Data Evaluation Report for a 10-Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 491648-02

DATA EVALUATION RECORD

WHOLE SEDIMENT ACUTE TOXICITY INVERTEBRATES, FRESHWATER

OPPTS Guideline 850.1735

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 and Growth Toxicity Test with 

the Freshwater Amphipod (Hyalella azteca) 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-113A

MRID No.: 491648-02

DP Barcode: D414359

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. Reviewed By: Mary Clock-Rust, Biologist, ERB4, EFED, OPP, OCSPP, EPA

Signature: Date: 1/14/15 

 

 

6. STUDY PARAMETERS:

Age of Test Organism: ≈7 days old

Definitive Test Duration: 10 days

Study Method: Flow-through
Data Evaluation Report for a 10-Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

2

Type of Concentrations: Mean-measured sediment (bulk and OCnormalized) 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 freshwater amphipod 

Hyalella azteca (~7 days old) at 10 /replicate and 8 replicates per treatment. An additional two

replicates per treatment were run for monitoring exposure concentrations at 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 (acetone; 0.01 mL/g) control, 10, 32, 103, 328 and 1050 μg a.i./kg dw sediment. 

Measured concentrations in sediment averaged < 2.0 (<LOQ), <2.0, 7, 20, 71, 220, and 693 μg a.i./kg

sediment in the negative, solvent, 10, 32, 103, 328 and 1050 μg a.i./kg dw sediment groups, 

respectively, representing between 63 and 70% of nominal. Measured pore water concentrations were 

<0.01 (<LOQ), <0.01, 0.210, 0.699, 2.81, 8.27 and 34.1 μg a.i./L in the negative, solvent, 10, 32, 103, 

328 and 1050 μg a.i./kg dw sediment groups, respectively. After 10 days, mean (± std dev) survival 

was significantly (p<0.05) different in the solvent control (5.6 ± 1.19) relative to the negative control 

(8.0 ± 1.9); however, not significant differences were detected between the tribufos-treated 

amphipods relative to the negative control with survival ranging between 74 – 93% across all 

treatments. Mean body weights were significantly different (p<0.05) in the two highest treatment 

groups relative to then negative control; however, the percent reduction in growth relative to the 

negative control was roughly similar (37 – 39%) in the two highest treatments. The study did not 

establish a definitive median effect or median lethal concentration for 50% of the species tested, i.e., 

the EC50 and LC50 are >1050 μg a.i./kg dw sediment (>34.1 μg a.i./L pore water). The NOAEC and 

LOAEC for the study are based on the decreased body weights and are 71 and 220 μg a.i./kg dw 

sediment, respectively (2.81 and 8.27 μg a.i./L pore water). The study is classified as scientifically 

sound and is consistent with guideline recommendations. 

Results Synopsis:

In terms of mean-measured sediment concentrations:

Survival:

LC50: >693 μg a.i./kg 95% C.I.: N/A

NOAEC: 693 μg a.i./kg

LOAEC: >693 μg a.i./kg

Growth:

EC50: 811 μg a.i./kg 95% C.I.: 311-2,114 μg a.i./kg

NOAEC: 71 μg a.i./kg

LOAEC: 220 μg a.i./kg
Data Evaluation Report for a 10-Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

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OC-normalized mean-measured sediment concentrations:

Survival:

NOAEC: 27,000 μg a.i./kg TOC

LOAEC: >27,000 μg a.i./kg TOC

Growth:

NOAEC: 2,700 μg a.i./kg TOC

LOAEC: 8,500 μg a.i./kg TOC

In terms of mean-measured pore water concentrations:

Survival:

NOAEC: 34.1 μg a.i./L

LOAEC: >34.1 μg a.i./L

Growth:

NOAEC: 2.81 μg a.i./L

LOAEC: 8.27 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 the freshwater invertebrate Hyalella azteca.

9. MAJOR GUIDELINE DEVIATIONS: 

 Nominal concentration levels were spaced by a factor of 3.2; whereas guidance 

recommends a maximum factor of 2X. 

 Survival averaged only 56% in the solvent control group, which did not meet the 

minimum guideline criteria (of ≥80%). The cause of the low survival rate was 

unknown. Given that survival in the tribufos treatments is relatively similar to that 

of the negative control, the poor solvent control performance is considered an 

artifact.

