Azole Fungicides as Synergists in the Aquatic Environment: An Assessment of how the Magnitude of Synergistic Interactions Depends on Bioaccessibility, Azole Concentration and Time

TY - BOOK

T1 - Azole Fungicides as Synergists in the Aquatic Environment

T2 - An Assessment of how the Magnitude of Synergistic Interactions Depends on Bioaccessibility, Azole Concentration and Time

AU - Bjergager, Maj-Britt Andersen

PY - 2015

Y1 - 2015

N2 - Despite contaminants occurring as mixtures in the aquatic environment, aquatic risk assessment is basedon single compounds. Mixture effects are estimated by means of additive models, most often able ofpredicting mixture effects within a factor two of observed effects. While this may be a reasonableapproach for non-interacting contaminants, synergizing compounds, enhancing the toxicity of others, maycompromise the predictive ability of the models. Though only rarely occurring, the phenomenon ofsynergism is of great concern, making apparently low and non-toxic contaminant concentrations apotential hazard.This PhD thesis evaluates the role of the so called azole fungicides as synergists in the aquaticenvironment through an assessment of the effect of sorption, time and azole concentration on theoccurrence and magnitude of synergistic interactions with pyrethroid insecticides towards the aquaticcrustacean Daphnia magna in both laboratory experiments and natural-like environments. In the PhDthesis, synergy is defined as happening in mixtures where either EC50 values decrease more than two-foldbelow the prediction by the model of Concentration Addition (horizontal assessment of synergy) or wherethe frequency of immobile animals increase more than two-fold above the prediction by the model ofIndependent Action (vertical assessment of synergy). The pesticides investigated are: the three azolesprochloraz, propiconazole and epoxiconazole and the three pyrethroids bifenthrin, esfenvalerate andalpha-cypermethrin. Though differing in physicochemical properties and toxic potential, the compoundsare comparable within their chemical class and the results obtained can most likely be extrapolatedwithin the two pesticide groups.Azoles and pyrethroids enter surface waters through particle facilitated transport in stormwater runoff ordrain water and in the aquatic environment, the pesticides mainly occur in sorbed form. Sorption istraditionally considered to limit bioaccessibility and toxicity of hydrophobic compounds, hence,synergistic interactions may be limited in natural environments compared to laboratory studies. It washypothesized that the toxicity of sorbed compounds depends on the quality of the sorbent and thestrength of the sorption. This was tested using a passive dosing and sampling system and six differentsuspended and/or dissolved sorbents (montmorillonite, goethite, the green algae Pseudokirchneriellasubcapitata, yeast (Saccharomyces cerevisiae), Aldrich Humic Acid (AHA) and soot). At the tested sorbentconcentrations of 25 mg L-1, yeast and soot significantly increased 48 h aquatic equilibrium concentrationsfor propiconazole while AHA showed significantly increased 48 h aquatic equilibrium concentrations forbifenthrin. Subsequent experiments indirectly assessed sorption strength by measuring the bioaccessiblefraction of the sorbed pesticides using poly(dimethylsiloxane) (PDMS) rods. Bifenthrin bioaccessibilitywas significantly reduced in an algae suspension of approximately 17,000 cells mL-1 while a tendency ofreduced bioaccessibility was observed in a suspension of AHA. Dry yeast and soot significantly reducedpropiconazole bioaccessibility. In general, however, reductions of bioaccessibility were low, reaching amaximum of 15-20%, and extractable fractions of sorbed pesticides were similar to those of freelydissolved pesticides. Acute toxicity of bifenthrin alone and in combination with propiconazole to theaquatic crustacean