Data Requirement:			PMRA Data Code	 	{............}
                     EPA DP Barcode			461050
                     OECD Data Point		{............}
                     EPA MRID 				51398104
                     EPA Guideline			850.1300

Test Material:	Chlorthal-dimethyl (DCPA)					Purity: 98.3%
Common name:	DCPA
            IUPAC: dimethyl 2,3,5,6-tetrachlorobenzene-1,4-dicarboxylate
            CAS name: Not reported
            CAS No.: 1861-32-1
            Synonyms: Not reported
            
Primary Reviewer: Elizabeth Krupka							Signature:  
Environmental Scientist, CDM/CSS-Dynamac JV				Date:  03/24/2021

Secondary Reviewer: Julie Burns  								Signature:  
Environmental Scientist, CDM/CSS-Dynamac JV				Date:  04/15/2021

Primary Reviewer:	Christina M. Wendel						Signature: 
EPA/OPP/EFED/ERB2/Biologist 								Date:  12/01/2021

Secondary Reviewer(s): Michael Wagman						Signature:
EPA/OPP/EFED/ERB2/Senior Scientist						Date: 11/30/2021

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

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

Date Evaluation Completed: 01-12-2021

CITATION: Manson, P.S., 2004. Chlorthal-dimethyl (DCPA): Reproduction study with Daphnia magna. Study performed by Covance Laboratories Ltd, North Yorkshire, England. Study number 1708/035. Study sponsored by AMVAC Chemical UK Ltd, Surrey, England. Study initiated September 19, 2003 and completed April 6, 2004.


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 Chlorthal-dimethyl (Technical DCPA) to Daphnia magna was studied under static renewal conditions at nominal concentrations of 0 (negative and solvent controls), 0.031, 0.098, 0.313, 1.0, and 3.2 mg a.i./L. The reviewer-calculated mean-measured concentrations based on the filtered samples of the test substance were <0.005 (<LOD, negative and solvent controls), 0.00250, 0.00310, 0.0135, 0.0793, and 0.328 mg a.i./L. Solubility and stability concerns were present in this study with the coefficient of variation for the four highest test concentrations in the mean-measured filtered samples ranged from 43 to 105%. Likewise, analytical verification of these concentrations represented 3 to 10% of nominal values in all treatment groups. Even though filtered samples have low recoveries in most test levels, these are preferred to unfiltered because they represent the bioavailable portion. The potentially erroneous value measured on Day 17 for the nominal 0.098 mg a.i./L test concentration was excluded from calculation of mean-measured test concentration for this level. Likely due to the solubility issues described above, the two lowest reviewer calculated mean-measured concentrations (0.0025 and 0.0031 mg a.i./L, nominal concentrations of 0.031 and 0.098 mg a.i/L, respectively) were very close together (~1.2 spacing). More importantly, both of these doses are below the Level of Detection (LOD, 0.005 mg ai/L), indicating that confidence in their measurements should be considered low and it is unclear whether daphnids were exposed to any bioavailable DCPA at these concentrations.

Mortality of parental daphnids was 30% in the negative control, and 0% in the solvent control. Although this is a validity requirement of both OCSPP, and OECD guidance is that mortality remain of <=20% in the control groups. The third dead daphnid (3/10) in the negative control was noted on the final day of the study (day 21), and it was also observed that this female was producing young as late as the day before (day 20). Therefore, although this is considered not to meet a validity requirement, based on these considerations, and since all of the remaining OCSPP 850.1300 validity criteria were met, this study can still be used for risk characterization only. 

All analyzed endpoints, except survival, were significantly affected in this study. The most sensitive endpoints were number of live offspring, successful birthrate, and time to first brood  The LOAEC is based on 22% reduction of number of live offspring, 27% reduction of successful birthrate and a 34% increase on time to first brood. The analytical measurements indicate that test organisms in the two lowest concentrations may not have been exposed to any bioavailable DCPA during part of the study, and the third concentration is the LOAEC which exhibited reproductive effects and also had analytical recovery issues below the Level of Detection (LOD) during part of the study (see reviewer comments below) and no Level of Quantification (LOQ) was reported. Therefore, a NOAEC could not be determined, and there is considerable uncertainty as to the actual doses the test organisms were exposed to for the three lowest test concentrations (including the LOAEC). The LOAEC is considered to be 0.0135 mg a.i./L (13.5 ug a.i./L), but given the analytical recovery issues, there is uncertainty surrounding the accuracy of this test concentration.

This study is scientifically sound and is classified as supplemental and may be used for risk characterization.  