10. SUBMISSION PURPOSE: Registration Review

11. MATERIALS AND METHODS:

A. Test Organisms

Guideline Criteria Reported Information
Data Evaluation Report for a 10-Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

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Guideline Criteria Reported Information

Species:

H. azteca or Chironomus tentans Hyalella azteca

Life Stage:

For C. tentans: third instar (9-11 days old). 

The instar stage of midges must be confirmed 

by head capsule width (approx. 0.38 mm).

For H. azteca: 7- to 14-day old amphipods 

must be produced. If growth is also an 

endpoint, a narrower range, such as 1- to 2-

day old amphipods should be used.

≈7 days old

At study initiation, the dry weight of a subpopulation of 20 amphipods averaged 0.02

mg dw/amphipod. 

Supplier

Brood stock can be obtained from laboratory, 

commercial, or government sources (sources 

obtained from the wild should be avoided 

unless cultured through several generations in 

the laboratory).

Laboratory culture (originally obtained from 

Aquatic BioSystems, Inc., Fort Collins, CO). 

All organisms from the same source? Yes
Data Evaluation Report for a 10-Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

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B. Source/Acclimation

Guideline Criteria Reported Information

Acclimation Period:

The required culture and testing temperature is 

23°C. The test organisms should be cultured 

in the same water to be used for testing. 

Seven days prior to testing, adult amphipods 

were removed from the main culture and held 

overnight. Neonates produced the following 

day (<24-hours old) were held for 7 days in 2-

L glass beakers. 

The organisms were held in water from the 

same source as the water used during the test. 

During the 14-day holding period 

immediately preceding the test, water 

temperature ranged from 23.2 to 24.4°C, pH 

ranged from 8.2 to 8.6, and dissolved oxygen 

ranged from 7.3 to 8.4 mg/L. 

Feeding:

Amphipods were fed a TetraMin®flake food 

suspension. 

Pretest Mortality:

A group of organisms should not be used if 

they appear unhealthy, discolored (e.g. <20% 

mortality 48 h before the beginning of a test).

Amphipods appeared normal during the 

holding period. 
Data Evaluation Report for a 10-Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

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C. Test System

Guideline Criteria Reported Information

Source of dilution water (overlying water) 

and sediment:

Soft reconstituted water or water from a 

natural source. Tap water is acceptable if it is 

dechlorinated, deionized, and carbon filtered, 

but its use is not encouraged. 

Uncontaminated natural sediment is 

recommended. 

Moderately-hard freshwater was obtained 

from an on-site laboratory well and sandfiltered and aerated. Prior to use, the water 

was again filtered (0.45 μm). 

Formulated sediment was prepared similar to 

that described in OECD Guideline 218 by 

mixing the following components: ca. 1% 

humic acid and dolomite, 5% alpha-cellulose, 

14% silt and clay (kaolin clay), and 80% 

industrial quartz sand. The dry constituents 

were mixed in a PK Twinshell mixer for ca.

20 minutes, and were stored at ambient 

conditions until needed. 

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). 

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.
Data Evaluation Report for a 10-Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

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Guideline Criteria Reported Information

Water Temperature

23°C for both species. The mean and 

instantaneous temperatures should not vary 

from the desired temperature by more than 1°C 

and 3°C, respectively. 

22.0 to 22.8C (daily monitoring)

22.0 to 23.5C (continuous monitoring)

pH

Should not vary more than 50%. Survival is 

best at pH >6.5 for C. tentans. 

7.6 to 8.2

Dissolved Oxygen

Maintained between 40 and 100%. 5.9 to 8.6 mg/L (≥69% saturation) 

Total Hardness

Should not vary more than 50%. H. azteca are 

sensitive to hardness (e.g., they are not found 

in waters with calcium at <7 mg/L and DO at 

<2 mg/L). 

128 to 148 mg/L as CaCO3

Conductivity

Should not vary more than 50%. 389 to 432 μS/cm

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 Textural Class – Loamy sand

Organic matter content – 4.5%

Organic carbon content – 2.6%

Moisture at 1⁄3 Bar – 10.6%

pH – 6.5

CEC – 2.4 meq/100 g

Bulk density – 1.26 g/cm3

Ammonia concentration of pore water – not 

reported
Data Evaluation Report for a 10-Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

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Guideline Criteria Reported Information

Additional Sediment Analysis

BOD, COD, cation exchange capacity, Eh, pE, 

total inorganic carbon, total volatile solids, 

acid volatile sulfides, total ammonia, metals, 

synthetic organic compounds, oil and grease, 

petroleum hydrocarbons, and interstitial water 

analysis.

