Data Evaluation Report for a 10

- Day Toxicity Test with Marine Amphipod Leptocheirus

plumu losus Using Sediment Spiked with Tribufos EPA MRID Number 491648

- 01

DATA EVALUATION RECORD
WHOLE SEDIMENT ACUTE TOXICITY INVERTEBRATES , MARINE
OPPTS Guideline 850.1740

1. CHEMICAL : Tribufos PC Code : 074801

2. TEST MATERIAL : Tribufos Purity: 98.2%

3. CITATION : Authors : Thomas, S .T., et al.

Title : Tribufo s: A 10

- Day Survival Toxicity Test with the Marine Amphipod ( Leptocheirus plumulosus ) Using Spiked

Sediment. Study Completion Date : June 21, 2013

Laboratory : Wildlife International, Ltd.

8598 Commerce Drive Easton, MD 21601 Sponsor : Amvac Chemica l Corporation

4100 E. Washington Blvd. Los Angeles, CA 90023 Laboratory Report ID : 246A

- 114

MRID No. : 491648

- 01

DP Barcode : D 414357

4. REVIEWED BY : Christie E. Padova, Staff Scientist, CSS

- Dynamac Corporation

Signature: Date: 0 8 / 1 6 / 1 3

APPROVED BY : Teri S. Myers, Environmental Scientist, CDM Smith

Signature: Date: 11/25/13

5. S ECONDARY REVIEWER : Mary Clock

- Rust, Biologist, ERB4, EFED, OPP, OCSPP, EPA Signature: Date: 1 /14 /1 5

6. STUDY PARAMETERS :

Age /s ize of Test Organism: 2

- 4 mm

Definitive Test Duration: 10 days

Study Method: Static , with aeration Type of Concentrations: Mean

- measured sediment ( bulk and OC

-Data Evaluati on Report for a 10

- Day Survival Toxicity Test with Marine Amphipod

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

2 normalized ) and pore water

7. EXECUTIVE SUMMARY :

In a 10

- day study, technical grade trib ufos (98.2% active ingredient) was used to spike formulated sediment and allowed to equilibrate for 32 days prior to introduction of the marine amphipod Leptocheirus plumulosus at 20 /replicate and 5 replicates per treatment. An additional 3 replicates pe r treatment were run for monitoring exposure concentrations at study initiation, Day 0 and Day 10; only the beaker used for monitoring 10

- day exposure had amphipods as well. Test concentrations were measured in sediment, overlying water and sediment pore water. Nominal exposure concentrations were control, solvent control (acetone 0.01 mL/ g sediment ) 10, 32, 103, 328 and 1050 μg a.i./kg dw sediment; measured concentrations were < 2.02 (LOQ), <2.02 6.9, 20, 64, 207, 773 μg a.i./kg dw, respectively; measure d sediment concentrations as a percent of nominal ranged between 62

– 74%. Measured pore water concentrations were <0.1, <0.1, 0.176, 0.381, 1.86, 7.10 and 22.7 μg a.i./L in the control, solvent control, 10, 32, 103, 328 and 1050 μg a.i./kg dw sediment gro ups. According to the study authors, mean survival (± std dev) at the highest treatment level (74±21.6) was significantly different (p<0.05) from pooled controls (92±7.89%) ; however, the reviewer’s analysis compared treatment groups to the negative control and did not indicate any statistically significant differences. Since the study author’s analysis results in a more conservative estimate of effects in terms of a NOAEC, the more conservative endpoints are used. The study is classified as scientifically sound and is consistent with guideline recommendations.

Results Synopsis:

In terms of mean

- measured sediment concentrations : L C 50 : > 773 μ g a.i./ kg 95% C.I.: N/A

NOAEC: 207 μg a.i./kg

LOAEC: 773 μg a.i./kg

In terms of mean

- measured pore water concentrations : L C 50 : > 22.7 μ g a.i./L 95% C.I.: N/A

NOAEC: 7.10 μg a.i./L

LOAEC: 22.7 μg a.i./L

8. ADEQUACY OF THE STUDY :

This stu dy is classified as scientifically sound and satisfies the guideline requirement for a whole sediment acute toxicity study with a marine invertebrate ( Leptocheirus plumulos us ).Data Evaluati on Report for a 10

- Day Survival Toxicity Test with Marine Amphipod

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

3 9. MAJOR GUIDELINE DEVIATIONS :

Nominal concentration levels were spaced by a factor of 3.2, whereas guidance recommends

a maximum factor of 2X.

