Data Requirement:			PMRA Data Code	 		{............}
                     EPA DP Barcode				420868
                     OECD Data Point			{............}
                     EPA MRID					49307510
                     EPA Guideline				850.1300
                     
Test material:	Dacthal(R) technical herbicide				Purity:  99.3%
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:  Christie E. Padova 					Signature:  
Staff Scientist, CDM/CSS-Dynamac JV					Date:  07/29/2016

Secondary Reviewer:  John Marton, Ph.D.					Signature:  
Environmental Scientist, CDM/CSS-Dynamac JV			Date:   07/31/2016

Primary Reviewer:	Christina M. Wendel					Signature: 
EPA/OPP/EFED/ERB2/Biologist 							Date:  11/29/2021

Secondary Reviewer(s): Michael Wagman					Signature:
EPA/OPP/EFED/ERB2/Senior Scientist 					Date: 11/29/2021

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

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

Date Evaluation Completed: 29-11-0

CITATION:  Shaw, A.C.  2013.  Dacthal  -  Full Life-Cycle Toxicity Test with Water Fleas, Daphnia magna, Under Static Renewal Conditions.  Unpublished study performed by Smithers Viscient, Wareham, Massachusetts.  Laboratory Study No. 11857.6114.  Study sponsored by AMVAC Chemical Corporation, Newport Beach, California.  Study initiated May 22, 2013 and completed November 4, 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. The CDM/CSS-Dynamac Joint Venture role does not include 
establishing Agency policies.

EXECUTIVE SUMMARY:

The 21-day chronic toxicity of Dacthal(R) DCPA to Daphnia magna was studied under static renewal conditions.  Daphnids were exposed to Dacthal(R) technical herbicide (99.3% ai) at nominal concentrations of 0 (negative control), 0 (0.1 mL/L DMF solvent control), 0.031, 0.063, 0.13, 0.25 and 0.50 mg a.i./L. Mean-measured concentrations were <0.0028 (<LOQ, controls), 0.030, 0.065, 0.14, 0.27 and 0.54 mg a.i./L, representing 98 to 110% of nominal concentrations.  The overall (mean-measured) NOAEC and LOAEC were 0.14 and 0.27 mg a.i./L (140 and 270 ug a.i./L), respectively, based on treatment-related effects on dry weight at the 0.27 mg a.i./L (270 ug a.i./L) treatment level. Additional treatment related effects on reproduction (cumulative number of offspring per surviving adult and successful birth rate) and total length were observed at the 0.54 mg a.i./L treatment level. However, no treatment-related effect was apparent regarding survival or time to first brood release.

Mortality (number of immobilized adults) and observations of abnormal behavior were recorded daily. Survival averaged 80 to 100% for all control and treatment levels, with no statistically-significant differences indicated.  In addition, no treatment-related effect on the time to first brood release was indicated; first brood release occurred on Days 7 and 8 for all levels.  

Following 21 days of exposure, the cumulative number of offspring produced averaged 136, 145, 165, 192, 188, 199 and 56 for the mean-measured 0 (negative control), 0 (solvent control), 0.030, 0.065, 0.14, 0.27 and 0.54 mg a.i./L levels, respectively.  The difference was statistically-significant compared to the negative control at the 0.54 mg a.i./L treatment level (p<0.05, ANOVA, Dunnett's).  For the mean-measured 0 (negative control), 0 (solvent control), 0.030, 0.065, 0.14, 0.27 and 0.54 mg a.i./L treatment levels, total body lengths of surviving daphnia at study termination averaged 4.74, 4.80, 4.84, 4.94, 4.96, 4.90 and 4.56 mm, respectively, and dry body weights averaged 1.15, 1.11, 1.19, 1.07, 1.12, 0.97 and 0.90 mg, respectively. For total length, the difference was statistically-significant compared to the negative control at the 0.54 mg a.i./L (540 ug a.i./L) treatment level (p<0.05, Mann-Whitney), and the for dry weight the difference was statistically significant to the negative control at the 0.27 mg a.i./L (270 ug a.i./L) treatment level (p<0.05, ANOVA, Williams).  

It should be noted that 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.

This study is scientifically sound and is classified as acceptable.

