Data Requirement:				PMRA Data Code	 		{............}
                        EPA DP Barcode				420874
                        OECD Data Point			{............}
                        EPA MRID 					49307514
                        EPA Guideline				850.1010


Test material:	Dacthal(R) technical herbicide			Purity:	99.857%
Common name:    DCPA
Chemical name:	IUPAC: Dimethyl tetrachloroterephthalate
            CAS name: 1,4-Dimethyl 2,3,5,6-tetrachloro-1,4-benzenedicarboxylate
            CAS No.: 1861-32-1
            Synonyms: Chlorthal; Chlorthal-dimethyl
            

Primary Reviewer: Mary Samuel								Signature:  
Environmental Scientist, CDM Smith/CSS-Dynamac JV		Date:   10/06/2016

Secondary Reviewer: John Marton, Ph.D.						Signature:  
Environmental Scientist, CDM Smith/CSS-Dynamac JV		Date:   10/27/2016

Primary Reviewer:	Christina M. Wendel						Signature: 
EPA/OPP/EFED/ERB2/Biologist 								Date: 10/25/2021

Secondary Reviewer(s): Michael Wagman						Signature: 
EPA/OPP/EFED/ERB2/Senior Scientist						Date: 11/10/2021

Reference/Submission No.:  {.....................}

Company Code 		{............}	[For PMRA]
Active Code			{............}	[For PMRA] 
Use Site Category:	{............}	[For PMRA]
EPA PC Code 		078701

Date Evaluation Completed: 10-11-2021


CITATION: Shaw, A.C.  2013.  Dacthal - Acute Toxicity of Water Fleas, Daphnia magna, Under Static Conditions, Following OCSPP Guideline 850.1010. Unpublished study performed by Smithers Viscient, Wareham, Massachusetts, USA. Laboratory Study No. 11857.6107. Study sponsored by AMVAC Chemical Corporation, Newport Beach, California, USA. Study initiated May 22, 2013 and completed November 15, 2013.

This Data Evaluation Record may have been altered by the Environmental Fate and Effects Division subsequent to signing by CDM/CSS-Dynamac JV personnel.



EXECUTIVE SUMMARY:

The 48-hour acute toxicity of Dacthal(R) (DCPA) to Daphnia magna was studied under static conditions. Daphnids were exposed to Dacthal(R) (DCPA) at nominal concentrations of 0 (negative and solvent controls), 0.031, 0.063, 0.13, 0.25 and 0.50 mg a.i./L. The mean-measured concentrations were <0.0026 (<LOQ, controls), 0.032, 0.066, 0.13, 0.27 and 0.55 mg a.i./L.  

Mortality and sublethal effects were observed daily. No immobility (mortality) or sublethal effects were observed in the control or treatment groups in this experiment.  The 48-hour EC50 was >0.55 mg a.i./L (>550 ug a.i./L), and the observed NOAEC was 0.55 mg a.i./L (550 ug a.i./L) using mean-measured concentrations.

Based on the results of this study, Dacthal(R) (DCPA) would be classified as not toxic up to the highest mean-measured concentration (and/or the solubility limit of DCPA) to Daphnia magna in accordance with the classification system of the U.S. EPA. 

It should be noted that the highest nominal concentration is at/above the solubility limit for DCPA (0.5 mg/L). However, both the stock solutions and test concentrations were mixed by inversion and were clear and colorless with no undissolved test substance throughout the test. This indicates that the test material was in solution, and the analytical measurements are deemed to be reliable.

This study is scientifically sound and is classified as acceptable.


