Data Evaluation Report for a 10-Day Toxicity Test with Marine Amphipod Leptocheirus 

plumulosus 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: Tribufos: 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 Chemical Corporation

4100 E. Washington Blvd. 

Los Angeles, CA 90023

Laboratory Report ID: 246A-114

MRID No.: 491648-01

DP Barcode: D414357

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

Signature: Date: 08/16/13

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

Signature: Date: 11/25/13

5. SECONDARY REVIEWER: Mary Clock-Rust, Biologist, ERB4, EFED, OPP, OCSPP, EPA

Signature: Date: 1/14/15 

 

6. STUDY PARAMETERS:

Age/size 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 Evaluation Report for a 10-Day Survival Toxicity Test with Marine Amphipod 

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

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normalized) and pore water

7. EXECUTIVE SUMMARY: 

In a 10-day study, technical grade tribufos (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 per 

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; measured 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 groups. 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:

LC50: >773 μg 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:

LC50: >22.7 μg 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 study is classified as scientifically sound and satisfies the guideline requirement for a 

whole sediment acute toxicity study with a marine invertebrate (Leptocheirus plumulosus).
Data Evaluation Report for a 10-Day Survival Toxicity Test with Marine Amphipod 

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

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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: Registration Review

11. MATERIALS AND METHODS:

A. Test Organisms

Guideline Criteria Reported Information

Species:

Ampelisca abdita, Eohaustorius estuarius, 

Leptocheirus plumulosus, or Rhepoxynius

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 organisms from the same source? Yes
Data Evaluation Report for a 10-Day Survival Toxicity Test with Marine Amphipod 

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

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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

Guideline 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, maintained 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 at 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 UVsterilized. 

Formulated sediment was prepared similar to 

that described in OECD Guideline 218 

(2004), 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% alphacellulose, 14% silt and clay (kaolin clay), and 

80% industrial quartz sand. The dry 

constituents 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 formulated sediment (sampled 

12/26/12). 
Data Evaluation Report for a 10-Day Survival Toxicity Test with Marine Amphipod 

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

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Guideline 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 μg/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 NH3) were <0.17 mg/L.

Water Temperature

15°C for E. estuarius and R. abronius, 20°C 

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

24.1 to 25.2C (daily monitoring)

23.7 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 (≥95% saturation)

Salinity

Overlying water salinity should 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 NH3
Data Evaluation Report for a 10-Day Survival Toxicity Test with Marine Amphipod 

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

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Guideline Criteria Reported Information

Sediment Characterization

All sediment must be characterized for: pH, 

ammonia concentration of pore water, organic 

carbon content (total organic carbon (TOC)), 

particle size distribution, and percent water 

content. 

Particle distribution – 83% sand, 5% silt, and 

12% clay

USDA classification – loamy sand

Organic matter 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 – 1.24 g/cm3

Ammonia concentration of pore water – not 

reported

Additional Sediment Analysis

BOD, COD, cation exchange capacity, Eh, 

total inorganic carbon, total volatile solids, 

acid volatile sulfides, metals, synthetic organic 

compounds, oil and grease, petroleum 

hydrocarbons, and interstitial water analysis.

None reported

Effect of Grain Size

Grain size may adversely affect some species 

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 sediments 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 Evaluation Report for a 10-Day Survival Toxicity Test with Marine Amphipod 

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

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Guideline Criteria Reported Information

Laboratory Spiked Sediment Common name: tribufos

IUPAC name: S,S,S-tributyl phosphorotrithioate 

CAS name: S,S,S-tributyl phosphorotrithioate

CAS No.: 78-48-8

Description: liquid

Lot No.: NX81AX0015

Purity: 98.2%

Storage: ambient conditions

Octanol-water partition coefficient (Kow): 

3.13 x 105

Freundlich soil adsorption coefficient (KF): 

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 acetone. 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 Evaluation Report for a 10-Day Survival Toxicity Test with Marine Amphipod 

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

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Guideline Criteria Reported Information

Test Concentrations For Spiked Sediment

For LC50 calculation, test concentrations 

should bracket the predicted LC50; 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. 

An 18-mL volume of the appropriate dosing 

solution was added to 150 g of sediment, and 

hand-mixed 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 600 

g of untreated sediment and mixed on a 

rotary mixer for 1 hour. Finally, 1050 g of 

untreated sediment was added to achieve a 

final weight of 1800 g, and the 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) was selected to 

establish the LC50 and NOAEC/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 beakers containing ca. 175 mL of 

sediment (ca. 2.0-cm depth) and ca. 775 mL

(ca. 8.6-cm depth) of overlying filtered 

seawater. 
Data Evaluation Report for a 10-Day Survival Toxicity Test with Marine Amphipod 

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

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Guideline 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 lux. 

Continuous lighting was provided. 

At test initiation, light 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 water. 

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 sediment
Data Evaluation Report for a 10-Day Survival Toxicity Test with Marine Amphipod 

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

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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 chambers; 

Overlying water is added to chambers in a 

manner that minimizes suspension of sediment.

