	DATA EVALUATION RECORD
                CHRONIC SEDIMENT Hyalella azteca TOXICITY TEST
          (FOLLOWING EPA TEST METHOD 100.4, draft guideline 850.1770)

1.  CHEMICAL:  Dacthal					PC Code No.:  078701

2.  TEST MATERIAL:  Dacthal Technical		Purity:  99.3%

3.  CITATION:

	Authors:	Picard, C.R.
	Title:	Dacthal Technical - 42-Day Toxicity Test Exposing Freshwater Amphipods (Hyalella azteca) to a Test Substance Applied to Sediment Under Static-Renewal Conditions Following EPA Test Methods.
	Study Completion Date:	March 10, 2016
	Laboratory:	Smithers Viscient
		790 Main Street
		Wareham, Massachusetts 02571-1037, USA
	Sponsor:	AMVAC Chemical Corporation
		4695 MacArthur Court, Suite 1200
		Newport Beach, California 92660, USA
	Laboratory Report ID:	11857.6111
	MRID No.:	49865801
	DP Barcode:	432679

4.  REVIEWED BY:  David A. McEwen, Staff Scientist, CDM Smith/CSS-Dynamac JV 
     Signature:  		Date:  11/25/2016

     APPROVED BY:  Moncie V. Wright, Environmental Scientist, CDM Smith/CSS-Dynamac JV
	 
	Signature: 	Date:  12/15/2016

5.  REVIEWED BY:  Christina M. Wendel, Biologist, OPP/EFED/ERB2

        	Signature:  				Date: 10/26/2021

		Susan Thomas, Biologist, OPP/EFED/ERB4

        	Signature:  		Date: 05/17/2021

		Michael Wagman, Senior Scientist, OPP/EFED/ERB2

     Signature:	Date: 11/22/2021
6.  DISCLAIMER:  This Data Evaluation Record may have been altered by the Environmental Fate and Effects Division subsequent to signing by CDM/CSS-Dynamac JV personnel.

7.  STUDY PARAMETERS:

     	Scientific Name of Test Organism:	Hyalella azteca
     	Age of Test Organism:	8 days old
     	Definitive Test Duration:	42 day
     	Study Method:	Intermittent flow-through
     	Type of Concentrations:	Time-weighted average (TWA) sediment (bulk and OC-normalized), TWA pore water and TWA overlying water concentrations 
     
8.  EXECUTIVE SUMMARY:

The 42-day chronic toxicity of Dacthal (Technical DCPA) to Hyalella azteca was studied under static renewal conditions at nominal concentrations of 0 (negative and solvent controls), 6.3, 13, 25, 50 and 100 mg a.i./kg-sediment. The reviewer-calculated time-weighted (TWA) concentrations were <0.42 (<LOD, negative and solvent controls), 5.2, 12, 22, 42 and 93 mg a.i./kg in bulk sediment (OC-normalized treatment concentrations equivalent to 248, 548, 1024, 1976 and 4405 mg a.i./kg OC, based on 2.1% organic matter content), <0.0029 (<LOD, negative and solvent controls), 0.065, 0.15, 0.18, 0.28, and 0.36 mg a.i./L in pore water and <0.0029 (<LOD, negative and solvent controls), 0.0049, 0.013, 0.015, 0.022, and 0.026 mg a.i./L in overlying water.  

Results Synopsis

Endpoint
                                 Bulk Sediment
                                 (mg a.i./kg)
                    OC-Normalized Sediment (mg a.i./kg OC)
                                  Pore Water
                                  (mg a.i./L)
                                Overlying Water
                                  (mg a.i./L)
Day 28 Survival*
                                   NOAEC: 42
                                   LOAEC: 93
                                  NOAEC: 1976
                                  LOAEC: 4405
                                  NOAEC: 0.28
                                  LOAEC: 0.36
                                 NOAEC: 0.022
                                 LOAEC: 0.026
Days 35 and 42 Survival
                                   NOAEC: 93
                                 LOAEC: >93
                                  NOAEC: 4405
                                LOAEC: >4405
                                  NOAEC: 0.36
                                LOAEC: >0.36
                                 NOAEC: 0.026
                               LOAEC: >0.026
Day 28 length*
                                   NOAEC: 22
                                   LOAEC: 42
                                  NOAEC: 1024
                                  LOAEC: 1976
                                  NOAEC: 0.18
                                  LOAEC: 0.28
                                 NOAEC: 0.015
                                 LOAEC: 0.022
Days 35 and 42 Offspring/female
                                   NOAEC: 93
                                 LOAEC: >93
                                  NOAEC: 4405
                                LOAEC: >4405
                                  NOAEC: 0.36
                                LOAEC: >0.36
                                 NOAEC: 0.026
                               LOAEC: >0.026
Days 35 and 42 Reproduction
                                   NOAEC: 93
                                 LOAEC: >93
                                  NOAEC: 4405
                                LOAEC: >4405
                                  NOAEC: 0.36
                                LOAEC: >0.36
                                 NOAEC: 0.026
                               LOAEC: >0.026
Day 42 Length
                                   NOAEC: 93
                                 LOAEC: >93
                                  NOAEC: 4405
                                LOAEC: >4405
                                  NOAEC: 0.36
                                LOAEC: >0.36
                                 NOAEC: 0.026
                               LOAEC: >0.026
Male to female ratio**
                                   NOAEC: 93
                                 LOAEC: >93
                                  NOAEC: 4405
                                LOAEC: >4405
                                  NOAEC: 0.36
                                LOAEC: >0.36
                                 NOAEC: 0.026
                               LOAEC: >0.026
     *Statistically different (4%); and biologically meaningful (p = 0.02, survival and p <0.05, D28 length)
     ** There were significant differences between the negative and solvent control, this would result in a NOAEC and LOAEC of 42 and 93 mg a.i./kg-sediment in bulk sediment (OC-normalized 1976 and 4405 mg a.i./kg OC);0.28, and 0.36 mg a.i./L in pore water; and 0.022, and 0.026 mg a.i./L in overlying water, respectively.  
     Endpoint(s) Affected:  Day 28 Survival and Length
     Most Sensitive Endpoint(s): Day 28 Length
     
9.  ADEQUACY OF THE STUDY: 

	A.  Classification:  This study is scientifically sound and is classified as acceptable.

      B. Rationale:  This study followed methods described in the "Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates", 2[nd] Edition, Test Method 100.4 (U.S. EPA, 2000).  
      
