UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON D.C., 20460 

OFFICE OF 

CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

PC Code: 122990 

D422848 

October 3, 2014 

SUBJECT: Reclassification of 6 Mesotrione Aquatic Ecotoxicology Studies 

From: 

Reviewed: 

Through: 

To: 

Michael Wagman, Biologist 

Environmental Risk Branch 6 

Environmental Fate and Effects Division (7507P) 

Amy Blankinship, Chemist ~ (' fl( feY A 1) \ v ·)-\ Y 

Environmental Risk Branch 6 \A 

Environmental Fate and Effects Division (7507P) 

Mark Corbin, Branch Chief . 1 ~ d ( ~ Environmental Risk Branch 6 ·· ~ ~ 

Environmental Fate and Effects Division (7507P) -

Jolene Trujillo, Chemical Review Manager 

Linda Arrington, Branch Chief 

Risk Management and Implementation Branch V 

Pesticide Re-evaluation Division (7508P) 

As part of the problem formulation for the ecological risk and drinking water exposure 

assessments for mesotrione (PC Code 122990; D417229), a review of the available 

ecotoxicological data was performed. While the classification for many of the submitted studies 

did not change after review (still deemed Acceptable), the classification for a number of aquatic 

toxicity studies did change. The reason(s) for the change in the study classifications varied but 

included: the occurrence of strongly acidic test conditions in treatments and/or strongly basic 

conditions in controls, presence of precipitate in samples without removal 

(filtration/centrifugation) prior to analytical analysis, and/or lack of ability to discern a doseresponse. These study limitations introduce uncertainty in the ability to adequately discern a 

treatment-related effect. With the exception of the study on mesotrione's degradate, MNBA, 

effects on green algae (MRID 4490171 0; this study was already considered supplemental) EFED 

has reclassified the studies from "Acceptable" to "Supplemental". Further, most of these studies 

(with the exception ofMRID 44505121, aquatic plant test using the freshwater diatom, Navicula 

pelliculosa) should only be used qualitatively as they are not of suffiCient quality for quantitative 

use in risk assessments. A list of the studies and rationale for reclassification of each study is 

provided below. In general, while these studies were reclassified, additional data is not 

requested at this time. The reclassification of the chronic daphnid life cycle test (MRID 

4450501 0) results in a lack of chronic data for any aquatic invertebrate; however, since the acute 
mysid toxicity endpoint was much more sensitive than the acute daphnid toxicity endpoint, 

EFED recommends that a new chronic aquatic invertebrate study (850.1350) be requested using 

mysids. EFED intends to then use the acute and chronic estuarine/marine invertebrate data in an 

acute-to-chronic (ACR) approach to estimate a chronic freshwater invertebrate endpoint. 

Table 1: Ecotoxicological data requirements for Mesotrione

MRID Study Classification1 Remarks

44505121 Supplemental

Study was previously identified as acceptable. 

Study may be used quantitatively in risk 

assessment. See below for rationale for 

downgrade.

44901710, 44505007, 

44505010, 44505124, 

44505122

Supplemental

Studies were previously identified as 

acceptable (or in the case of MRID 44901710, 

could have been used quantitatively in risk 

assessment). These studies may not be used 

quantitatively in risk assessment. See below 

for rationale for downgrades.

1OPPIN Classifications:

Acceptable; Acceptable/Guideline; Acceptable/Non-Guideline; Cited; Confirmatory; Decision 

Deferred; Extraneous submission; Guideline; In Review; minimum; No Decision; Partially 

Acceptable; Satisfactory; Screened-acceptable for review; Screened-not acceptable; Supplemental; 

Supplemental/Non-Guideline; Unacceptable; Unacceptable/Guideline; Unacceptable/Non-Guideline; 

Upgradeable. 

3EPA Accession Number; No MRID number was assigned for this study.

EFED has reviewed and reclassified the following studies submitted for Mesotrione (PC Code 

122990):

Kent, S.J., N. Shillabeer, J.E. Caunter, S.J. Wallace. ZA1296: Acute Toxicity to the Sheepshead 

Minnow (Cyprinodon variegatus). Unpublished study performed by Brixham Environmental 

Laboratory, Brixham Devon, UK. Laboratory Report No. BL5592/B. Study sponsored by 

ZENECA Ag Products. Study Completed on February 16, 1996. MRID 44505007.

This study on sheepshead minnow (Cyprinodon variegatus) was initially considered to meet the 