Results Synopsis

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

	NOAEC: could not determined
	LOAEC: 0.0135 mg ai/L (13.5 ug a.i./L)

   Endpoint(s) Affected: Length, number of live offspring, successful birthrate, and time to first brood
   Most Sensitive Endpoint: Number of live offspring, successful birthrate, and time to first brood

I. MATERIALS AND METHODS

	GUIDELINE FOLLOWED:		The study protocol was based on procedures outlined in OECD Guideline No. 211, Daphnia magna Reproduction Test (1998) and U.S. EPA OCSPP Guideline No. 850.1300, Daphnid Chronic Toxicity Test (1996). The reviewer assessed the study methods according to the most recent versions of the OCSPP 850.1300 (2016) and OECD 211 (2012) guidance, noting similarities and/or differences where they occurred. The following deficiencies and deviations were noted: 

 Mortality of parental daphnids was 30% in the negative control, and 0% in the solvent control. A validity requirement of both OCSPP and OECD guidance is that mortality remain <=20% in  control groups. The third dead daphnid (3/10) in the negative control was noted on the final day of the study (day 21), and it was also observed that this female was producing young as late as the day before (day 20). Therefore, although this is considered not to meet a validity requirement, based on these considerations, and since all of the remaining OCSPP 850.1300 validity criteria were met, this study can still be used for risk characterization only. 
 Solubility and stability concerns were present in this study with the coefficient of variation (CV) for the four highest test concentrations in the mean-measured filtered samples ranged from 43 to 105%. Likewise, analytical verification of these concentrations represented 3 to 10% of nominal values in all treatment groups. Even though filtered samples have low recoveries in most test levels, these are preferred to unfiltered because they represent the bioavailable portion Although two solubility trials were conducted as recommended by OCSPP, issues were not resolved. OCSPP 850.1000 recommends experimental design modifications (e.g. use of saturator columns, environmental controls, or flow-through conditions) to aid in solubility. This is considered a major source of uncertainty in the study.
 Likely due to the solubility issues described above, the two lowest reviewer calculated mean-measured concentrations (0.0025 and 0.0031 mg a.i./L, nominal concentrations of 0.031 and 0.098 mg a.i/L, respectively) were very close together (~1.2 spacing). OCSPP 850.1000 recommends dose spacing of 1.5--3.2 between doses. The study author used geometric mean-measured concentrations, and for the lowest concentration the geometric mean measured concentration at the nominal concentrations of 0.031 mg/L has been calculated by applying a mean of the % recoveries at the higher concentrations (21.5 %) to the nominal concentration. More importantly, both of these concentrations are below the Level of Detection (LOD; 0.005 mg ai/L), indicating that confidence in their measurements should be considered low and it is unclear whether daphnids were exposed to any bioavailable DCPA at these concentrations. Further, for all of the analytical measurements at the three lowest concentrations (reviewer calculated arithmetic mean measured 0.002, 0.0031, and 0.0135 mg a.i./L), values reported by the study authors that were below the LOD (0.005 mg ai/L) and not included in the study author's calculation of the geometric mean measured concentration, but were converted to (1/2) LOD, and included in the mean measured concentration calculations by the reviewer. For the analytical measurements of the third concentration--mean measured 0.0135 mg a.i./L (nominal 0.313 mg ai/L)-- in the Day 7 new and Day 10 old samples were identical to the two lower doses (all three lower test concentrations measured 0.0025 mg a.i./L in Day 7 new and Day 10 old samples). Taking into account that this is (1/2) the LOD, suggests that daphnids may not have actually been exposed to any bioavailable DCPA at least during these three days (next analytical measurement was on D14 and was above the LOD).  Further, the null concentration (0) is within 1 standard deviation of the overall mean measured concentration (standard deviation of 0.014) for this dose (Appendix 1 below shows the measured concentrations during each renewal period, mean measured concentrations, standard deviations and CVs). In addition, no level of quantitation was reported for the study. Therefore, since this third dose was the LOAEC based on effects to reproductive endpoints, there is considerable uncertainty as to the actual dose needed to elicit these effects.  This is considered a major study deficiency.
 Several endpoints were not evaluated by the study author, including time to first brood, successful birthrate, length, and survival. However, sufficient information was available for the reviewer to evaluate these endpoints and therefore this is not considered a major deviation.  
 Dry weight at test termination was not determined as recommended by OCSPP guidance. OECD guidance does not provide such a recommendation.
 The health of the culture was not reported. OCSPP guidance recommends that no more than 5% of the culture stock die or show signs of stress during the 48 hours preceding the test. OECD guidance recommends that the culture used in the test be derived from a healthy stock (i.e. showing no signs of stress such as high mortality, presence of males and ephippia, delay in the production of the first brood, discolored animals, etc.).
 The physicochemical properties were not reported, as required by both OCSPP and OECD guidance.
 Incomplete dilution water parameters were reported. OCSPP guidance requires that dilution water be analyzed, and these properties be recorded. OECD guidance does not have such a requirement.

