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 : Tr ibufos: 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 : A mvac Chemical Corporation

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

- 113A

MRID No. : 491648

- 02

DP Barcode : D 414359

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. Reviewed By : Ma ry Clock

- Rust, Biologist, ERB4, EFED, OPP, OCSPP, EPA

Signature: Date : 1/ 14 /1 5

6. STUDY PARAMETERS :

Age of Test Organism: ≈ 7 days old

Definitive Test Duration: 10 days

Study Method: Flow

- throughData 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 OC

normalized) and pore water

7. EXECUTIVE SUMMARY : In a 10

- day study, t echnical 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 repli cates per treatment were run for monitoring exposure con centrations 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. No minal 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 negativ e, 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, solven t, 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 signific ant 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 concentrati on for 50% of the species tested, i.e.,

the EC 50 and LC 50 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 (2.8 1 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:

LC 50 : >693 μg a.i./ kg 95% C.I.: N/A NOAEC: 693 μg a.i./ kg LOAEC: >693 μg a.i./ kg

Growth:

EC 50 : 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./ kgData Evaluation Report for a 10

- Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

3 OC

- normalized mean

- measured sediment concentrations:

Survival:

NOAEC: 27

, 000 μg a.i./ kg TOC

LOA EC: >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 :

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

recommends a maximum factor of 2X.

 Survival averaged only 56% in the sol vent control group, which did not me e t 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

artifa ct.

10. SUBMISSION PURPOSE : R egistration R eview

1 1

. MATERIALS AND METHODS :

A. Test Organisms

Guideline Criteria Reported InformationData Evaluation Report for a 10

- Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

4 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 sub

population of 20 amphipods averaged 0.0 2 mg dw/amphipod.

Supplier Brood stock can be obtained from laboratory, commercial, or government sources ( s ources obtained from the wild should be avoide d 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? YesData Evaluation Report for a 10

- Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

5 B. Source/Acclimation

Guideline Criteria Reported Inf ormation

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 hel d overnight. Neonates produced the following day (<24

- hours old) were held for 7 days in 2

- L glass beakers.

T he 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 t o 8. 4 mg/L.

Feeding: Amphipods were fed a TetraMin ® flake food suspension.

Pretest Mortality: A group of organisms should no t 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

6 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

- h ard freshwater was obtained

from an on

- site laboratory well and sand

filtered 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 c omponents: 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 stre ss? 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.Data Evaluation Report for a 10

- Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

7 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. 2 2

. 0 to 2 2 .8  C (daily monitoring)

2 2

. 0 to 2 3

. 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 ( ≥ 6 9 % 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). 1 28 to 14 8 mg/L as CaCO 3

Conductivity Should not vary more than 50%. 3 89 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 distribu tion

– 8 3 % sand, 5 % silt, and

1 2 % clay

USDA T extur al Class

– L oam y 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/cm 3
Ammonia concentration of pore water

– not

r eportedData Evaluation Report for a 10

- Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

8 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 interst itial water analysis. No ne 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

solub ility, estimated toxicity, precision and bias of analytical method, handling and disposal procedures. 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: li quid

Lot No.: NX81AX0015 Purity: 98.2% Storage: ambient conditions Aqueous solubility: 2.3 mg/L at 20°C

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 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

9 Guideline Criteria Reported Information

Test Concentrations For Spiked Sedi ment 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 mix er 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

sedim ent was added to achieve a final weight

of 1 5 00 g , and t he batch sediments were mixed on a rotary mixer ca. 25.5 hour s

.

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

the LC 50 and NOAEC/LOAEC.

Test Aquaria

1. Mater ial : 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 m L of treated sediment ( depth of ca. 3. 0 cm) and approximately 150

- 175 m L 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

10 Guideline Criteria Reported Information

Type of Dilution System

D aily 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 wa ter 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 init iation was 5 04 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 sedimentData Evaluation Report for a 10

- Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

11 D. Test Design

Guideline Criteria Reported Information

Sedime nt 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 su spension 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. 1 0 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 flow

through systems, the flow rates should not vary by more than 10% between any two chambers at any time. Proper operation

shou ld 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 initia tion

.

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

water interface. Amphipods were impartially assigned one and

two at a time into transfer chambers until each

chamber c ontained 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 requi red 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

12 Guideline Criteria Reported Information

Mo nitoring 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 1 0 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

assi gned to test vessels? Yes

Feeding

C. tentans in each test chamber are fed 1.5 ml of a 4 g/L Tetrafin 7 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 over

feeding 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 te mperatures should not vary from the desired temperature by more than 1 and 3°C, respectively. H ardness, alkalinity, specific conductance and ammonia were measured in composite overlying water samples collected from each control level and the highest treat ment 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 o r 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, an d 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

14 1 2

. REPORTED RESULTS :

A. General Results

Guideline Criteria Reported Information

Quality assurance an d 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, No tification No. 6283, 1999), with the following exception s : periodic analyses of water and sediment for potential contaminants

.

