Acute Aquatic Toxicity of Metofluthrin Metabolites in the Environment

J. Pestic. Sci. 38(4), 173–180 (2013) DOI: 10.1584/jpestics.D13-009

Original Article

Acute aquatic toxicity of metofluthrin metabolites in the environment

Mitsugu Miyamoto,* Akiko Fujiwara, Hitoshi Tanaka and Toshiyuki Katagi

Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 4–2–1 Takatsukasa, Takarazuka, Hyogo 665–8555, Japan (Received February 14, 2013; Accepted June 29, 2013)

Acute aquatic toxicity of eight major metabolites of the pyrethroid insecticide metofluthrin, potentially formed via oxidation and ester cleavage in the environment, was examined using three representative species, fathead minnow (Pimephales promelas), Daphnia magna and green alga (Pseudokirchneriella subcapitata). All metabolites showed a wide range of toxicity but were more than a hundredfold and tenfold less toxic than metofluthrin to pyrethroid-sensitive (fish and daphnid) and -insensitive (algal)

taxa, respectively; 0.44 to >120 mg/L (fish 96-hr LC50), 6.3 to >120 mg/L(daphnid 48-hr EC50), and 2.6 to >110 mg/L (algal

96-hr EyC50). The structural modification via ester cleavage and/or oxidation was found to significantly control the acute aquatic toxicity of the metabolites. The decreased lipophilicity in the metabolites generally resulted in much less acute toxicity, the extent of which was dependent on an introduced functional group such as formyl as a toxicophore and carboxyl causing a higher acidity. © Pesticide Science Society of Japan Keywords: fathead minnow, Daphnia magna, alga, metofluthrin, ecotoxicity, metabolite.

ester linkage, as reported for 4′-OH-bifenthrin.10) Introduction Metofluthrin (I) (SumiOne®, Eminence®) [2,3,5,6- Pyrethroid is one of the most important chemical classes of in- tetrafluoro-4-(methoxymethyl)benzyl(1 R,3R)-2,2-dimethyl-3- secticide for both agricultural and public hygiene uses; it is not ((1EZ)-prop-1-enyl)cyclopropanecarboxylate] is a new pyre- only exhibits an excellent biological activity but also readily de- throid insecticide with an extremely high knockdown activ- grades biotically and abiotically in the environment.1–4) It can ity, especially against mosquitoes.11,12) Similarly to other pyre- be well understood from its mode of action that pyrethroid is throids, I exhibits high acute toxicity to common carp (Cyprinus highly toxic to fish and arthropods (crustaceans and insects) in carpio) and rainbow trout (Oncorhynchus mykiss) with 96-hr general, but extremely less toxic to other invertebrates such as LC50 of 0.0012 and 0.00306 mg/L, respectively, and to Daphnia 5) mollusk and aquatic plants including algae. Rapid degradation magna with 48-hr EC50 of 0.0047 mg/L, while it is much less of pyrethroid in the environment makes its aquatic risks under toxic to green algae (Pseudokirchneriella subcapitata) with 72-hr 12–14) practical uses acceptable and manageable, while ecological risk EbC50 of 0.16 mg/L. Considering the typical use pattern of I, assessment of corresponding metabolites is very limited at the which is distributed into the air by vaporization as a mosquito present. In general, metabolic transformation of a pesticide ei- adulticide, its direct emission into the aquatic environment is ther destroys a toxicophore structure or introduces a new func- most unlikely. Even if emitted into the aquatic environment, I tional group, generally leading to increased molecular hydro- degrades via either hydrolysis to the corresponding acid and al- philicity, and the changes in the mode of action or the uptake cohol moieties or sunlight photolysis with successive oxidation potential result in less toxicity of metabolites than the parent.6) at the prop-1-enyl side chain with no remarkable change in an Most metabolites formed via ester cleavage show far less toxicity E/Z isomer ratio because of similar degradation rates and no 15) (LC50, EC50) to sensitive taxa by 2–6 orders of magnitude than isomerization. Furthermore, when I is distributed in the ter- do corresponding pyrethroids,7–9) while limited information is restrial environment, aerobic microbes in soil rapidly metabolize available on the aquatic toxicity of metabolites having an intact it via ester cleavage followed by successive oxidation16) or it is photodegraded by sunlight on the soil surface.17) Eight major * To whom correspondence should be addressed. metabolites (II–IX) detected through environmental fate stud- E-mail: [email protected] ies, as shown in Fig. 1, have unique chemical structures different Published online September 9, 2013 from other pyrethroids. © Pesticide Science Society of Japan The objective of this study has been to determine basic 174 M. Miyamoto et al. Journal of Pesticide Science