10. SUBMISSION PURPOSE : R egistration R eview

1 1

. MATERIALS AND METHODS :

A. Test Organisms

Guideline Criteria Reported Information

Species:

A mpelisca abdit a, E ohaustorius estuarius,

L eptocheirus plumulosus, or R hepoxynius

abronius Leptocheirus plumulosus

Life Stage:

2

- 4 mm for L. plumulosus to 3

- 5 mm for the three remaining species. No mature male or

female A. abdita or L. plumulosus should be

used for testing. 2

- 4 mm

The supplier verified the identity and size of the species.

Supplier The methods for collection of test organisms are species

- specific. Organisms obtained from

the wild are permissible. Chesapeake Cultures Hayes, VA

All organism s from the same source? YesData Evaluati on Report for a 10

- Day Survival Toxicity Test with Marine Amphipod

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

4 B. Source/Acclimation

Guideline Criteria Reported Information

Acclimation Period : The organisms were used in the test the same day they were received.

Feeding: None

Pretest Mortality: Not reported

C. Test System

Gu ideline Criteria Reported Information

Source of dilution water (overlying water)

and sediment: Natural sea water is preferable, but reconstituted water is acceptable. Natural sea

water should be from an uncontaminated source, covered, mai n tained at 4°C, and used within 2 days of collection.

Uncontaminated natural sediment is

recommended , but formulated sediment is acceptable

. Natural sediments should be

stored at 4°C in the dark and used within 2 to

8 weeks of collection. Natural seawater collected a t Indian River Inlet, DE was passed through a sand filter (25 μm) , diluted to a salinity of approximately 20 ‰ with on

- site laboratory well water, and aerated. Prior to use, the water was again filtered (0.45 μm) and UV

sterilized

.

Formulated sediment was prepared similar to

that described in OECD Guideline 2 18 (200 4 ), but used alpha

- cellulose as the source of organic matter instead of peat moss. The sediment was composed of ca

. 1% humic acid and dolomite, 5% alpha

cellulose, 14% silt and clay (kaolin clay), and 80% industrial quartz sand. The dry

constituen ts were mixed in a PK Twinshell ®

mixer for 20 minutes and stored under

ambient conditions until use.

The results of periodic analyses to measure selected organic and inorganic constituents were provided for dilution water (sampled

12/26/12 ) and formulat ed sediment (sampled 12/26/12).Data Evaluati on Report for a 10

- Day Survival Toxicity Test with Marine Amphipod

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

5 Gu ideline Criteria Reported Information

Does water support test animals without observable signs of stress? Yes

Quality Of Water If problems are observed in culturing or testing of organisms, it is desirable to test water quality. Particulate, TOC, COD should be <5 mg/L and residual chlorine <11 μ g/L

. There were no apparent problems with water quality. Measurements of particulate matter, TOC, COD, and residual chlorine were not provided.

During the study, measured ammonia levels (as NH 3 ) were <0.17 mg/L.

Water Temperature
15°C for E. estuari us and R. abronius , 20°C

for A. abdita , and 25°C for L. plumulosus. 2 4

. 1 to 2 5

. 2  C (daily monitoring)

2 3 .7 to 25.1  C (continuous monitoring)

pH 8.1 to 8. 2

Dissolved Oxygen Results are unacceptable if DO falls to <60%

saturation. 7.0 to 7 .5 mg/ L ( ≥ 9 5 % saturation)

Salinity Overlying water salinity s hould be 28 ppt for

A. abdita and R. abronius and 20 ppt for E.

estuarius and L. plumulosus. 20 to 22 ‰

Ammonia <0.17 mg/L (<LOD) as NH 3Data Evaluati on Report for a 10

- Day Survival Toxicity Test with Marine Amphipod

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

6 Gu ideline Criteria Reported Information

Sediment Characterization

All sediment must be characterized fo r: pH, ammonia concentration of pore water, organic carbon content (total organic carbon (TOC)), particle size distribution, and percent water

content. Particle distribution

– 8 3 % sand, 5 % silt, and

1 2 % clay

USDA classification

– loam y sand Organic matt er content

– 4.0 % Organic carbon content

– 2.3 %
Moisture at 1/3 Bar

– 10.6 % pH

– 6.5

CEC

– 2.8 meq/100 g Bulk density

– 1. 2 4 g/cm 3 Ammonia concentration of pore water

– not reported

Additional Sediment Analysis BOD, COD, cation exchange capacity, Eh,

tot al inorganic carbon, total volatile solids, acid volatile sulfides, metals, synthetic organic compounds, oil and grease, petroleum hydrocarbons, and interstitial water analysis. No ne reported