Daphnia magna was assessed in suspensions of AHA, P. subcapitata andmontmorillonite. All sorbents decreased acute toxicity of both bifenthrin and the mixture compared topure aquatic media. Reduced toxicity may, however, not exclusively be caused by decreased bifenthrinbioaccessibility but also at least partly by faster dissipation of bifenthrin from the aquatic phase in thepresence of sorbents. In all sorbent suspensions, synergistic interactions were of greater magnitude thanin pure aquatic media. The results indicate that azoles and pyrethroids transported to surface waterssorbed to suspended matter at the tested concentrations may be more bioaccessible to pelagic organismsthan previously considered, may increase exposure concentrations via additional exposure routes and caninteract synergistically.In aquatic microcosms, the 8–14 fold synergy observed after 2 and 7 days of exposure of D. magnaneonates to mixtures of 90 μg L−1 prochloraz and 0.17-0.83 μg L−1 esfenvalerate was equivalent to orgreater than the 3–7 fold synergy found in 2-days laboratory tests without sorbents. Incubating newneonates in situ 7 and 14 days after pesticide addition gave EC50 values of 0.012±0.001 and <0.005 μg L−1esfenvalerate in the mixture treatments, based on measured water column concentrations. As thedetection limit of 0.005 μg L−1 is more than ten-fold lower than the lowest esfenvalerate concentrationobserved to cause ecologically significant effects across other long term studies under field-likeconditions, results indicate that also on a longer time scale, prochloraz synergizes the effect ofesfenvalerate under field-like conditions in the microcosms.Lower threshold concentrations for synergistic effects were assessed using three different experimentalset-ups focusing on the effect of test duration and the way of dosing on the capability to capturesynergistic interactions between the alpha-cypermethrin and one of the three azoles measured on D.magna immobilization. Similar lower threshold concentrations were observed in all three tests anddecreased with increasing test duration from 9.8±4.9 μg L-1, 145.4±30.5 μg L-1 and 249.7±83.4 μg L-1 instandard 48 h toxicity tests for prochloraz, propiconazole and epoxiconazole, respectively, to 5.7±1.5 μg L-1, 49.6±8.6 μg L-1 and 40.2±13.8 μg L-1, respectively, in the 14-days tests. Testing synergy in relation to themodel of Concentration Addition provided the most conservative values. The threshold values for thevertical assessments in tests where the assessment approaches could be compared were in general 1.2 to4.7 fold higher than the horizontal assessments. No synergy was observed on neonate number and sizewithin the 14 days tested.In conclusion, the results presented in this PhD thesis show that synergistic interactions found in thelaboratory also take place under field like conditions at quantitatively similar levels, and that the effectlasts for several weeks. Also, it has been shown that the magnitude of synergistic interactions increasedwith time and the results emphasize the importance of test duration when assessing synergy. Azoleconcentrations within the typically monitored range of up to 0.5 μg L-1 are not likely to cause severesynergy concerning D. magna immobilization, though effects in other more sensitive species or withrespect to more sensitive endpoints than immobilization might occur. In addition, synergizing potentialappears to be a general feature across the group of azoles, increasing the risk of cumulative effects ofsynergistically acting azoles in the environment. As a consequence of sorbents acting as vectors andpotential accumulation within exposed organisms, aquatic organisms may experience larger exposureconcentrations, leading to greater synergistic effects, than expected based on single azole concentrationsmeasured in the aquatic environment. Altogether, the thesis recommends performing risk assessment of

