UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON D.C., 20460 

OFFICE OF 

CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

PC Codes: 112602, 125601, 128976 

DP Barcode: D426420 

Date: May 6, 20 15 

MEMORANDUM 

SUBJECT: Review of Three Ecotoxicity Studies in Support of Paclobutrazol 

Registration Review 

TO: 

FROM: 

Khue Nguyen, Chemical Review Manager 

Tom Moriarty, Team Leader 

Neil Anderson, Branch Chief 

Risk Management and Implementation Branch 1 

Pesticide Re-Evaluation Division (7508P) 

Office of Pesticide Programs 

111' k . I/I lk- ~I & Its' Meghan Radtke, Ph.D., Biologist , ·y -- fl«.tA,/I 

Environmental Risk Branch I 

Environmental Fate and Effects Division (7507P) 

Office of Pesticide Programs 

BY: 

APPROVED 

Sujatha Sankula, Ph.D., Branch Chief /

1 \J \ , 

~ 

. ~ ~~ -= ;.;=--

Environmental Risk Branch l · 

Environmental Fate and Effects Division (7507P) 

Office of Pesticide Programs 

The Environmental Fate and Effects Division (EFED) has reviewed three ecotoxicity 

studies that were submitted by Syngenta Crop Protection in support of Registration 

Review. The studies were from the scientific peer-reviewed literature and address 

transpo11 and uptake of paclobutrazol, uniconazole (a structurally similar plant growth 

regulator) and trinexapac-ethyl in grass and sapling trees. Open literature review 

summaries have been prepared for each study that outline the study's findings, 

limitations, and potential use for risk assessment. See Table I for more details. 
TABLE 1. Details for the Paclobutrazol, Uniconazole, and Trinexapac-etbyl Studies 

Study Type Orga nism MRID Study Cilation 

Study Summary Classification 

Metabolism Kentucky bluegrass 49577 101 Beasley, J.S., and B.E. Qualitative Paclobutrazol uptake was 

and uptake of (Poa pratensis) Branham. 2005. Analysis higher in creeping 

paclobutrazol of paclobutrazol and bentgrass than Kentucky 

and trinexapac Creeping bentgrass trinexapac acid in turfgrass bluegrass, regardless of 

acid (Agrostis clippings. !111ernatio11a/ temperature. Trinexapac 

(degradate of stolonifera) Tur/grass Society Research acid uptake did not vary 

trinexapac- Journal, I 0: 1170-1175. between species, 

ethyl) regardless of temperature. 

Open Half-lives were shorter at 

Literature higher temperatures. 

Trinexapac acid half-lives 

ranged trom 3.1 to 6.4 

days and paclobutrazol 

trom 6 to 15 days, 

depending on species and 

temperature. 

Rationale: Limited 

information available on 

number of plants tested 

and replication. 

Growth Bean 49577102 Sterrett, J.P. 1985. Qualitative Growth inhibition 

inhibition, (Phaseolus Pac lobutrazol: a promising observed for most species, 

translocat ion. vulgar is) growth inhibitor for but timing and doses 

and injection into woody plants. differed. 

metabolism of California privet J. Amer. Soc. Hort. Sci., 

paclobutrazol (Ligustrium 11 0(1): 4-8. Movement of 

ovalifolium) paclobutrazol documented 

Open from injection site to 

Literature Red maple shoots and roots. Little 

(Acer rubrum) degradation observed with 

a 27-day interval inside of 

White ash the plant. 

(Frai:inus 

americana) Rationale: Quality 

control data for the 

Yellow poplar analytical methods was 

(Liriodendron not presented. 

t ul ipifera) 

American 

sycamore 

(Platanus 

occident a/is) 

Apple 

(A,fa=lus domestica) 

2 
Uniconazole Silver maple 49577 103 Arron, G.P. 1990. Qualitative Uniconazole reduced 

translocat ion (Acer Translocation of shoot length. 

and saccharinu111) uniconazole after trunk 

metabolism injection of silver maple For up to two growing 

saplings. Journal of Plant seasons, the majority of 

Open Growth Regulation, 9: 141- uniconazole stayed near 

Literature 146. the injection site; only a 

sma ll percent was 

translocated to leaves, 

where it was observed to 

accumulate until leaf fa ll. 

Uniconazole metabolism 

was slow and did not 

appreciably occur in the 

stems and/or near the 

injection site of the plant. 