Effect of Grain Size Grain size may adversely affect some spec ies of amphipod:

A. abdita : survival may be impacted in sediments containing 95% or more

sand. L. plumulosus : survival should not be impacted in clean sediments containing 100% sand to 100% sand + clay.

E

. estuarius : survival is unaffected by

clean sedime nts containing 0.6 to 100% sand. However, increased mortality may be associated with increased proportions of fine

- grained

sediment.

R

. abronius : very fine grains, particularly silts and clays, may reduce survival. As 89 and 95% survival was observed in the negative and solvent control group, respectively, sediment grain size did not adversely affect survival of this species.Data Evaluati on Report for a 10

- Day Survival Toxicity Test with Marine Amphipod

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

7 Gu ideline Criteria Reported Information

Laboratory Spiked Sediment Common name: tribufos

IUPAC name: S,S,S

- tributyl phosphoro

trithioate

CAS name: S,S,S

- tributyl phosphoro

trithioate

CAS No.: 78

- 48

- 8 Description: liquid

Lot No.: NX81AX0015 Purity: 98.2% Storage: ambient conditions

Octanol

- water partition coefficient (K ow ):

3.13 x 10 5 Freundlich soil adsorption coefficient (K F ): 0.26 L/kg Aqueous solubility: 2.3 mg/L at 20°C

Stock Solutions

Test material should be dissolved in a solvent prior to mixing into test sediment; If solvent is used, both solvent control and negative control are required. A 1050 μg a.i./ mL primary stock solution was prepared in acet one. The primary stock solution was then serially diluted with acetone to prepare the four secondary stocks (1.0, 3.2, 10.3, 32.8 and 105 μg a.i./ mL).

A solvent control and negative control group

were included in the test.Data Evaluati on Report for a 10

- Day Survival Toxicity Test with Marine Amphipod

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

8 Gu ideline Criteria Reported Information

Test Concentrations For Spik ed Sediment

For LC 50 calculation, test concentrations should bracket the predicted LC 50 ; sediment concentrations may be normalized to factors other than dry weight ( e.g

. , organic content, acid volatile sulfides); Sediment may be mixed using rolling mill, feed mixer or hand mixer. Nominal test concentrations were prepared on a dry weight (dw) sediment basis and were corrected for the percent active ingredient

.

A n 1 8

- mL volume of the appropriate dosing solution was added to 150 g of sediment, and

hand

- mi xed with a stir rod. The 150

- g premixes were then placed in a fume hood

for ca. 1 hour to allow the solvent to partially dissipate. The premix was then added to 6 00 g of untreated sediment and mixed on a rotary mixer for 1 hour. Finally, 1050 g of

untre ated sediment was added to achieve a final weight of 1 8 00 g , and t he batch sediments were mixed on a rotary mixer ca. 1 hour.

For concurrent pore water equilibration trials, an additional 1000 g of sediment were prepared for the 10 (low) and 1050 (high) μg a.i./ kg levels using the same procedure as previously described with the exception of the weights of sediment and volume of stock (10 mL was added).

The range of concentrations ( 10 to 1050 mg/kg dw sediment) w as selected to

establish the LC 50 and NO AEC/LOAEC.

Test Aquaria

1. Material : Glass or stainless steel or perfluorocarbon plastics.

2. Size : 1 L chambers containing 175 ml (2 cm) of sediment and 800 ml of overlying

water. 1. Glass beakers with loose covers

2. 1 L beak ers containing ca. 175 m L of sediment ( ca. 2.0

- cm depth ) and ca. 775 m L ( ca. 8. 6

- cm depth) of overlying filtered

sea water

.Data Evaluati on Report for a 10

- Day Survival Toxicity Test with Marine Amphipod

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

9 Gu ideline Criteria Reported Information

Type of Dilution System

Static Static

During the test, reverse osmosis water was added to the chambers as needed to replace

the small amounts of water lost due to evaporation.