	Results Synopsis

   Test Organism Age (e.g., 1[st] instar):  neonates, <24 hours old
   Test Type (Flow-through, Static, Static Renewal):  static renewal
   
   NOAEC:  0.14 mg a.i./L (140 ug a.i./L)
   LOAEC:  0.27 mg a.i./L  (270 ug a.i./L)
   
      Endpoint(s) affected: reproduction (cumulative number offspring and successful birth rate), length, and dry weight 
   Most sensitive endpoint(s):  dry weight

I. MATERIALS AND METHODS

   GUIDELINE FOLLOWED:		The study protocol was based upon procedures outlined in U.S. EPA OCSPP (formerly OPPTS) Guideline 850.1300, Daphnid Chronic Toxicity Test (Draft, 1996) and U.S. EPA OCSPP (form. OPPTS) Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies (Draft, 1996).
   
	Deficiencies from U.S. EPA OCSPP 850.1300 include: 
 The reproductive health and pre-test mortality of the brood culture were not described. Additionally, no information is provided to determine if the culture used for testing is first-brood progeny. This is considered a minor study deficiency for the final OCSPP 850.1300 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 850.1300 guideline. 
 It was not reported if ephippia were produced by control animals during the test, although "adult daphnids used to produce offspring for this test showed no signs of stress such as high mortality, presence of males or ephippia." Otherwise, all validity requirements as delineated in both the draft OPPTS and final OCSPP 850.1300 guideline were fulfilled.  
         
   These deficiencies do not affect 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) and OECD (OECD, 1988) regulations with the following exceptions:  routine soil 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: 						99.3%
      
      Stability of compound 
      under test conditions:		Stable (reviewer-calculated CVs of <=12% from periodic analysis of the new and aged test solutions).
      
      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, neonates, <24 hours old
      							EPA and OECD recommend Daphnia magna
      Age of the parental stock:  	Not reported
                           EPA recommends that young daphnids <=24 hours old from a separate parental culture be used
		Source:  					Smithers Viscient laboratory cultures
                           EPA requires all test organisms must be produced from laboratory reared culture that has been maintained for at least 21 days at test conditions in dilution water with renewal of the culture medium at least three times per week.
   
   B. STUDY DESIGN:

   	1. Experimental Conditions  

         a. Range-finding Study: A preliminary 21-day 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. After 21 days, survival was 100% for the controls (negative and solvent) through 0.050 mg/L treatment levels, and 80% at the 0.50 mg/L treatment level.  
         
         The number of young produced per female (reproduction) during the study averaged 141 and 156 for the negative and solvent control levels, respectively, and 161, 145, 145, 210 and 122 for the nominal 0.000050, 0.00050, 0.0050, 0.050 and 0.50 mg/L treatment levels, respectively. At study termination, total lengths averaged 4.48, 4.48, 4.56, 4.49, 4.44, 4.60 and 4.55 mm and dry body weights averaged 0.77, 0.85, 0.76, 0.72, 0.80, 0.67 and 0.73 mg for the negative control, solvent control, 0.000050, 0.00050, 0.0050, 0.050 and 0.50 mg/L treatment levels, respectively. Nominal dacthal 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
Parental acclimation:
Period:

Conditions: (same as test or not)

Feeding:
















Health (any mortality observed):

Continuous 

Same as test

During the culture, daphnids were fed various amounts based on their age. Daphnids that were 0 to 6 days old were fed 1.0 mL of  an algal suspension (Ankistrodesmus falcatus, 4 x 10[7] cells/mL), and 0.5 mL of a suspension of yeast, cereal leaves and digested flaked fish food (YCT) per day. Daphnids that were 7 to 10 days old were fed 1.5 mL of algae and 0.5 mL of YCT suspension per vessel per day. Daphnids that were >10 days old were fed 2.0 mL of algae and 0.5 mL YCT suspension per vessel per day.

The adults used to produce offspring for this test showed no signs of disease or stress (such as high mortality), and no males or ephippia were observed during the holding period.  Pre-test mortality was not reported.
Brood daphnia were maintained in culture water (prepared same as for dilution water) at 20 +- 2°C under a 16-hour light photoperiod (650 to 830 lux at the surface of the culture solution).  

The culture water was prepared by fortifying well water based on the formula for hard water (U.S. EPA, 1975). The prepared water was passed through an Amberlite[(R)] XAD-7 resin column to remove any potential organic contaminants. It was characterized as having a total hardness of 160 mg/L as CaCO3, total alkalinity of 92 to 94 mg/L as CaCO3, pH of 7.9 to 8.5, dissolved oxygen concentration of 7.1 to 9.8 mg/L and a conductivity of 570 to 630 uS/cm.  