	The deficiencies and deviations do impact the acceptability of the study.

  	COMPLIANCE:		Signed and dated GLP, and Quality Assurance statements were provided.  A statement of No Data Confidentiality Claims was provided, but not signed. This study was conducted in accordance with the United Kingdom Statutory Instrument 1999 No. 3106, GLP Regulations 1999, OECD GLP (revised 1997, issued January 1998) ENV/MC/CHEM (98)17.

   A. MATERIALS:

   	1. Test Material: 		Chlorthal-dimethyl (DCPA)
   
      Description: 			Grey powder

      Lot No./Batch No.: 		010616-2 (Batch No.)

		Purity: 					98.3%
      Stability of compound 
      under test conditions:	Unstable. The coefficient of variation for the four highest test concentrations in the mean-measured filtered samples ranged from 43 to 105%. Likewise, analytical verification of these concentrations represented 3 to 10% of nominal values in all treatment groups. 
   
		Physicochemical properties of Chlorthal-dimethyl (DCPA).  
Parameter
Values
Comments
Water solubility 
0.2  -  0.5 mg/L
@ 25 °C
Vapor pressure
Not reported

UV absorption
Not reported

pKa
Not reported

Kow
Not reported

      
      Storage conditions of 
      test chemicals:				Not reported. All precautions required in the handling, storage and disposal of the test substance normally taken for a new chemical undergoing safety evaluation were outlined by the Sponsor. 
   	2. Test Organism: 
      
      Species:  					Daphnia magna
      								EPA and OECD recommend Daphnia magna. EPA also allows the use
      								of D. pulex.
      Age at test initiation:  		<24 hours old
      								EPA recommends that daphnids are in their first instar (<=24 hrs old)
      Source:  					In-house laboratory culture derived from a clone held at Sheffield University, Sheffield, UK		
                              EPA recommends all test organisms be from the same laboratory-reared culture. 
B. STUDY DESIGN:

   	1. Experimental Conditions  

 Range-finding Study: A range-finding study was not reported. However, a semi-static test design for the definitive test were selected based on the results of a series of stability trials. An initial stability trial was conducted to establish the most appropriate carrying / solubilizing agent for preparing the test media in the main growth test. The incorporation of Chlorthal-dimethyl (DCPA) into solution and its maintenance for the 24-hour period was most successful in the stock solutions and test media prepared using dimethylformamide (DMF).  
            
            A further stability trial was then conducted to establish the stability of DCPA in the test media and stock solutions over an extended period. Solvent stocks and test media were prepared at 10 mg/mL and 1.0 mg/L nominal concentrations, respectively, and maintained under test conditions for the longest expected exposure period during the test (72 hours - Friday to Monday). The nominal concentrations of DCPA in DMF solvent were achieved and maintained.
         
 Definitive Study:  
            
Table 1:  Experimental Parameters
                                   Parameter
                                    Details
                                    Remarks


                                   Criteria
Parental acclimation:
Period:


Condi
tions: (same as test or not)




Feeding:









                                       
                                       
Health (any mortality observed):

Continuous (cultured under test conditions).

Same as test (temperature of 20 +- 2°C, pH of 7.4 to 7.5, 94 to 103% air saturation, under fluorescent lighting for a 16-hour photoperiod).

Fed daily with a concentrated suspension of Chlorella
vulgaris at approximately 5 x 10[5] cells/mL (equivalent to approximately 0.1 mg carbon
per Daphnia magna per day). A seaweed extract (Marinure(R), Glenside Organics Ltd.,
Stirling, Scotland) was also added at each medium renewal as a dietary supplement.

Not reported



The recommended acclimation period for brood daphnids, in 100-percent dilution water at dilution temperature, is a minimum of 48 hours prior to start of test. Daphnids should be fed the same food as used for the definitive test [for automatic feeding devices, a suggested rate is 5 to 7 mg food (either solids or algal cells, dry weight) per liter of dilution water or test solution; and for manual once-a-day feeding, a suggested rate is 15 mg food (dry weight) per liter of dilution water or test solution].