Aeration Overlying sea water should be continuously aerated except when test organisms are being

added. DO should be maintained at approximately 90% saturation using gentle aeration without disturbing the sediment. Air was bubbled into the test chambers at a

rate of >1 bubble per second, but not so great

as to disturb the sediment.

Photoperiod Constant lighting to assure that the test organisms remain burrowed. Recommended intensity is 500 to 1000 lu x. Continuous lighting was provided.

At test initiation, l ight intensity was 947 lux

at the surface of the water over one representative test chamber

.

Solvents Use of a solvent should be avoided since they may influence the concentration in pore w ater. If used, it should not exceed 0.5 mL/L for

static tests or 0.1 mL/L for flow

- through tests. Acceptable solvents include triethylene glycol, methanol, ethanol, or acetone. Surfactants

should not be used. Acetone, 0.01 mL /g sed imentData Evaluati on Report for a 10

- Day Survival Toxicity Test with Marine Amphipod

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

10 D. Test Design

Guideline Criteria Reported Information

Sediment Into Test Chambers One day prior (Day

- 1) to start of test: test sediment, reference sediment, and negative control sediment should be thoroughly homogenized and added to test cha mbers; Overlying water is added to chambers in a manner that minimizes suspension of sediment. Based upon the results of a concurrently

run pore water equilibration trial (see Reviewer’s Comments), t he prepa red sediment/water systems were positioned in

a randomized order in an environmental

chamber and allowed to equilibrate for ca. 32 days prior to introduction of the test organisms . A pproximately three times per week during the equilibration period, 400 mL of overlying water was removed and replaced wit h clean dilution water.

Renewal of Overlying Water: The overlying water does not have to be

renewed. None during the definitive study

Placing Organisms in Test Chambers: Should be handled as little as possible and introduced into overlying water below t he air

water interface. Amphipods were sequentially assigned one

and two at a time into transfer chambers

until each chamber contained 20 organisms. Each group of 20 organisms was then impartially assigned and transferred to exposure chambers. All trans fers were

made beneath the water surface using wide

bore pipettes.

Range Finding Test A definitive test will not be required if no toxicity is observed at concentrations of 100 mg/kg dry weight of sediment

. Nominal test concentrations were selected

in consultation with the sponsor and were based on the results of exploratory range

finding toxicity data (not provided).Data Evaluati on Report for a 10

- Day Survival Toxicity Test with Marine Amphipod

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

11 Guideline Criteria Reported Information

Monitoring the test All test chambers should be checked daily and observations made to assess organism behavior

such as sediment avoid ance. Test vessels were observed daily for mortality and abnormal behavior.

Nominal Concentrations of Definitive Test Control(s) and at least 5 test concentrations; dilution factor not greater than 50%. Concentrations above aqueous solubility may be

u sed. 0 (negative control ), 0 (solvent control), 10, 32, 103, 328 and 1050 μg a.i./ kg dw

sediment

Aqueous solubility was 2.3 mg/L at 20°C

.

Number of Test Organisms

20 organisms per test chamber are

recommended. Five replicates per treatment

should be us ed. 100 amph i pods per level, with 20 amphipods per replicate beaker

, and five biological replicates per level

An additional three replicates per level

were maintained : one for test initiation water chemistry plus two chambers for analytical purposes (Days 0 and 10)

. The

chamber s prepared for Day

- 10 analytical

purposes contained organisms, while the ones prepared at test initiation for water chemistry and Day

- 0 analytical purposes

did not.

Test organisms randomly or impartially assigned to test vessels? Yes

Feeding Organisms do not have to be fed during the test. The amphipods were not fed during the test.Data Evaluati on Report for a 10

- Day Survival Toxicity Test with Marine Amphipod

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

12 Guideline Criteria Reported Information

Water Parameter Measurements Temperature should be measured daily from at least one replicate per treatment, and must b e monitored continuously in water bath or exposure chamber.

Salinity, DO, and pH should be measured in overlying water daily in one test chamber per treatment, and should be measured in all test chambers at study initiation and termination.

Ammonia s hould be measured near day 2 and day 8, and should be accompanied by pH and
temperature measurements.

pH, temperature, and ammonia concentration should be measured in pore water at the beginning of the test. Temperature and DO w ere measured daily

i n overlying water from one alternating replicate chamber of each group

. Temperature was also continuously in a beaker of water placed adjacent to the test

chambers. The pH was measured in overlying water from one replicate on Days 0, 3

, 7 and 10.

