Appendix H Summary of Toxicity Studies For DMDS

Birds

The effect of DMDS on the Bobwhite quail ( Colinus virginianus ) is used to represent the effect for all birds, as well as reptiles and terrestrial-phase amphibians. Two acute avian studies were received, one for acute oral and one for acute inhalation. The Bobwhite ( Colinus virginianus ) acute oral LD 50 =314 mg/kg (250.2-403.0mg/kg) (MRID 470528-13) was classified as acceptable. The ac ute oral study will not be used in the assessment because no dietary exposure is expected.

The acute oral toxicity of DMDS to 31-wk-old Northern Bobwhite quail ( Colinus virginianus ) was assessed over 21 days. DMDS was administered to the birds by lubricating an appropriately do sed gelatin capsule and orally inserting directly into the crop at nominal doses of 0 (negativ e control), 37, 62, 104, 172, 287, 496 and 798 mg ai/kg bw. The 21 day-acute oral LD 50 was 314 mg ai/kg bw. The 21 day NOAEL of DMDS to the Northern Bobwhites ( Colinus virginianus ), based on a 17% reduction in mean male feed consumption was 104 mg ai/kg bw. According to the US EPA classification, DMDS would be classified as moderately toxic to Northern Bobwhites ( Colinus virginianus ) on an acute oral basis.

By test termination, mortality was 0% in the control and 0, 0, 0, 10, 40, 80 and 100% in the nominal 37, 62, 104, 172, 287, 496 and 798 mg ai/kg bw, respectively. There were

treatment-related reduc tions in body weight change and mean feed consumption in the 172-496 mg ai/kg bw treatment groups. The foll owing symptoms of toxicity were noted: ruffled appearance, lesions, reduced reacti on to external stimuli (sound and movement), wing droop, loss of coordination, lower limb wea kness, loss of rightin g reflex, prostrate posture and anorexia.

Gross necropsy results revealed he morrhaging of the lungs, discolored ki dneys, lungs and spleens and a gelatinous f ilm covering the surface of the crop, which appeared to be

opaque.

In an acute inhalation toxicity study, 16- to 17-week old northern bobwhite quail (five per sex; 181-220 g) were exposed by whole-body inhalation to DMDS (purity

≥ 98.53%) for 4 hours at analytically-determine d concentrations of 0 (control), 304, 402, 504, 608, and 1512 ppm (equivalent to 0, 1.17, 1.55, 1.94, 2.34, and 5.82 mg ai/L, respectively). The animals were observed for up to 14 days post-exposure then subjected to gross pathological examination.

At the control, 304, 402, 504, 608, and 1512 ppm levels, cumulative mortality was 0, 0, 1, 2, 4, and 5 for males, 0, 0, 1, 4, 5, and 5 for females, and 0, 0, 2, 6, 9, and 10 for combined sexes, respec tively. Except for a single male from the 608 ppm exposure

level that was euthanized in extremis on day 5, all mortality oc curred during the exposure period. The 4-hour LC 50 (with 95% C.I.) was 512 (420- 625) ppm for males, 449 (403- 501) ppm for females, and 478 (426-536) ppm for combined sexes.Clinical signs of toxicity we re observed in birds from the ≥ 402 ppm levels during and immediately following exposure; effects included impaired equi librium, prostration, gasping, hyperactivity, convulsions, twitchi ng, labored respira tion, and increased respiration. Surviving birds from the 402 pp m level completely recovered by day 2, but effects (impaired equilibrium, labored respiration, and/or white material on the anogenital or right leg areas) pe rsisted in survivors from th e 504 and 608 ppm levels through 14 days.

No treatment-related effects on body wei ght changes or food consumption were observed, and there were no remark able findings at necropsy.

Fish

Fish are represented by a warm-water species and a cold-water species. An acceptable acute Zebra fish ( Danio rerio ) study resulted in an LC 50 =7.50mg/L (MRID 470528-15). However, this was not the most se nsitive reported endpoint. In a 96-h acute toxicity study, Zebrafish ( Danio rerio ) were exposed to DMDS at initial measured concentrations of <0.010 (<LOQ; control), 5.30, 10.6, 22.3, 40.2 and 89.1 mg ai/L under static-renewal conditions; nominal concentr ations were 0 (negative control), 6.25, 12.5, 25, 50 and 100 mg/L. The 96-h LC 50 was 7.50 mg ai/L. The NOAEC value, based on mortality and sub-lethal effect s, was 5.30 mg ai/L. Sub-lethal effects (i.e., lethargy, fish lying on the side, missing escape reflex, fi sh lying on abdomen and hyperventilation) were observed in the groups exposed to initial measured concentrations of 10.6, 22.3 and 40.2 mg ai/L of DMDS. Based on the result s of this study, DMDS would be classified as moderately toxic to Zebrafish ( Danio rerio ) in accordance with the classification system of the U.S. EPA.