   Results Synopsis

   Test Organism Age (e.g., 1[st] instar): <24-hours old
   Test Type (Flow-through, Static, Static Renewal): Static

   48-hour EC50: >0.55 mg a.i./L	(>550 ug a.i./L)		95% C.I.: N/A
   Probit Slope:  N/A								95% C.I.: N/A
   Observational NOAEC: 0.55 mg a.i./L (550 ug a.i./L) 
   
   Endpoint(s) Affected:  None
   
I. MATERIALS AND METHODS

   GUIDELINE FOLLOWED:		The study protocol was based upon procedures outlined in the U.S. EPA Ecological Effects Test Guideline (Draft, 1996), OCSPP (form. OPPTS) 850.1010, Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids. The following deficiencies were noted:
   
 Health of the parental daphnids (e.g., number of broods, presence of ephippia) was not described. This is an uncertainty and considered a minor study deficiency for both the draft OPPTS and final OCSPP 850.1010 guideline.
 The hardness, particulate matter, and chlorine content of the dilution water were not reported. OCSPP guideline recommend that these parameters are measured and that these water quality characteristics meet EPA specifications. The lack of reporting is considered a minor study deficiency for both the draft OPPTS and final OCSPP 850.1010 guideline.
 Results from the periodic screening analysis of the dilution water were not reported in the study report, although representative samples were analyzed periodically for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, MA. The study author reported that no contaminants were detected in levels considered toxic. However, the results of these analyses were not provided in the study report for reference, which is considered a minor study deficiency for both the draft OPPTS and final OCSPP 850.1010 guideline. 
   
   These study deficiencies do not have an impact on the acceptability of this study.
   
   COMPLIANCE:				Signed and dated GLP, Quality Assurance, and Data Confidentiality statements were provided.  This study was conducted in compliance with all pertinent U.S. EPA GLP (40 CFR, Part 160) regulations with the following exceptions: routine food and water contaminant screening analyses.  These analyses, however, were performed using certified laboratories and standard validated methods.

   A. MATERIALS:

   	1. Test material  				Dacthal(R) technical herbicide

      Description: 				Not reported

      Lot No./Batch No. :			120904-1

      Purity: 						99.857%

      Stability of compound 
      under test conditions:		Stable. The mean-measured concentrations ranged from 100 to 110% of nominal concentrations.  

      Storage conditions of 
      test chemicals: 				Dark, room temperature
   
		 Physicochemical properties of DCPA.
Parameter
Values
Comments
Water solubility at 20°C
Not reported

Vapor pressure
Not reported

UV absorption
Not reported

pKa
Not reported

Kow
Not reported

      (OECD recommends water solubility, stability in water and light, pKa, Pow, vapor pressure of test compound)
   	2. Test organism: 

		Species: 					Daphnia magna				
		Age at test initiation:			<24-hours old 									
		Source: 						Smithers Viscient laboratory culture

   B.  STUDY DESIGN:

      1. Experimental Conditions

         a. Range-finding Study:  A 48-hour static range-finding study was performed at nominal concentrations of 0 (negative and solvent (DMF) controls), 0.000050, 0.00050, 0.0050, 0.050 and 0.50 mg/L. After 48 hours, no immobility or sub-lethal effects were observed among daphnids exposed to any of the treatment levels tested or the controls.  
         
         Nominal concentrations selected for use in the definitive study were based on these results and in consultation with the sponsor.  
         
         b. Definitive Study
         
Table 1:  Experimental Parameters
                                   Parameter
                                    Details
                                    Remarks
                                       
                                       
                                   Criteria
Acclimation
Period and conditions:





Feeding:







Health:  (any mortality observed)

N/A- No acclimation period was necessary because the culturing and test conditions were the same (temperature, dilution water, and lighting).

Daphnids were fed unicellular green algae, Ankistrodesmus falcatus (4 x10[7] cells/mL), and supplemented with suspension of YCT (yeast, cereal leaves and flaked fish food). Daphnids were not fed during the test.

No signs of immobility or sub-lethal effects were observed.  
Adults were cultured in water from the same source as used in testing. 

Representative samples of the food source were analyzed periodically for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, MA. None of these compounds have been detected in the food samples at concentrations considered to be toxic in any of the samples analyzed. However, the results of these analyses were not provided in the study report for reference.


The recommended acclimation period is a minimum of 7 days. Organisms should not feed during the study. 
Pretest mortality should be <3% 48 hours prior to testing.
Duration of the test

48 hours



EPA requires 96 hours, except daphnids which are 48 hours.
Test condition

Static/flow-through

Type of dilution system for flow- through method.

Renewal rate for static renewal


Static 


N/A

N/A



The recommended flow rates are 5 - 10 volume additions/24 hours; meter systems should be calibrated before and after the study and checked twice daily during the test period.
Aeration, if any
No aeration was provided during the test.

Test vessel

Material: (glass/stainless steel)
Size:
Fill volume:


Glass beakers
250 mL
200 mL



EPA requires:  small organisms in 3.9 L (1 gallon) wide mouth jars with 2-3 L of solution or daphnids and midge larvae in 250 ml jars w/ 200 ml fill
Source of dilution water
Dilution water was prepared by fortifying well water and filtering through an Amberlite XAD-7 resin column. The dilution water had a hardness of 160 mg/L as CaCO3, total alkalinity of 92 mg/L, a conductivity of 580 uS/cm, a pH of 8.2, and a dissolved oxygen concentration of 8.1 mg/L.

Representative samples of the dilution water were analyzed periodically for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, MA. None of these compounds have been detected in the water samples at concentrations considered to be toxic in any of the samples analyzed, in agreement with ASTM (2002) standard practice.


Recommended source of dilution water is soft, reconstituted water or water from a natural, uncontaminated source.  EPA does not recommend the use of dechlorinated tap water; however, its use may be supportable if the biological responses for the organisms and chemical analyses of residual chlorine meet conditions in the Agency's 850.1010 guidelines for dilution water (http://www.epa.gov/opptsfrs/OPPTS_Harmonized/850_Ecological_Effects_Test_Guidelines/Draft/850.1010Opdf).  Dilution water should be intensely aerated before the study.  
Water parameters

Hardness
pH
Dissolved oxygen




Temperature
Total Organic Carbon (TOC)
Particulate matter
Metals
Pesticides
Chlorine


Not reported
8.1 to 8.3
7.1 to 8.2 (5.6, and 5.3 mg/L represent 60% saturation at 19ºC, and 21ºC, respectively; and 9.7, and 9.3 mg/L represent 105% saturation at 19ºC, and 21ºC, respectively)
18-21°C
1.8 mg/L (dilution water, July 2013)
Not reported
Not detected
Not detected
Not reported
Dilution water had a hardness of 160 mg/L as CaCO3 (same as culture water). 

In addition, representative samples of dilution water were analyzed monthly for TOC concentration. 


Hardness:
EPA recommends  40 - 48 mg/L as CaCO3 (OECD recommends 140 - 250 mg/L)
pH: 
EPA recommends: 7.2 - 7.6 (OECD recommends pH of 6-9); measured at start and end of test in control, high, medium, and low test concentrations
Temperature: 
EPA recommends: 20[o]C for Daphnia (measured hourly) in at least one test vessel or if water baths are used, every 6 hr, may not vary > 1[o]C; 
OECD recommends range of 18-22C (+-1C)
Dissolved oxygen:  
EPA recommends: Measured at start and every 48 hours thereafter in control, high, medium, and low test concentrations.
Static: 60-100% during 1[st] 48 hr and 40-100% during 2[nd] 48 hr
Flow-through: 60-100% at all times
Number of replicates 
Negative control:
Solvent control:
Treatments:

4
4
4 per level
Daphnids were impartially selected and distributed.


EPA requires 2 or more containers for each treatment group; individuals must be randomly assigned to test vessels

OECD recommends 4 groups of 5 animals for each test concentration and the controls
Number of organisms per replicate
Negative control:
Solvent control:
Treatments:

5
5
5 per level
20 daphnids per concentration and control.


EPA/OECD requires 5 treatment levels plus one or more control groups; no more than 10% or 5% of control organisms should die during a static or flow-through study, respectively

EPA requires a minimum of 20 daphnids in 2 or more containers per treatment; however, if a limit test is conducted, it must be shown that the LC50/EC50 is >100 mg/L by exposing 30 organisms to >=100 mg/L or greater. Biomass loading rate for static <= 0.8 g/L at <= 17[o]C and  0.5 g/L at > 17[o]C; flow-through:  10 g/L at <= 17[]C and <=5 g/L at > 17[]C.

OECD recommends a minimum of 20 animals, preferably with 4 groups of 5 animals for each test concentration. There should be at least 2ml of test solution for each animal.
Treatment concentrations
Nominal:



Mean-Measured:

0 (negative and solvent controls), 0.031, 0.063, 0.13, 0.25 and 0.50 mg a.i./L 

<0.0026 (<LOQ, controls), 0.032, 0.066, 0.13, 0.27 and 0.55 mg a.i./L



Treatment concentrations should include a geometric series of at least five concentrations plus a control with each recommended concentration being at least 60% of the next higher one. The variability of measured concentrations between replicates of the same concentration should not exceed 1.5.

OECD recommends that the highest test concentration should result in 100% immobilization and not be >=1 g/L, while the lowest concentration should have no observable effect.
Solvent (type, percentage, if used)
Dimethylformamaide (DMF)
0.1 mL/L



Solvents should not exceed 0.5 ml/L for static tests or 0.1 ml/L for flow-though tests. OECD recommends that the solvent not exceed 100 mg/L.
Lighting
16-hours light/8-hours dark photoperiod. Fluorescent lighting with an intensity 650 to 830 lux (60-77 footcandles) in the culture area; and 370 to 850 lux (34-79 footcandles) in the test area. 30-minute transition period of low light intensity was provided when lights went on and off.



EPA-recommended photoperiod is 16 hours of light and 8 hours of dark with a 15-30 minute transition period.
OECD: optional light-dark cycle or complete darkness.
Stability of chemical in the test system
The mean-measured concentrations were 100 to 110% of nominal concentrations.  
The highest nominal concentration is at the solubility limit for DCPA (0.5 mg/L). Stock solutions, and test concentrations were mixed by inversion and were clear and colorless w/ no undissolved test substance throughout test.
Recovery of chemical 
Level of Quantitation (LOQ)
Level of Detection
100-110% of nominal
0.0026 mg/L
Not reported
At exposure initiation (0 hour) and exposure termination (48 hours), a single sample was removed from each test solution and control and analyzed for dacthal concentration. Samples removed at exposure initiation were taken from the intermediate vessels prior to division into replicate test vessels. The samples at exposure termination were a composite of all replicates within each concentration.

The method validation was conducted prior to test initiation (24 June 2013) and established an average recovery of 97.9 +- 4.86% for dacthal (DCPA) from 20%. filtered seawater (Appendix 2; pg. 40 in the study report).
Positive control {if used, indicate the chemical and concentrations}
N/A

Other parameters, if any
None



2. Observations:  

Table 2:  Observations
                                   Criteria
                                    Details
                                    Remarks
Parameters measured including the sublethal effects
Immobility and sublethal effects (abnormal behavior or appearance)
Immobilization was defined as those animals not able to swim within 15 seconds after gentle agitation of the test vessel.
Observation intervals 
0, 24 and 48 hours after test initiation

Were raw data included?
Yes

Other observations, if any
None

         
      
II. RESULTS AND DISCUSSION
   
   A. MORTALITY:

   After 48 hours, no immobility was observed in the control or treatment groups. The EC50 was estimated as >0.55 mg a.i./L, based on the mean-measured concentrations.

Table 3:  Effect of Dacthal(R) (DCPA) on Mortality of Daphnia sp.[a] 
                                Mean-measured 
                         (and nominal) Concentrations 
                                  (mg a.i./L)
                               No. of organisms
                              Observation period


                                   24 hours
                                   48 hours


                                   No. Dead
                                  % Immobile
                                   No. Dead
                                  % Immobile
<LOQ[b] (Dilution water only Control)
                                      20
                                       0
                                       0
                                       0
                                       0
<LOQ[b] (Solvent Control)
                                      20
                                       0
                                       0
                                       0
                                       0
0.032 (0.031)
                                      20
                                       0
                                       0
                                       0
                                       0
0.066 (0.063)
                                      20
                                       0
                                       0
                                       0
                                       0
0.13 (0.13)
                                      20
                                       0
                                       0
                                       0
                                       0
0.27 (0.25)
                                      20
                                       0
                                       0
                                       0
                                       0
0.55 (0.50)
                                      20
                                       0
                                       0
                                       0
                                       0
EC50 (with 95% CI)
>0.55 mg a.i./L
a	Data were obtained from Table 3 on page 24 of the study report.
b	LOQ = 0.0026 mg a.i./L

   B. SUB-LETHAL TOXICITY ENDPOINTS:

   No sublethal effects were observed for any treatment group during the test.  

Table 4:  Effect of Dacthal(R) (DCPA) on Sublethal Effects  -  Daphnia magna
                                Mean-measured 
                         (and nominal) Concentrations 
                                  (mg a.i./L)
                              Observation Period

                                   24 hours
                                   48 hours

                                  % Affected
                                  % Affected
<LOQ[a] (Dilution water only Control)
                                       0
                                       0
<LOQ[a] (Solvent Control)
                                       0
                                       0
0.032 (0.031)
                                       0
                                       0
0.066 (0.063)
                                       0
                                       0
0.13 (0.13)
                                       0
                                       0
0.27 (0.25)
                                       0
                                       0
0.55 (0.50)
                                       0
                                       0
EC50
>0.55 mg a.i./L
Positive control, if used  % sublethal effect: EC50:
N/A
  a 	LOQ = 0.0026 mg a.i./L

   C. REPORTED STATISTICS: 
   
   Due to the lack of immobility in the control and treatment groups, no statistical analyses were performed.
	
   D. VERIFICATION OF STATISTICAL RESULTS:

   Statistical Method: The mortality data and mean-measured concentrations of the test material were entered into the program CETIS, (Version 1.8.7.12) with backend settings implemented by EFED on 10/20/15. There were no mortalities in the test, so statistical analyses were not conducted.

   48-hour EC50: >0.55 mg a.i./L	(>550 ug a.i./L)		95% C.I.: N/A	
   Probit Slope:	 N/A								95% C.I.: N/A

   E.  STUDY DEFICIENCIES: 

   Though minor deviations were noted, there were no major study deficiencies.

   F.  REVIEWER'S COMMENTS: 
   
   The reviewer's results are in complete agreement with those of the study author's.  
   
   The study was conducted following the draft OPPTS 850.1010 (1996) guideline, and the reviewer considered both the draft and the final OCSPP 850.1010 (2016) guideline in their evaluation of the data.
   
   The in-life phase of the definitive test was conducted from July 23 to July 25, 2013.
   
   Although the highest nominal concentration is at/above the solubility limit for DCPA (0.5 mg/L). Both the stock solutions, and test concentrations were mixed by inversion and were clear and colorless with no undissolved test substance throughout the test, indicating that the test material was in solution and deemed to be reliable. 
   
   The method validation (June 24, 2013) as documented in Appendix 2 (pg. 40-53 in the study report) used 20%. seawater for the recovery method. It was conducted prior to test initiation and established an average recovery of 97.9 +- 4.86% for DCPA from 20%. filtered seawater. Although tested in `seawater' this was usable and acceptable method for recovery of DCPA in aquatic environments.   
   
G. CONCLUSIONS: 

   This study is scientifically sound and is classified as acceptable. After 48 hours, no mortalities or sublethal effects were observed in the controls or treatment groups. Based on the lack of effects, the EC50 value was >0.55 mg a.i./L (>550 ug a.i./L) and the observed NOAEC was 0.55 mg a.i./L (550 ug a.i./L) based on the mean-measured concentrations.  
   
   48-hour EC50: >0.55 mg a.i./L	(>550 ug a.i./L)		95% C.I.: N/A
   Probit Slope:  N/A								95% C.I.: N/A
   Observational NOAEC: 0.55 mg a.i./L (550 ug a.i./L)
   
   Endpoint(s) Affected:  None
   

III. REFERENCES: 

ASTM, 2002. Standard practice for conducting acute toxicity tests with fishes, macroinvertebrates and amphibians. Standard E729-88a. American Society for Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, Pennsylvania.

Ives, M.  2013.  CETIS, Comprehensive Environmental Toxicity Information System (TM).  User's Guide.  Tidepool Scientific Software, McKinleyville, California.

U.S. EPA, 1996. Office of Chemical Safety and Pollution Prevention. Ecological Effects Test Guideline. OCSPP 850.1010. Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids, "Public Draft". EPA 712-C-96-114. April 1996. U.S. Environmental Protection Agency, Washington, DC.