Cultures should not contain ephippia and should produce young before Day 12 and produce at least 3 young per adult per day within 7 days prior to test.
Pretest mortality should be <20% 48 hours prior to testing.
Test condition:

static renewal/flow-through:

Type of dilution system- for flow through method:

Renewal rate for static renewal:


Static Renewal


N/A

Every 2 or 3 days (renewed on days 3, 5, 7, 10, 12, 14, 17 and 19)



EPA recommends consistent flow rate of >= 5 vol/24 hours, meter systems calibrated before study and checked twice daily during test period. Flow rates should not vary >10% between test chambers.
For static-renewal: test dilution water should be replaced at least once every 3 days.
Aeration, if any:
None reported



EPA recommends if aeration is needed to achieve DO level, it should be done before the addition of the test substance, and all treatment and control chambers should be given the same aeration treatment.
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:



Glass beakers


100 mL 

50 mL



EPA recommends for static tests: 250 ml jars
For flow-through tests: glass or stainless-steel containers with stainless steel or nylon screen bottoms and can be constructed using 250 - mL beakers or other suitable containers equipped with screened overflow holes, standpipes, or V-shaped notches. Daphnids should always be submerged in at least 5 cm of test solution. 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:
                                       

Quality of dilution water
Particulate matter:
TOC:
Un-ionized ammonia:
Residual chlorine:
Total organophosphorus pesticides:
Total organochlorine pesticides +
   PCBs:
Organic chlorine:
Hardness as CaCO3:
Specific conductivity:
pH:
Dissolved Oxygen:
Alkalinity:
Standard hard water prepared AnalaR grade reagents and reverse-osmosis (RO) water

Not reported
Not reported
Not reported
Not reported
Not reported

Not reported
Not reported
168 to 173 mg/L as CaCO3
Not reported
7.4 to 7.5
94 to 103% air saturation
102 to 107 mg/L as CaCO3



Recommended source of dilution water is: surface or ground water, reconstituted water or dechlorinated tap water if daphnids will survive in it for the duration of the culturing, acclimation, and testing periods without showing signs of stress. Maximum allowable concentrations for water quality parameters are provided in EPA's 850.1300 guideline (https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-850-ecological-effects-test-guidelines).
 
Water quality during testing

pH:

Dissolved oxygen:

Temperature:

Other measurements:


Photoperiod:

Interval of water quality measurements:


7.1 to 8.6

87 to 108%

19.0 to 21.5°C

Ambient Temperature 
19.1 to 20.3°C

16-hr L:8-hr D


The pH, temperature, and dissolved oxygen were determined in each freshly prepared test medium at each renewal. At the end of each exposure period, the pH, temperature, and dissolved oxygen were determined from pooled samples of the replicate test vessels for each test concentration.

Total hardness and alkalinity were determined at each
renewal.

The ambient temperature, and minimum and maximum temperatures of ASTM dilution media from a vessel adjacent to the test vessels were recorded daily.



EPA Recommendations:
pH: OECD recommends that pH range be 6-9 and does not vary more than 1.5 units in any one test.

Dissolved oxygen: 60-105% saturation.  

Temperature: 20+-1°C. OECD recommends a range of 18 - 22°C; temperature should not vary > +-2°C.

Photoperiod: 16 hours light and 8 hours darkness, with a 15 to 30 - min transition period.

DO, temperature, and pH should be measured at the beginning of the test and on days 7, 14, and 21 in at least two chambers of the high, middle, low, and control test concentrations.
Number of replicates 
Negative control:
Solvent control:
Treatments:

10
10
10/level



Static-renewal: 10 or more replicates of one daphnid each.
Flow-through: four replicates of equal number of daphnids.
Number of organisms per replicate:


1 organism for a total of 10 per treatment and control group




For each test concentration and control group (negative and solvent, if used);

For static tests, EPA and OECD recommend a minimum of 10 daphnids held individually. 
For flow-through tests, 20 daphnids total divided equally into four replicates at each concentration and control.
Treatment Concentrations:
nominal:



mean-measured: 




0 (negative and solvent controls), 0.031, 0.098, 0.313, 1.0, and 3.2 mg ai/L 

<0.005 (<LOD, negative and solvent controls), 0.00250, 0.00310, 0.0135, 0.0793, and 0.328 mg ai/L* 

*Calculated by the reviewer using filtered test concentrations per EFED technical direction. 
Treatment concentrations should include a geometric series at a separation factor of 1.5 to 2 of at least five concentrations plus a control/solvent control. The variability of measured concentrations between replicates of the same concentration should not exceed +-20%. 

Concentration of test substance in each test chamber should be measured at a minimum before the test and on days 7, 14 and 21, and in the appropriate chamber after a malfunction.