      C. Reparability:  N/A

10.  GUIDELINE DEVIATIONS:

   Although this study does not fulfill any current U.S. EPA guideline requirement, notable deviations from U.S. EPA Test Method 100.4 included:  
   
 Dissolved oxygen (DO) levels fell to 2.17 mg/L on Day 6 in the 50 and 100 mg/kg treatment levels, which is below the minimum recommendation limit of 2.5 mg/L. It was reported that the water renewal rate was increased (from 2x/day to 4x/day) to return dissolved oxygen levels to an acceptable range, and that this deviation had no impact on the results or interpretation of this study as the dissolved oxygen levels observed were within the acceptable range for the "majority of the exposure and the deviation was relatively short in duration". Although the range of the DO was provided, the time period at which a low DO (<2.5 mg/L) was experienced by test organisms was not provided, (i.e., daily DO measurements were not reported so that the length of low in DO could be confirmed). The controls appear to be healthy, and met all validity criteria, and therefore, this is considered to be a minor deviation. 
 There appeared to be a general trend for the reproductive endpoints that the solvent improved performance compared to the negative (water only) control. However, there were no significant differences between solvent and negative controls except for male to female ratio, where they were statistically significantly different with an approximate 40% difference in ratios (negative control mean of 1.1 males to female compared to 0.70 males to female in the solvent control). This does introduce some uncertainty into the reproduction endpoints.
   
11.  MATERIALS AND METHODS:

   A.  Test Organisms/Acclimation

                              Guideline Criteria
                             Reported Information
Species
Hyalella azteca 
Amphipods
Hyalella azteca
Age of Test Organisms
7- to 8-days old

8-days old at test initiation
Source
Smithers Viscient Laboratory cultures 
All organisms from the same source?
Yes
Culture Conditions

Amphipod cultures were maintained under flow-through conditions in 20-L glass aquaria containing ca. 15 L of culture water (same source as overlying water used in definitive study).  Nine days prior to test initiation, a sub-culture of reproducing adults was transferred to 9.5-L aquaria (isolation tanks) containing 8 L of culture water. Juvenile amphipods (<24 hours old) produced from the isolated adults were pipetted into 1-L beakers containing ca. 0.80 L of laboratory well water. Juvenile amphipods were reared under static conditions for 8 days with gentle oil-free aeration. 
  
During the holding period, the DO ranged from 6.5 to 7.9 mg/L and temperature was maintained at 23 to 25°C. 
Feeding

During holding and acclimation, amphipods were fed daily with a combination of yeast, cereal leaves and flaked fish food suspension (YCT, 100 mg/mL). A small amount (2.5 mL) of Ankistrodesmus falcatus and ca. 3 drops of flaked fish food suspension were added at the start of isolation to provide a supplemental food source. 
Pre-test Mortality

Test organisms appeared healthy, with no mortality observed in the juvenile population during the 48 hours prior to test initiation.
      

      
      B.  Test System

                              Guideline Criteria
                             Reported Information
Dilution water (overlying water)

Laboratory well water, which was unadulterated water from a 100-m bedrock well supplemented with untreated Town of Wareham well water.
The water was characterized as having a total hardness and total alkalinity of 34 to 72 and 20 to 31 mg/L as CaCO3, respectively, a pH range of 6.8 to 7.3, and a conductivity range of 300 to 480 uS/cm. The TOC was 0.85 and 0.26 mg/L in September and October 2015, respectively.
Representative samples of the overlying water were analyzed periodically for pesticides, PCBs, and toxic metals by GeoLabs, Inc., Braintree, MA. None of these compounds were detected in any of the samples analyzed in at concentrations that are considered toxic in agreement with ASTM (2007) standard practice. The results of these analyses were not provided in the study report for reference.
Does water support test animals without observable signs of stress?

Yes
Water Temperature
Mean daily:  23 +- 1°C
Instantaneous:  23 +- 3°C
Daily monitoring:  21 to 24°C 
Continuous monitoring:  20 to 24°C 
pH
Overall range: 6.3 to 7.4 (overlying water)
Overall range: 6.6-7.5 (in pore water)
Overall range: 6.7 (in the sediment)
Dissolved Oxygen
>2.5 mg/L
Overall range: 2.2 to 7.9 mg/L (see Reviewer's Comments)
Ammonia
Should not vary more than 50%.  
Overall range: <=0.10 to 0.21 mg/L as N (overlying water)
Overall range:  <=0.10 to 0.21 mg/L as N (in pore water)
Hardness
Should not vary more than 50%.  
Overall range: 52 to 84 mg/L as CaCO3 (overlying water)
Alkalinity
Should not vary more than 50%.  
Overall range: 16 to 26 mg/L as CaCO3 (overlying water)
Conductivity
Overall range: 340 to 590 μS/cm (overlying water)
Test Sediment

Artificial sediment prepared according to OECD Guideline 218 
(SMV Batch No. 072915). The sediment consisted of 5% sphagnum peat, 20% kaolin clay, and 75% fine sand; 2.4 kg sphagnum peat, 9.6 kg kaolin clay and 36 kg fine sand (based on a dry weight basis). While blending the sediment components using a large-scale laboratory mixer, a total of 9.6 L of laboratory well water was also added.