None reported

Laboratory Spiked Sediment

Material should be reagent grade unless prior 

evaluations dictate formulated materials, etc.; 

Must know the test material's identity, quantity 

of major ingredients and impurities, water 

solubility, estimated toxicity, precision and 

bias of analytical method, handling and 

disposal procedures.

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

Aqueous solubility: 2.3 mg/L at 20°C

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 (acetone) control and negative 

control group were included in the test.
Data Evaluation Report for a 10-Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

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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. 

A 15-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, 750 g of untreated 

sediment was added to achieve a final weight 

of 1500 g, and the batch sediments were 

mixed on a rotary mixer ca. 25.5 hours.

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: 300 ml high-form lipless beakers 

containing 100 ml of sediment and 175 ml of 

overlying water.

1. Glass and stainless steel mesh

2. 300-mL beakers (compartments) with two 

stainless steel mesh-covered holes on either 

top side. Test compartments contained 

approximately 100 mL of treated sediment 

(depth of ca. 3.0 cm) and approximately 150-

175 mL of overlying water (depth of ca. 6.7

cm). 
Data Evaluation Report for a 10-Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

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Guideline Criteria Reported Information

Type of Dilution System

Daily renewal or a flow-through system may 

be used. 

Flow-through

The test compartments were indiscriminately 

placed into a temperature-control water bath 

within a diluter tank (with one control or 

treatment group per tank). The water level in 

the test compartments (i.e., beakers) was 

maintained by the water levels in the diluter 

tanks. 

Flow Rate

2 volume changes/day Ca. 2 volume additions/day

Aeration

Dilution water should be vigorously aerated 

prior to use so that dissolved oxygen in the 

overlying water remains above 40% saturation.

None reported

Photoperiod

16 hours light, 8 hours dark at 500 to 1000 lux. 16 hours light/8 hours dark, with 30-minute 

transition periods. 

Light intensity at test initiation was 504 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 Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

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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 pore water 

equilibration trial (see Reviewer’s 

Comments), the prepared sediment/water 

systems were allowed to acclimate for ca. 10

days prior to introduction of the test 

organisms. 

Renewal of Overlying Water:

Renewal of the overlying water should be 

conducted on day -1 prior to the addition of 

organisms or food on day 0. For flowthrough systems, the flow rates should not 

vary by more than 10% between any two 

chambers at any time. Proper operation 

should be verified by calibration prior to test 

initiation. 

The dilution water was delivered directly into 

the test compartments, passively forcing out 

the water through the holes in the sides of the 

beakers. The diluter was calibrated prior to 

test initiation. 

Placing Organisms in Test Chambers:

Should be handled as little as possible and 

introduced into overlying water below the airwater interface.

Amphipods were impartially assigned one and 

two at a time into transfer chambers until each 

chamber contained ten organisms. Each 

group of ten organisms was then impartially 

assigned and transferred to a test 

compartment. All transfers 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 Evaluation Report for a 10-Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

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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 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 sediment

Number of Test Organisms

10 organisms per test chamber are 

recommended. 8 replicates per treatment 

should be used.

80 amphipods per level, with 10 amphipods 

per replicate chamber and eight biological 

replicates per level

An additional two replicates per level were 

maintained for analytical purposes (Days 0 

and 10). The chambers prepared for Day-10 

analytical purposes contained organisms. 

Test organisms randomly or impartially 

assigned to test vessels? Yes

Feeding

C. tentans in each test chamber are fed 1.5 ml 

of a 4 g/L Tetrafin7 suspension daily. H. 

azteca may be fed with a mixture of yeast, 

Cerophy, and trout chow (YCT) at a rate of 

1.5 mL daily per test chamber. A drop in DO 

levels below 2.5 mg/L may indicate overfeeding and feeding should be suspended in 

all treatments until DO levels increase.

Amphipods were fed 1.5 mL of a 4.0-g/L 

TetraMin®flake food suspension on Days 0 to 

9.
Data Evaluation Report for a 10-Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

13

Guideline Criteria Reported Information

Water Parameter Measurements

Conductivity, hardness, pH, alkalinity, and 

ammonia should be measured in all 

treatments at the beginning and end of the 

test. 

DO should be measured daily. 

Temperature should be measured daily in one 

test chamber from each treatment. The mean 

and instantaneous temperatures should not 

vary from the desired temperature by more

than 1 and 3°C, respectively.

