﻿Data Requirement:PMRA Data Code      {............}
                        EPA DP Barcode420869
                        OECD Data Point{............}
                        EPA MRID 49307511
                        EPA Guideline850.1075


Test material:Dacthal(R) technical herbicidePurity: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 SamuelSignature:  
Environmental Scientist, CDM Smith/CSS-Dynamac JVDate: 10/06/2016

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

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

Secondary Reviewer(s): Michael WagmanSignature: 
EPA/OPP/EFED/ERB2/Senior ScientistDate: 11/12/2021

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

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

Date Evaluation Completed: 12-11-2021


CITATION: Shaw, A.C.  2013. Dacthal  -  Acute Toxicity of Sheepshead Minnow (Cyprinodon variegatus) Under Static-Renewal Conditions. Unpublished study performed by Smithers Viscient, Wareham, Massachusetts, USA. Laboratory Study No. 11857.6109. Study sponsored by AMVAC Chemical Corporation, Newport Beach, California, USA. Study initiated May 22, 2013 and completed September 30, 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:

In a 96-hour acute toxicity study, sheepshead minnow (Cyprinodon variegatus) 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 under static-renewal conditions. Mean-measured concentrations were <0.0027 (<LOQ, controls), 0.024, 0.047, 0.091, 0.18 and 0.44 mg a.i./L. 

Mortality and sublethal effects were observed daily. After 96 hours of exposure, no mortalities or sublethal effects were observed in the control or treatment groups in this experiment. Based on the lack of effects, the 96-hour LC50 value was >0.44 mg a.i./L (>440 ug a.i./L), and the observed NOAEC was 0.44 mg a.i./L (440 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 to Cyprinodon variegatus in accordance with the classification system of the U.S. EPA.  

It should be noted that although the highest nominal concentration is near 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. Additionally, prior to exposure the two highest test concentrations (0.25 and 0.5 mg/L) were `unltrasonicated' for approximately 5 minutes for maximum solubilization. In the end all solutions were clear and colorless with no visible undissolved test substance, and so based on these factors, the test material was in solution, and deemed to be reliable. 

This study is scientifically sound and is classified as acceptable.
   
   
   Results Synopsis

   Test Organism Size/Age (mean weight or length): wet weight: 0.20 (0.12-0.31) g; total length 22 (17-25) mm (juvenile)
   Test Type (Flow-through, Static, Static Renewal): Static-renewal
   
   96-hour LC50: >0.44 mg a.i./L(>440 ug a.i./L)95% C.I.:  N/A
   Probit Slope:  N/A95% C.I.:  N/A
   Observational NOAEC: 0.44 mg a.i./L (440 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 (1996) Ecological Effects Test Guideline (Draft) OCSPP (form. OPPTS) 850.1075, Fish Acute Toxicity Test, Freshwater and Marine. The following deficiencies were noted:
   
The hardness, particulate matter, and chlorine content of the dilution water were not reported. OCSPP guidance 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 850.1075 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, even though they were referenced as being in Appendix 4 (this was not provided; only up to Appendix 2 was provided in the study report), this is considered a minor study deficiency for both the draft OPPTS and final OCSPP 850.1075 guideline. 

These study deficiencies do not impact the acceptability of the 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) and OECD (OECD, 1988) regulations with the following exceptions: routine food and water 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:Measured concentrations were variable, with recoveries ranging from 69.6 to 87.5% of nominal. The associated coefficients of variation ranged from 31.0 to 37.7%. Since the renewal period was conducted at the half-way point of the test, the time-weighted average concentrations were identical to the mean-measured 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: Sheepshead Minnow (Cyprinodon variegatus)
      Age at test initiation:Juvenile
      Weight at study initiation: Mean wet weight: 0.20 g (range: 0.12 to 0.31 g); (N = 30)
      Length at study initiation: Mean total length: 22 mm (range: 17 to 25 mm); (N = 30)
      Source:  Smithers Viscient culture

   B.  STUDY DESIGN:

      1. Experimental Conditions

         a. Range-finding Study:  A 96-hour static renewal range-finding study was performed at nominal concentrations of 0 (negative and solvent controls), 0.000050, 0.00050, 0.0050, 0.050 and 0.50 mg/L. One test vessel containing ten fish was established for each treatment level and the control. After 96 hours, no mortality or sub-lethal effects were observed among sheepshead minnow 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 study sponsor.  
         
         b. Definitive Study

Table 1:  Experimental Parameters
Parameter
Details
Remarks


Criteria
Acclimation 

Period:

Conditions: (same as test or not)




Feeding:




Health: (any mortality observed)


>14 days.