Fig. 1. Structures of metofluthrin metabolites in the environment with their route of formation. aquatic ecotoxicological profiles of the major metabolites identi- on a standard commercial fish food (TetraMin®, Tetra Werke, fied in environmental fate studies of metofluthrin, using three Germany), and juveniles (total length, 1.5–2.9 cm) were used standard aquatic species (fish, daphnid, algal), in relation to the for bioassays. D. magna cultures, held at ca. 20°C with a 16-hr structural modification from metofluthrin. daylight photoperiod, regularly fed on commercially available chlorella (Chlorella V12, Chlorella Industry Co., Ltd., Tokyo, Materials and Methods Japan), and <24-hr-old neonates were used. Elendt M4 medium 1. Chemicals referenced in OECD guideline 20218) or ASTM Hard Reconsti- The metabolites of I (II–VI and VIII) were prepared as test tuted Fresh Water19) was used as culture water. Pure water with substances in our laboratory according to the reported meth- an electrical resistivity of more than 17 MΩ cm, provided by a ods.11,15–17) The chemical purity of each metabolite was deter- Barnstead E-pure D4643 (4Module E-pure, Barnstead Ther- mined by HPLC to be greater than 98%. VII (99.4%) and IX molyne Co., Iowa, USA), was used to prepare the culture media. (>97%) were purchased from Showa Denko K.K. (Tokyo, Japan) Fathead minnows and D. magna were not fed during the bioas- and Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan), respectively, says. Precultures of P. subcapitata were prepared prior to each and used without further purification. All other chemicals were bioassay from stocks in a refrigerator and incubated at 23–26°C of a reagent grade and purchased from commercial suppliers in the medium referenced in OECD guideline 20118) under con- unless otherwise noted. tinuous shaking (Multishaker MMS-310, Tokyo Rikakikai Co., Ltd.) and illumination (fluorescent bulbs). 2. Test organisms Fathead minnow (P. promelas), D. magna, and the unicellular 3. Bioassays green algal species, P. subcapitata (ATCC22662), were chosen The acute toxicity of each metabolite on three species was ex- as test species because of recommendations in international test amined basically in accordance with the corresponding inter- guidelines, such as those of the OECD (Organisation for Eco- national guidelines of the OECD.18) The same types of media for nomic Co-operation and Development).18) Parental organisms culturing were used for the bioassays. The aqueous solution of a were originally obtained from the National Institute for Envi- metabolite (V–IX) was prepared at a desired concentration by ronmental Studies (Ibaraki, Japan) for fathead minnows and its direct dissolution, followed by serial dilution with the media. from Sumika Technoservice Corporation (Hyogo, Japan) for Otherwise, a metabolite (II–IV) was first dissolved inN ,N- D. magna and P. subcapitata. The cultures of fathead minnows dimethylformamide (DMF), followed by dilution with the media were held in tap water dechlorinated with activated charcoal at at 0.1 mL DMF/L. When a test substance was partly dissolved ca. 25°C with a 16-hr daylight photoperiod. They regularly fed in the media, the supernatant by decantation was used for the Vol. 38, No. 4, 173–180 (2013) Acute aquatic toxicity of metofluthrin metabolites in the environment 175 exposure and chemical analysis. The chemical analysis of each and the limited buffering capacity of the OECD medium, the metabolite was regularly conducted in each test, together with pH effect on algal toxicity was conveniently examined for VI at measurements of temperature (Multi-thermometer, JAPAN PET 100 mg/L by readjusting the pH of the exposure OECD medium DRUGS Co., Ltd.) and pH (Model B-212, Horiba Ltd., Japan). to ca. 8 with 0.1 N NaOH. Except in the algal tests, the dissolved oxygen (DO) concentration in the test solution was also measured (SevenGo pro, Met- 4. Chemical analysis tler Toledo, Columbus, Ohio, USA). At least at the initiation and termination of exposure, the con- 3.1. Fish acute toxicity test centration of each metabolite was determined by direct HPLC Groups of seven fathead minnows were exposed without rep- analysis of each test solution after an appropriate dilution. A Shi- lication in 1000 mL of each test solution for 96 hr under static madzu HPLC system (LC-10AD pump, SCL-10A system con- conditions using 1-L glass beakers. In the case of III, 20-L size troller and SPD-10A UV detector at 230 nm) equipped with an stainless steel vessels (27×27×32 cm) filled with a 10-L test L-column ODS (5 µm, 4.6mmϕ×150 mm; Chemicals Evaluation solution were used instead under static-renewal (every 48 hr) and Research Institute, Japan, Tokyo) was operated at a flow rate conditions to maintain exposure concentrations. The test vessels of 1.0 mL/min under the isocratic condition. The mixing ratio were partly immersed in a temperature-controlled water bath (v/v) of acetonitrile/0.05% trifluoroacetic acid water as a mobile maintained at 25±1°C under a photoperiod of 16 hr/day using phase and the typical retention time of each metabolite in paren- fluorescent bulbs (ca. 500–1000 lx). Except in the cases of III thesis are as follows: II, 35/65 (13.3, 16.2, 17.3, and 18.5 min); and VI, the test solutions were mildly aerated to maintain the III, 3/2 (6.8 min); IV, 2/3 (17.5 min); V, 2/3 (8.1 min); VI, 1/9 DO levels. Biological observations including mortality and sub- (8.1 min); VII, 1/3 (11.5 min); VIII, 1/3 (8.5 min); and IX, 5/95 lethal toxic symptoms (e.g., loss of equilibrium) were made daily, (5.2 min). and mortality was defined as the lack of movement after gentle stimulation. 5. Statistical analysis