Hardness, alkalinity, specific conductance and 

ammonia were measured in composite 

overlying water samples collected from each 

control level and the highest treatment level at 

test initiation and termination. 

pH was measured in overlying water from one 

alternating replicate from all levels on Days 0, 

3, 7, and 10.

DO and temperature were measured in 

overlying water from one alternating replicate 

from all levels daily. Temperature was also 

continuously monitored in the negative 

control diluter tank. 

Chemical Analysis

Needed if solutions were aerated, if chemical 

was volatile, insoluble, or known to absorb, if 

precipitate formed, if containers were not 

steel or glass, or if flow-through system was 

used.

Concentrations should be measured in bulk 

sediment, interstitial water, overlying water, 

and stock solution.

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 Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

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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 water and sediment for 

potential contaminants.

Control Criteria 

Was control mortality <20%?

Were control C. tentans an average size of 

≥0.6 g?

Survival averaged 80% in the negative control 

group, but only 56% in the solvent control

N/A

Percent Recovery of Chemical: Sediment: 101 ± 9.71% 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

- Dry weight (determined by pooling all living 

organisms from a replicate and drying at 60 to 

90°C to a constant weight)

- Body length (amphipod only) 

- Survival

- Dry weight

Raw data included? Yes
Data Evaluation Report for a 10-Day Toxicity Test with Freshwater Amphipod Hyalella Using Sediment Spiked with Tribufos

EPA MRID Number 49164802

15

Effects Data

The concentrations of tribufos and survival and growth of Hyalella are presented in Table 1 below. Dry weight was the most sensitive 

endpoint. The study author identified the NOAEC and LOAEC as 71 ug a.i./kg and 220 ug a.i./kg sediment (mean measured 

concentrations). Based on the mean measured concentrations of tribufos in pore water, the NOAEC was 2.81 ug a.i./L and the LOAEC 

was 8.27 ug a.i./L.

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

Hyalella azteca.

Toxicant Concentration

Mean No. ± SD

Surviving 

Amphipods

(% survival)

Percent 

Reduction

in 

Survivalb

(%)

Mean 

Individual Dry 

Body Weights

(mg ± SD)

Percent 

Reductionb

in Weights

(%)

Nominal

(μg a.i./kg)

Mean-Measured(a)

Sediment

(μg a.i./kg)

(% nominal)

Pore Water

(μg a.i./L)

Overlying

Water

(μg a.i./L)

Negative 

Control <LOQ <LOQ <LOQ 8.0 ± 1.9(80%) --- 0.12 ± 0.02 ---

Solvent Control <LOQ <LOQ <LOQ 5.6 ± 1.19*

(56%) 30 0.11 ± 0.02 ---

Pooled Control --- --- --- --- --- 0.11 ± 0.02 ---

10 7.0

(70%) 0.210 <LOQ 8.0 ± 1.77(80%) 0 0.10 ± 0.02 11
Data Evaluation Report for a 10-Day Toxicity Test with Freshwater Amphipod Hyalella Using Sediment Spiked with Tribufos

EPA MRID Number 49164802

16

Toxicant Concentration

Mean No. ± SD

Surviving 

Amphipods

(% survival)

Percent 

Reduction

in 

Survivalb

(%)

Mean 

Individual Dry 

Body Weights

(mg ± SD)

Percent 

Reductionb

in Weights

(%)

Nominal

(μg a.i./kg)

Mean-Measured(a)

Sediment

(μg a.i./kg)

(% nominal)

Pore Water

(μg a.i./L)

Overlying

Water

(μg a.i./L)

32 20

(63%) 0.699 <LOQ 7.4 ± 1.41(74%) 7.8 0.16 ± 0.06 -38

103 71

(69%) 2.81 <LOQ 7.9 ± 1.36(79%) 1.6 0.11 ± 0.03 2.7

328 220

(67%) 8.27 <LOQ 9.3 ± 0.71(93%) -16 0.07 ±0.03* 39

1050 693

(66%) 34.1 0.08

8.1 ± 1.36

(81%) -1.6 0.07 0.01* 37

aThe LOQ for sediment was 2.00 μg a.i./kg; the LOQ for overlying and pore water was 0.100 μg a.i./L.

bPercent reduction in survival is expressed relative to the negative control.