Prior to use, the peat was pre-soaked in dilution water for 1 week. During this time, the peat was amended with 160 g of powdered CaCO3 to increase the pH from 3.3 to 6.0.  
Representative samples of the sediment were analyzed periodically for pesticides, PCBs, and toxic metals by GeoLabs, Inc., Braintree, MA. None of these compounds were detected at concentrations that are considered toxic in any of the samples analyzed in agreement with ASTM (2007) standard practice. The results of these analyses were not provided in the study report for reference.
Sediment Characterization
 
Characterized by Agvise Laboratories, Northwood North Dakota
Particle distribution  -  83% sand, 4% silt, 13% clay
TOC  -  2.1%
Percent solids  -  68.78% 
pH  -  6.7
Test Material

  Identity:  Dacthal[(R)] technical herbicide (DCPA a.i.)
IUPAC name: Tetrachloroterephthalic acid
CAS name: 2,3,5,6-tetrachloro-1,4-benzenedicarboxylic acid
CAS No.:  1861-32-1
Description:  Not reported
Batch No.:  120904-1
Purity:  99.3%
Storage:  Room temperature, in a dark ventilated cabinet 
Aqueous solubility: 0.5 mg/L 

A 25 mg/mL primary stock solution was prepared by dissolving 0.6299 g of test substance in 25 mL with acetone. Five individual dosing stock solutions were then prepared (at 1.23 to 19.7 mg/mL) by diluting the appropriate volume of primary stock into 10 mL acetone. All dosing stock solutions were clear and colorless with no visible undissolved test substance.  
Solvent

Acetone, 9 mL/1.770 kg dw

The acetone was allowed to completely evaporate during the mixing procedure.  

Both solvent control and negative control groups were included in the study.   
Sediment Spiking

A jar-rolling technique was used to apply the test substance to the sediment. A 9-mL volume of the appropriate prepared dosing stock solution (in acetone) was applied to 0.050 kg of fine silica sand and the solvent was allowed to evaporate off for 70 minutes. The dry sand was then added to 2.75 kg of wet sediment (total of 1.770 kg dw sand + sediment based on a percent solids value of 68.78%) in individual glass jars (e.g., 4-L). Each jar was sealed and placed on a rolling mill, then rolled at room temperature for 4 hours at ca. 15 rpm. The solvent and negative controls were prepared in a similar manner, only without the addition of test material (solvent control) or solvent (negative control).

The range of nominal concentrations (nominally 6.3 to 100 mg/kg dw) was selected based on preliminary testing and in consultation with the Sponsor.  
Sediment Conditioning

A 28-day preliminary study was performed prior to the definitive study to determine the appropriate equilibration time for sediment toxicity testing with dacthal (Smithers Viscient Study No. 11857.6112; Appendix 2; pg. 78-80).
The results demonstrated that pore water concentrations on Days 14 and 21 were statistically similar and suggestive of pore water equilibrium. Based on these results, and consultation with the study sponsor, an approximate 14-day equilibration time of spiked sediments was used in the sediment toxicity testing with dacthal.  

The jars containing treated sediment were stored upright in a refrigerator at 2 to 8°C in the dark for a 14-day equilibration period.   

Once a week during the 14-day equilibration period and prior to being added into the replicate exposure vessels, the jars were mixed on the rolling mill at room temperature for 2 hours to ensure the sediment was homogeneous.
Test Vessels
300 mL high-form lipless glass beakers containing 100 mL of sediment and 175 ml of overlying water
300-mL glass vessels, with a slot cut on the top edge covered with 40-mesh screen for drainage.  

Each vessel contained 100 mL (ca. 4.0-cm layer) of sediment (equivalent to 104 g dw) and 175 mL of overlying water. The total overlying water plus sediment volume was maintained at approx. 275 mL.  
Type of Dilution System
Continuous or intermittent

Intermittent flow-through
Flow Rate
2 volume additions/day
Days 0-4:  2 volume additions/day 
Days 5-42:  4 volume additions/day
(increased due to low DO levels observed on Days 0-4)
Aeration
None, unless DO in overlying water drops <2.5 mg/L

None reported
Photoperiod
16 hours light, 8 hours dark using wide-spectrum fluorescent lights; intensity of 100 to 1000 lux

16-hour light/8-hour dark photoperiod using fluorescent bulbs at an intensity range of 230 to 940 lux.
Feeding

Each replicate test vessel received 1.5 mL of YCT (yeast, cereal leaves and flaked fish food suspension) and 0.50 mg of flaked fish food in an aqueous suspension daily. Representative samples of the food source were analyzed periodically for pesticides, PCBs, and toxic metals by GeoLabs, Inc., Braintree, MA. None of these compounds have been detected at concentrations that are considered toxic in any of the samples analyzed in agreement with ASTM (2007) standard practice.  The results of these analyses were not provided in the study report for reference.
   	
   C.  Test Design

                              Guideline Criteria
                             Reported Information
Duration
42 days

42 days
Sediment into Test Chambers
One day prior (Day -1) to start of test:  each sediment should be thoroughly homogenized and added to test chambers; Overlying water is added to chambers in a manner that minimizes suspension of sediment.
Test systems were established on Day -1. Each vessel contained 100 mL (ca. 4.0-cm layer) of sediment (equivalent to 151 g wet weight or 104 g dw based on unrounded wet weight per vessel) and 175 mL of overlying water. Overlying water was gently added using a turbulence reducer (a modified plastic disk), and each vessel was placed under the renewal system.  
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 should be verified by calibration prior to test initiation.  
The overlying water was renewed by adding two volume additions of water per test vessel per day via an intermittent delivery system in combination with a calibrated water-distribution system. The test system was calibrated before test initiation and visually inspected at least twice daily for proper functioning. The water system cycled approx. seven times per day and increased to 14 times per day on test day 5 (two and four overlying volume replacements per vessel per day, respectively).
Monitoring the test
All test chambers should be checked daily and observations made to assess organism behavior such as sediment avoidance.
Test systems were observed daily for organism behavior (e.g., adverse effects) and physical characteristics of test solutions (sediment:water system).  
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.
A 42-day range-finding test was conducted at nominal concentrations of 0.39, 1.6, 6.3, 25, and 100 mg/kg (dry weight). A control and a solvent (acetone) control were also included.  