3.2. Daphnid acute immobilization test Acute toxicity values, fish 96-hr LC50, daphnid 48-hr EC50 and

Groups of five D. magna were exposed in 50 mL (for VI) or algal 96-hr EC50, were determined on the basis of the arithmetic 100 mL (in the other cases) of each test solution for 48 hr with- mean measured test concentrations. When the measured con- out aeration under static conditions using 100-mL glass bea- centration at the end of exposure decreased by >20% as com- kers, similarly to the fish study but at 20±2°C. Four replicates pared with the initial concentration, a time-weighted mean were established at each test concentration and for the control. concentration was used instead. Among similar response vari- Biological observations including immobilization and sublethal ables on algal test, such as “biomass” (area under the growth toxic symptoms (e.g., erratic swimming) were made daily. Im- curve, EbC50) and “yield” (final cell density minus the initial one, mobilization was defined as the lack of free swimming within EyC50), the latter one was selected as a representative sensitive 15 sec after gentle agitation. one for the algal evaluation.18) The probit or linear interpolation 3.3. Algal growth inhibition test method was applied. The former method was used for sufficient P. subcapitata at the estimated initial population of 104 cells/mL datasets for fish and daphnids (III for fish and II for daphnids). was exposed in 5 mL of each test solution for 96 hr under stat- Computer programs of PROBIT (ver. 1.5, U.S. EPA) and ICP ic shaking at 200 rpm using 10-mL glass vials loosely covered (ver. 2.0, U.S. EPA) were used for these regression analyses. If with a transparent film. Three replicates were established at each the dataset was sufficient, their 95% confidence intervals were test concentration and twice for the control (water and solvent) also estimated. group. The test vessels were placed on a horizontal shaking incu- Results bator (Multishaker MMS-310, Tokyo Rikakikai Co., Ltd.) maintained at 24±2°C under continuous illumination with fluores- 1. Fish acute toxicity test cent bulbs (1700–2500 lx measured with the Digital Illuminom- The water temperature, DO, and pH were mostly within the eter T-1H, Minolta Camera). The test vessels were repositioned normal ranges: 24–26°C, 5.0–8.9 ppm, and 5.8–8.4, respectively. daily in order to reduce/normalize bias between replicates and Some low DO values of 2.8–4.8 ppm, sporadically observed in test groups. Algal biomass (cell density) was estimated daily by the exposures of II, IV, and VIII, had no impact on the fish, as measuring the fluorescence of chlorophyll-a in algal cells using no abnormal behavior, such as a loss of equilibrium, was ob- a fluorescence spectrophotometer (LS-55 luminescence spec- served in any test group. Exposure to V–IX, formed via ester trometer, PerkinElmer, Inc.). Each sample (0.05–0.5 mL) re- cleavage, resulted in no mortality even at the highest nominal moved from the test vessels was diluted with acetone and the concentration of 100 mg/L, as listed in Table 1. Since dead fish fluorescence in a quartz cuvette (1 cm pathlength) was measured were observed only at 24 hr, the cumulative mortality was found at excitation and emission wavelengths of 430 and 667 nm, re- to be independent of an exposure period of up to 96 hr. Among spectively.20) the metabolites having an intact ester linkage, II and IV showed Since a remarkable pH decrease below 5 was observed for no and slight toxicity at the nominal concentration of 100 mg/L, the test solutions of VI and IX at 100 mg/L due to their acidity respectively. In contrast, a clear dose-response was observed for 176 M. Miyamoto et al. Journal of Pesticide Science