Control Criteria Was control mortality <2 0%?

Were control C. tentans an average size of ≥0.6 g? Survival averaged 80% i n the n egative 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

- D ry weight

Raw data in cluded? YesData 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 s ediment (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 LO AEC was 8.27 ug a.i./L

.

Table 1

. Nominal and measured concentrations of tribufos i n 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

Survival b
(%) Mean

Individual Dry Body Weights (mg ± SD) Percent

Reduction b in Weights (%) Nominal
(μg a.i./ kg) Mean

- Measured (a)

Sediment (μ g a.i./ kg)

(% nominal) Pore Water

( μ g a.i./ L) Overlying

Water ( μ g 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 11Data 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

Survival b
(%) Mean

Individual Dry Body Weights (mg ± SD) Percent

Reduction b in Weights (%) Nominal
(μg a.i./ kg) Mean

- Measured (a)

Sediment (μ g a.i./ kg)

(% nominal) Pore Water

( μ g a.i./ L) Overlying

Water ( μ g 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 0.0 8 8.1 ± 1.36 (81%) - 1.6 0.07 0.01* 37

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

b Percent reduction in survival is expressed relative to the negative control. * Statistically

- significant difference (p<0.05) from the negative control (sur vival) 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, a ll organisms appeared normal throughout the study. There were a few o bservations of organ i sms 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 w ere few in number, observed in all treatment groups and

controls

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

treatment

- related.

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

and solvent (acetone) control level s , respectively, and 80, 74, 79, 93 and 81% in the mean

measured 7.0, 20, 71, 220 and 693 μg a.i./ kg sedi ment levels, respectively ( Table 1 )

. The re

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 LC 50 was > 693 μ g a.i./ kg, and the NOAEC and LOAEC

were 693 and >693 μ g a.i./ kg , respectively.

According to the study authors, m ean dry weigh ts 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/amphi p od for the mean

- measured 7.0, 20, 71, 220 and 693 μ g a.i./ kg s ediment levels, respectively ( Table 1 )

. Differences wer e statistically

- significant (p<0.05) compared to the pooled control (0.11 mg/amphipod) at the 220 and 693 μ g a.i./ kg level s

. The authors concluded based on mean body weight, the 10

- day EC 50 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, c oncentrations remained predominantly associated with the sediment, and were generally consistent dur ing the 10

- day study , with recoveries of 53.4 to 78.6% of nominal for all levels (both intervals). Overall mean

measured concentrations in sediment ranged from 63 t o 70% of nominal levels ( Table 1 )

.

In overlying water, tribufos was only above the LOQ o nce, on Day 0 at the 1050 μg a.i./ kg level, when tribufos was detected at 0.1 15 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 w ater 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. Th ere 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

.

S urvival 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 s tatistically 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

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

The 10

- day LC 50 and EC 50 value s w ere 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./ kgData Evaluation Report for a 10

- Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

19 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

1 3

. 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 Compari son 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 si gnificant effects were observed for survival. The LC 50 for survival

and EC 50 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 assum ing equal variances; a significant difference was detected (p>0.05) for the survival endpoint.

S ubsequent treatment level comparisons were made to the negative control response only. Given that survival in the treatment groups was roughly similar to that i n 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. 1.8.7.1 2 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./ kgData Evaluation Report for a 10

- Day Toxicity Test with Freshwater Amphipod Hyalella

Using Sediment Spiked with Tribufos EPA MRID Number 49164802

20 1 4

. 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 gr oups 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 In ternational. 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 chro matography 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 pres sure 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 concentra tions 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 a s 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, respecti vely, 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 statistically

significant compared to the pooled control at all treatment levels (p<0.05) with a flat dose

res ponse. 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

21 2013. The study is classified as scientifically sound and is consistent with guideline

recomme ndations.

1 5

. 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 Fresh w ater
Invertebrates

.

U .S. Environmental Prot ection 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 Contr ol Materials in Whole Sediment Toxicity Tests. Environ. Toxicol. Chem.

18:222

- 230.

OECD Guideline 218. 200 4

. Sediment

- Water Chironomid Toxicity Test Using Spiked Sediment. Organization for Economic Cooperation and Development. Adopted 13 April 200 4.

APHA, AWWA, WPCF. 199 8

. Standard Methods for the Examination of Water and

Wastewater

. 20 th Edition, American Public Health Association. American Water Works Association. Water Pollution Control Federation, New York.

West, Inc., and D.D. Gulley. 19 9 6. TOXSTAT Version 3.5. Western EcoSystems Technology, Inc., Cheyenne, WY.

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

London.