OECD recommends that at least 5 test concentrations and a control be used in a geometric series with a separation factor not exceeding 3.2.
Solvent (type, percentage, if used):
DMF (dimethylformamide)
It is unclear what the DMF concentration used in the study was.


Solvent concentration should not exceed 0.1 ml/L.  Recommended solvents include dimethylformamide and triethylene glycol, but acetone and ethanol can be used if necessary.
OECD recommends <0.1 ml/L of solvent.
Recovery of the chemical: 




Frequency of determination:

Level of Quantitation:
Level of Detection:
Recoveries ranged from 3 to 10% of nominal, based on the reviewer-calculated mean-measured (filtered) test concentrations. * 
Days 0, 3, 7, 10, 14, and 17** 

Not reported
0.005 mg ai/L
*Recoveries ranged from 5 to 71% of nominal, based on study author-calculated geometric measured concentrations of the filtered samples.

**Day 17 measured concentrations of filtered sample were excluded from the reviewer's calculation of  mean-measured test concentrations due to a potentially erroneous reported result.
Positive control {if used, indicate the chemical and concentrations} 
N/A

Other parameters, if any
None 

   

   2. Observations:  

Table 2:  Observations
                                  Parameters
                                    Details
                                    Remarks


                                   Criteria
Parameters measured including the sublethal effects/toxicity symptoms
F0 survival
F0 length
Number of offspring
Appearance of first brood




Recommended endpoints measured:
- survival of first-generation daphnids (F0);
- number of offspring produced per female;
- time to first brood;
- dry weight and length (optional) of each first
     generation daphnid alive at the end of the
     test (F0);
- survival of offspring (F1) and successful
     birthrate;
- incidence and description of morphological
     abnormalities and behavioral effects;
- observations of other effects or clinical signs.
Observation intervals 
All test vessels were examined daily. Growth measurements were determined on Day 21.    
The number of immobilized daphnids in each chamber should be recorded on day 21 of the test. After offspring are produced, they should be counted and removed from the test chambers every 2 or 3 days.
Were raw data included?
Yes

Other observations, if any
None



II. RESULTS AND DISCUSSION

   A. MORTALITY AND SUB-LETHAL EFFECTS:

   Adult survival at 21 days averaged 70% and 100% in the negative and solvent control groups, respectively, and ranged from 80 to 100% in the groups exposed to the test material.
   

Table 3:  Effect of Chlorthal-dimethyl (DCPA) on Survival, Growth, and Reproduction of Daphnia sp. 
                                 Mean-Measured
                           (Nominal) Concentration,
                                   mg a.i./L
                                Adult Survival 
                                   (Day 21)
                           Mean Time to First Brood
                                   (Days)[a]
                        Mean Cumulative Number of Live
                               Juveniles After 
                                  21 Days [b]
                        Length of Surviving Adults [c]
                                     (mm)
                        Dry Weight of Surviving Adults 
                                     (mg)

                                     No. 
                                       %




Negative Control 
                                       7
                                      70
                                      8.0
                                      131
                                     4.98
                                      NR
Solvent Control
                                      10
                                      100
                                      7.9
                                      138
                                     4.79
                                      NR
0.00250 (0.031)
                                      10
                                      100
                                      8.3
                                      140
                                     4.92
                                      NR
0.00310 (0.098)
                                       9
                                      90
                                      8.4
                                      180
                                     5.30
                                      NR
0.0135 (0.313)
                                       9
                                      90
                                     10.7
                                      102
                                     5.21
                                      NR
0.0793 (1.0)
                                       9
                                      90
                                     17.0
                                     7.5*
                                     4.98
                                      NR
0.328 (3.2)
                                       8
                                      80
                                     16.8
                                     1.4*
                                     4.55
                                      NR
NOAEC
                                      NA
                                      NA
                               0.246 mg a.i./L 
                             (unfiltered samples) 
                               0.0144 mg a.i./L
                             (filtered samples)**
                                      NA
                                      NA
LOAEC
                                      NA
                                      NA
                               0.679 mg a.i./L 
                             (unfiltered samples)
                               0.0579 mg a.i./L 
                             (filtered samples)**
                                      NA
                                      NA
EC50
                                      NA
                                      NA
                                     0.35 
                           (95% C.I.: 0.18 to 0.62) 
                                  mg a.i./L 
                             (unfiltered samples)
                                0.05 mg a.i./L 
                          (95% C.I.: Not calculable) 
                             (filtered samples)**
                                      NA
                                      NA
   [a] Calculated by the reviewer based on appearance of first brood as reported in Table 9, pp. 39-42 of the MRID. 
   b Calculated by the reviewer in CETIS. Averages differ from the study author's results on p. 14 of the MRID as the study author only included juveniles from surviving adults. 
   c Data obtained from Table 13, pp. 55-56 of the MRID. 
 * Significantly different from the solvent control, based on the study author's results (Dunnett's test, p < 0.001). 
 ** Based on the study author's analysis using geometric mean measured concentrations.
 NR = Not reported
 NA = Not analyzed