EPA recommends that prior to testing, daphnids that are at least 10-12 days old (those that have had at least one brood) should be separated from the culture, put in separate container and maintained for at least 21 days to insure that good health conditions are present
Test condition:
Static renewal/flow-through:

Type of dilution system- for flow through method.

Renewal rate for static renewal

Static renewal


N/A

Three times per week (at 48- to 72-hour intervals)
 


(EPA requires consistent flow rate of 5-10 vol/24 hours, meter systems calibrated before study and checked twice daily during test period)
Aeration, if any

None reported



EPA recommends test chambers should not be aerated
Duration of the test
21 days



Recommended duration is 21 days.
Test vessel
Material (glass/stainless steel):

Size (for growth and reproduction/survival test):

Fill volume:

Clear glass beakers


100 mL

80 mL 



1. Recommended Material:  Glass, No.      316  stainless steel, or 
   perfluorocarbon plastics
2. Recommended Size:  250 ml with 200 ml fill volume; 100 ml with 80 ml fill volume 
OECD guideline recommends that parent animals be maintained individually; one per vessel, with 50 - 100 ml of medium in each vessel.
Source of dilution water
Culture and dilution water were prepared in 1900-L batches by fortifying well water according to the formula for hard water (U.S. EPA, 1975).  The prepared water was passed through an Amberlite(R) XAD-7 resin column to remove any potential organic contaminants. 

Water quality parameters were measured on each batch prior to use; the prepared water had a total hardness of 160 mg/L as CaCO3, alkalinity of 90 to 94 mg/L as CaCO3, pH of 8.1 to 8.3, DO of 7.4 to 8.7 mg/L, and a conductivity of 580 to 600 μS/cm.
Fortified water was discarded if not used within 14 days.

Periodic analysis of representative samples of the dilution water source for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, MA; indicated that none of these compounds were detected at concentrations that were considered toxic in any of the water samples analyzed in agreement with ASTM (2002) standard practice. However, the results of these analyses were not provided in the study report for reference.


Recommended source of dilution water includes unpolluted well or spring water that has been tested for contaminants, or appropriate reconstituted water (see ASTM for details).
Water parameters:
Hardness
pH
Dissolved oxygen


Temperature


Total Organic Carbon

Particulate matter
Metals
Pesticides
Chlorine

Frequency of measurements
During 21-day exposure period
160 to 170 mg/L as CaCO3
7.9 to 8.4
7.3 to 9.3 mg/L (>=81% of saturation)

Daily:  19 to 21°C 
Continuous:  17 to 21°C

1.8 and 0.64 mg/L (dilution water, July and august 2013, resp.)
Not reported
Not reported
Not reported
Not reported

DO, temperature, and pH were measured in each test and control solution at the beginning (new solutions) and end (composite sample of aged solutions) of each renewal period. Temperature was also continuously monitored in the environmental chamber. Total hardness, alkalinity, and conductivity were measured in the freshly-prepared solutions of the negative control and highest nominal test concentration with surviving organisms at exposure initiation and weekly throughout the test. On Day 21, samples were taken from aged solutions. 
Total alkalinity ranged from 94 to 96 mg/L as CaCO3 and conductivity ranged from 600 to 640 uS/cm.


Recommended hardness:  160 to 180 mg/L as CaCO3; OECD recommends > 140 mg/L as CaCO3
Recommended pH:  7.6 to 8.0, pH should not deviate by more than 1.0 unit for more than 48 hours.  OECD recommends that pH range be 6 - 9 and does not vary more than 1.5 units in any one test.
Recommended dissolved oxygen:    renewal should not drop below 50% for more than 48 hours.
Recommended flow-through: >= 60% throughout test.
Recommended temperature:
20°C +- 2°C.; should not deviate from 20+-C by more than 5°C for more than 48 hours. OECD recommends a range of 18 - 22[o]C; temperature should not vary more than +- 2[o]C
OECD guideline recommends that total organic carbon < 2 mg/L
Number of replicates:
For growth and reproduction:

For survival test:

10 per level

(Same)

There were five toxicant levels spaced by a separation factor of 2.