Same (fish were maintained in dilution water near the test temperature).

Commercially prepared flake fish (Zeigler Brothers Prime Flakes) food, ad libitum, provided daily except during the 48 hours prior to testing. 

No mortality was observed during the 48-hour period prior to test initiation.
Prior to testing fish were held in a 55-L glass tank under a photoperiod of 16L:8D. Seawater that flowed into this tank had a salinity of 19 to 20 ppt. Temperature 22 to 28°C; and maintained at 22 to 24°C the 7-day period prior to exposure.
Representative samples of the food source were analyzed periodically for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, MA.


The recommended acclimation period is a minimum of 14 days; OECD guideline recommends a minimum of 12 days.  Pretest mortality should be < 3% 48 h. prior to testing.  OECD pretest mortality criteria: >10% = rejection of entire batch; > 5 and < 10% = continued acclimation for 7 days; <5% = acceptable.
Duration of the test
96 hours



The recommended test duration is 96 hours.
Test condition

Static/flow-through

Type of dilution system - for flow- through method.

Renewal rate for static renewal


Static-renewal

N/A


Once, at 48- hours



A reproducible supply of toxicant is recommended.   Consistent flow rate is usually 5-10 vol/24 hours; meter systems should be calibrated before and after study and checked twice daily during test period. 
Aeration, if any
No aeration reported.



Aeration is not recommended; OECD guideline recommends aeration.  If aeration is necessary, test solutions must be analyzed periodically to verify exposure.
Test vessel

Material: (glass/stainless steel)


Size:

Fill volume:


Aquarium constructed of glass and silicone adhesive.

39 x 20 x 25 cm (L x W x H)

15 L 
Test aquaria were impartially placed in a temperature-controlled water bath designed to maintain exposure solution temperatures at 22 +- 1°C.


Test vessel size is usually 19 L (5 gal) or  30 x 60 x 30 cm.
Fill volume is usually 15-30 L of solution.
Source of dilution water
Quality:
Dilution water was natural seawater obtained from the Cape Cod Canal, Bourne, and Massachusetts. Prior to use, the seawater was passed through a series of polypropylene core filters (20- and 5-micron) and recirculated within an epoxy-lined concrete reservoir. The dilution water was prepared by adjusting the salinity to 20 +- 3%. with the laboratory well water. 

The prepared dilution water had a salinity of 20%., and pH of 7.9 to 8.0.



Recommended source of dilution water is soft, reconstituted water or water from a natural 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/850.1010.pdf)  Dilution water should be intensely aerated before the study.  OECD permits dechlorinated tap water.
Water parameters:
Hardness

pH

Dissolved oxygen

Total Organic carbon

Particulate Matter

Metals

Pesticides

Chlorine

Temperature

{Salinity for marine or estuarine species}

Intervals of water quality measurement

Not reported

7.7 to 8.0

5.6 to 7.1 mg/L (>=60% saturation)

1.7 mg/L (dilution water, July 2013)

Not reported

Not detected

Not detected

Not reported

22 to 23°C

20%.


Dissolved oxygen concentration, temperature, and pH were measured once daily in all test chambers throughout the exposure period.  TOC were measured monthly.  
Representative samples of the dilution water source were analyzed periodically for the presence of pesticides, PCBs and toxic metals (Appendix 4, which was not provided in the study report) by GeoLabs, Inc., Braintree, MA. 
None of these compounds were detected in any of the water samples analyzed, in agreement with ASTM (2002) standard practices.


Hardness:
EPA recommends  40 - 48 mg/L as CaCO3 (OECD recommends 10 - 250 mg/L)
pH:
EPA recommends  7.2 - 7.6; 8.0-8.3 for marine-stenohaline fishes, 7.7-8.0 for estuarine-euryhaline fishes, monthly range < 0.8); (OECD recommends  pH 6.0 - 8.5)
Dissolved Oxygen:
EPA recommends:  Static: $ 60% during  first 48 hrs and $ 40% during second 48 hrs; flow-through: $ 60%; (OECD guideline recommends at least 80% saturation value).
Temperature:  
EPA recommends 12 EC  for coldwater species, 17 or 22 EC  for warmwater species, and  22 + 1 EC  for  estuarine/marine organisms. (OECD recommends 21 - 25oC for bluegill and 13 - 17oC for rainbow trout).
Salinity:  
EPA recommends 30-34%. (parts per thousand) for marine, 10-17%. for estuarine fish, weekly range < 6%..  