* Statistically-significant difference (p<0.05) from the negative control (survival) or pooled control (dry weight).
Data Evaluation Report for a 10-Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

17

Other Significant Results: 

Biological: According to the study authors, all organisms appeared normal throughout the 

study. There were a few observations of organisms leaving the sediment, on the surface of 

the sediment, climbing the walls of the test chamber or swimming in the water column; 

however, these observations were few in number, observed in all treatment groups and 

controls, and did not appear to be dose responsive and were therefore not considered to be 

treatment-related. 

The study authors reported that survival after 10 days averaged 80 and 56% for the negative 

and solvent (acetone) control levels, respectively, and 80, 74, 79, 93 and 81% in the meanmeasured 7.0, 20, 71, 220 and 693 μg a.i./kg sediment levels, respectively (Table 1). There 

was a statistically-significant difference in survival between the solvent control compared to 

the negative control (p<0.05). The study authors reported it was unclear what caused the 

low percent survival in the solvent control as 1) the sediment preparation procedure should 

have removed most of the solvent from the test sediment, 2) the same concentration of 

solvent was used in all treatments, and 3) the percent survival in the other treatment groups 

were close to or above the guideline criterion indicating the solvent wasn't affecting the 

organisms' survival. The 10-day LC50 was >693 μg a.i./kg, and the NOAEC and LOAEC 

were 693 and >693 μg a.i./kg, respectively. 

According to the study authors, mean dry weights were 0.12 and 0.11 mg/amphipod for the 

negative and solvent control levels, respectively, and 0.10, 0.16, 0.11, 0.07 and 0.07 

mg/amphipod for the mean-measured 7.0, 20, 71, 220 and 693 μg a.i./kg sediment levels, 

respectively (Table 1). Differences were statistically-significant (p<0.05) compared to the 

pooled control (0.11 mg/amphipod) at the 220 and 693 μg a.i./kg levels. The authors 

concluded based on mean body weight, the 10-day EC50 was >693 μg a.i./kg, and the 

NOAEC and LOAEC were 71 and 220 μg a.i./kg, respectively. 

Analytical: Measured concentrations in stock solutions ranged from 85.5 to 116% 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 53.4 to 78.6% of nominal for all levels (both intervals). Overall meanmeasured concentrations in sediment ranged from 63 to 70% of nominal levels (Table 1). 

In overlying water, tribufos was only above the LOQ once, on Day 0 at the 1050 μg a.i./kg 

level, when tribufos was detected at 0.115 μg a.i./L. According to the study authors, pore 

water concentrations remained generally consistent (with 0- to 10-Day percent differences of 

-15 to +22%). Mean-measured pore water concentrations were 0.210, 0.699, 2.81, 8.27 and 

34.1 μg a.i./L for the nominal 10, 32, 103, 328 and 1050 μg a.i./kg sediment levels, 

respectively (Table 1). 
Data Evaluation Report for a 10-Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

18

B. Statistical Results

Method: The negative and solvent control survival responses were compared using a t-test. 

Since there was a significant difference found between the control groups for survival data 

(p<0.05), the study authors compared treatment groups to the negative control to identify 

levels that were statistically-significant. There were no significant differences found 

between the control groups for growth data, and thus the study authors compared treatment 

groups to the pooled control for these data.

Survival data were evaluated for normality using the Chi-square test and for homogeneity of 

variance using Bartlett’s test. The data were deemed normal with homogeneous variance by 

the study authors, and the data were analyzed using a Dunnett’s multiple means test to 

identify those treatment levels that were statistically different (p<0.05) from the negative 

control. Growth 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 by the study authors, and these data were analyzed using a 

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 and growth data. 

The 10-day LC50 and EC50 values were 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. The study is classified as 

scientifically sound and is consistent with guideline recommendations. 

In terms of mean-measured sediment concentrations:

Survival:

NOAEC: 693 μg a.i./kg

LOAEC: >693 μg a.i./kg

Growth:

NOAEC: 71 μg a.i./kg

LOAEC: 220 μg a.i./kg
Data Evaluation Report for a 10-Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

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In terms of mean-measured pore water concentrations:

Survival:

NOAEC: 34.1 μg a.i./L

LOAEC: >34.1 μg a.i./L

Growth:

NOAEC: 2.81 μg a.i./L

LOAEC: 8.27 μg a.i./L

13. VERIFICATION OF STATISTICAL RESULTS: 

Statistical Method: The reviewer tested for normality using the Shapiro-Wilk test for 

normality and for homogeneity of variance using Bartlett’s test for equality of variance. 