Table 1. Fish acute toxicity of metofluthrin metabolites

Test concentration (mg/L) Cumulative mortality (%) 96-hr LC50 (mg/L) ECOSAR estimated LC50 Metabolite f Nominal Mean measured 96 hrd [95% C.I.] (mg/L) II 0 (Solvent)a NAc (<1.0) 0 >48 13 (ester) 100a,b 48 0 36 (neutral) III 0 (Solvent) a NA (<0.040) 0 0.44e 4.4 (aldehyde) 0.25 0.16 0 [0.30–0.65] 8.6 (ester) 21 (neutral) 0.50 0.29 29 1.0 0.56 57* 2.0 1.1 100 4.0 2.2 100 IV 0 (Solvent) a NA (<10) 0 >77 64 (ester-acid) 100b 77 14 13 (neutral) V 0 NA (<2.0) 0 >92 180 (neutral organic acid) 100b 92 0 VI 0 NA (<2.0) 0 >94 40000 (neutral organic acid) 100a 94 0 VII 0 NA (<1.0) 0 >95 140 (benzyl alcohol) 100 95 0 370 (neutral) VIII 0 NA (<20) 0 >120 7400 (neutral organic acid) 100 120 0 IX 0 NA (<1.0) 0 >99 58000 (neutral organic acid) 100a 99 0 *: Survived fish showed toxic symptoms (loss of equilibrium or hyperactive swimming). a: The results at the lower test concentrations or of negative control were omitted because of their insignificance. b: Since undissolved test substance was observed in the test solution, the exposure and chemical analysis were conducted using a supernatant solution by decantation. c: Not applicable (The limit of detection was indicated in the parenthesis). d: Since cumulative mortalities at 24, 48 and 72 hr were identical with those at 96 hr, those values were omitted. e: LC50 value was estimated by probit analysis. f: Chemical class used for estimation in the parentheses.