3 
Open Literature Review 

Chemical Name: Paclobutrazol and Trinexapac-ethyl 

PC Codes: 12560 l, I 12602 

CAS Nos. 76738-62, 95266-40-3 

MRID: 49577101 

ECOTOX Record Number and Citation: 

Beasley, J.S., and B.E. Branhan1. 2005. Analysis of paclobutrazol and trinexapac acid in 

turf grass clippings. International Twfgrass Society Research Journal, 10: 11 70 - 1175. 

Purpose of Review : Paclobutrazol Registration Review 

Date of Review: 4/24/ 15 

Description of Use: Qualitative 

Summary of Study Findings: 

Executive Summary 

Paclobutrazol and trinexapac acid (active ingredient derived from trinexapac-ethyl) are systemic 

plant growth regulators. The objective of the study was to evaluate the effect of temperature on 

the metabolism of paclobutrazol and trinexapac ac id in cool season turf grasses. Ha lf-lives were 

calculated for each temperature/species combination, based on residue measurements collected at 

5 different time points. 

Methods 

Kentucky bluegrass cv 'Moo.nlight' and creeping bentgrass cv 'L-93' were seeded into 5 x 20 cm 

containers. At I 0 weeks, an equal number of each species (the number was not specified) was 

transferred into 18 or 30° growth chambers and allowed to acclimate to the environmental 

conditions for 2 weeks. After acclimation, equal numbers of both species in each growth 

chamber ( 18 or 30° C) were treated with either 0.27 kg ai/ha of trinexapac-ethyl (0.24 lb ai/ A) or 

0.42 kg ai/ha of paclobutrazol (0.37 lb ai/A). The plants were returned to their respective growth 

chambers and remained there until harvesting. Trinexpac-ethyl treated plants were harvested at 

2, 5, 8, l l , and 14 days after application. Paclobutrazol treated plants were harvested at 2, 10, 

18, 26, and 34 days after application. The experiment was repeated once. 

1 
Residues were analyzed using high performance liquid chromatography with ultraviolet 

detection (HPLC-U V) for paclobutrazol or trinexapac acid in leaves. Detai Is of the extraction 

and analysis process can be found in the journal article. 

Plant growth regulator metabolism was analyzed as a complete block design with repeated 

measures with SAS (2003). Turfgrass species and temperature regime were analyzed as fixed 

factors over time and sampling time was transformed to a logarithmic scale for the linear 

regression analysis. For trinexapac acid, only four harvest dates were incorporated into the 

analysis because the fifth date yielded residues below the detection limit. 

Results 

Trinexapac acid uptake was similar for both species of grass at both temperatures, based on the 

residue measurements at 2 days after application. Metabolism rates increased at the higher 

temperatures (see Table 1 for the half-lives). 

Paclobutrazol uptake varied by species with creeping bentgrass taking up more paclobutrazol than 

Kentucky bluegrass, regardless of the temperature. Metabolism rates also varied with creeping 

bcntgrass metabolizing paclobutrazol faster at 18° C, but not at 30° C (see Table I for the halflives). 

Table 1. Half-lives for trinexapac acid and paclobutrazol 

Kentuckv Blueerass Creeoine Benterass 

18° c 30° c 18° c 30° c 

Trinexpac acid 5.3 days 3.4 days 6.4 days 3.1 days 

Paclobutrazol 11-1 5 days 7-9 days 9-1 1 days 6-8 days 

Data Quality Evaluation 

Recovery from fo11ificd samples was 78% ± 6% for trinexapac acid and 84% ± 7% for 

paclobutrazol. Recoveries are generally reasonable, although the trinexapac acid residue 

measurements may somewhat underestimate the true residues in plants. 

Rationale for Use: 

The study presents information about the half-lives of paclobutrazol and trinexapac acid in two 

species of turf grass and how these are affected by different temperatures. In addition, it shows 

that paclobutrazol uptake may vary by species whereas trinexapac-ethyl uptake was similar for 

the two species that were tested. 

Limitations of Study: 

The study did not specify the number of plants that were analyzed for each treatment level. The 

study acknowledged that the extraction method used for trinexapac acid may not have accurately 

estimated the amount of trinexapac taken up by the plants. The method measured trinexapac 

2 
acid; however, the initial chemical taken into the plant is trinexapac-ethyl. A better approach 

would have been to measure both trinexapac-ethyl and trinexapac acid at the first harvest date 

and add these two measurements together. Raw data were not included in the study report. 

Description of Use in Document: 

Qualitative. The half-lives should be treated as qualitative information because the study did not 

provide details about the number of plants that were analyzed per treatment group or the number 

of times that the study \Vas replicated. Variation appeared to be high for some of the sampled 

time points and it is important to understand the extent of the sampling effort. 

References: 

SAS Institute. 2003. The SAS system for Windows. Release 9.1 . SAS Inst., Cary, NC. 