   B. EFFECT ON REPRODUCTION AND GROWTH:  The mean day to first brood release was 8.0 and 7.9 days in the negative and solvent controls, respectively, and ranged from 8.3 to 17.0 days in the treatment groups. Effects were generally dose responsive, with the delay in first brood corresponding to increases in treatment concentration.  
   
   The mean number of live juveniles after 21 days was 131 and 138 in the negative and solvent controls, compared to 140, 180, 102, 7.5, and 1.4 juveniles in the mean-measured 0.00250, 0.00310, 0.0135, 0.0793, and 0.328 treatment groups, respectively. The study author noted significant effects in the two highest treatment groups, compared to the solvent control. However, the study author's analysis excluded surviving juveniles from adults that died during the study. 

   Mean total body length averaged 4.98 and 4.79 mm in the negative and solvent control groups, respectively, and ranged from 4.55 to 5.30 mm in the groups exposed to the test material. The length of fish at test termination did not follow a clear dose-responsive pattern. 
   
   C. REPORTED STATISTICS: 
   
   The data were square root transformed prior to analysis. Levene's test for equality of variances among the concentrations was also performed and showed no evidence of heterogeneity (P >= 0.01).
   
   The total numbers of juveniles from surviving adults produced at each exposure concentration were compared to the solvent control using a one-sided Dunnett's test for pairwise comparisons. A two-sided two-sample t-test was also performed to compare the solvent and negative controls. 
   
   The mean number of live juveniles was used to estimate the EC50. The solvent control was used as a baseline, and a value for each concentration was calculated as a percentage of the solvent control value. 
   
   Mean Cumulative Number of Live Juveniles After 21 Days:
   Based on unfiltered Samples
   NOAEC: 0.246 mg ai/L 
   LOAEC: 0.679 mg ai/L 
   EC50: 0.35 mg ai/L 			95% C.I.: 0.18 to 0.62 mg ai/L
   
   Based on filtered Samples
   NOAEC: 0.0144 mg ai/L 
   LOAEC: 0.0579 mg ai/L 
   EC50: 0.05 mg ai/L 		  	95% C.I.: Could not be calculated
   
   D. VERIFICATION OF STATISTICAL RESULTS:

   Statistical Method: The reviewer analyzed the study endpoints using CETIS statistical software version 1.9.6.12 with database backend settings implemented by EFED on 7/25/17. Even though filtered samples have low recoveries in most test levels, these are preferred to unfiltered because they represent the bioavailable portion. Per EFED direction provided via email on 3/25/2021, the potentially erroneous value measured on Day 17 for the nominal 0.098 mg ai/L test concentration was excluded from calculation of mean-measured test concentration for this level. Mean-measured concentrations calculated by the reviewer based on the filtered samples were used for analysis and reporting. 
   
   Negative and solvent control data for all endpoints were compared using an Equal Variance t Two-Sample test (α = 0.05) and no significant differences were noted, though length, mortality and survival data only narrowly passed (p =~0.065). All further hypothesis testing was conducted comparing treatment data to negative control data only. 
   
   The data were tested for normality and homogeneity of variance using Shapiro-Wilk's and Bartlett's test, respectively. Length data fit the normal distribution and were homoscedastic. The length data were not monotonically decreasing so were analyzed using a Dunnett Multiple Comparison test (α = 0.05). The number of live offspring and successful birthrate data did not meet either ANOVA assumption, lacked monotonicity, and were subsequently analyzed using a Mann-Whitney U Two-Sample test (α = 0.05). Time to first brood data had a non-normal distribution and the results of variance testing were indeterminate. Hypothesis testing was conducted with both an increasing and decreasing trend. As these data were monotonically increasing, this endpoint was analyzed using the Jonckheere-Terpstra Step-Down Test. Survival data were analyzed using the Fisher Exact/Bonferroni-Holm Test. 
   