Amm onia and s alinity w ere measured in the overlying water of an additional replicate prepared at each level at test initiation and

from a composite sample from each biological replicate at test termination.

Pore water was not monitored.

Chemical Analysis Concentrations should be measured in sediment, interstitial water, and overlying water at the beginning and end of the test. Degradation products should also be measured where appropriate. The test concentrations were measured in the sediment, overlying test water, and the pore water of the sediment on Days 0 and

10.

Pore water was isolated from sediment using centrifugation at ca. 3000 rpm for ca. 10 minutes.Data Evaluati on Report for a 10

- Day Survival Toxicity Test with Marine Amphipod

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

13 1 2

. REPORTED RESULTS :

A. General Results

Guideline Criteria Repo rted Information

Quality assurance and GLP compliance statements were included in the report? Yes. This study was performed in compliance with GLP standards as published by the U.S. EPA (40 CFR Parts 160 and 792), OECD [ENV/MC/CHEM( 98)17], and Japan MAFF (11 NohSan, Notification No. 6283, 1999), with the following exception s : periodic analyses of salt water and sediment for potential contaminants

.

Control Criteria Was recovery of organisms from control sediment should equal or exc eed 90% in a 10

- day test or 80% in a 28

- day test? Yes, survival averaged 8 9 and 95 % for the negative and solvent control groups

, respectively

Percent Recovery of Chemical: Sediment: 88.6 ± 5.74 % of nominal (based on QC samples fortified at 10

. 0 , 100 o r 1100

μ g a.i./ kg and analyzed with the sample set)

No further QC samples were generated.

Data Endpoints

- Survival

- Reburial, optional for E. estuarius, L. plumulosus, and R. abronius. - Survival

Raw data included? YesData Evaluati on Report for a 10

- Day Survival Toxicity Test with Marine Amphipod

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

14 Eff ects Data
The concentrations of tribufos and survival of Leptocheirus are presented in Table 1 below. The study author identified an effect on survival at the highest dose level (LOAEC=773 ug a.i./kg) by making a comparison to the pooled controls and using Bonferroni’s t

- test. The reviewer made all comparisons to the negative control and identified a LOAE C of >773 ug a.i./kg.

Table 1 . Nominal and measured concentrations of tribufos in whole sediment, pore water and overlying water and 10

- day survival of Leptocheirus plumulosus

.

Toxicant Concentration

Percent
Survival ± SD Nominal
( μ g a.i./ kg) Mean

- Measured (a)

Sediment ( μ g a.i./ kg)
(% of nominal) Pore Water

( μ g a.i./ L) Overlying

Water
( μ g a.i./ L)

Negative Control <LOQ <LOQ <L OQ 89 ± 10.8

Solvent Control <LOQ <LOQ <LOQ 95 ± 0.00

Pooled Control

--- --- --- 92 ± 7.89

10 6.9

(69%) 0.176 <LOQ 93 ± 5.70

32 20

(63%) 0.381 <LOQ 93 ± 4.47

103 64

62%) 1.86 <LOQ 93 ± 9.75

328 207

(63%) 7.10 <LOQ 90 ± 6.12

1050 773

(74%) 22.7 0.09 74 ± 21.6*

(a) The LOQ for sediment was 2.00

- 2.02 μ g a.i./ kg ; the LOQ for overlying and pore water was 0.100 μg a.i./ L. * Statistically

- significant diffe r ence (p < 0.05) from the pooled control using Bonferroni’s t

- test

.Data Evaluati on Report for a 10

- Day Survival Toxicity Test with Marine Amphipod

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

15 Other Significant Results:

Biological: According to the study authors, s urvival afte r 10 days averaged 89 and 95% for the negative and solvent control levels, respectively, compared to 93, 93, 93, 90 and 74 %
for the mean

- measured 6.9, 20, 64, 207 and 773 μ g a.i./ kg sediment levels, respectively ( Table 1 )

. Bonferroni’s t

- test indicated a statistically

- significant difference (p<0.05) compared to the pooled control at the 773 μ g a.i./ kg level. The 10

- day LC 50 was > 773 μ g a.i./ kg, and the NOAEC and LOAEC were 207 and 773 μ g a.i./ kg, respectively.