III at 0.16–1.1 mg/L, and the lowest LC50 value among the me- tested metabolites with the EyC50 value of 2.6 mg/L. While V and tabolites was estimated to be 0.44 mg/L. VII partly inhibited (ca. 60%) at the highest concentrations, VI and IX showed very steep dose-responses from 0 to 100% inhi- 2. Daphnid acute immobilization test bition at the highest two concentrations, with significantly acidic Similarly to the fish tests, the water temperature, DO, and pH pH values (4.0–5.9) being recorded. The additional bioassay of were almost within the normal ranges: 19–23°C, 7.8–10.6 ppm, VI at the readjusted pH of ca. 8 showed insignificant growth in- and 7.0–8.9, respectively. No or negligible immobilization hibition even at 100 mg/L (EyC50>100 mg/L). In the other cases, (≤10%), even at 100 mg/L, was observed for VI–IX (Table 2), the measured pH values of the test solutions were in the range of while the exposure to V at 100 mg/L caused 90% cumulative im- 6.2–8.9, reflecting OECD medium conditions and algal photo- mobility after 48 hr, resulting in the EC50 value of 71 mg/L. IV synthesis. The yE C50 value of each metabolite was thus estimated showed no immobilization at up to a 100 mg/L difference from as listed in Table 3. a slight fish toxicity, while the dose-response toxicity was clearly Discussion observed for II at 13–55 mg/L (EC50=52 mg/L). III was most toxic to daphnids among the tested metabolites with the EC50 Although the chemical structure prerequisite for aquatic toxicity (48-hr) value of 6.3 mg/L. is not clearly defined for pyrethroids, modification at a molecular end may cause a slight change in the toxicity of a metabo- 3. Algal growth inhibition test lite. In fact, 4′-OH-bifenthrin, only hydroxylated at the 4′-posi- The exponential increase of a mean cell density, >16 times per tion of the biphenyl moiety, still showed high toxicities to fish 10) 72 hr, showed excellent algal growth in the control groups at (LC50, 0.0039 mg/L) and daphnids (EC50, 0.0012 mg/L). Fur- 23–26°C without any unusual variation, indicating the validity thermore, ester cleavage at the side chain in the acid moiety of of all exposures. Any remarkable growth inhibition, ≥25% for acrinathrin and/or hydration of its α-benzyl cyano group greatly 5 96 hr, was not observed for II, IV, and VIII, even at 100 mg/L, reduced its toxicity by a factor of 55 to >5×10 in fish (LC50, 21) while the other metabolites exhibited clear dose-responses. Sim- 0.33–0.39 mg/L) and daphnids (EC50, 0.548 to >10 mg/L). ilarly to the fish and daphnid tests,III was most toxic among the These observations may indicate the relevance of molecular hy- Vol. 38, No. 4, 173–180 (2013) Acute aquatic toxicity of metofluthrin metabolites in the environment 177

Table 2. Daphnid acute immobilization by metofluthrin metabolites

Test concentration (mg/L) Cumulative immobility (%) 48-hr EC50 (mg/L) ECOSAR estimated EC50 Metabolite f Nominal Mean measured 24 hr 48 hr [95% C.I.] (mg/L) II 0 (Solvent) a NAc (<1.0) 0 0 52d 23 (ester) 13a 13 0 0 [44–67] 23 (neutral) 25 24 0 5* 50 49 0 40* 100 55 0* 60* III 0 (Solvent) a NA (<0.040) 0 0 6.3e 3.8 (aldehyde) 0.25 0.16 0 0 [5.5–6.6] 16 (ester) 13 (neutral) 0.50 0.32 0* 0* 1.0 0.60 10* 5* 2.0 1.2 0* 0* 4.0 2.4 0* 10* 8.0 4.7 5* 25* 16 9.4 45* 100 32 19 95* 100 IV 0 (Solvent) a NA (<10) 0 0 >76 110 (ester-acid) 100b 76 0 0 8.6 (neutral) V 0 NA (<1.0) 0 0 71e 110 (neutral organic acid) 13a 12 0 0 [66–77] 25 24 0 10* 50 47 0 0* 100 92 15* 90* VI 0 NA (<2.0) 0 0 >92 19000 (neutral organic acid) 100a 92 0 0 VII 0 NA (<1.0) 0 0 >93 100 (benzyl alcohol) 100 93 0 0 210 (neutral) VIII 0 NA (<10) 0 0 >120 4000 (neutral organic acid) 100 120 0 0 IX 0 NA (<1.0) 0 0 >95 29000 (neutral organic acid) 25a 24 0 0 50 50 0 0* 100 95 0 10