Primary Reviewer: Meghan Radtke, Ph.D., Biologist 

Date: 4/24/ 15 ~ /lu,jj/I 

Secondary Reviewer: Edward Odenkirchen, Ph.D., Senior Science Advisor 

Date: 5/4/ 15 fZ.A. OJ- t/))-1,_ 

3 
Open Literature Review 

Chemical Name: Paclobutrazol 

PC Code: l 2560 I 

CAS No. 76738-62 

MRID: 49577102 

ECOTOX Record Number and Citation: 

Sterrett, J.P. 1985. Paclobutrazol: a promising growth inhibitor for injection into woody plants. 

J. Amer. Soc. Hort. Sci., 110 (I): 4 - 8. 

Purpose of Review: Paclobutrazol Registration Review 

Date of Review: 4/27115 

Description of Use: Quali tative 

Summary of Study Findings: 

Executive Summary 

The objectives of the study were to determine I) if injected paclobutrazol slowed the growth of 

bean, shade trees, and apple trees; and 2) the extent of translocation and metabolism in apple 

trees after injection. The study demonstrated that paclobutrazol suppresses growth in a number 

of different species and that it is translocated to different parts of the plant, away from the 

injection site. Metabolism of paclobutrazol is slow to occur. 

Methods 

Growth Inhibition 

Beans (Phaseo/us vu/garis) were grown from seeds and were injected with paclobutrazol at 

doses or 0, 0.0 l , 0.1 , I , I 0, 100, and 1000 μg/plant. Stem height was measured immediately 

before the treatment and at 7 days following the treatment, along with shoot weight and leaf area. 

There were 5 replicates per treatment level and the entire experiment was repeated once. 

Two-year old California privet (Ligustrium ovalifolium) seedlings with a single 1 cm diameter 

stem were injected with 0. I, 10, l 00, and I 000 ~tg ai/plant. Immediate ly following the injection, 

the stem of the plant was cut off at 10 cm above the soil. The number and length of new stump 

sprouts was measured during a 64-day observation period. Each treatment was replicated 5 

times in a randomized block design and the entire experiment was repeated once. 

1 
Trunks of several species of saplings were injected with paclobutrazol emulsion. In 198 l, red 

maples (Acer rubrum) and white ash (Fraxinus americana) saplings ranging 3 to 4 cm in 

diameter were injected at bud break in April with 4 ml of paclobutrazol. Branches were then 

pruened to a single lateral of two-year old wood following the treatment. Data were collected on 

height increase from three tagged branches per plant throughout the growing season. There were 

5 replicates per treatment. In 1983, red maple (4.1 cm diameter), white ash (4.3 cm diameter), 

and yellow poplar (Liriodendron tulipifera) (3 .6 cm diameter) were injected with 4 ml of 

paclobutrazol and Ameri can sycamore trees (P!atanus occidentalis) (3.0 cm diameter) were 

injected with 2 ml of paclobutrazol. The tops of the main stems were pruned before the 

injection. Injections were made 100 cm below the pruned part of the main stem at bud break and 

one month after bud break. Data on height were collected from three tagged branches per 

sapling during the growing season. The treatments were replicated 5 times in a randomized 

complete block design and linear regression analysis included the growth of untreated trees. 

Translocation and Metabolism 

One-year old golden delicious apple trees (Mazlus domestica) were potted and immediately 

injected with 0 or 500 μg paclobutrazol and 0.5% azo sulfamide dye. The trees were pruned to 

85 cm above the injection site immediately following the injection. The growth of the upper I 0 

shoots was measured weekly over a period of 46 days after injection. The treatments were 

replicated 4 times in a randomized complete block design. 

Another set of one-year old apple trees were used to determine the metabolism of paclobutrazol 

in different plant parts. Dormant trees were injected with 500 14C paclobutrazol (c-2 labeled) 5 

cm above the root/stem transition zone. The tops of the trees were pruned to 85 cm above the 

injection site. One group of 4 trees was harvested immediately after the injection while a second 

group of 4 trees was harvested 27 days after the injection, when signs of growth inhibition were 

apparent. The trees were separated into 4 parts: root stock containing the roots and injection 

site (Section I), 20 cm long section above the graft (Section 2), and two additional 20 cm 

sections up the main stem (Sections 3 and 4). The fibrous roots from tap roots, wood (xylem), 

bark (phloem), buds, and new shoots from each section were analyzed separately. Details of the 

extraction method can be found in the study. Treatments were replicated 4 times and the 

experiment was repeated once. 

Results 

Growth Inhibition 

Growth inhibition was observed for most of the species that were tested (Table 1 ). 

2 
Table 1. Growth inhibition for plants treated with paclobutrazol 

Species Observation 

Bean Statistically significant decrease in height, fresh weight, and leaf size 

after 7 days at the 0.1, I, I 0, I 00, and I 000 μg ai/plant dose levels (p S 

0.05) 

California privet Statistically significant reduction in sprout growth after 64 days at all 

dose levels - 1, I 0, I 00, and I 00 μg ai/plant (p S 0.05) 

Red maple Statistically significant suppression of shoot growth after 4 months for 

0.4 and 8 mg ai/plant (p S 0.05). A statistically significant difference was 

observed in June after trees were injected at bud break (April) and 30 

days after bud break (May). 

White ash No statistically significant reductions in shoot growth 4 months after 

application for any dose. A statistically significant difference was 

observed in June after trees were injected at bud break (April) and 30 

days after bud break (May). By early August, there was no statistically 

significant difference between injected plants and the negative control. 

Yell ow poplar A statistically significant difference was observed in June after trees were 

injected at bud break (April) and 30 days after bud break (May). By early 

August, there was no statistically significant difference between injected 

plants and the negative control. 

American sycamore A statistically significant difference was observed in June after trees were 

injected at bud break (April) and 30 days after bud break (May). By early 

August, there was no statistically signifi cant difference between injected 

plants and the negative control. 

Trans/ocation and Metabolism 

Immediately after injection, the paclobutrazol was located in the xylem and phloem of the stem 

and roots within 20 cm of the injection site. At 27 days, 23% of the 14C activity was in the shoots, 

11 % in the xylem and phloem tissue between the graft and apex, 58% in the xylem and phloem 

tissue near the injection site, and 8% in the roots. This indicates that paclobutrazol moved through 

the plant to tissues away from the initial injection site. Over 90% of the 14C activity in the xylem 

and phloem and over 85% in the shoot tissue after 27 days was determined to be paclobutrazol, 

indicating that little degradation occurred. 

Data Quality Evaluation 

Reference samples were run for the chromatograph analysis, for comparison with samples from 

the treated trees. All experiments were replicated. Other data quality information regarding the 

analyses were not presented in the study. 

Rationale for Use: 

The study presents information about the translocation of paclobutrazol in trees and documents 

that it reduces plant growth on a variety of species. It also presents information regarding the 

degradation of paclobutrazol, upon inj ection into the plant. 

3 
Limitations of Study: 

The study did not present quality control data regarding the analytical methods. 

Description of Use in Document: 

Qualitative. The translocation and metabolism data should be treated as qualitative information 

because the study did not provide details about the verification of the performance of the 

analytical methods. 

Primary Reviewer: Meghan Radtke, Ph.D., Biologist 

Oatc: 4/27/15 ~ ~ 

Secondary Reviewer: Edward Odenkirchen 

Date: 5/4/ 15 tf,(A.A/ OJ4 
Open Literature Review 

Chemical Name: Uniconazole 

PC Code: 128976 

CAS No. 83657-22-1 

MRID: 49577103 

ECOTOX Record Number and Citation: 

Arron, G.P. 1990. Translocation of uniconazole after trunk injection of silver maple saplings. 

Journal of Plant Growth Regulation, 9: 141-1 46. 

Purpose of Review: Registration Review 

Date of Review: 4/28115 

Description of Use: Qualitative 

Summary of Study Findings: 

Executive Summary 

The objectives of the study were to determine 1) the effect of trunk-inj ected uniconazole on 

silver maple growth saplings 18 months after application; 2) movement of uniconazole in the 

trees; 3) the proportion of uniconazole in fo liage during the first and second fall after application; 

and 4) the amount of unmetabolized uniconazole in leaves. The study shows that most of the 

uniconazole remained in the stem of the trees, near the injection site during two growing seasons. 

A higher percentage of uniconazole, compared to its metabolites, was detected in foliage at the 

end of each growing season, thus suggesting that metabolism is not occurring in the stems of 

trees. 

Methods 

Four-year old silver maple trees (Acer saccharinum) were used for the experiment. Prior to 

injection with uni conazole, the trees were trimmed such that approximately one third of the 

canopy was removed. For the "foliage" experiment, leaves were harvested over two growing 

seasons to track translocation of uniconazole. For the "harvest" experiment, the distribution of 

uniconazole in trees was measured by harvesting the whole sapling at different time periods. 

Trees were injected with 18 mg 14

C-uniconazole approximately 40 cm above the soil surface. 

Controls were injected with I .4 ml of isopropyl alcohol. For the foliage experiment, 5 trees were 

initially injected with uniconazole, but one of these was not a successful injection (very little 

1 
uniconazole activity was detected in the tree); there were 5 control trees. For the harvest 

experiment, 12 trees were injected with uniconazole and 6 with alcohol (controls). 

Shoot growth (extension growth of all shoots and sprouts originating at the root collar) was 

measured to the nearest 0.5 cm in the fall of 1987 and 1988 for all plants (foliage and harvest 

experiments). The median shoot lengths were compared using a Mann-Whitney non-parametric 

test. 

For the foliage experiment, 30 leaves were randomly sampled inunediately after injection (day 0) 

and then at 7, 17, 25, 35, 45, 65, 85, 101 , 122, 133, 375, 416, 444, 472, and 497 days later. In 

addition, all of the remaining foliage was removed from the trees on days 133 and 497. For the 

harvest experiment, tlu-ee saplings injected with uniconazole were dug up on day 0, 17, 134, and 

500 and separated by plant part: fibrous roots, tap root, main stem samples ( 15 cm sections), 

branched stem sections, 1986 stem growth sections, and foliage. All samples were frozen and 

stored at -1 6° C until analysis. See the study for details on the methodology used to measure 14Ccontent and the metabolism of uniconazole. 

Results 

Growth Inhibition 

Uniconazole did not affect the number of shoots or sprouts per sapling in either experiment. There 

was a significant reduction in the mean shoot length per sampling for the harvest experiment in 

both years. In addition, there was a significant reduction in median shoot length in 1998 for the 

foliage experiment. 

Trans/ocat ion 

During the growing seasons of 1987 and 1988, the 14C activity in the leaves collected in the foliage 

experiment linearly increased from late May to early October, suggesting that uniconazole is 

collecting in the foliage of the trees. 14C measurements at 0, 17, 134, and 500 days after injection 

indicated that most of the injected uniconazole was staying near the injection site in the main stem 

of the tree. A small amount of 14C was detected in branched stem sections and foliage at the 17. 

134, and 500 day measurement points. Table I shows the 14C-activity in several tissue types from 

the foliage experiment. 

2 
Table 1. •.ic-activity in tissue of silver maple saplings harvested at 0, 17, 134, and 500 days 

after trunk injection with uniconazole 

Days after 14C-activity % of total 14C-activity injected (as % of total 14C-activity 

injection retri eved retrieved) 

Whole saplings Stem sample (45 cm section Foliage 

bracketing injection site) 

0 86.7 ± 7.1 8 84.1 ± 8.8 (97.0 ± 3.5) 0.0 ± 0.0 (0.0 ± 0.0) 

17 67.6 ± 25 .6 49. I ± 18.0 (72.9 ± 4.8) 1.5 ± 0.7 (2.5 ± 1.6) 

134 62.6 ± 5.3 51.1 ± 4.5 (8 1.2 ± 4.2) 7.1 ± 1.7 (11.3 ± 2.5) 

500 52.7 ± 17.8 45.0 ± 18.7 (84.2 ± 8.0) 2.3 ± 0.6 ( 4.6 ± 1.8) 

avalues are given as mean ± SD (N = 3) 

Metabolism 

To determine the proportion of 14C-uniconazole versus metabolites, extracts were analyzed from 

samples collected on days 134 and 497 of the fol iage experiment. Uniconazole made up 53% and 

71 % of the 14C-activity detected in the leaves in the 134 and 497 day collections, respectively. 

This suggests that metabolism of uniconazole is not occurring in the stems, as a decrease in 

uniconazole was not seen at the 497 day sampling period. 

Data Quality Evaluation 

Reference sampl es were run for the chromatograph analysis, for comparison with samples from 

the treated trees. The number of replicates was limited (n = 3 for harvest study and n = 4 for 

foliage study), but usually the standard deviations were small. 

Rationale for Use: 

The study presents information about the translocation of uniconazole in trees and its 

accumulation in leaves during the growing season. Metabolism of uni conazole does not appear 

to take place in the stems/trunks of trees. Concentrations of uni conazole are reported for foliage 

samples. 

Limitations of Study: 

The study shows a peak foliage activity of 7. I +/- l.7 % at day 134; however, it should be noted 

that I I 7 days between the previous measurement had transpired so the study cannot reliably be 

used to determine the actual peak value. 

Description of Use in Document: 

Qualitative. The translocation and metabolism data should be treated as qualitative information 

because of limited repli cation and long intervals between sampling. 

3 
Primary Reviewer: Meghan Radtke, Ph.D., Biologist 

Date: 4/28/ 15 ~~ 

:::: :~ ~ev dGk~ ,: Science dvisor 

4