Analytical: Results of tribufos from dosi ng stock solutions ranged from 104 to 124% of nominal concentrations. In the test systems, c oncentrations remained predominantly associated with the sediment, and were generally consistent during the 10

- day study , with
recoveries of 60 to 79% of nominal f or all levels ( both intervals )

. Overall mean

- measured concentrations in sediment ranged from 62 to 74% of nominal levels ( Table 1 )

.

In overlying water, tribufos was only above the LOQ once, on Day 10 at the 1050 μg a.i./ kg level, when tribufos was detected at 0.130 μg a.i./ L. In pore water, concentrations generally increased (with 0

- to 10

- Day percent differences of +18 to +85%), except at the 1050 μg

a.i./ kg level, where the concentration of tribufos decreased 10% between 0 and 10 Days.

Mean

- measured pore water concentrations were 0.176, 0.381, 1.86, 7.10 and 22.7 μg a.i./ L for the nominal 10, 32, 103, 328 and 1050 μg a.i./ kg sediment levels, respectively ( Table 1 )

.

B. Statistical Results

Method: The study authors compared n egative and solvent control survival responses using

a T

- test. N o differences were observed. The study authors pooled the two sets of data.

P ercent survival data were evaluated for normality using the Ch i

- square test and for

h omogeneity of variance using Levene’s test. The data were deemed normal with homogeneous variance, and were analyzed using Bonferroni t

- test to identify those

t reatment levels that were statistically different (p<0.05) from the pool ed control.

The NOAEC and the LOAEC were determined by visual interpretation of the dose

- respo n se pattern and statistical analysis of the survival data.

The 10

- day LC 50 value was observed to be greater than the highest concentration tested.

All st atistical analyses were conducted using TOXSTAT Version 3.5 , and reported in terms

of mean

- measured sediment and pore water concentrations.Data Evaluati on Report for a 10

- Day Survival Toxicity Test with Marine Amphipod

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

16 In terms of mean

- measured sediment concentrations : L C 50 : > 773 μ g a.i./ kg 95% C.I.: N/A

NOAEC: 207 μg a.i./ kg

LOAEC: 773 μg a.i./ kg

In terms of mean

- measured pore water concentrations : L C 50 : > 22.7 μ g a.i./ L 95% C.I.: N/A

NOAEC: 7.10 μg a.i./ L

LOAEC: 22.7 μg a.i./ L

1 3

. VERIFICATION OF STATISTICAL RESULTS :

Statistical Method: The reviewer statistically verified survival results by first testing for normality and homogeneity of the data using the Shapiro Wilk’s and Bartlett’s tests, respectively. The data were found to be normally distributed with h omoge ne ous variance. The data did not follow a linear decrease, but the response at the highest levels appeared to

be dose

- dependent. The negative and solvent control groups were compared using the

Wilcoxon Rank Sum Two

- Sample T

- test assuming un equal variances an d nonparametric

distribution ; no significant difference was detected (p>0.05) and subsequent treatment level comparisons were made to the negative control response only. These analyses were conducted using CETIS v. 1.8.7.12 with backend settings implemente d by EFED on

5/29/13.

1 4

. REVIEWER'S COMMENTS :

The reviewer’s conclusions differed from the study author’s. T he reviewer identified no significant effects at any treatment level relative to the negative control using the Dunnett Multiple Comparison Te st , resulting in NOAEC and LOAEC values of 773 μ g a.i./ kg and >773 μ g a.i./ kg , respectively. These differed from the study author’s NOAEC and LOAEC

values of 207 μ g a.i./ kg and 773 μ g a.i./ kg , respectively . Simi lar results were observed for

OC

- normalized s ediment and pore water. Comparison of the treatment levels to the negative control only, rather than a pooled control, may have contributed to the reviewer’s differing conclusion from the study author, who identified an effect for survival at the highest d ose only using Bonferroni’s t

- test (compared to the pooled control)

.

Results were prov ided in terms of mean

- measured sediment and pore water concentrations

. In addition, the reviewer provided results in terms of OC

- normalized sediment

concentrations.