An acceptable acute Rainbow trout ( Oncorhynchus mykiss ) study (MRID 470528- 14) resulted in an LC 50 =0.96 mg/L that will be used in this assessment. In a 96-h acute toxicity study, Rainbow Trout ( Oncorhynchus mykiss ) were exposed to DMDS at initial measured concentrations of <0.010 (<LOQ; control), 0.303, 0.563, 1.36, 3.44 and 7.55 mg ai/L under static-renewal conditions; nominal concentrations were 0 (negative control), 0.625, 1.25, 2.5, 5 and 10 mg/L. The 96-h LC 50 was 0.960 mg ai/L. The NOAEC value, based on mortality and sub-leth al effects, was 0.563 mg ai/L. Sub-lethal effects (i.e., lethargy, staggering, slow escap e reflex, missing escape reflex, lying on the bottom of the test vessel and hyperventilation) were observed in the groups exposed to

initial measured concentrati ons of 1.36, 3.44 and 7.55 mg ai/L. Based on the results of this study, DMDS would be classified as hi ghly toxic to Rainbow Trout in accordance with the classification system of the U.S. EPA.

Aquatic Invertebrates

The 48-hour acute toxicity of DMDS to Daphnia magna was studied under staticrenewal conditions. Daphnids were exposed to nominal concentrations of 0 (negative control), 0.970, 2.13, 4.70, 10.3, 22.7, and 50 mg/L. Mortality and sub-lethal effects were observed daily. There wa s no control mortality over the 48h test period. The 48-hour LC 50 /EC 50 was 1.61 mg ai/L. The 48-hr NOAEC based on immobility was 0.621 mg ai/L. With the exception of immobility, no other biological effects were reported.

Estuarine/marine fish

In an acceptable 96-h acute toxicity study (MRID 473075-04), Sheepshead Minnow ( Cyprinodon variegates ) were exposed to DMDS at nominal concentrations of 0 (negative control), 2.6, 4.3, 7.2, 12 and 20 mg a. i./L under static renewal conditions. The

mean measured concentrati ons were <2.00 (<LOQ; nega tive control), 2.3, 4.0, 6.9, 10 and 18 mg a.i./L . The 96-h LC 50 (95% C.I.) was 5.6 (4.0-6.9) mg a.i/L. The NOAEC value was <2.3 mg a.i/L, based on sub-lethal effects observed at all levels; the EC 50 was determined to be 3.7 (2.3-6.9) mg a.i. /L, based on sub-lethal effects.

Cumulative mortality was 0% in the negative control, 2.3 and 4.0 mg a.i./L treatment groups. Cumulative mortality was 90% in the 6.9 mg a.i./L treatment group, and 100% in the 10 and 18 mg a.i./L treatmen t groups. Based on the results of this study, dimethyl disulfide would be classified as moderately toxic to Sheepshead Minnow ( Cyprinodon variegatus ) on an acute toxicity basis in accordance with the classification system of the U.S. EPA.

Signs of toxicity (lethargy, loss of equilibrium, lying on the bottom and surfacing) were observed during the test and were considered treatment related in all treatment groups. There were no sub-lethal effects obs erved in the control treatment group. Sublethal effects were noted in all subsequent treatment levels. Loss of equilibrium was observed in the 2.3 mg a.i./L treatment group at 72 hours. Loss of equilibrium was observed in the 4.0 mg a.i./L treatment group at 1.5, 24, 48, 72 and 96 hours. Surfacing and lying on the bottom was also observed in the 4.0 mg a.i./L treatment group at 72 hours. Surfacing and lethargy was additionally no ted in the 4.0 mg a.i./L treatment group at 96 hours. Loss of equilibrium was observed in the 6.9 mg a.i./L treatment group at 48 and 72 hours. Lying on the bottom was also obser ved in the 6.9 mg a.i./L treatment group at 72 hours. Lethargy was additi onally observed in the 6.9 mg a.i./L treatment group at 96 hours. Surfacing was observed in the 10 mg a.i./L treatment group at 24 hours. Lying on the bottom was noted in the 10 mg a.i./L treatment group at 48 hours.