	
	F0 Length:
	NOAEC: 0.0793 mg ai/L
	LOAEC: 0.328 mg ai/L

	F0 Survival:
	NOAEC: 0.328 mg ai/L
	LOAEC: >0.328 mg ai/L

	No. of Live Offspring:
	NOAEC: could not determined
	LOAEC: 0.0135 mg ai/L
	
	Successful Birth Rate:
	NOAEC: could not determined
	LOAEC: 0.0135 mg ai/L

	Time to First Brood:
	NOAEC: could not determined
	LOAEC: 0.0135 mg ai/L

   Endpoint(s) Affected: Length, number of live offspring, successful birthrate, and time to first brood
   Most Sensitive Endpoint: Number of live offspring, successful birthrate, and time to first brood
   
   E.  STUDY DEFICIENCIES:  
   
 Mortality of parental daphnids was 30% (3/10) in the negative control, and 0% in the solvent control. A validity requirement of both OCSPP, and OECD guidance is a mortality of <=20% in the control groups. 
 Solubility and stability concerns were present in this study with the coefficient of variation for the four highest test concentrations in the mean-measured filtered samples ranged from 43 to 105%. Likewise, analytical verification of these concentrations represented 3 to 10% of nominal values in all treatment groups. Even though filtered samples have low recoveries in most test levels, these are preferred to unfiltered because they represent the bioavailable portion. Although two solubility trials were conducted as recommended by OCSPP, issues were not resolved. OCSPP 850.1000 recommends experimental design modifications (e.g. use of saturator columns, environmental controls, or flow-through conditions) to aid in solubility. This is considered a major source of uncertainty in the study.
 Likely due to the solubility issues described above, the two lowest reviewer calculated mean-measured doses (0.0025 and 0.0031 mg a.i./L, nominal concentrations of 0.031 and 0.098 mg a.i/L, respectively) were very close together (~1.2 spacing). OCSPP 850.1000 recommends dose spacing of 1.5--3.2 between doses. The study author used geometric mean-measured doses, and for the lowest dose the geometric mean measured concentration at the nominal concentrations of 0.031 mg/L has been calculated by applying a mean of the % recoveries at the higher concentrations (21.5 %) to the nominal concentration. However, more importantly, both of these doses are below the Level of Detection (0.005 mg ai/L), indicating that confidence in their measurements should be considered low and it is unclear whether daphnids were exposed to any bioavailable DCPA at these doses.  Further, for all of the analytical measurements at the three lowest doses (reviewer calculated mean measured 0.002, 0.0031, and 0.0135 mg a.i./L), values reported by the study authors that were <Level of Detection (LOD) (0.005 mg ai/L) and not included in the calculation of the geometric mean measured concentration, were converted to (1/2) LOD, and included in the calculation of the mean measured concentrations by the reviewer. For the analytical measurements of the third dose--mean measured 0.0135 mg a.i./L (nominal 0.313 mg ai/L)-- in the Day 7 new and Day 10 old samples were identical to the two lower doses (all three lower test concentrations measured 0.0025 mg a.i./L in Day 7 new and Day 10 old samples). Taking into account that this is (1/2) the LOD, suggests that daphnids may not have actually been exposed to any bioavailable DCPA at least during these three days (next analytical measurement was on D14 and was above the LOD).  Further, the null concentration (0) is within 1 standard deviation of the overall mean measured concentration (standard deviation of 0.014) for this dose (Appendix 1 below shows the measured concentrations during each renewal period, mean measured concentrations, standard deviations and CVs). In addition, no level of quantitation was reported for the study. Therefore, since this third dose was the LOAEC based on effects to reproductive endpoints, there is considerable uncertainty as to the actual dose needed to elicit these effects. This is considered a major study deficiency.
 Several endpoints were not evaluated by the study author, including time to first brood, successful birthrate, length, and survival. However, sufficient information was available for the reviewer to evaluate these endpoints and therefore this is not considered a major deviation.  

   F.  REVIEWER'S COMMENTS:  
   
   The reviewer determined a more sensitive NOAEC than the study author for the number of live offspring. This result is likely due to the following: 1) the reviewer compared treatment data to the negative control whereas the study author used the solvent control for analysis, 2) the reviewer used the Mann-Whitney U Two-Sample test for hypothesis testing, whereas the study author used Dunnett's Test, 3) the reviewer's results are based on the mean-measured concentrations for filtered samples (which reflected EFED technical direction to exclude one erroneous value), compared to the study author's results based on the geometric mean measured filtered or unfiltered samples, and 4) the reviewer included data for live offspring produced by parental daphnids that died after the reproductive period began (on Day 7) whereas the study author excluded these offspring. The study author did not evaluate the time to first brood, successful birthrate, length, and survival endpoints. The reviewer's results are presented in the Executive Summary and Conclusions sections of this DER. 
   