The analytical methods used to analyze the test samples were based upon methodsData Evaluati on Report for a 10

- Day Survival Toxicity Test with Marine Amphipod

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

17 developed by the Wildlife International. Aliquots of overlying water and isolated pore

water were centrifuged at 14,500 rpm for 5 minutes

, diluted to within the range of the calibration curve with 50:50 (v:v) acetonitrile:HPLC

- grade water , and analyzed for tribufos using liquid chromatography coupled with tandem mass spectroscopy ( LC

- MS/MS )

. Portions of isolated sediment were extracted twice by sonicating with acetonitrile fo r 10 minutes per extraction. Samples were centrifuged, and t he combined extracts were diluted

with high pressure liquid chromatography ( HPLC )

- grade water . Secondary dilutions were performed, as necessary, with 50:50 (v:v) acetonitrile:HPLC

- grade water to bring the nominal concentration within the calibration range, and aliquots were analyzed for tribufos using LC

- MS/MS.

Prior to the definitive study, a 14

- day non

- GLP test was conducted in order to establish the time required to reach equilibration in t he treated sediment. In the first trial, sediment was dosed at 0.01 and 100 μg a.i./ kg and held in test chambers under test conditions. Samples were collected after 48 hours, 7 days, 9 days and 14 days of equilibration, and

concentrations of tribufos in isolated pore water from the 100 μg a.i./ kg samples were 9.01, 6.13, 6.97 and 8.45 μg a.i./ L, respectively. All pore water results from the 0.01 μg a.i./ kg samples were <LOQ (<0.100 μg a.i./ L). Since concentrations in the pore water did not appear to be in equilibrium after 14 days, a second non

- GLP trial was conducted in conjunction with the definitive test. In the second trial, an additional four sacrificial replicates of sediment per level were dosed at the low and high treatment concentrations ( i.e.

, 10 and 1050 μg a.i./ kg sediment) and maintained for up to 28 days. After 14, 17, 21 and 28 days, concentrations of tribufos in pore water from the 10

- μg a.i./ kg sediment spikes were 0.115, 0.312, 0.439 and 0.310 μg a.i./ L, respectively, and from the 1050

- μg a.i./ kg sediment spikes were 15.1, 22.6, 28.6 and 28.0 μg a.i./ L, respectively. The results of the second trial indicated that equilibration had been achieved by 28 days.

The experimental phase of the definitive study was conducted from March 19

- 30

, 201 3

.

Given that the study authors derived a more sensitive endpoint based on comparisons to the pooled control, the reviewers defer to the more sensitive endpoints reported by the study

authors.

In terms of mean

- measured sediment concentrations : L C 50 : > 773 μ g a.i./ kg 95% C.I.: N/A

NOAEC: 207 μg a.i./kg

LOAEC: 773 μg a.i./kgData Evaluati on Report for a 10

- Day Survival Toxicity Test with Marine Amphipod

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

18 In terms of mean

- measured pore water concentrations : L C 50 : > 22.7 μ g a.i./L 95% C.I.: N/A

NOAEC: 7.10 μg a.i./L

LOAEC: 22.7 μg a.i./L

1 5

. REFERENCES :

A merican Society fo r Testing and Materials. 2008. A STM Standard E 1367

- 03

. Standard Test Method for Measuring the Toxicity of Sediment

- Associated Contaminants with Estuarine

and Marine Invertebrates.

U.S. Environmental Protection Agency. 1996. S eries 850

- Ecological Effects Test Guidelines ( draft ), OPPTS 850.1740: Whole Sediment Acute Toxicity Invertebrates , Marine

.

U.S. Environmental Protection Agency. 2008. Preliminary Problem Formulation for Ecological Risk, Endarngered Species, and Dri nking Wter Exposure Assessments for Tribufos. Office of Prevention, Pesticides and Toxic Substances, Washington DC.

Kemble, N.E., et al. 1999. Tolerance of Freshwater Test Organisms to Formulated Sediments for use as Control Materials in Whole

- Sedime nt Toxicity Tests. Environ. Toxicol. Chem.

18:222

- 230.

OECD . 2004. OECD Guideline 218. Sediment

- Water Chironomid Toxicity Test Using Spiked

Sediment. Adopted 13 April.

West, Inc., and D.D. Gulley. 1996. TOXSTAT Version 3.5. Copyright 1996. We stern EcoSystems Technology, Inc., Cheyenne, WY.

U.S. Environmental Protection Agency. 1996. Series 850

- Ecological Effects Test Guidelines ( draft ), OPPTS 850.1735: Whole Sediment Acute Toxicity Invertebrates , Freshwater.