Results for survival on Days 28, 35, and 42 ranged from 84 to 99% in all treatment groups, including controls. Length on Day 28 was 5.53 and 5.41 mm in the negative and solvent control groups, respectively, and 5.25, 5.10, 5.41, 5.17, and 5.20 mm per amphipod in the nominal 0.39, 1.6, 6.3, 25, and 100 mg a.i./kg treatment levels, respectively. Length on Day 42 was 6.10 and 6.01 mm in the negative and solvent control groups, respectively, and 6.05, 6.04, 6.07, 5.95, and 5.97 mm per amphipod in the nominal 0.39, 1.6, 6.3, 25, and 100 mg a.i./kg treatment levels, respectively. No treatment-related effects on growth were observed at any treatment level compared to the negative control at any time point. Reproduction on Day 35 averaged 5.9 and 6.8 offspring per female for the negative and solvent control groups, respectively, and 7.3, 7.6, 5.1, 2.9, and 5.8 offspring per female for the nominal 0.39, 1.6, 6.3, 25, and 100 mg a.i./kg treatment levels, respectively. Reproduction on Day 42 averaged 13 and 17 offspring per female for the negative and solvent control groups, respectively, and 16, 18, 14, 10, and 14 offspring per female for the nominal 0.39, 1.6, 6.3, 25, and 100 mg a.i./kg treatment levels, respectively. No treatment-related effects on reproduction were observed at any treatment level compared to the negative control at either time point. Male:Female ratios averaged 1.0, 0.80, 1.0, 1.4, 1.2, 1.0, and 2.4 for the nominal 0 (negative control), 0 (solvent control), 0.39, 1.6, 6.3, 25, and 100 mg a.i./kg treatment levels, respectively.  No treatment-related effects on Male:Female ratio was observed at any treatment level compared to the negative control. Based on these results, and in consultation with the Sponsor, nominal sediment concentrations were selected for the definitive test.
Nominal Sediment Concentrations
0 (negative control), 0 (solvent control), 6.3, 13, 25, 50, and 100 mg a.i./kg dw sediment
Number of Test Organisms
10 organisms per chamber are recommended; 12 replicates per treatment should be used (4 for 28-day survival and 8 for 35- and 42-day survival, growth, and reproduction)
10 organisms per test chamber

12 biological replicates per level; 120 organisms per test concentration. 

An additional three replicates (containing organisms) were established in the solvent control and treatment groups, while six replicates were established for the negative control for chemical analysis and monitoring water quality of the pore water.  
Test organisms randomly or impartially assigned to test vessels?

Yes
Water Parameter Measurements
Conductivity, hardness, alkalinity, and ammonia should be measured in all treatments at the beginning and end of the sediment exposure (Days 0 and 28).  Conductivity should also be determined weekly.

DO and pH should be measured at least three times per week.    

Temperature should be measured daily in at least one chamber from each level, and monitored continuously in the water bath.
Overlying water:
For all levels, total hardness, alkalinity, conductivity, and total ammonia (as N) concentrations were measured in a composite sample from all available biological replicates on Days 0, 28, 29, and 42. 

DO, temperature, and pH were measured in the overlying water of each replicate vessel used for biological monitoring on Days 0, 28, 29, and 42.  On the remaining days, DO and temperature were measured daily in one alternating replicate from each control and treatment group.  Temperature was also continuously monitored in an auxiliary vessel in the water bath.

Pore water: 
The pH and ammonia (as N) were measured in isolated pore water from the negative control on Days 0, 14 and 28.  
Chemical Analysis

Pre-test analyses:  
Treated sediment from all levels (prior to allocation into the replicate vessels) was analyzed for dacthal.  

In-life analyses:  
Concentrations of dacthal were determined in sediment, pore water and overlying water from surrogate test vessels collected on Days 0, 14, and 28 (termination of sediment phase of exposure).  

The overlying water was decanted and the sediment was centrifuged at ca. 10,000 g for 30 minutes to isolate the sediment and pore water matrices.  Sediment samples were then mixed well prior to analysis.   

Aqueous samples were analyzed using high performance liquid chromatography with ultraviolet detection (HPLC/UV) and sediment samples were analyzed using gas chromatography with micro-electron capture detection (GC/uECD) based on methodology validated at Smithers Viscient (see Reviewer's Comments).

12.  REPORTED RESULTS:

   A.  General Results
                              Guideline Criteria
                             Reported Information
Quality assurance and GLP com - pliance statements were in - cluded in the report?
Yes. This study was conducted in accordance with GLP Standards as specified in OECD (1998) and U.S. EPA (40 CFR, Part 160) with the following exceptions: routine water, sediment and food contaminant screening analyses for pesticides, PCBs, and toxic metals. It was reported however that these analyses were conducted following standard validated methods.
Control Criteria
Was average survival on Day 28 >=80%

Was average growth on Day 28 >=0.15 mg (dry weight) and/or >=3.2 mm (length) per amphipod? 