Estuarine/marine invertebrates

The effect of DMDS on marine inverteb rates is represented by the mysid. An acceptable acute mysid toxicity test resu lted in an LC50=4.9mg/L (3.9-6.1) (MRID 473075-05). A NOEAC =2.5m g/L was also reported. Based on the LC 50 in this study (4.9 mg ai/L), dimethyl disulfide would be categorized as moderately toxic to mysid shrimp on an acute toxicity basis. This value will be used in the assessment.

A 96h static renewal test was conducted with juveniles <24h old. Nominal concentrations were 0 (negative cont rol), 1.1, 2.3, 4.5, 9.0 and 18 mg ai/L. Mean measured concentrations were <0.5, 1.1, 2.5, 4.3, 10.0, and 19 mg a.i./L. Twenty organisms per treatment level were used. There was no control mortality.Terrestrial Plants

The effect of DMDS on the vegetative vigor of monocot (corn, Zea mays ; onion, Allium cepa ; oat, A vena sativa; and ryegrass, Lolium perenne ) and dicot (cabbage,

Brassica oleracea ; soybean, Glycine max ; sugarbeet, Beta vulgaris ; lettuce, Lactuca

sativa ; tomato, Lycopersicon esculentum ; and radish, Rapahnus sativus ) crops was

studied at nominal concentrations of 18.8, 37.5, 75, 150, and 300 lbs a.i./A.

This study was conducted in a greenhouse. Th e controls and treatments were covered with plastic to retain the DM DS gas. After application, th e plants were set outside to reduce cross contamination. The plastic was cu t within 48h and the plan ts returned inside the greenhouse. On Day 21 the surviving plants per pot were recorded, cut, and dried for measuring the plant dry weight. Plant he ight was measured on days 7, 14, and 21 by measuring from the soil surface to the tip of the apical meristem or tip of the tallest leaf.

No measured concentrations were ma de for this study. Therefore, nominal concentrations were used in the analysis. Th e highest application ra te used in the study was 300lb/A. This is below the proposed a pplication rate of 455lb/A. The most sensitive dicot for both height and weight was to mato. An acceptable study resulted in an EC 25 =82 lbs/A for dry weight and an EC 25 =55 lbs/A for height. The tomato EC 25 value for height

will be used in this assessment. The NOAEC=37.5lb/A for tomato.

The study authors used a standard phytotoxic ity rating system. There were cases of necrosis, leaf curl, adventitious growth, mo rtality, and chlorosis across all species and most concentrations; there were generally less effects observed at the two lowest

treatment levels.

Terrestrial Invertebrates

The effect of DMDS on terrestrial invertebrates is represented by the Honey bee ( Apis mellifera ). The young adult worker bees were exposed for 48h. This acceptable study resulted in an LD 50 >100μg/bee (MRID 474712-03). This classifies DMDS as practically nontoxic to bees. This va lue will be used in the assessment.

Aquatic Plants

The effect of DMDS on vascular plants is represented by toxicity results for

Lemna . An acceptable study resulted in a Frond EC 50 =28mg/L. In a 7-day acceptable toxicity study (MRID 474712-04), the freshwat er floating aquatic vascular plants Duckweed ( Lemna gibba ) were exposed to dimethyl disulfide at nominal concentrations of 0 (negative control), 3.1, 6.3, 13, 25, 50, and 100 mg a.i./L under static renewal conditions. Time-weighted aver age concentrations were <2 .00 (<LOQ, control), 3.2, 5.6, 12, 24, 48, and 95 mg a.i./L.The 7-Day NOAEC and EC 50 values for frond density, th e most sensitive endpoint, were 3.2 and 28 mg a.i./L, respectively. The % growth inhibition in frond density, in the treated algal culture as compared to the negative control, ranged from 2.6 to 86%.

Chlorosis was observed at all treatment levels and was more widespread in the highest two treatment levels. Necrosis was observed in all treatment levels except the lowest test concentration, and was more prevalent in the three highest treatment levels. Mortality was mostly observed at the highest treatment level.

There were also three freshwater nonvascul ar aquatic plant studi es received. An acceptable study (MRID 474712-05) for the fres hwater algae (Anabaena flos-aquae) resulted in a NOAEC <0.17mg/L and a 96h EC50=0.32mg/L (0.11-0.90 mg/L) for cell density.

In a 96-hour acute toxicity study, cultures of the freshwater blue-green algae Anabaena flos-aquae were exposed to Dimethyl Disulfid e at nominal concentrations of 0 (negative control), 0.15, 0.38, 0.96, 2.4, 6.0, and 15 mg a.i./L under static conditions.

Mean-measured concentrations were <0.100 (<LOQ; control), 0.17, 0.34, 0.80, 1.9, 5.5, and 14 mg a.i./L.