*: Survived daphnids showed toxic symptoms (hypoactive swimming). a, b, c, f: Same as Table 1. d: EC50 value was estimated by probit analysis. e: EC50 value was estimated by linear interpolation. drophobicity of pyrethroids with their aquatic toxicity. Among elimination of these metabolites as one of the reasons for their the metabolites with modification at the prop-1-enyl side chain lower toxicity. of I, significantly low toxicity was observed for II and IV in The daphnid EC50/NOEC ratio of III was calculated to be 39 13) three tested species (LC50 and EC50, >48 mg/L). In contrast, III (6.3 mg/L vs. 0.16 mg/L), while the corresponding values of I showed a higher toxicity than II and IV to all species, but much and twenty pyrethroids13) were much smaller, 1.6 and 3.8±2.8, less than I by two to three orders of magnitude to pyrethroid- respectively. Such a moderate concentration-response pattern in sensitive taxa (fish and daphnid) and one order of magnitude the sublethal effect of III, different from pyrethroids, indicated to pyrethroid-insensitive ones (alga). Gobas et al. applied the that the observed toxicity did not originate from pyrethroid- diffusion rate of a hydrophobic chemical through membrane- specific mode of action but from other mechanisms. Since al- diffusion layer barriers to express the rates of chemical uptake dehydes are well known as toxic chemicals,24) the formyl group and elimination in fish,22) and they suggested the constant up- in III is most likely to be a toxicophore. This is supported by the take and proportional decrease in elimination with an increas- toxicity of aldehyde metabolites of fluridone and glyphosate.6) ing log Kow (n-octanol/water partition coefficient) above 3–4. Therefore, the prop-1-enyl side chain at a molecular end is con-

The log Kow values of II–IV are estimated to be 3.1–3.5 by the sidered to be one of the key structures relating to aquatic toxicity EPI-Suite23) and much lower than I (5.0),14) suggesting higher of I. 178 M. Miyamoto et al. Journal of Pesticide Science

Table 3. Algal growth inhibition by metofluthrin metabolites

Test concentration (mg/L) Mean cell density,g ×104 cells/mL 96 hr EyC50 (mg/L) ECOSAR estimated Metabolite 96 hr f Nominal Mean measured 24/48/72 hr [95% C.I.] EC50 (mg/L) (yield inhibition %) II 0 (Solvent)a NAc (<2.0) 4.7/22/86 300 (−) >61 8.4 (ester) 25a 22 4.7/23/88 310 (−2) 25 (neutral) 50 37 5.0/22/82 280 (7) 100 61 5.0/20/70 230 (23) III 0 (Solvent)a NA (<0.20) 4.6/26/120 360 (−) 2.6d 7.6 (aldehyde) 1.3 0.84 4.6/25/100 340 (6) [2.5–2.7] 5.4 (ester) 16 (neutral) 2.5 1.7 5.0/25/93 250 (30) 5 3.5 4.3/21/56 120 (68) 10 7.8 3.7/8.0/10 24 (94) 20 15 3.7/7.9/6.9 6.1 (99) IV 0 (Solvent)a NA (<10) 4.6/26/120 360 (−) >74 38 (ester-acid) 100b 74 4.3/25/110 340 (6) 11 (neutral) V 0 NA (<0.10) 5.1/30/140 440 (−) 84d 120 50a 55 5.1/28/120 450 (−2) [82–85] (neutral organic acid) 100 95 3.6/17/57 160 (64) VI 0 NA (<2.0) 5.3/24/96 310 (−) 75d 7600 50a 51 6.4/25/93 330 (−4) [75–76] (neutral organic acid) 100 100 1.0/1.2/1.0 0.58 (100) VI (pH 8) 0 NMe 5.4/38/125 509 (−) >100 100 NM 5.0/24/88 315 (38) VII 0 NA (<1.0) 4.0/25/120 390 (−) 97d 40 (benzyl alcohol) 25a 26 3.8/25/110 400 (−4) [92–100] 130 (neutral) 50 53 3.8/25/98 360 (5) 100 110 3.7/19/45 150 (62) VIII 0 NA (<10) 4.0/25/120 390 (−) >110 2300 100 110 3.7/24/95 390 (−1) (neutral organic acid) IX 0 NA (<1.0) 4.5/23/95 290 (−) 75d 11000 50a 51 5.1/23/95 300 (−2) [74–75] (neutral organic acid) 100 99 0.63/0.69/1.7 2.2 (99)

a, b, c, f: Same as Table 1. d: EyC50 value was determined by linear interpolation. e: Not measured. g: Estimated by measuring fluorescence of chlorophyll- a in algal cells.