AB - Despite contaminants occurring as mixtures in the aquatic environment, aquatic risk assessment is basedon single compounds. Mixture effects are estimated by means of additive models, most often able ofpredicting mixture effects within a factor two of observed effects. While this may be a reasonableapproach for non-interacting contaminants, synergizing compounds, enhancing the toxicity of others, maycompromise the predictive ability of the models. Though only rarely occurring, the phenomenon ofsynergism is of great concern, making apparently low and non-toxic contaminant concentrations apotential hazard.This PhD thesis evaluates the role of the so called azole fungicides as synergists in the aquaticenvironment through an assessment of the effect of sorption, time and azole concentration on theoccurrence and magnitude of synergistic interactions with pyrethroid insecticides towards the aquaticcrustacean Daphnia magna in both laboratory experiments and natural-like environments. In the PhDthesis, synergy is defined as happening in mixtures where either EC50 values decrease more than two-foldbelow the prediction by the model of Concentration Addition (horizontal assessment of synergy) or wherethe frequency of immobile animals increase more than two-fold above the prediction by the model ofIndependent Action (vertical assessment of synergy). The pesticides investigated are: the three azolesprochloraz, propiconazole and epoxiconazole and the three pyrethroids bifenthrin, esfenvalerate andalpha-cypermethrin. Though differing in physicochemical properties and toxic potential, the compoundsare comparable within their chemical class and the results obtained can most likely be extrapolatedwithin the two pesticide groups.Azoles and pyrethroids enter surface waters through particle facilitated transport in stormwater runoff ordrain water and in the aquatic environment, the pesticides mainly occur in sorbed form. Sorption istraditionally considered to limit bioaccessibility and toxicity of hydrophobic compounds, hence,synergistic interactions may be limited in natural environments compared to laboratory studies. It washypothesized that the toxicity of sorbed compounds depends on the quality of the sorbent and thestrength of the sorption. This was tested using a passive dosing and sampling system and six differentsuspended and/or dissolved sorbents (montmorillonite, goethite, the green algae Pseudokirchneriellasubcapitata, yeast (Saccharomyces cerevisiae), Aldrich Humic Acid (AHA) and soot). At the tested sorbentconcentrations of 25 mg L-1, yeast and soot significantly increased 48 h aquatic equilibrium concentrationsfor propiconazole while AHA showed significantly increased 48 h aquatic equilibrium concentrations forbifenthrin. Subsequent experiments indirectly assessed sorption strength by measuring the bioaccessiblefraction of the sorbed pesticides using poly(dimethylsiloxane) (PDMS) rods. Bifenthrin bioaccessibilitywas significantly reduced in an algae suspension of approximately 17,000 cells mL-1 while a tendency ofreduced bioaccessibility was observed in a suspension of AHA. Dry yeast and soot significantly reducedpropiconazole bioaccessibility. In general, however, reductions of bioaccessibility were low, reaching amaximum of 15-20%, and extractable fractions of sorbed pesticides were similar to those of freelydissolved pesticides. Acute toxicity of bifenthrin alone and in combination with propiconazole to theaquatic crustacean Daphnia magna was assessed in suspensions of AHA, P. subcapitata andmontmorillonite. All sorbents decreased acute toxicity of both bifenthrin and the mixture compared topure aquatic media. Reduced toxicity may, however, not exclusively be caused by decreased bifenthrinbioaccessibility but also at least partly by faster dissipation of bifenthrin from the aquatic phase in thepresence of sorbents. In all sorbent suspensions, synergistic interactions were of greater magnitude thanin pure aquatic media. The results indicate that azoles and pyrethroids transported to surface waterssorbed to suspended matter at the tested concentrations may be more bioaccessible to pelagic organismsthan previously considered, may increase exposure concentrations via additional exposure routes and caninteract synergistically.In aquatic microcosms, the 8–14 fold synergy observed after 2 and 7 days of exposure of D. magnaneonates to mixtures of 90 μg L−1 prochloraz and 0.17-0.83 μg L−1 esfenvalerate was equivalent to orgreater than the 3–7 fold synergy found in 2-days laboratory tests without sorbents. Incubating newneonates in situ 7 and 14 days after pesticide addition gave EC50 values of 0.012±0.001 and <0.005 μg L−1esfenvalerate in the mixture treatments, based on measured water column concentrations. As thedetection limit of 0.005 μg L−1 is more than ten-fold lower than the lowest esfenvalerate concentrationobserved to cause ecologically significant effects across other long term studies under field-likeconditions, results indicate that also on a longer time scale, prochloraz synergizes the effect ofesfenvalerate under field-like conditions in the microcosms.Lower threshold concentrations for synergistic effects were assessed using three different experimentalset-ups focusing on the effect of test duration and the way of dosing on the capability to capturesynergistic interactions between the alpha-cypermethrin and one of the three azoles measured on D.magna immobilization. Similar lower threshold concentrations were observed in all three tests anddecreased with increasing test duration from 9.8±4.9 μg L-1, 145.4±30.5 μg L-1 and 249.7±83.4 μg L-1 instandard 48 h toxicity tests for prochloraz, propiconazole and epoxiconazole, respectively, to 5.7±1.5 μg L-1, 49.6±8.6 μg L-1 and 40.2±13.8 μg L-1, respectively, in the 14-days tests. Testing synergy in relation to themodel of Concentration Addition provided the most conservative values. The threshold values for thevertical assessments in tests where the assessment approaches could be compared were in general 1.2 to4.7 fold higher than the horizontal assessments. No synergy was observed on neonate number and sizewithin the 14 days tested.In conclusion, the results presented in this PhD thesis show that synergistic interactions found in thelaboratory also take place under field like conditions at quantitatively similar levels, and that the effectlasts for several weeks. Also, it has been shown that the magnitude of synergistic interactions increasedwith time and the results emphasize the importance of test duration when assessing synergy. Azoleconcentrations within the typically monitored range of up to 0.5 μg L-1 are not likely to cause severesynergy concerning D. magna immobilization, though effects in other more sensitive species or withrespect to more sensitive endpoints than immobilization might occur. In addition, synergizing potentialappears to be a general feature across the group of azoles, increasing the risk of cumulative effects ofsynergistically acting azoles in the environment. As a consequence of sorbents acting as vectors andpotential accumulation within exposed organisms, aquatic organisms may experience larger exposureconcentrations, leading to greater synergistic effects, than expected based on single azole concentrationsmeasured in the aquatic environment. Altogether, the thesis recommends performing risk assessment of

UR - https://soeg.kb.dk/permalink/45KBDK_KGL/fbp0ps/alma99122228654805763

M3 - Ph.D. thesis

BT - Azole Fungicides as Synergists in the Aquatic Environment

PB - Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen

ER -