Survival met assumptions of normality and equal variance, while growth (as dry weight) 

proved to be nonparametric and of unequal variance. Neither endpoint exhibited a linear 

dose-dependent response. The reviewer determined the NOAEC and LOAEC for survival 

using the Dunnett’s Multiple Comparison test, while growth data were analyzed using the 

non-parametric Mann-Whitney Wilcoxon Test (at a 95% level of certainty). Significant 

decreases relative to the negative control were detected for the two highest treatment groups 

for growth, though no significant effects were observed for survival. The LC50 for survival

and EC50 for growth could not be determined given that all responses were <40% relative to 

controls.

The negative and solvent control groups were compared using a Student’s t-test assuming 

equal variances; a significant difference was detected (p>0.05) for the survival endpoint. 

Subsequent treatment level comparisons were made to the negative control response only. 

Given that survival in the treatment groups was roughly similar to that in the negative 

control, the aberrant performance of the solvent control was not deemed to be due to the 

solvent. These analyses were conducted using CETIS v. 1.8.7.12 with backend settings 

implemented by EFED on 5/29/13.

Based upon mean-measured sediment concentrations:

Survival:

NOAEC: 693 μg a.i./kg

LOAEC: >693 μg a.i./kg

Growth (dry weight):

NOAEC: 71 μg a.i./kg

LOAEC: 220 μg a.i./kg
Data Evaluation Report for a 10-Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

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14. REVIEWER'S COMMENTS: 

The reviewer’s conclusions agreed with the study author’s. Both the study author and the 

reviewer compared treatment survival data to the negative control only, as a significant 

difference had been found between the negative and solvent control groups for survival data 

(p<0.05). Results were reported in terms of mean-measured sediment and pore water 

concentrations. In addition, the reviewer also provided results in terms of organic carbon 

(OC)-normalized sediment concentrations. 

The analytical methods used to analyze the test samples were based upon methods 

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. Sediment was dosed at 0.01 

and 100 μg a.i./kg and held in test chambers under test conditions. Samples were collected 

after 2, 8, 9 and 14 days of equilibration, and concentrations of tribufos in isolated pore 

water from the 100 μg a.i./kg samples were 5.67, 9.67, 9.96 and 8.69 μg a.i./L, respectively. 

All pore water results from the 0.01 μg a.i./kg samples were <LOQ (<0.500 μg a.i./L). The 

results of the trial indicated that equilibration had been achieved by 8 days. 

A definitive study attempt was conducted from February 18-28, 2013, but was repeated in 

attempt to achieve a NOAEC. The same nominal treatment levels were tested and the same 

experimental design was followed as was used in the final definitive test. Survival ranged 

from 83 to 95% for all levels, with no treatment-related effect observed for any level. The 

average dry weight per amphipod in the negative and solvent control levels was 0.15 and 

0.13 mg, respectively, compared to 0.06, 0.06, 0.07, 0.07 and 0.07 mg for the nominal 10, 

32, 103, 328 and 1050 μg a.i./kg levels, respectively. Differences were statisticallysignificant compared to the pooled control at all treatment levels (p<0.05) with a flat doseresponse. As a NOAEC and LOAEC were not determined, the test was repeated. 

The experimental phase of the definitive study was conducted from April 22 to May 7, 
Data Evaluation Report for a 10-Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

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2013. The study is classified as scientifically sound and is consistent with guideline 

recommendations. 

15. REFERENCES:

American Society for Testing and Materials. 2005. ASTM Standard E 1706-05. Standard Test 

Methods for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater 

Invertebrates. 

U.S. Environmental Protection Agency. 1996. Series 850 – Ecological Effects Test Guidelines 

(draft), OPPTS 850.1735: Whole Sediment Acute Toxicity Invertebrates, Freshwater. 

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 Guideline 218. 2004. Sediment-Water Chironomid Toxicity Test Using Spiked Sediment. 

Organization for Economic Cooperation and Development. Adopted 13 April 2004. 

APHA, AWWA, WPCF. 1998. Standard Methods for the Examination of Water and 

Wastewater. 20th Edition, American Public Health Association. American Water Works 

Association. Water Pollution Control Federation, New York. 

West, Inc., and D.D. Gulley. 1996. TOXSTAT Version 3.5. Western EcoSystems Technology, 

Inc., Cheyenne, WY.

Finney, D.J. 1971. Statistical Methods in Biological Assay. Second Edition. Griffin Press, 

London.