Did controls produce >=2 offspring per female between Days 28 and 42?
All control criteria met:
Negative control:  97% 
Solvent control:  98%

Negative control:  6.04 mm per amphipod
Solvent control:  5.93 mm per amphipod

Negative control:  9.8 offspring per female
Solvent control:   12 offspring per female
Percent Recovery of Chemical

Procedural recoveries (from QC samples) conducted concurrently with sample analysis:  

Sediment:
Spiked at 3.00, 25.0, and 100 mg a.i./kg
Recovery range of 84.5 to 108% (n=9)
LOQ sediment = 0.40 to 0.42 mg a.i./kg

Aqueous:
Spiked at 0.00500, 0.100, and 1.00 mg a.i./L
Recovery range of 84.9 to 110% (n=9)
LOQ water = 0.0023 to 0.0029 mg a.i./L 
Data Endpoints

Day 28:
- Survival 
- Length 
Day 35:
- Survival
- Offspring per female
Day 42:
- Survival
- Length 
- Offspring per female
- Male: female ratio
Raw data included?
Yes 
	
Effects Data:

     28-Day Endpoints:  
     
     Survival on Day 28 averaged 97 and 98% in the negative and solvent control groups, respectively, and ranged from 91 to 99% for all treatment levels, with no statistically-significant differences from the negative control indicated at any level. Using mean-measured sediment concentrations, the reported NOAEC and LOAEC for Day 28 survival were 92 and >92 mg a.i./kg, respectively. In terms of mean-measured pore water concentrations, the reported NOAEC and LOAEC were 0.34 and >0.34 mg ai./.L, respectively.
     
     Amphipod lengths at Day 28 averaged 6.04 and 5.93 mm per amphipod for the negative and solvent control groups, respectively, and 5.91, 6.00, 6.08, 5.82, and 5.79 mm per amphipod for the mean-measured 5.3, 11, 21, 41, and 92 mg a.i./kg treatment levels, respectively. No statistically-significant differences from the negative control were reported at any level. The reported NOAEC and LOAEC for Day 28 growth were 92 and >92 mg a.i./kg, respectively. In terms of mean-measured pore water concentrations, the reported NOAEC and LOAEC were 0.34 and >0.34 mg a.i./L, respectively.
     
     
     
                                  Test Conc.
                                Mean-measured 
                            (and Nominal) Sediment,
                                  mg a.i./kg
                                    Day 28
                                    Day 35
                                    Day 42
                                       
                                   Survival
                                   (% +- SD)
                        Length per Amphipod (mm +- SD)
                                   Survival
                                   (% +- SD)
                        No. Offspring per Female +- SD
                                   Survival
                                   (% +- SD)
                        Length per Amphipod (mm +- SD)
                        No. Offspring per Female +- SD
                           Male: Female Ratio +- SD
                               Negative control
                                    97 +- 5
                                 6.04 +- 0.068
                                    96 +- 5
                                  5.3 +- 4.0
                                    94 +- 5
                                 6.11 +- 0.20
                                  9.8 +- 8.8
                                  1.1 +- 0.54
                                Solvent control
                                    98 +- 6
                                 5.93 +- 0.27
                                    95 +- 8
                                  7.2 +- 4.9
                                   90 +- 12
                                 6.08 +- 0.10
                                   12 +- 10
                                 0.70 +- 0.94
                                   5.3 (6.3)
                                    99 +- 3
                                 5.91 +- 0.28
                                    95 +- 8
                                  9.1 +- 4.5
                                   91 +- 15
                                 6.26 +- 0.17
                                   14 +- 7.7
                                 0.94 +- 0.59
                                    11 (13)
                                    98 +- 5
                                 6.00 +- 0.13
                                    96 +- 7
                                  5.0 +- 2.2
                                    98 +- 5
                                 6.16 +- 0.28
                                  8.2 +- 4.7
                                 0.56 +- 0.36
                                    21 (25)
                                    93 +- 8
                                 6.08 +- 0.12
                                   93 +- 10
                                   11 +- 5.3
                                   80 +- 22
                                 6.28 +- 0.24
                                   16 +- 9.0
                                 0.43 +- 0.12*
                                    41 (50)
                                    95 +- 8
                                 5.82 +- 0.14
                                    95 +- 8
                                  6.5 +- 4.4
                                    95 +- 8
                                 6.37 +- 0.21
                                  9.4 +- 4.8
                                 0.68 +- 0.37
                                   92 (100)
                                    91 +- 9
                                 5.79 +- 0.052
                                    94 +- 7
                                  4.5 +- 2.4
                                   89 +- 11
                                 6.29 +- 0.23
                                   14 +- 14
                                  1.7 +- 2.2
*	Significantly different compared to the negative control using Wilcoxon's Test with Bonferroni's Adjustment; however, not considered to be treatment-related due to the lack of a dose-response.
LOQ = 0.40 to 0.42 mg a.i./kg dw for sediment samples and 0.0023-0.0029 mg a.i./L for aqueous samples.

     
     35-Day Endpoints:  
     
     After 35 Days, survival averaged 96 and 95% for the negative and solvent control groups, respectively, and ranged from 93 to 96% for all treatment levels, with no statistically-significant differences from the negative control indicated at any level.  Using mean-measured sediment concentrations, the reported NOAEC and LOAEC for Day 35 survival were 92 and >92 mg a.i./kg, respectively. Using mean-measured pore water concentrations, the reported NOAEC and LOAEC for Day 35 survival were 0.34 and >0.34 mg a.i./L, respectively.    
     
     Reproduction on Day 35 averaged 5.3 and 7.2 offspring per female for the negative and solvent control groups, respectively, and 9.1, 5.0, 11, 6.5, and 4.5 offspring per female for the mean-measured 5.3, 11, 21, 41, and 92 mg ai/kg treatment levels, respectively. No statistically-significant differences from the negative control were indicated at any level. The reported NOAEC and LOAEC for Day 35 reproduction were 92 and >92 mg ai/kg, respectively. In terms of mean-measured pore water concentrations, the reported NOAEC and LOAEC were 0.34 and >0.34 mg a.i./L, respectively.   
     