The toxicity of the metabolites (VII, VIII, and IX), originat- their uptake, and, as a result, it is supposed to alter their algal 28) ing from the alcohol moiety of I, in fish and daphnids (LC50 or toxicity. The pKa values of VI and IX can be respectively esti-

EC50, >93 mg/L) resembles that of 2,3,5,6-tetrafluoro-4-meth- mated to be 6.73/4.26 and 1.96/0.95 by ACD/pKa DB (ver. 4.56, 25) 27) ylbenzoic acid and 2,3,5,6-tetrafluorobenzoic acid (LC50 Advanced Chemistry Development Inc., Toronto, Canada). By or EC50, >100 mg/L), independent of a number of a carboxylic comparing these pKas and the observed pH values (4.0–4.1) at group. The very weak toxicity of V and VI (LC50 or EC50, 71 to the toxic level, 78% of VI should be present in an undissoci- >94 mg/L) was found to be common to the chrysanthemic acid ated form, but IX is fully ionized. Since both metabolites ex- 7,26) 7) derivatives of cypermethrin, lambda-cyhalothrin, tefluth- hibited the same EyC50 value of 75 mg/L irrespective of different 25) 21) rin, and acrinathrin (LC50 or EC50; 3 to 180, >16 to 100, ionized fractions, factors other than carboxylic acid dissocia- >15.8 to >182 and 69 to >110 mg/L, respectively). The last one, tion are likely to control their toxicity. Such a low pH results in − cyclopropyl dicarboxylic acid from acrinathrin, only shows an an extremely small fraction of HCO3 as a bio-available carbon algal toxicity similar to VI. source.29) Therefore, this may be a key factor in the observed The algal toxicity of dicarboxylic acid derivatives (VI and IX) algal growth inhibition of these highly acidic metabolites. The with steep dose–response curves can be accounted for by lower inhibitory effect on algal growth is known for highly acidic tere- pH values (4.0–5.9) of the corresponding solutions. The disso- phthalic acid30) and benzoic acid drivatives.31) The medium pH ciation of carboxylic groups in these metabolites may control effect on algal toxicity was further confirmed by the greatly -re Vol. 38, No. 4, 173–180 (2013) Acute aquatic toxicity of metofluthrin metabolites in the environment 179

Table 4. A worst-case assumption of PEC values of each metabolite in water and resulting TER values

Metabolite I II III IV V VI VII VIII IX Molecular weight (g/mol) 360 394 348 364 154 158 224 238 238 Water solubility (mg/L)a 0.5 8.06 10.43 12.61 557.9 87320 4055 1904 13620 a Koc (L/kg) 6184 43.51 220.2 87.3 38.79 2.24 21.94 6.57 2.26 PEC (ng/L)b from I in water 0.76 0.83 0.73 0.76 0.32 0.33 0.47 0.50 0.50 from I in soilc 0.41 3.4 2.5 3.0 1.4 1.5 2.0 2.2 2.2 Lowest TER, ×106 Fish 0.0016 14 0.18 26 66 63 48 55 45 Daphnid 0.0062 15 2.5 25 51 61 47 55 43 Algae 0.21 18 1 25 59 67 49 50 34 a: Estimated by EPI-Suite (Lower value as the worst-case) except I (measured value). b: Estimated by assuming 100% degradation of I as the worst-case. c: Estimated by EU FOCUS STEP 2 simulation (ver. 1.1), based on the soil PEC of I (2.6×10−5 mg/kg14) converted using an application rate of 0.02 g/ha, under the scenario of no drift and crop interception) with the worst-case assumption (DT50=1000 days, 100% occurrence and 5% runoff).