   Even though filtered samples have low recoveries in most test levels, these are preferred to unfiltered because they represent the bioavailable portion. Per EFED determination on 03/25/2021, this potentially erroneous value from the nominal 0.098 mg a.i./L test concentration was excluded from analysis. Likely due to the solubility issues described above, the two lowest reviewer calculated mean-measured concentrations (0.0025 and 0.0031 mg a.i./L, nominal concentrations of 0.031 and 0.098 mg a.i/L, respectively) were very close together (~1.2 spacing). However, more importantly, both of these doses are below the Level of Detection (LOD, 0.005 mg ai/L), indicating that confidence in their measurements should be considered low and it is unclear whether daphnids were exposed to any bioavailable DCPA at these concentrations.
   
   The following OCSPP 850.1300 validity requirements were not met:
   
 Mortality of parental daphnids was 30% in the negative control, and 0% in the solvent control. A validity requirement of both OCSPP, and OECD guidance is a mortality of <=20% in the control groups. However, the third dead daphnid (3/10) in the negative control was noted on the final day of the study (day 21), and it was also observed that this female was producing young as late as the day before (day 20). Therefore, based on the combination of all of these considerations and the fact that all the other validity criteria were met as outlined below, this study can be used for risk characterization only.

   Additionally, all of the remaining OCSPP 850.1300 validity criteria were met including:

 Each surviving control daphnid produced an average of >=60 young (131 and 138 in negative and solvent control groups, respectively). 
 The coefficient of variation around the mean number of living offspring produced per parent animal in the control(s) was <=25% (% CVs were 16% and 13% in negative and solvent control groups, respectively).
 Test vessels were identical.
 Organisms were randomly assigned to test vessels.
 No ephippia were produced by control animals
 Daphnids were < 24 hours old at test initiation.
 Surfactants or dispersants were not used in the preparation of the test solutions. 

	The in-life phase of the test was conducted sometime between October 3 to November 14, 2003.


G. CONCLUSIONS: 

This study is scientifically sound and is classified as supplemental and may be used for risk characterization. All analyzed endpoints, except survival, were significantly affected in this study. The most sensitive endpoints were number of live offspring, successful birthrate, and time to first brood . The LOAEC is based on 22% reduction of number of live offspring, 27% reduction of successful birthrate and a 34% increase on time to first brood. The analytical measurements indicate that test organisms in the two lowest concentrations may not have been exposed to any bioavailable DCPA during part of the study, and the third concentration is the LOAEC which exhibited reproductive effects. Therefore, a NOAEC could not be determined, and there is considerable uncertainty as to the actual doses the test organisms were exposed to. However, reproductive effects were observed at low doses as noted in the noted LOAEC. The LOAEC is 0.0135 mg a.i./L (13.5 ug a.i./L) .

III. REFERENCES:

Dunnett, C.W. 1955. A multiple comparison procedure for comparing several treatments with a control. J. Amer. Stat. Assoc. 50:1096-1121.

All other references were standard guidelines. 


Appendix I. Analytical Measured Concentrations

                        Nominal Concentration (mg ai/L)
                       Measured Concentration ( mg ai/L)
                     Mean-Measured Concentration (mg ai/L)
                                    Std Dev
                                     %CVs
                                       

                                  Day 0 (new)
                                  Day 3 (old)
                                  Day 7 (new)
                                 Day 10 (old)
                                 Day 14 (new)
                                 Day 17 (old)



% of nominal
                                     0.031
                                    0.00250
                                    0.00250
                                    0.00250
                                    0.00250
                                    0.00250
                                    0.00250
                                    0.00250
                                      0.0
                                       0
                                       8
                                     0.098
                                    0.00551
                                    0.00250
                                    0.00250
                                    0.00250
                                    0.00250
                                    0.872*
                                    0.00310
                                    0.0013
                                      43
                                       3
                                     0.313
                                    0.0263
                                    0.00603
                                    0.00250
                                    0.00250
                                    0.00758
                                    0.0358
                                    0.0135
                                     0.014
                                      105
                                       4
                                      1.0
                                    0.0759
                                    0.0774
                                    0.0225
                                    0.0139
                                     0.146
                                     0.140
                                    0.0793
                                     0.056
                                      71
                                       8
                                      3.2
                                     0.238
                                     0.224
                                    0.0479
                                    0.0133
                                     0.622
                                     0.820
                                     0.328
                                     0.32
                                      99
                                      10
Data obtained from Table 3, p. 33 of the MRID








Values reported by the study author that were <LOD were converted to 1/2 the LOD.





LOD = 0.005 mg/L









* Value appears to be erroneous and is excluded from calculation of mean-measured test concentration.