     42-Day Endpoints:  
     
     Survival at Day 42 averaged 94 and 90% for the negative and solvent control groups, respectively, and ranged from 80 to 98% for all treatment levels, with no statistically-significant differences from the negative control indicated at any level.  Using mean-measured sediment concentrations, the reported NOAEC and LOAEC for Day 42 survival were 92 and >92 mg ai/kg, respectively. Using mean-measured pore water concentrations, the reported NOAEC and LOAEC were 0.34 and >0.34 mg a.i./L, respectively.    
     
     Amphipod lengths at Day 42 averaged 6.11, 6.08, 6.26, 6.16, 6.28, 6.37, and 6.29 mm per amphipod for the mean-measured 0 (negative control), 0 (solvent control), 5.3, 11, 21, 41, and 92 mg a.i./kg treatment levels, respectively. No statistically-significant differences from the negative control were observed at any level. The reported NOAEC and LOAEC for Day 42 growth were 92 and >92 mg a.i./kg, respectively.  In terms of mean-measured pore water concentrations, the NOAEC and LOAEC were 0.34 and >0.34 mg a.i./L, respectively.   
     
     Reproduction on Day 42 averaged 9.8 and 12 offspring per female for the negative and solvent control groups, respectively, and 14, 8.2, 16, 9.4, and 14 offspring per female for the mean-measured 5.3, 11, 21, 41, and 92 mg a.i./kg treatment levels, respectively. No statistically-significant differences from the negative control were indicated at any level. The reported NOAEC and LOAEC for Day 42 reproduction were 92 and >92 mg a.i./kg, respectively. In terms of mean-measured pore water concentrations, the reported NOAEC and LOAEC for Day 42 reproduction were 0.34 and >0.34 mg a.i./L, respectively.   
     
     Male: female ratios averaged 1.1, 0.70, 0.94, 0.56, 0.43, 0.68, and 1.7 for the mean-measured 0 (negative control), 0 (solvent control), 5.3, 11, 21, 41, and 92 mg a.i./kg treatment levels, respectively. The value at 21 mg ai/kg was significantly different compared to the negative control; however, due to the lack of a dose-response this finding was not considered to be treatment-related. The reported NOAEC and LOAEC for male: female ratio were 92 and >92 mg a.i./kg, respectively, in terms of mean-measured sediment concentrations and 0.34 and >0.34 mg a.i./L, respectively, in terms of mean-measured pore water concentrations. 
     
	Analytical Data:  
     
     Treated sediment from all levels (prior to allocation into the replicate vessels) was analyzed for dacthal. Recoveries ranged from 94 to 110% of nominal.  During testing, concentrations of dacthal were determined in sediment, overlying water, and pore water on Days 0, 14 and 28.  
     
     Concentrations of dacthal in sediment were relatively constant to slightly declining during the 28-day period (with reviewer-calculated coefficients of variation of 5 to 29%; see Appendix 1). Overall mean-measured sediment concentrations were 5.3, 11, 21, 41, and 92 mg ai/kg, representing 83 to 92% of the nominal test concentrations.
     
     Concentrations of dacthal were slightly variable in pore water during the 28-day exposure period (with reviewer-calculated coefficients of variation of 18 to 36%; see Appendix 1).  
     
     In overlying water, concentrations of dacthal decreased over time during the 28-day exposure period (with reviewer-calculated coefficients of variation of 48 to 90%). 
     
     B.  Statistical Results (From Study Report)
     
     Endpoints that were statistically-analyzed included survival and growth (length) on Day 28; survival and reproduction (offspring per female) on Day 35; and survival, growth, reproduction, and male: female ratio on Day 42. Analyses were performed with CETIS[TM] (version 1.8, 2013) statistical software.  CETIS(TM) was also used to calculate any relevant LC/IC50 values. Results were provided in terms of mean-measured sediment concentrations.
     
     An Equal Variance Two-Sample t-Test or Wilcoxon's Rank Sum Two-Sample Test were used to compare the performance of the negative control and solvent control data. No significant differences between the control groups were observed for any endpoint. For all endpoints, treatment-level data were compared to the negative control data.
     
     Data for each endpoint were tested for normality using the Shapiro-Wilks' Test and for homogeneity of variance using Bartlett's Test.  Data pertaining to 28- and 35-Day survival, 42-day reproduction, and 42-Day male: female ratio did not meet the assumptions for normality and were subsequently analyzed using the non-parametric Wilcoxon's Test with Bonferroni's Adjustment. Data for all endpoints met the assumption for homogeneity of variance with the exception of 42-day male: female ratio. Data pertaining to 28- and 42-day growth, 35-day reproduction, and 42-day survival were analyzed with Bonferroni's Adjusted t-Test.  The NOAEC and LOAEC values were assigned based on significance.  All statistical analyses were conducted at the 95% level of certainty except in the case of the qualification tests (i.e., Shapiro-Wilks' and Bartlett's test), in which a 99% level of certainty was applied.  
     