−5 14) duced 96-hr EyC50 of >100 mg/L in the additional bioassay of VI and 2.6×10 mg/kg in freshwater and soil, respectively. The at pH 8. Considering the unlikelihood of pH change by dilution PEC value of each metabolite was conveniently estimated from in natural bodies of water related to its buffering capacity, the that of I by assuming its complete degradation to the corre- toxicity of VI and IX observed on a laboratory scale should be sponding metabolite with runoff introduction from soil, as an an unrealistic overestimate, and the toxic profile under standard unrealistic worst case. The surface water PEC of each metabo- pH conditions (>100 mg/L for VI) is appropriate for risk assess- lite in 5% run-off scenario was estimated with FOCUS STEP2 ments as prescribed in the UK technical guidance.32) simulation37) by assuming a formation of 100%. The water solu-

Metabolites generally show less aquatic toxicity than does bility and soil Koc value of each metabolite were conveniently 23) pesticide itself, but unexpected toxicity is not ruled out. Instead estimated by EPI-Suite, and the worst-case DT50 of 1000 days of conducting any toxicity study, QSAR approaches33,34) using in soil was used. The TER values in the three species range from the relationship of toxicity with physico-chemical parameters 1.8×105 to 2.5×106 for the most toxic metabolite (III); they are such as log Kow have been applied to predict the ecotoxicity of a much higher for the other metabolites by one or two orders of chemical. The ECOSAR program (ver. 1.11),35) one of the most magnitude (Table 4). These TER values are more than a hun- popular models,34) estimates aquatic toxicity based on regression dredfold higher than those for I (1.6×103–2.1×105). Based on from training sets of experimentally obtained ecotoxicity data these large margins of safety, the risks to the aquatic ecosystem with a physico-chemical property (mainly estimated log Kow) by from all the metabolites as well as the parent under typical usage taking account of 111 chemical classes, such as aldehydes and were considered to be negligible. esters. ECOSAR predicted the LC50 and EC50 values of each me- References tabolite mostly within one order of magnitude, but a few outli- ers were found (Tables 1–3). ECOSAR underestimated the fish 1) J. Gan, F. Spurlock, P. Hendley and D. P. Weston (eds.): “Synthetic Py- toxicity of III when it was classified as ester and neutral organ- rethroids Occurrence and Behavior in Aquatic Environments,” Ameri- ics, while the reasonably close value to that in our study was es- can Chemical Society, Washington D.C., 2008. 2) T. Katagi: Top. Curr. Chem. 314, 167–202 (2012). timated when an aldehyde class was selected. This supports a 3) T. R. Roberts, D. H. Hutson, P. W. Lee, P. H. Nicholls and J. R. Plim- formyl group as the dominant toxicophore in III, as mentioned mer (eds.): “Metabolic Pathways of Agrochemicals Part II: Insecticides above. Much greater underestimation of toxicity to three species and Fungicides,” The Royal Society of Chemistry, Cambridge, 1999. was obtained by ECOSAR for acidic metabolites (VI, VIII, and 4) J. P. Leahey (ed.): “The Pyrethroid Insecticides,” Taylor & Francis, IX), which may be accounted for by the limited maximum con- London & Philadelphia, 1985. centration taken in the toxicity tests (nominal 100 mg/L) as well 5) S. J. Maund, P. J. Campbell, J. M. Giddings, M. J. Hamer, K. Henry, as an acidity effect in the algal toxicity. E. D. Pilling, J. S. Warinton and J. R. Wheeler: Top. Curr. Chem. 314, As a further ecotoxicological profiling of the metabolites in 137–165 (2012). relation to a typical usage of the parent, an ecological risk as- 6) C. J. Sinclair and A. B. A. Boxall: Environ. Sci. Technol. 37, 4617–4625 sessment for each metabolite of I was conveniently conducted in (2003). 7) I. R. Hill: Pestic. Sci. 27, 429–465 (1989). terms of acute toxicity exposure ratio (TER), defined as the tox- 8) V. Zitko, W. G. Carson and C. D. Metcalfe: Bull. Environ. Contam. icity (LC50, EC50) value divided by its predicted environmental Toxicol. 18, 35–41 (1977). 36) concentration (PEC), a so-called quotient method. In the case 9) K. E. Day and R. J. Maguire: Environ. Toxicol. Chem. 9, 1297–1300 ® of SumiOne Liquid vaporizer, a typical use pattern in the EU (1990). region, the PEC value of I was calculated to be 7.6×10−7 mg/L 180 M. Miyamoto et al. Journal of Pesticide Science

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