Number of replicates should include a control(s) and at least 5 test concentrations; dilution factor should not be greater than 50%. 
OECD recommends that at least 5 test concentrations be used in a geometric series with a separation factor not exceeding 3.2.
Number of organisms:
For growth and reproduction:

For survival test:

1 organism per replicate

(Same)
Daphnids were impartially distributed to each of the 10 replicates for each test concentration and control


Recommended number of organisms include 22 daphnids/test concentration;
7 test chambers should contain 1 daphnid each, and 3 test chambers contain 5 daphnids each.
OECD recommends holding a minimum of 10 daphnids individually for static tests. 
For flow-through tests, 40 animals should be divided into 4 groups of 10 animals at each test concentration.
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

<0.0028 (<LOQ, controls), 0.030, 0.065, 0.14, 0.27 and 0.54 mg a.i./L


Test samples were collected from freshly-prepared test solutions (all levels) on Days 0, 2, 16 and 19 and from a composite sample (of all available replicates) of aged solutions on Days 2, 5, 19 and 21. Samples were analyzed for Dacthal using high performance liquid chromatograph with ultraviolet detection (HPLC/UV). 

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.

Measured concentrations were consistent between sampling intervals for both newly prepared and aged solutions. Concentration of dacthal in new solutions ranged from 100 to 120% of nominal, concentrations in aged solutions ranged from 82 to 110% of nominal. The mean-measured concentrations represented 98 to 110% of nominal levels, with minimal analytical variation observed (reviewer-calculated CVs of <=12% for all levels). 
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. 63-75 in the study report).       
Solvent (type, percentage, if used)

Dimethylformamaide (DMF)
0.1 mL/L



Solvent concentration should not exceed 0.5 ml/L for static tests or 0.1 ml/L for flow-through tests. Recommended solvents include dimethylformamide, triethylene glycol, methanol, acetone and ethanol.
OECD recommends <=0.1 ml/L of solvent.
Lighting
16-hours light, 8-hours dark; the intensity ranged from 2.6 to 11 μEm[-2]s[-1] (~192 to 814 lux) at the surface of the exposure solutions was provided by fluorescent bulbs.  
Sudden transitions from light to dark and vice versa were avoided


Recommended photoperiod is 16 hours light and 8 hours of dark.
Recovery of the chemical: 

Frequency of determination

LOD:
LOQ:
106 to 116% of nominal (n=17)

Days 0, 2, 5, 16, 19 and 21

Not reported
0.0025 to 0.0028 mg a.i./L
Based on QC samples fortified at 0.0188, 0.125 and 1.00 mg/L and analyzed concurrently with test samples.  Excludes one outlier with no recovery due to a stock preparation error.
Positive control {if used, indicate the chemical and concentrations} 

N/A

Other parameters, if any
During the culture, daphnids were fed various amounts based on their age. Daphnids that were 0 to 6 days old were fed 1.0 mL of an algal suspension (Ankistrodesmus falcatus, 4 x 10[7] cells/mL), and 0.5 mL of a suspension of yeast, cereal leaves and digested flaked fish food (YCT) per day. Daphnids that were 7 to 10 days old were fed 1.5 mL of algae and 0.5 mL of YCT suspension per vessel per day. Daphnids that were >10 days old were fed 2.0 mL of algae and 0.5 mL YCT suspension per vessel per day. During the test, daphnids were fed once daily at a rate of 200 uL of algal suspension(Ankistrodesmus falcatus, 4 x 10[7] cells/mL), and 50 uL of a YCT suspension once daily. 
For the months of July and August (2013), the quantity of algal suspension (200 uL) was equivalent to ca. 0.43 and 0.53 mg Carbon/daphnid/day, respectively.

      2. Observations:  

Table 2:  Observations
                                  Parameters
                                    Details
                                    Remarks


                                   Criteria
Data endpoints measured (list)
- Parental survival (immobility)
- Clinical signs of toxicity
- Time to first brood release
- Neonate production
- Terminal length and dry weight of surviving first-generation daphnia
The time to first brood release was reported, but not statistically-evaluated by the study author. 
Immobilization was defined as the inability of daphnids to swim within 15 seconds of gentle agitation of the test vessel.
Numbers of offspring were determined upon the first brood release in any vessel, day 7, and daily throughout the remainder of the test.