                                   Endpoint
                                    Methods
                           Mean-measured Sediment, 
                                  mg a.i./kg
                           Mean-measured Pore Water,
                                   mg a.i./L
28-Day survival
Wilcoxon's Test with Bonferroni's Adjustment
                                  NOAEC:  92
                                LOAEC:  >92
                                 NOAEC:  0.34
                               LOAEC:  >0.34
28-Day length
Bonferroni's Adjusted t-Test
                                  NOAEC:  92
                                LOAEC:  >92
                                 NOAEC:  0.34
                               LOAEC:  >0.34
35-Day survival
Wilcoxon's Test with Bonferroni's Adjustment
                                  NOAEC:  92
                                LOAEC:  >92
                                 NOAEC:  0.34
                               LOAEC:  >0.34
35-Day offspring per female
Bonferroni's Adjusted t-Test
                                  NOAEC:  92
                                LOAEC:  >92
                                 NOAEC:  0.34
                               LOAEC:  >0.34
42-Day survival
Bonferroni's Adjusted t-Test
                                  NOAEC:  92
                                LOAEC:  >92
                                 NOAEC:  0.34
                               LOAEC:  >0.34
42-Day length
Bonferroni's Adjusted t-Test
                                  NOAEC:  92
                                LOAEC:  >92
                                 NOAEC:  0.34
                               LOAEC:  >0.34
42-Day offspring per female
Wilcoxon's Test with Bonferroni's Adjustment
                                  NOAEC:  92
                                LOAEC:  >92
                                 NOAEC:  0.34
                               LOAEC:  >0.34
42-Day male: female ratio
Wilcoxon's Test with Bonferroni's Adjustment
                                  NOAEC:  92
                                LOAEC:  >92
                                 NOAEC:  0.34
                               LOAEC:  >0.34
     
     Endpoint(s) Affected:  None

13.  VERIFICATION OF STATISTICAL RESULTS

     Statistical Method: 
     
     The reviewer analyzed the data using CETIS statistical software version 1.8.7.12 with database backend settings implemented by EFED on 10/20/2015. The negative and solvent control data were compared using a two-sample t-test. No significant differences were detected, except for male to female ratio, and for all other endpoints the subsequent analyses were conducted by comparing treatment data to the negative control only. For male to female ratio, given the observed significant difference between the negative and solvent controls (solvent 37% more biased towards female Hyallela), the reviewer conducted the analysis both against the negative control alone and against the solvent control alone. CETIS summary and analytical output files for each analysis are attached to this DER.
     
     Data were then tested for normality using Shapiro-Wilk's test (α = 0.01) and for homogeneity of variance using Bartlett's test (α = 0.01). The Day 28 and Day 42 length, Day 35 number offspring/female, and Day 35 and Day 42 reproduction data met the assumptions and were therefore analyzed using Dunnett's and Williams tests.  The Day 28, 35, and 42 survival, male:female ratio, and Day 42 offspring/female data did not meet both assumptions and were analyzed using the Mann-Whitney U Two-Sample Test and the Jonckheere-Terpstra Step Down test. All endpoints were analyzed using one-sided decreasing test assumptions (alternate hypothesis of control > treatment), except for the comparison on the solvent control to treatments for male to female ratio, which was analyzed using one-sided increasing test assumptions (alternate hypothesis of control < treatment).  
     
     Results were expressed as time-weighted average (TWA) bulk and OC-normalized sediment, overlying, and pore water concentrations. 

Endpoint
                                 Bulk Sediment
                                 (mg a.i./kg)
                    OC-Normalized Sediment (mg a.i./kg OC)
                                  Pore Water
                                  (mg a.i./L)
                                Overlying Water
                                  (mg a.i./L)
Day 28 Survival*
                                   NOAEC: 42
                                   LOAEC: 93
                                  NOAEC: 1976
                                  LOAEC: 4405
                                  NOAEC: 0.28
                                  LOAEC: 0.36
                                 NOAEC: 0.022
                                 LOAEC: 0.026
Days 35 and 42 Survival
                                   NOAEC: 93
                                 LOAEC: >93
                                  NOAEC: 4405
                                LOAEC: >4405
                                  NOAEC: 0.36
                                LOAEC: >0.36
                                 NOAEC: 0.026
                               LOAEC: >0.026
Day 28 length*
                                   NOAEC: 22
                                   LOAEC: 42
                                  NOAEC: 1024
                                  LOAEC: 1976
                                  NOAEC: 0.18
                                  LOAEC: 0.28
                                 NOAEC: 0.015
                                 LOAEC: 0.022
Days 35 and 42 Offspring/female
                                   NOAEC: 93
                                 LOAEC: >93
                                  NOAEC: 4405
                                LOAEC: >4405
                                  NOAEC: 0.36
                                LOAEC: >0.36
                                 NOAEC: 0.026
                               LOAEC: >0.026
Days 35 and 42 Reproduction
                                   NOAEC: 93
                                 LOAEC: >93
                                  NOAEC: 4405
                                LOAEC: >4405
                                  NOAEC: 0.36
                                LOAEC: >0.36
                                 NOAEC: 0.026
                               LOAEC: >0.026
Day 42 Length
                                   NOAEC: 93
                                 LOAEC: >93
                                  NOAEC: 4405
                                LOAEC: >4405
                                  NOAEC: 0.36
                                LOAEC: >0.36
                                 NOAEC: 0.026
                               LOAEC: >0.026
Male to female ratio**
                                   NOAEC: 93
                                 LOAEC: >93
                                  NOAEC: 4405
                                LOAEC: >4405
                                  NOAEC: 0.36
                                LOAEC: >0.36
                                 NOAEC: 0.026
                               LOAEC: >0.026
 *Statistically different (4%); and biologically meaningful (p = 0.02, survival and p <0.05, D28 length)
 ** There were significant differences between the negative and solvent control, this would result in a NOAEC and LOAEC of 42 and 93 mg a.i./kg-sediment in bulk sediment (OC-normalized 1976 and 4405 mg a.i./kg OC);0.28, and 0.36 mg a.i./L in pore water; and 0.022, and 0.026 mg a.i./L in overlying water, respectively.  
     
     Endpoint(s) Affected:  Day 28 Survival and Length
     Most Sensitive Endpoint(s): Day 28 Length

14.  REVIEWER'S COMMENTS:

The reviewer's statistical analyses and interpretation thereof differed from those of the study author's. The study author found no effects for any endpoint, whereas the reviewer found statistically significant differences between the negative control and the high treatment group for Day 28 length and survival; however, the difference was only 4%, but the effect was biologically meaningful (p = 0.02, survival and p <0.05, D28 length). Additionally, the reviewer calculated and used the time-weighted average concentrations for the matrices in this study, and the reviewer estimated toxicity values for all matrices. The reviewer calculated TWA concentrations given the length of time between measurements of DCPA in the various sample matrices (sediment, and/or water), on test days 0, 14 and 28, respectively. The study author used the mean-measured concentrations, and only assessed two matrices (bulk sediment and pore water). The reviewer's results are presented in the Conclusions section of this DER. 