850.1075 guideline requirement. However, mesotrione concentrations were above solubility at 

the two highest treatment levels, no co-solvent was used and precipitate was formed, although 

samples were centrifuged prior to chemical analysis. Even at the lowest concentration (100 

mg/L (ppm)), the solution was pale yellow and was progressively darker in each higher test 

concentration. Small amounts of precipitate were observed in the 180 and 320 mg/L nominal 

treatment levels which dissolved after 24 hours, while precipitate in the higher treatments 

remained throughout the duration of the test. Initial concentrations were not included in the 

mean measurements as the study authors suggested that during the majority of the study the 

measured concentrations were closer to those values obtained at 48 and 96 hours than the 0-hour 

concentrations. Additionally, the pH in the two highest treatment levels was very low (pH<4.5, 

likely due to the addition of the test substance), and may have caused the toxicity in those 

treatment levels, rather than the inherent toxicity of the test compound. Sublethal effects 

including sounding, loss of balance, weakness, rapid respiration and ceased swimming were 

observed in all treatment groups except the lowest concentration (100 ppm). Previously, the 
LC50 from this study was reported as 520 ppm. Due to the issues discussed, this LC50 value is 

not considered valid. However, since no mortality or sublethal effects were observed at the 100 

ppm concentration level, the observed NOAEC is considered to be 100 ppm, and the 96-Hr LC50 

is considered to be greater than 100 ppm. Since these values are approximately 3-4 orders of 

magnitude above the expected maximum EECs from all recently conducted assessments (US 

EPA, 2014; D415369), acute risk is not anticipated to be likely to this taxa and a new acute 

mortality study for an estuarine/marine fish is not requested at this time. 

Morris, D.S., H. Kelso, N. Shillabeer, N.J. Williams, and S.J. Wallace. ZA1296: Chronic 

Toxicity to Daphnia magna. Unpublished study performed by Brixham Environmental 

Laboratory, Brixham Devon, UK. Laboratory Report No. BL5832/B. Study sponsored by 

ZENECA Ag Products. Study Completed on January 31, 1996. MRID 44505010.

This study on the waterflea, Daphnia magna, was initially classified as acceptable. During the 

re-evaluation of the study, several deficiencies were noted with this study including that the three 

highest treatment solutions (≥ 320 mg/L) had extremely acidic conditions (pH 3.8—4.7) which 

likely caused the toxicity observed in those treatment levels, rather than any inherent toxicity of 

the test compound. Additionally, those concentrations also had precipitates in their solutions (it 

appears that the undissolved material was separated from the solutions prior to sample analysis, 

but the study does not state how the separation was accomplished). However, a more serious 

problem with this study is that the most sensitive endpoint, dry weight, did not exhibit a doseresponse relationship whereas at the lowest treatment concentration (97 mg/L), mean dry weight 

was significantly reduced by 46% compared to controls, but at the next highest treatment group 

of 180 mg/L, mean dry weight was reduced 18% compared to controls and was not considered 

statistically significantly different than control daphnids. Since mortality was 100% at all higher 

treatment levels, the dose-response relationship for dry weight in the study cannot be determined 

from only those two treatment levels that had surviving daphnids and which had highly 

inconsistent results. Therefore, the classification of this study is now supplemental and may only 

be used qualitatively in a risk assessment. 

As a result of this reclassification, there are no data available for mesotrione’s effects to aquatic 

invertebrates. The lack of this data is a major data gap. However, since mesotrione was much 

more acutely toxic (~250x) to mysids than to daphnids, EFED is requesting that an 

estuarine/marine life cycle test be conducted with the mysid, Mysidopsis bahia (OPPTS 

850.1350). A new chronic freshwater invertebrate test is not requested at this time. EFED 

intends to use the data generated from the chronic estuarine/marine life cycle test to calculate an 

Acute-to-Chronic Ratio (ACR) and derive a freshwater invertebrate chronic value from this ACR 

using the available acute daphnid data.

Smythe, D.V., S.J. Kent, and N. Shillabeer. ZA1296: Toxicity to the Blue-Green Alga 

Anabaena flos-aquae. Unpublished study performed by Brixham Environmental Laboratory, 

Brixham Devon, UK. Laboratory Report No. BL5801/B. Study sponsored by ZENECA Ag 

Products. Study Completed on July 13, 1996. MRID 44505122.

This study on the blue-green algae, Anabaena flos-aquae was initially classified as acceptable. 

However, precipitate formed in the highest test concentration (175 mg/L) and there was no 

filtration or centrifugation of test samples prior to chemical analysis. Additionally, the pH in the 
two highest treatment concentrations (96 and 175 mg/L) declined to very acidic conditions over 

the course of the test (pH 3.6—4.1) which is likely to have caused the cell death at these 

concentrations, rather than any inherent toxicity of the test chemical. For these reasons, the 

classification of the study is changed to supplemental, for qualitative use only for the IC50 

endpoint. However, since pH levels remained within test guideline conditions at all other 

concentrations, the calculated NOAEC for this study remains at 56 ppm, and the IC50 is reported 

as > 56 ppm. As these endpoint values are much higher (≥ 3 orders of magnitude) than the 

maximum EECs predicted from any previous assessment for mesotrione uses and the available 

data (including this study) indicate that nonvascular aquatic plants do not appear to be 

particularly sensitive to mesotrione, a new study is not requested at this time.

Shillabeer, S.J. Kent, and D.V. Smyth. ZA1296: Toxicity to the Green Alga Selenastrum 

capricornutum. Unpublished study performed by Brixham Environmental Laboratory, Brixham 

Devon, UK. Laboratory Report No. BL6113/B. Study sponsored by ZENECA Ag Products. 