Based upon the results of a concurrentlyrun pore water equilibration trial (see 

Reviewer’s Comments), the prepared 

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. Approximately three times per 

week during the equilibration period, 400 

mL of overlying water was removed and 

replaced with 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 the airwater 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 transfers were 

made beneath the water surface using widebore 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 rangefinding toxicity data (not provided). 
Data Evaluation Report for a 10-Day Survival Toxicity Test with Marine Amphipod 

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

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Guideline Criteria Reported Information

Monitoring the test

All test chambers should be checked daily and 

observations made to assess organism behavior 

such as sediment avoidance.

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 

used.

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 used.

100 amphipods 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 

chambers 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 Evaluation Report for a 10-Day Survival Toxicity Test with Marine Amphipod 

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

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Guideline Criteria Reported Information

Water Parameter Measurements

Temperature should be measured daily from at 

least one replicate per treatment, and must be 

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 should 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 were measured daily 

in 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. 

Ammonia and salinity were 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 Evaluation Report for a 10-Day Survival Toxicity Test with Marine Amphipod 

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

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12. REPORTED RESULTS:

A. General Results

Guideline Criteria Reported 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 exceptions: 

periodic analyses of salt water and sediment 

for potential contaminants.

Control Criteria 

Was recovery of organisms from control 

sediment should equal or exceed 90% in a 10-

day test or 80% in a 28-day test?

Yes, survival averaged 89 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 or 1100

μg 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? Yes
Data Evaluation Report for a 10-Day Survival Toxicity Test with Marine Amphipod 

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

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Effects 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 LOAEC 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 

(μg a.i./kg)

Mean-Measured(a)

Sediment

(μg a.i./kg)

(% of nominal)

Pore Water

(μg a.i./L)

Overlying

Water

(μg a.i./L)

Negative Control <LOQ <LOQ <LOQ 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 μg a.i./kg; the LOQ for overlying and pore water was 0.100 μg a.i./L.

* Statistically-significant difference (p<0.05) from the pooled control using Bonferroni’s t-test.
Data Evaluation Report for a 10-Day Survival Toxicity Test with Marine Amphipod 

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

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Other Significant Results:

Biological: According to the study authors, survival after 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 μg 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 μg a.i./kg level. The 10-day LC50 was >773 μg 

a.i./kg, and the NOAEC and LOAEC were 207 and 773 μg a.i./kg, respectively. 

Analytical: Results of tribufos from dosing stock solutions ranged from 104 to 124% of 

nominal concentrations. In the test systems, concentrations remained predominantly 

associated with the sediment, and were generally consistent during the 10-day study, with 

recoveries of 60 to 79% of nominal for 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 negative and solvent control survival responses using 

a T-test. No differences were observed. The study authors pooled the two sets of data. 

Percent survival data were evaluated for normality using the Chi-square test and for 

homogeneity of variance using Levene’s test. The data were deemed normal with 

homogeneous variance, and were analyzed using Bonferroni t-test to identify those 

treatment levels that were statistically different (p<0.05) from the pooled control. 

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

pattern and statistical analysis of the survival data. 

The 10-day LC50 value was observed to be greater than the highest concentration tested. 

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

of mean-measured sediment and pore water concentrations. 
Data Evaluation Report for a 10-Day Survival Toxicity Test with Marine Amphipod 

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

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In terms of mean-measured sediment concentrations:

LC50: >773 μg 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:

LC50: >22.7 μg a.i./L 95% C.I.: N/A

NOAEC: 7.10 μg a.i./L

LOAEC: 22.7 μg a.i./L

13. 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 homogeneous 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 unequal variances and 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 implemented by EFED on 

5/29/13.

14. REVIEWER'S COMMENTS: 

The reviewer’s conclusions differed from the study author’s. The reviewer identified no 

significant effects at any treatment level relative to the negative control using the Dunnett 

Multiple Comparison Test, resulting in NOAEC and LOAEC values of 773 μg a.i./kg and 

>773 μg a.i./kg, respectively. These differed from the study author’s NOAEC and LOAEC 

values of 207 μg a.i./kg and 773 μg a.i./kg, respectively. Similar results were observed for 

OC-normalized sediment 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 dose 

only using Bonferroni’s t-test (compared to the pooled control).

Results were provided 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 methods 
Data Evaluation Report for a 10-Day Survival Toxicity Test with Marine Amphipod 

Leptocheirus plumulosus Using Sediment Spiked with Tribufos EPA MRID Number 49164801

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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 for 10 

minutes per extraction. Samples were centrifuged, and the 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 the 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, 2013. 

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:

LC50: >773 μg a.i./kg 95% C.I.: N/A

NOAEC: 207 μg a.i./kg

LOAEC: 773 μg a.i./kg
Data Evaluation 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:

LC50: >22.7 μg a.i./L 95% C.I.: N/A

NOAEC: 7.10 μg a.i./L

LOAEC: 22.7 μg a.i./L

 

15. REFERENCES:

American Society for Testing and Materials. 2008. ASTM 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. Series 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 Drinking 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-Sediment 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. Western 

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.