence of chlorpyrifos and alachlor or metolachlor in surface waters also may pose great risks to the midge larvae.
In a separate study, AChE activity was determined for midge larvae (C. tentans) exposed to either OP insecticides alone or OP insecticides in binary combination with atrazine (200 µg/L). Although atrazine by itself did not reduce the level of AChE activity, atrazine in combination with chlorpyrifos significantly decreased AChE activity as compared to chlorpyrifos only treatments. Although similar trends existed for malathion and methyl parathion, differences were not statistically significant. These results match previously published toxicity data where atrazine, although not acutely toxic even at much higher levels, decreased EC50 values for chlorpyrifos by a magnitude of 4, decreased methyl parathion values by a magnitude of 2, and did not decrease values for malathion.
Cytochrome P450 activity was characterized in third instar larvae of the aquatic midge, C. tentans. Optimal in vitro assay conditions with two different substrates (methoxy resorufin, ethoxy resorufin, and aldrin) were determined. The activity was concentrated in the microsomal fraction of whole body homogenates and was nicotinamide adenine dinucleotide phosphate dependent. Comparisons of control and atrazine-exposed midges indicated increased cytochrome P450 activity as a result of atrazine exposure. This activity was determined by difference spectroscopy and by oxidative transformation of the substrates. The molecular weight of this protein was similar in size (45 kDa) to cytochrome P450 enzymes reported for other insects. Immunochemical studies using a Drosophila melanogaster anti-P450 polyclonal antiserum further support the cytochrome P450 nature of this inducible protein. Finally, the cytochrome P450 family 4 (CYP4) gene expression in C. tentans was analyzed in third instar larvae exposed to atrazine using degenerate family 4 (CYP4) primers to amplify P450 fragments and Northern analysis to assess mRNA transcript abundance. These results indicate increased CYP4 transcript abundance in midges exposed to atrazine.
To understand the molecular basis of the observed synergistic effect of a representative triazine herbicide (i.e., atrazine) on the toxicity of chlorpyrifos, we started to use a new molecular technique known as restriction fragment differential display polymerase chain reaction to directly compare the gene-expression profiles of two mRNA samples that were isolated simultaneously from the atrazine-treated and untreated midge larvae. Our initial study has revealed 12 genes either over-expressed or under-expressed significantly in the atrazine-treated midges. The overexpressed genes putatively include cytochrome c oxidase polypeptide III, ecdysone-induced protein, cell-to-cell movement protein, elongation factor 1-gamma type 1, glucokinase, and voltage-dependent P/Q-type calcium channel-alpha; the underexpressed genes include myosin VIIa, basic endochitinase, ephrin-B2 precursor, glycogen phosphorylase, protocadherin alpha-10 precursor, and an unidentified protein. The ultimate objective of this part of the project is to create complete differential gene expression profiles, and identify and characterize the genes responsible for the synergistic toxicity in atrazine-treated midges.
Journal Articles on this Report: 5 Displayed | Download in RIS Format
| Other project views: | All 37 publications | 5 publications in selected types | All 5 journal articles |
| Type | Citation | ||
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Anderson TD, Lydy MJ. Increased toxicity to invertebrates associated with a mixture of atrazine and organophosphate insecticides. Environmental Toxicology And Chemistry. 2002;21(7):1507-1514. |
R827589E04 (Final) |
not available |
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Belden JB, Lydy MJ. Impact of atrazine on organophosphate insecticide toxicity. Environmental Toxicology and Chemistry 2000;19(9):2266-2274. |
R827589E04 (Final) |
not available |
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Belden JB, Lydy MJ. Effects of atrazine on acetylcholinesterase activity in midges (Chironomus tentans) exposed to organophosphorous insecticides. Chemosphere 2001;44(8):1685-1689. |
R827589E04 (Final) |
not available |
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Jin-Clark Y, Lydy MJ, Zhu KY. Effects of atrazine and cyanazine on chlorpyrifos toxicity in Chironomus tentans (Diptera: Chironomidae). Environmental Toxicology and Chemistry 2002;21(3):598-603. |
R827589E04 (Final) |
not available |
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Miota F, Siegfried BD, Scharf ME, Lydy MJ. Atrazine induction of cytochrome P450 in Chironomus tentans larvae. Chemosphere 2000;40(3):285-291. |
R827589E04 (Final) |
not available |
pesticides, atrazine, mixture toxicity, synergistic response, Chironomus tentans, organophosphorus, OP, cytochrome P450, CYP4. , Ecosystem Protection/Environmental Exposure & Risk, Scientific Discipline, RFA, Aquatic Ecosystems & Estuarine Research, Analytical Chemistry, Ecological Risk Assessment, Aquatic Ecosystem, Environmental Chemistry, toxicity studies, environmental policy impact, contaminant input, contaminant exposure, synergistic toxicity, aquatic ecosystems, bioassessment, ecosystem stress, ecosystem monitoring
Progress and Final Reports:
Original Abstract