Recommended endpoints measured:
- Survival of first-generation daphnids,
- Number of young produced per
  female,
- Dry weight (required) and  length (optional) of each  first generation daphnid alive at the end of the  test,
- Observations of other effects or clinical signs.
Observation intervals 
Daily    

Were raw data included?
Yes, sufficient

Other observations, if any
N/A


II. RESULTS AND DISCUSSION

	A. MORTALITY:

   Survival at 21 Days was 100 and 90% for the negative and solvent control levels, respectively, and 100, 100, 100, 100 and 80% for the mean-measured 0.030, 0.065, 0.14, 0.27 and 0.54 mg a.i./L treatment levels, respectively.  No statistically-significant differences were indicated at any level compared to the pooled control (95% survival).  The NOAEC and LOAEC were 0.54 and >0.54 mg a.i./L, respectively.
   
Table 3:  Effect of Dacthal(R) (DCPA) on Survival, Reproduction, and Growth of Daphnia sp.[a] 
                                   Treatment
                                 Mean-measured
                        (and Nominal) conc. (mg a.i./L)
                            Mean % Adult Survival 
           Mean No. of Offspring Released per Surviving Female +- SD
                       Mean Total Body Length (mm) +- SD
                          Mean Dry Weight (mg) +- SD
Negative control
                                      100
                                   136 +- 18
                                 4.74 +- 0.15
                                 1.15 +- 0.20
Solvent control
                                      90
                                   145 +- 16
                                 4.80 +- 0.15
                                 1.11 +- 0.13
Pooled control
                                      95
                                   140 +- 18
                                 4.77 +- 0.15
                                 1.14 +- 0.17
0.030 (0.031)
                                      100
                                   165 +- 18
                                 4.84 +- 0.11
                                 1.19 +- 0.12
0.065 (0.063)
                                      100
                                   192 +- 24
                                 4.94 +- 0.09
                                 1.07 +- 0.17
0.14 (0.13)
                                      100
                                   188 +- 15
                                 4.96 +- 0.10
                                 1.12 +- 0.10
0.27 (0.25)
                                      100
                                   199 +- 33
                                 4.90 +- 0.14
                                 0.97 +- 0.15
0.54 (0.50)
                                      80
                                   56 +- 23*
                                 4.56 +- 0.23*
                                 0.90 +- 0.30*
NOAEC, mg a.i./L
                                     0.54
                                     0.27
                                     0.27
                                     0.27
LOAEC, mg a.i./L
                                   >0.54
                                     0.54
                                     0.54
                                     0.54
a Data were obtained from Table 12 and 13 on page 42 and 43 of the study report.
* Statistically-reduced (p<0.05) compared to the pooled control.  
  
   B. EFFECTS ON REPRODUCTION AND GROWTH:  
   
   First brood release occurred on test Day 8 for all treatment levels tested, with the exception of the 0.14 and 0.27 mg/L treatment levels, where the first brood release occurred on Day 7.  Following 21 days of exposure, the cumulative number of offspring produced averaged 136, 145, 165, 192, 188, 199 and 56 for the mean-measured 0 (negative control), 0 (solvent control), 0.030, 0.065, 0.14, 0.27 and 0.54 mg a.i./L levels, respectively.  The difference was statistically-significant compared to the pooled control (140 offspring) at the 0.54 mg a.i./L treatment level (p<0.05, Bonferroni's Adjusted t-Test).  The subsequent NOAEC and LOAEC for reproduction were 0.27 and 0.54 mg a.i./L, respectively.  
   
   For the mean-measured 0 (negative control), 0 (solvent control), 0.030, 0.065, 0.14, 0.27 and 0.54 mg a.i./L treatment levels, total body lengths of surviving daphnia at study termination averaged 4.74, 4.80, 4.84, 4.94, 4.96, 4.90 and 4.56 mm, respectively, and dry body weights averaged 1.15, 1.11, 1.19, 1.07, 1.12, 0.97 and 0.90 mg, respectively.  For both growth endpoints, the difference was statistically-significant compared to the pooled control (4.77 mm and 1.14 mg) at the 0.54 mg a.i./L treatment level (p<0.05, Bonferroni's Adjusted t-Test).  The NOAEC and LOAEC for total length and dry weight were 0.27 and 0.54 mg a.i./L, respectively.  
   
   C. REPORTED STATISTICS: 
   
   Data endpoints that were statistically-analyzed included organism survival, reproduction (cumulative number of offspring produced per surviving female), and growth (as total body length and dry body weight).  Data obtained for the time to first brood release were not statistically-evaluated.  All analyses were conducted at the 95% level of certainty except in the case of the Shapiro-Wilks' and Bartlett's Tests, in which the 99% level of certainty was applied.  Data were analyzed using CETIS(TM) (version 1.8; 2013) statistical software.  
   