Study Completed on August 27, 1997. MRID 44505124

This study conducted with the green algae, Pseudokirchneriella subcapitata (formerly described 

as Selenastrum capriocornutum) was initially classified as acceptable. However, the pH in the 

control and lowest two treatments (0.39 and 0.82 mg/L) reached very basic conditions (pH 9.9---

10.0) by the end of the test, which may indicate that the algal cultures had grown to the extent 

they were at the upper bound of the test systems carrying capacity. These high pH levels are 

unlikely to represent actual conditions in the environment and may have inhibited performance 

in each of these groups, most importantly in the control group. This leads to uncertainty around 

the IC50 estimate. As such, the classification of this study is now supplemental and the IC50 

estimate should not be used quantitatively in risk assessment. While controls may be 

compromised in these suboptimal conditions, as the pH was similar in the two lowest treatments 

and controls and growth rates remained similar between these two lowest treatments compared to 

controls, it is expected that these treatment levels would still be similar to controls under the 

optimal conditions suggested by the guideline recommendations. Therefore, the NOAEC of 0.39 

mg/L is still considered valid and the IC50 is now considered to be > 0.82 mg/L. Since there is 

still a large difference between these endpoints and either the most sensitive aquatic plant data 

(duckweed IC50 of 17.7 ppb) or the maximum EECs predicted from past assessments (USEPA, 

2014, D415369), a new study is not requested at this time. 

Smythe, D.V., S.J. Kent, and N. Shillabeer. ZA1296: Toxicity to the Freshwater Diatom 

Navicula pelliculosa. Unpublished study performed by Brixham Environmental Laboratory, 

Brixham Devon, UK. Laboratory Report No. BL5780/B. Study sponsored by ZENECA Ag 

Products. Study Completed on June 29, 1996. MRID 44505121

This study conducted with the freshwater diatom, Navicula pelliculosa was initially classified as 

acceptable. However, the dose-response relationship observed in the study was not what is 

typically expected in algal toxicity studies. The test substance appeared to elicit progressively 

increasing stimulation of algal colonies with concentration (11—46% stimulation compared to 

controls) except for the very highest concentration of 92 mg/L which experienced a 96% 

decrease in cell density compared to the control plants. Due to this lack of a dose-response 

relationship and the resulting lack of confidence around the IC50 estimate (for which 95% 

confidence intervals could not even be defined), the study is classified as Supplemental, but may 
be used for quantitative purposes in risk assessment. The study’s NOAEC and EC25 are 46 mg/L 

and 68 mg/L, respectively. These endpoints are significantly higher than other available 

endpoints for aquatic plants. A new study with freshwater diatoms is not requested at this time. 

Smythe, D.V., S.J. Kent, and N. Shillabeer. MNBA: Toxicity to the Green Alga Selenastrum 

capricornutum. Unpublished study performed by Brixham Environmental Laboratory, Brixham 

Devon, UK. Laboratory Report No. BL6066/B. Study sponsored by ZENECA Ag Products. 

Study Completed on July 31, 1997. MRID 44901710. 

This study conducted with the green algae, Pseudokirchneriella subcapitata (formerly described 

as Selenastrum capriocornutum) was initially classified as Supplemental due to it only being 

conducted for 72 hours (following OECD’s TG 201 guideline), rather than the EPA 850.4500 

guideline recommendation of 96 hours. Although the study is still considered Supplemental, this 

review is meant to indicate that the data contained within should no longer be considered of 

quantitative value, but may only be used for qualitative purposes in risk assessment. This 

clarification is due to the fact that the pH in the control and four lowest treatment groups rose to 

very high basic levels (pH 9.5—10.0) and declined to very acidic levels (<4.5) in the three 

highest treatment doses. These high and low pH’s are unlikely to represent relevant conditions 

in the environment and are likely to have impacted both control performance and each treatment 

level’s performance. It is likely that the 100% inhibition exhibited in the three highest treatment 

doses were due to the acidity caused by the test compound and may not reflect any inherent 

toxicity of the test chemical. The combined effects of these pH issues result in a high degree of 

uncertainty around the IC50 and NOAEC endpoints and make comparison with the green algae 

study (MRID 44505124, see above) with parent mesotrione difficult. However, in this study 

since the pH of the control and three lowest concentrations (3.3—10 mg/L) was similarly high 

and only minimal inhibition (<3%) was observed in these concentrations compared to controls. 

While the green algae study with parent mesotrione had significant inhibition at concentrations 

as low as 1.5 mg/L, it does not appear that MNBA is likely as toxic as parent mesotrione to nonvascular aquatic plants. Although there is still some uncertainty as to the comparison of 

MNBA’s toxicity to vascular aquatic plants (which are much more sensitive to parent mesotrione 

than non-vascular aquatic plants) compared to parent mesotrione, new data for this taxa is not 

requested at this time. In the absence of additional data, conservative assumptions may be made 

in the risk assessment, such as considering MNBA as toxic to vascular aquatic plants as parent 

mesotrione.

References

USEPA, 2014. Mesotrione: Drinking Water Exposure Assessment for New Use on 

Mesotrione-Tolerant Soybean. (DP Barcode 415369). April, 2014. 

USEPA/OPPTS/OPP.