The % growth inhibition in cell density, in the treated algal culture as compared to the negative control, ranged from 36 to 98%. The % growth i nhibition in biomass, in the treated algal culture as compared to the negati ve control, ranged from 13 to 94%. The % growth inhibition in growth rate, in the treate d algal culture as compared to the negative control, ranged from 9.5 to 72%.

There was no adherence to cells to any of the test chambers and aggregations were not observed in any of the treatment gr oups. Enlarged cells were observed at the three highest test concentrations.

The other two freshwater nonvascular plant studies were for one freshwater diatom and one freshwater alga. An acceptable nonvascular aquatic plant study (MRID 474712- 06) for the freshwater diatom ( Navicula pelliculosa ) resulted in an EC 50 =22 mg/L (20-23 mg/L) for biomass. In a 96-hour acute toxicity study, cult ures of the freshwater diatom Navicula pelliculosa were exposed to Dimethyl Disulfid e at nominal concentrations of 0 (negative control), 2.3, 3.9, 6.5, 11, 18, and 30 mg a.i./L under static conditions. Initialmeasured concentrations were <1.00 (<LOQ; control), 2.76, 5.10, 7.05, 11.9, 18.8, and 32.4 mg a.i./L.

The % growth inhibition in cell density, in the treated algal culture as compared to the negative control, ranged from -54 to -88% in the first five treatment levels, and was 94% at the highest treatment level. The % growth inhibition in biomass, in the treated algal culture as compared to the negative control, ranged from -28 to -32% in the first four treatment groups, and was 8.2 to 96% in the highest two treatment groups. The % growth inhibition in growth rate, in th e treated algal culture as comp ared to the negative control, ranged from -10 to -14% in all but the highest treatment group, which was 63%.A study (MRID 474712-07) received for the green algae ( Pseudokirchneriella subcapitata) was classified as supplemental and will only be used as a Tier I study (MRID 470528-17).

In a 72-hour acute toxicity study, cultures of Pseudokirchneriella subcapitata were exposed to DMDS at serial di lution factors of 1:16, 1:8, 1:4, 1:2 and 1:1 of an initial 100 mg/L stock solution under static conditions; a negative control was also included. The measured values at 0-hours were 5.73, 11.6, 21.9, 44.0 and 86.7 mg ai/L. The measured values at 72-hours were <LOQ, 7.2, 19.2, 39.8 and 78.9 mg ai/L. A mean measured

concentration could not be determined for th e 1:16 dilution factor and the mean measured

concentrations for the serial dilutions of 1:8, 1:4, 1:2 and 1:1 were 9.40, 20.6, 41.9 and 82.8 mg ai/L, respectively. The NOAEC and EC 50 /IC 50 values based on cell density were 5.73 and 17 mg ai/L, respectivel y. The % growth inhibition, based on cell densities at 72 hours, in the treated al gal culture as compared to the control ranged from -5 to 99%.

Biomass was the most sensit ive endpoint, with an EC 50 of 15 mg ai/L; the NOAEC (and

EC 05 ) for biomass was lower than the lowest treatment level (i.e., 5.73 mg ai/L), which inhibited growth 10%, rela tive to the control.

Cells in the 44.0 and 86.7 mg ai/L treatment groups were observed to be enlarged. No major pH shifts were observed.

In addition to the freshwater aquatic pl ant studies an estuarine/marine diatom study (MRID 474712-07) was classified as acceptable. This study with Skeletonema

costatum resulted in a biomass EC 50 =1.2 mg/L (0.55-2.6 mg/L).

In a 96-hour acute toxicity study, cultures of the marine diatom Skeletonema costatum were exposed to Dimethyl Disulfide at nomi nal concentrations of 0 (negative control), 0.58, 1.3, 2.8, 6.2, 14, and 30 mg a.i./L under static conditions. Mean-measured concentrations were <0.300 (<LOQ; c ontrol), 0.48, 0.95, 2.6, 5.7, 12, and 27 mg a.i./L.

The % growth inhibition in cell density, in the treated algal culture as compared to the negative control, ranged from 22 to 98%. The % growth i nhibition in biomass, in the treated algal culture as compared to the negati ve control, ranged from 29 to 96%. The % growth inhibition in growth rate, in the treate d algal culture as compared to the negative control, ranged from 5.8 to 94%.

The most sensitive endpoint was biomass, with NOAEC and EC 50 values of <0.48 and 1.2 mg a.i./L, respectively.

There was no aggregation/flo cculation of algae in the c ontrol or treatment groups.

Adherence of cells to the test chambers was observed in the negative control and the first

three test concentrations.