   Data for the negative and solvent control groups were compared for each endpoint using an Equal Variance Two-Sample Test.  No statistically-significant differences were indicated between the two controls for any endpoint, and subsequent comparisons were made to the pooled controls.  
   
   Survival data were analyzed using Fisher's Exact Test with Bonferroni-Holm's Adjustment.  Reproduction and growth data were checked for normality using the Shapiro-Wilks' Test and for homogeneity of variance using Bartlett's Test.  Reproduction and growth data met both assumptions and were analyzed using Bonferroni's Adjusted t-Test.  
   
   Survival
   NOAEC:  0.54 mg a.i./L 
   LOAEC:  >0.54 mg a.i./L
   
   Reproduction
   NOAEC:  0.27 mg a.i./L
   LOAEC:  0.54 mg a.i./L
   
   Length
   NOAEC:  0.27 mg a.i./L
   LOAEC:  0.54 mg a.i./L
   
   Dry Weight
   NOAEC:  0.27 mg a.i./L
   LOAEC:  0.54 mg a.i./L
   Endpoint(s) affected:  reproduction, length, and dry weight 
   Most sensitive endpoint(s):  reproduction, length, and dry weight
   
   D. VERIFICATION OF STATISTICAL RESULTS:

   Statistical Method:  The reviewer analyzed F0 survival, time to first brood, reproduction (total offspring, offspring per surviving female per reproductive day), and F0 growth (dry weight, total length). Negative and solvent control data for each endpoint were compared using a two-sample t-test assuming equal variance. No differences were detected, and all subsequent analyses were conducted by comparing treatment data to the negative control only. Data were tested for normality using the Shapiro-Wilk's test (α = 0.01) and for homogeneity of variance using Bartlett's test (α = 0.01). Dry weight, total offspring, and successful birth rate met these assumptions and were analyzed using analysis of variance followed by Dunnett's multiple comparison test. Additionally, dry weight was also analyzed using analysis of variance followed by Williams multiple comparison test. Survival was analyzed using Fisher's Exact test, and total length, and time to first brood, which did not meet assumptions of normality and equal variance, were analyzed using the non-parametric Mann-Whitney U two-sample test. All analyses were conducted at α = 0.05 unless specified otherwise, and all toxicity values are based on the mean-measured concentrations.
   
   Survival
   NOAEC:  0.54 mg a.i./L (540 ug a.i./L)
   LOAEC:  >0.54 mg a.i./L (>540 ug a.i./L)
   
   Time to Frist Brood
   NOAEC:  0.54 mg a.i./L (540 ug a.i./L)
   LOAEC:  >0.54 mg a.i./L (>540 ug a.i./L)
   
   Total Offspring
   NOAEC:  0.27 mg a.i./L (270 ug a.i./L)
   LOAEC:  0.54 mg a.i./L (540 ug a.i./L)
   
   Successful Birth Rate
   NOAEC:  0.27 mg a.i./L (270 ug a.i./L)
   LOAEC:  0.54 mg a.i./L (540 ug a.i./L)
   Length
   NOAEC:  0.27 mg a.i./L (270 ug a.i./L)
   LOAEC:  0.54 mg a.i./L (540 ug a.i./L)
   
   Dry Weight
   NOAEC:  0.14 mg a.i./L  (140 ug a.i./L)
   LOAEC:  0.27 mg a.i./L  (270 ug a.i./L)
   
      Endpoint(s) affected: reproduction (cumulative number offspring and successful birth rate), length, and dry weight 
   Most sensitive endpoint(s): dry weight
   
   E.  STUDY DEFICIENCIES: 
   
   This study is scientifically sound; however, the following deviations and/or deficiencies from OCSPP (form. OPPTS) 850.1300 guidance were observed:  the reproductive health and pre-test mortality of the brood culture were not described, and it was not reported if ephippia were produced by control animals during the test, although "adult daphnids used to produce offspring for this test showed no signs of stress such as high mortality, presence of males or ephippia." 
   
   F.  REVIEWER'S COMMENTS:
   
   The reviewer's conclusions were comparable to those reported by the study author; however the study author compared the treatment-level data to the performance of the pooled controls for all endpoints, whereas the reviewer compared the results to the negative control only. Additional endpoints not analyzed by the study author but analyzed by the reviewer included time to first brood, and successful birth rate (offspring per surviving female per reproductive day; calculated in Appendix I below). The reviewer also determined that overall the terms of monotonicity were met for dry weight, and so it was also analyzed using analysis of variance followed by Williams multiple comparison test, resulting in a lower endpoint. 
   