On test day 6, dissolved oxygen (DO) levels fell to 2.17 mg/L in the nominal 50 and 100 mg/kg treatment levels, below the minimum recommended guideline limit of 2.5 mg/L. It was reported that the water renewal rate was increased (from 2x/day to 4x/day) to return dissolved oxygen levels to an acceptable range, and that this deviation had no impact on the results or interpretation of this study as the dissolved oxygen levels observed were within the acceptable range for the "majority of the exposure and the deviation was relatively short in duration". Although the range of the DO was provided, the time period at which a low DO (<2.5 mg/L) was experienced by test organisms was not provided, (i.e., daily DO measurements were not reported so that the length of low in DO could be confirmed). The controls appear to be healthy, and met all validity criteria, and therefore, this is considered to be a minor deviation.

There appeared to be a general trend for the reproductive endpoints that the solvent improved performance compared to the negative (water only) control. However, there were no significant differences between solvent and negative controls except for male to female ratio, where they were statistically significantly different with an approximate 40% difference in ratios (negative control mean of 1.1 males to female compared to 0.70 males to female in the solvent control). However, this does introduce some uncertainty into the reproduction endpoints.

Time-weighted average (TWA) concentrations were reviewer-calculated for all matrices (refer to copy of Excel worksheet in Appendix I) and are reported in the Conclusions sections of the DER.  TWA concentrations were calculated by the reviewer using the following equation:


     where:
     C TWA is the time-weighted average concentration,
     C j is the concentration measured at time interval j (j = 0, 1, 2...n)
     t j is the number of hours (or days or weeks, units used just need to be consistent in the equation) of the test at time interval j (e.g., t 0 = 0 hours (test initiation), t 1 =24 hours, t 2 =96 hours).
     
The NOAEC and LOAEC values were also reported in terms of OC-normalized sediment concentrations in the Conclusion section of the DER. The TOC of the sediment was 2.1%. The conversion table is reported below:

                          Bulk sediment (mg a.i./kg)
                                     % OC
                         OC-normalized (mg a.i./kg OC)
                                      5.2
                                      2.1
                                      248
                                      12
                                      2.1
                                      548
                                      22
                                      2.1
                                     1024
                                      42
                                      2.1
                                     1976
                                      93
                                      2.1
                                     4405

Amphipods were inadvertently not added to replicate C of Control and replicate K of the 6.3 mg/kg treatment group at exposure initiation. Consequently, these replicates were omitted from all statistical analyses of both lethal and sub-lethal endpoints. It was reported that three replicates for day 28 endpoints and seven replicates for day 42 endpoints were deemed sufficient to define the overall response for all endpoints.

Biological observations were made but not recorded for offspring recovery on day 42 for replicate J in the 50 and 100 mg/kg treatment levels. Consequently, these replicates were omitted from statistical analysis of the day 42 reproduction endpoint. It was reported that this deviation had a minimal impact on the interpretation of this study as the observations made and recorded on all other replicates at day 42 provided sufficient data for calculating reproductive endpoints.  

On test days 0 and 33, the water bath minimum temperature recorded was below the acceptable range at 20°C and on test days 0, 1, 2, 32, 33 and 34, the lowest water temperature within the test vessels was below the acceptable temperature with a low temperature of 21°C.  It was reported that since the temperatures observed were within the acceptable tolerance of the species, changes would have occurred at a gradual rate, and the temperature was within the acceptable range for the remainder of the exposure, this deviation did not have a negative impact on the results or interpretation of the study.  

Aqueous samples were analyzed for dacthal technical concentration using high performance liquid chromatography with ultraviolet detection (HPLC/UV) and sediment samples were analyzed using gas chromatography with micro-electron capture detection (GC/uECD) based on methodology validated at Smithers Viscient. The method validations were conducted prior to the initiation of the definitive study and established average recoveries of 102+-3.90% from artificial sediment and 97.9+-4.86% from filtered seawater. It was reported that conditions and procedures used throughout the analysis of exposure solutions and QC samples during this study were similar to those used in the method validation studies with the following exception related to analysis of the aqueous samples only:  the calibration curve from the validated method was prepared at concentrations from 0.00200 to 0.0500 mg/L. Three additional standards at concentrations of 0.100, 0.250 and 0.500 mg/L were added to the calibration curve to better enable the quantitation of the aqueous samples.  

The ammonia (as N) and pH were measured in isolated pore water from the negative control level on Days 0, 14, and 28. Ammonia levels ranged from <=0.10 to 1.8 mg/L, and the pH ranged from 6.6 to 7.5.  

The results from the 42-day preliminary range-finding study showed Male:Female ratios averaged 1.0, 0.80, 1.0, 1.4, 1.2, 1.0, and 2.4 for the nominal 0 (negative control), 0 (solvent control), 0.39, 1.6, 6.3, 25, and 100 mg a.i./kg treatment levels, respectively. A similar pattern between the negative control (average of 1), solvent control (<1), and highest treatment level (>> 1) was observed in the definitive study. This suggests that both the solvent effect on M:F ratios and the high treatment-related effect are real effects and not just noise based on the high variability of this endpoint.

The experimental phase of the definitive test was conducted from September 16 to October 28, 2015.  

15.  REFERENCES:

	None, other than standard guidelines and methodologies.

APPENDIX I. Copy of Excel Worksheet with TWA Concentrations