Water quality should be measured at beginning of test and every 48 hours.
Number of replicates/groups:
control:
solvent control:
treated ones:

2
2
2 per level



Recommended number of replicates include a control and five treatment levels. Each concentration should be 60% of the next highest concentration; concentrations should be in a geometric series.
Number of organisms per replicate / groups:
control:
solvent control:
treated ones:


10 
10
10 per level
20 fish per controls and each treatment level


Number of organisms per replicate should be   $ 10/concentration; OECD guideline recommends at least 7 fish/concentration.
Biomass loading rate
0.13 g fish tissue/L test solution



Recommended static conditions are # 0.8 g/L at # 17EC and # 0.5 g/L at > 17EC.  Recommended flow-through conditions are # 1 g/L/day.  OECD recommends a maximum of 1 g fish/L for static and semi-static, while higher rates are recommended for flow-through.
Test concentrations:
nominal:



mean-measured:

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

<0.0027 (<LOQ, controls), 0.024, 0.047, 0.091, 0.18 and 0.44 mg a.i./L
The study author calculated geometric mean-measured concentrations to be 0.023, 0.044, 0.084, 0.17, and 0.42 mg a.i./L.

The highest nominal concentration is at the solubility limit for DCPA (0.5 mg/L).

Both stock solutions as well as the test concentrations were clear and colorless throughout test. In addition, the material, prior to exposure the highest test concentration was `unltrasonicated' for approx. 5 minutes for maximum solubilization. In the end all solutions were clear and colorless with no visible undissolved test substance.
Solvent (type, percentage, if used)
Dimethylformamaide (DMF)
0.1 mL/L



The solvent should not exceed 0.5 ml/L for static tests or 0.1 ml/L for flow-through tests; OECD recommends that the solvent not exceed 100 mg/L.
Lighting
16-hours light/8-hours dark photoperiod. Light intensity was 516 to 1010 lux (48 to 94 footcandles) at the solutions surface. 
Sudden transitions from light to dark and vice versa were avoided.
Photoperiod was maintained with an automatic timer. 


The recommended photo period is 16 hours of light and 8 hours of dark with a 15-30 minute transition period.  OECD recommends a photo period of 12 -16 hours.
Feeding
Fish were not fed during the study exposure.



Fish should not feed during the study.
Recovery of chemical 

Frequency of determination



Level of quantitation

Level of detection

69.6 to 87.5% of nominal

Samples were collected at 0, 6, 24, 48, 72, and 96 hours and analyzed via HPLC/UV detection. 

0.0027mg/L

Not reported
The method validation was conducted prior to test initiation (24 June 2013) and established an average recovery of 97.9 +- 4.86% for dacthal from 20 ppt filtered seawater (Appendix 2; pg. 40-52 in the study report).

Measured concentrations of freshly prepared solution (0- and 48-hours) ranged from 79 to 110% of nominal concentration. Measured concentrations of the aged solution (48- and 96-hours) ranged from 45 to 65% of nominal concentration. Refer to copy of Excel worksheet in Appendix I.
Positive control {if used, indicate the chemical and concentrations} 
N/A

Other parameters, if any
None


   2. Observations:

Table 2:  Observations
Parameter
Details
Remarks


Criteria
Parameters measured including the sublethal effects/toxicity symptoms
Mortality
Sublethal effects (behavioral abnormalities, i.e., lethargy)

Observation intervals 
0, 6, 24, 48, 72, and 96 hours



Observation intervals should be a minimum  of every 24 hours.
Were raw data included?
Yes

Other observations, if any
None



II. RESULTS AND DISCUSSION:
   
   A. MORTALITY:

   After 96 hours of exposure, no mortalities were observed in the controls or treatment groups.  The LC50 value was reported to be >0.42 mg a.i./L using the geometric mean-measured concentrations.



Table 3:  Effect of Dacthal(R) (DCPA) on Mortality of Sheepshead Minnow (Cyprinodon variegatus).a
Mean-measuredb
(and nominal) Concentrations
(mg a.i./L)
No. of fish at start of study
Observation period


6 Hrs
24 Hrs
48 Hrs
72 Hrs
96 Hrs


No. Dead
% Mortality
No. Dead
% Mortality
No. Dead
% Mortality
No. Dead
% Mortality
No. Dead
% Mortality
<LOQc 
(Dilution water Control)
20
0
0
0
0
0
0
0
0
0
0
<LOQc
(Solvent Control)
20
0
0
0
0
0
0
0
0
0
0
0.024 (0.031)
20
0
0
0
0
0
0
0
0
0
0
0.047 (0.063)
20
0
0
0
0
0
0
0
0
0
0
0.091 (0.13)
20
0
0
0
0
0
0
0
0
0
0
0.18 (0.25)
20
0
0
0
0
0
0
0
0
0
0
0.44 (0.50)
20
0
0
0
0
0
0
0
0
0
0
LC50 (95% CI)
>0.42 mg a.i./Ld
aData were obtained from Table 3 on page 24 of the study report.
bBased on reviewer calculated mean-measured concentrations (refer to copy of Excel worksheet in Appendix I)
cLOQ = 0.0027 mg a.i./L
d The study author used the geometric mean-measured concentrations; whereas the reviewer used mean-measured concentrations 


   B. NON-LETHAL TOXICITY ENDPOINTS:

   No sublethal effects were observed in the controls or treatment groups during the study.  

Table 4:  Sublethal Effects of Dacthal(R) (DCPA) on Sheepshead Minnow (Cyprinodon variegatus).a
Mean-measuredb
(and nominal) Concentrations
(mg a.i./L)
Observation period

6 Hrs
24 Hrs
48 Hrs
72 Hrs
96 Hrs

% affected
% affected
% affected
% affected
% affected
< LOQc 
(Dilution water Control)
AN
AN
AN
AN
AN
< LOQc (Solvent Control)
AN
AN
AN
AN
AN
0.024 (0.031)
AN
AN
AN
AN
AN
0.047 (0.063)
AN
AN
AN
AN
AN
0.091 (0.13)
AN
AN
AN
AN
AN
0.18 (0.25)
AN
AN
AN
AN
AN
0.44 (0.50)
AN
AN
AN
AN
AN
EC50 (95% CI)
Not reported
  aData were obtained from p. 19 of the study report.
  bBased on reviewer calculated mean-measured concentrations (refer to copy of Excel worksheet in Appendix I)
  c LOQ = 0.0027 mg a.i./L
  AN = All normal

   C. REPORTED STATISTICS: 
   
   No statistical analyses were performed due to a lack of mortality in the controls and all treatment groups. 

   D. VERIFICATION OF STATISTICAL RESULTS:

   Statistical Method: Mortality data and corresponding mean-measured concentrations were entered into CETIS statistical software version 1.8.7.12 with database backend settings implemented by EFED on 10/20/15. There were no mortalities in the test, so statistical analyses were not conducted.

   96 hour LC50: >0.44 mg a.i./L95% C.I.: N/A
   Probit Slope:N/A95% 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 were based on the mean-measured concentrations (refer to copy of Excel worksheet in Appendix I) , whereas the study author's results were based on the geometric mean-measured concentrations. The reviewer's results are reported in the Executive Summary and Conclusions sections of this DER.

   The study was conducted following the draft OPPTS 850.1075 (1996) guideline, ASTM Standard E729-96 (2007), and the reviewer considered both the draft and the final OCSPP 850.1075 (2016) guideline and ASTM E729-96 in their evaluation of the data.
   
   The in-life phase of the definitive test was conducted from July 29 to August 2, 2013.
   
   Although the highest nominal concentration is at 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. Additionally, prior to exposure the two highest test concentrations (0.25 and 0.5 mg/L) were `unltrasonicated' for approximately 5 minutes for maximum solubilization. In the end all solutions were clear and colorless with no visible undissolved test substance, and so based on these factors, the test material was in solution, and deemed to be reliable.

   G. CONCLUSIONS:

   This study is scientifically sound and is classified as acceptable. After 96 hours of exposure, no mortalities or sublethal effects were observed in the control or treatment groups. Based on the lack of effects, the 96-hour LC50 was >0.44 mg a.i./L (>440 ug a.i./L), and the observed NOAEC was 0.44 mg a.i./L (440 ug a.i./L) based the on mean-measured concentrations.  
   
   96 hour LC50: >0.44 mg a.i./L(>440 ug a.i./L)95% C.I.: N/A
   Probit Slope:N/A95% C.I.: N/A
   Observational NOAEC: 0.44 mg a.i./L (440 ug a.i./L) 
   
   Endpoint(s) Affected:  None


III.  REFERENCES:


ASTM, 2002. Conducting acute toxicity tests with fishes, macroinvertebrates and amphibians. Standard E729-96. American Society of Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428.

Ives, M.  2011.  CETIS, Comprehensive Environmental Toxicity Information System (TM).  User's Guide.  Tidepool Scientific Software, McKinleyville, California.

U.S. EPA. 1996. Office if Chemical Safety and Pollution Prevention. Ecological Effects Guideline, OCSPP 850.1075. Fish Acute Toxicity Test, Freshwater and Marine. "Public Draft". EPA712-C-96-118. April 1996. U.S. Environmental Protection Agency, Washington, D.C.

APPENDIX I. Copy of Excel Worksheet with Measured Concentrations