   It was not reported if ephippia were produced by control animals during the test, although "adult daphnids used to produce offspring for this test showed no signs of stress such as high mortality, presence of males or ephippia."  Otherwise, all validity requirements as delineated in OCSPP (form. OPPTS) 850.1300 guidance (draft, 1996) for a daphnid chronic toxicity test were fulfilled (i.e., controls >= 80% survival and >= 60 offspring per female). Although the controls met this, the highest test concentration had 80% survival and produced <60 offspring per female, resulting in significant effects compared to the negative control. 
   
   All exposure and QC samples were analyzed for dacthal using high performance liquid chromatography with ultraviolet detection (HPLC/UV) based on methodology validated at Smithers Viscient (Appendix 2, pg 63-75 of the study report). The method validation was conducted prior to the initiation of the definitive exposure and established an average recovery of 97.9 +- 4.86% for dacthal from filtered seawater. It was reported that conditions and procedures used throughout the analysis of exposure solutions and QC samples during this study were the same as those used in the method validation study.  
   
   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. 
   
   On test Day 9, the minimum temperature on the minimum/maximum thermometer was recorded at 17°C.  It was reported that since the minimum/maximum thermometer was inadvertently not reset after calibration, the temperature reading was not representative of the true temperature of the test chamber or test solutions and this deviation did not have a negative impact on the results or interpretation of the study.
   
   The study was conducted following the draft OPPTS 850.1300 (1996) and 850.1000 (1996) guideline, and the reviewer considered both the draft and the final OCSPP 850.1300 (2016) and 850.1000 (2016) guidelines in their evaluation of the data.
   
   The in-life phase of the definitive test was conducted from July 24 to August 14, 2013.
   
   G. CONCLUSIONS:  
   
   This study is scientifically sound and is classified as acceptable. Based on treatment-related effects on dry weight at the 0.27 mg a.i./L treatment level, the overall NOAEC and LOAEC were 0.14 and 0.27 mg a.i./L (140 and 270 ug a.i./L), respectively (mean-measured concentrations). Additional treatment related effects on reproduction (cumulative number of offspring per surviving adult and successful birth rate) and total length were observed at the 0.54 mg a.i./L treatment level. However, no treatment-related effect was apparent regarding survival or time to first brood release.
   
III.  REFERENCES: 

Biesinger, K.E., R.W. Andrew and J.W. Arthur.  1974.  Chronic toxicity of NTA (nitrilotriacetate) and metal-NTA complexes to Daphnia magna.  J. Fish. Res. Board Can.  31: 486-490.

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

Kenaga, E.E. 1978.  Test organisms and methods useful for early assessment of acute toxicity of chemicals.  Env. Sci. Technol. 12: 1322-1329.  M

Macek, K.J., et al.  1976a.  Chronic toxicity of lindane to selected aquatic invertebrates and fishes. EPA-660/3-76-046. U.S. Environmental Protection Agency. Washington, D.C.

Macek, K.J., et al.  1976b.  Chronic toxicity of atrazine to selected aquatic invertebrates and fishes.  EPA-600/3-76-047.  U.S. Environmental Protection Agency. Washington, D.C.

Maki, A.W. and H.E. Johnson.  1975.  Effects of PCB (Arochlor 1254) and p,p 'DDT on production and survival of Daphnia magna Strauss. Bull. Environ. Contam. Toxicol. 13: 412-416.

Nebeker, A.V. F.A. Puglisi.  1974.  Effect of polychlorinated biphenyls (PCB's) on survival and reproduction of Daphnia, Gammarus and Tanytarsus. Trans. Amer. Fish. Soc. 103: 722-728.

Schober, U. and W. Lampert.  1977.  Effects of sublethal concentrations of the herbicide atrazine on growth and reproduction of Daphnia pulex. Bull. Environ. Contam. Toxicol. 17:269-277.

Winner, R.W. and M.P. Farrell.  1976.  Acute and chronic toxicity of copper to four species of Daphnia.  J. Fish. Res. Board. Can.  33:  1685-1691.  



APPENDIX I.  COPY OF EXCEL SPREADSHEET CALCULATION OF SUCCESSFUL BIRTH RATE:

