Document ID: EPA-HQ-OPP-2011-0374-0030
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2022-04-28T04:00Z

Data Requirement:				PMRA DATA CODE {............}
                        EPA DP Barcode		420823
                        OECD Data Point	{............}
                        EPA MRID 			49307507
                        EPA Guideline		850.4550

Test material:	Dacthal Technical					Purity:	98.3%
Common name:	DCPA
Chemical name:	IUPAC:			Not reported
            CAS name:	Not reported
            CAS No.:		1861-32-1
				Synonyms:		Not reported

Primary Reviewer: Kindra Bozicevich							Signature:  
Environmental Scientist, CDM/CSS-Dynamac JV 				Date: 5/16/2016
Secondary Reviewer: Adrian Graff							Signature:  
Environmental Scientist, CDM/CSS-Dynamac JV 				Date: 10/21/2020

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

Secondary Reviewer(s): Michael Wagman						Signature: 
EPA/OPP/EFED/ERB2/Senior Scientist						Date: 11/18/2021

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

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

Date Evaluation Completed: 18-11-2021

CITATION: Arnie, J.R., Martin, K.H., and J.R. Porch. 2013. Dacthal: A 96-Hour Toxicity Test with the Freshwater Alga (Anabaena flos-aquae). Study performed by Wildlife International, Ltd., Easton, Maryland. Project ID 246P-101. Study sponsored by Amvac Chemical Corporation, Los Angeles, CA. Study initiated August 21, 2013 and completed December 5, 2013.

DISCLAIMER: This Data Evaluation Record may have been altered by the Environmental Fate and Effects Division subsequent to signing by CDM/CSS-Dynamac JV personnel. The CDM/CSS-Dynamac Joint Venture role does not include establishing Agency policies.

EXECUTIVE SUMMARY:

In a 96-hour acute toxicity study, cultures of freshwater blue-green alga, Anabaena flos-aquae (strain not reported), were exposed to Dacthal Technical (DCPA) at nominal concentrations of 0 (negative and solvent controls), 31, 63, 125, 250, and 500 ug a.i./L under static conditions. The test substance was unstable under the test conditions with 96-hour measured recoveries ranging from 62 to 68% of the 0-hour concentrations. The reviewer based toxicity values on the initial-measured concentrations, which were <10.0 (<LOQ, controls), 29.9, 63.5, 123, 261, and 535 ug a.i./L.

The % growth inhibition of cell density in the treated algal culture as compared to the control ranged from -79 to 25%. No endpoints were significantly affected and therefore the IC50 and NOEC values for all endpoints (yield, growth rate, and area under the growth curve) were >535 and 535 ug a.i./L, respectively, based on the initial-measured concentrations. Although the results should be approached with some caution as there is high variability in the study endpoints, including control replicates, limiting the ability to discern any potential significant differences. However, there were no effects observed at the solubility limit.

No morphological abnormalities were observed and there were no compound-related phytotoxic effects. The pH on Day 0 was 7.4 and 7.5 in the negative and solvent control groups, respectively, increasing to 8.3 and 8.9, respectively, by Day 4. The pH of the treatment groups on Day 0 ranged from 7.3 to 7.5, increasing to a range of 8.3 to 8.7 on Day 4. The greatest pH increase from Day 0 to 4 was observed in the solvent control (from 7.5 to 8.9).

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 sonication and were clear and colorless throughout the test, indicating that the test material was in solution, even though there was low recovery of the test material. Overall this study can still be deemed to be reliable and utilize the initial-measured concentrations for estimating the endpoints.

This study is scientifically sound and is classified as supplemental and the results for the growth endpoint may be used to calculate risk quotients, while the results for the yield and area under the curve endpoints may be used for risk characterization. 

Results Synopsis

   Test Organism: Freshwater blue-green alga, Anabaena flos-aquae (strain not reported)
   Test Type (Flow-through, Static, Static Renewal): Static
   
   Yield, Growth rate, and Area under the curve 
   IC05: Not calculable			95% C.I.: N/A
   IC50: >535 ug a.i./L			95% C.I.: N/A
   NOEC: 535 ug a.i./L
   
   Endpoint(s) Effected: None
   Most Sensitive Endpoint: N/A

I. MATERIALS AND METHODS

   GUIDELINE FOLLOWED:		This study was designed to comply with U.S. EPA OCSPP 850.4550, OECD Guideline 201, and EU Directive 92/69/EEC, Method C.3. The following deficiency and deviations from U.S. EPA OCSPP 850.4550 (2012), OCSPP 850.4000 (2012), OCSPP 850.1000 (2016), and OECD 201 were noted:

 The test substance was unstable under the test conditions, with 96-hour measured concentrations ranging from 62 to 68% of the initial-measured concentrations. According to the current OCSPP/EFED policy with regards to measured concentrations for algae studies, if the chemical is stable throughout the test period, then mean-measured concentrations are used for evaluation of endpoints. If the chemical degrades rapidly then the initial (Day 0) test concentrations are used for evaluation of endpoints. The use of the initial or Day 0 test concentrations are more appropriate for current EFED models. This is the guidance set forth in the EPA Rejection Rate Analysis Ecological Effects handbook (EPA 738-R-94-035, 1994). However, the instability of the test chemical does introduce uncertainty into the degree to which the initial exposures represent the exposure concentrations that elicited the observed effects in the study.
 The strain of the test organism was not reported. This is considered to be a minor deficiency.
 The physicochemical properties of the test item were not reported. This is considered to be a minor deficiency.
 It was noted that the stock cultures were maintained in-house (actively growing) for at least 2 weeks prior to test initiation. According to OCSPP 850.4450 guideline, newly obtained cultures should be maintained in-house for at least 6 weeks prior to use in testing.
 All characteristics of the dilution water (excluding presence of pesticides and metals) were not reported. OCSPP guidance recommend that these parameters are measured and that these water quality characteristics meet EPA specifications. However, the lack of this information is considered to be a minor deficiency as the Kow and solubility of DCPA, (4.3 and 0.5 mg/L, respectively), in water would not result in an underestimation of toxicity. 
 The coefficient of variation (CV) based on yield and area under the curve (AUC) for the negative control was 52% and 38%, respectively, which exceeds the OCSPP guideline recommendation of yield CV<15%. The CV based on growth rate for the negative control was 9%, which meets the OCSPP guideline recommendation of growth rate CV<15%. The PMSD for AUC was 56-85% and cell density were 89-179%, indicating there is too much variability in the study compared to its power to discern significant differences. This is considered a major study deficiency.
 The minimum light intensity (1960 lux) was below the OCSPP 850.4550 recommended target of 2150+-10% lux. Only the upper limit of 4440 lux is reported for OECD guidance and the lower limit is not described. OECD guidance does note that Anabaena flos-aquae grow well at lower light intensities and may be damaged at high intensities. The study reported a range of 1960-2230 lux, with a mean of 2133 +- 90.1 (SD); this was within the range of 2150+-10% lux. This is a minor deficiency. 

   The deficiency and deviations do affect the validity of the study. Specifically, the high control CVs make interpretation of potential statistical differences difficult for all but the growth rate endpoint. Additionally, the instability of the test compound in the test system introduces uncertainty regarding the actual exposure concentrations that elicited the observed effects. Although the study may be used quantitatively in risk assessment for the growth rate endpoint, it may be used for risk characterization for the yield and area under the curve endpoints. 

   COMPLIANCE:			Signed and dated GLP, Quality Assurance, and No Data Confidentiality statements were provided.  The study was conducted in compliance with the GLP standards of the U.S. EPA (40 CFR parts 160 and 792), OECD Principles of GLP ENV/MC/CHEM (98)17, and Japan MAFF 11 NohSan Notification No. 6283 with the following exceptions: periodic analyses of well water for potential contaminants and the characterization of the test substance and the stability of the test substance under the conditions of storage at the test site.

   A. MATERIALS:

   	1. Test material  				Dacthal Technical (DCPA)
      Description: 				Solid
      Lot No./Batch No. : 			090614-2
      Purity: 						98.3%
      Stability of compound 
      under test conditions:		Unstable. The 96-hour measured concentrations ranged from 62 to 68% of the initial-measured concentrations.
      Storage conditions of 
      test chemicals: 				Stored under ambient and dark conditions.
      
      Physicochemical properties of Dacthal Technical.
Parameter
Values
Comments
Water solubility at 20°C
Not reported

Vapor pressure
Not reported

UV absorption
Not reported

pKa
Not reported

Kow
Not reported

   2. Test organism: 
   
         Name:					Freshwater blue-green alga (cyanobacteria), Anabaena flos-aquae
         Strain:					Not reported
         Source: 					In-house cultures (maintained at Wildlife International, Easton, MD) originally obtained from the University of Toronto Culture Collection.
         Age of inoculum:		3 days
         Method of cultivation:	Cultured in freshwater AAP algal medium under continuous fluorescent light (2150  10% lux) at 24  2C.
         
   B.  STUDY DESIGN:

      1. Experimental Conditions

      a. Range-finding study:  A non-GLP, preliminary, range-finding toxicity test was conducted at 5.0, 50, and 500 ug a.i./L and yielded inhibitions of -102, -65, and -86% of mean cell density relative to the negative control, respectively, after 96 hours of exposure. The test solutions were clear and colorless, with no precipitation noted. Test concentrations of the definitive study were selected in consultation with the Sponsor and based on the results of the non-GLP, preliminary, range-finding toxicity test.

         b. Definitive Study

Table 1:  Experimental Parameters
                                   Parameter
                                    Details
                                    Remarks
                                       
                                       
                                   Criteria
Acclimation period:

Culturing media and conditions:  (same as test or not)

Health:  (any mortality observed)
N/A  -  cultured under test conditions

Same as test; AAP medium (Appendix 3)

Exponential growth phase. No further health observations or mortality were described. 
Algal cells used in this test were obtained from cultures actively growing in culture medium for at least two weeks prior to test initiation. Algal cells were transferred to fresh medium three days prior to test initiation.

EPA recommends two week acclimation period.

OECD recommends an amount of algae suitable for the inoculation of test cultures and incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about 3 days. When the algal cultures contain deformed or abnormal cells, they must be discarded.
Test system
Static/static renewal

Renewal rate for static renewal

Static

N/A

EPA expects the test concentrations to be renewed every 3 to 4 days (one renewal for the 7 day test, 3-4 renewals for the 14 day test).  
Incubation facility
Temperature-controlled environmental chamber at a temperature of 24 +-2°C equipped with a mechanical shaker
Test flasks were manually shaken (swirled) once each day.
Duration of the test
96 hours

EPA requires:  96-120 hours
OECD:  72 hours  
Test vessel
Material: (glass/stainless steel)
Size:
Fill volume:

Glass
250 mL
100 mL
Erlenmeyer flasks plugged with sterile foam stoppers

OECD recommends 250 ml conical flasks are suitable when the volume of the test solution is 100 ml or use a culturing apparatus.
Details of growth medium (freshwater AAP medium)
pH at test initiation:
pH at test termination:
Chelator used:
Carbon source:
Salinity (for marine algae):

7.4 (negative control)
8.3 (negative control)
Na2EDTA::2H2O
NaHCO3
N/A

The pH of the medium was adjusted to 7.5+-0.1 using 10% HCl and 0.1N sodium hydroxide.

The medium was sterilized by filtration (0.22 um) prior to use.
Appendix 3 contains the list of AAP algal medium constituents.
Stock nutrient solutions were prepared by adding reagent-grade chemicals to NANOpure(R) water (purified Wildlife International well water).
Appendix 4 contains the results of the most recent analyses (December 26, 2012) performed to measure concentrations of contaminants in well water used to prepare the algal medium. 

OECD recommends the medium pH after equilibration with air is ~8 with less than .001 mmol/l of chelator if used.

EPA recommends 20X-AAP and chelating agents (e.g. EDTA) in the nutrient medium for optimum cell growth. Lower concentrations of chelating agents (down to one-third of the normal concentration recommended for AAP medium) may be used in the nutrient medium used for test solution preparation if it is
suspected that the chelator will interact with the test material. ASTM reference, E1415-91and D 3978-80 (reapproved 1987).
If non-standard nutrient medium was used, detailed composition provided (Yes/No)
N/A

Dilution water used to prepare media 
Source of dilution water:

Quality of dilution water
Hardness:
Alkalinity:
pH:
Specific conductivity:
Salinity (for marine algae):
Water pretreatment (if any):
TOC:
COD:
Particulate matter:
Metals:
Pesticides/PCBs:
Chlorine:

Wildlife International, Ltd. well water

Not reported
Not reported
Not reported
Not reported
Not reported
None
Not reported
Not reported
Not reported
See remarks
All < Reporting Limit
Not reported
Water analyses were performed on samples collected on December 26, 2012 (Appendix 4).

Metals (mg/L)
Calcium  -  33.5
Chloride  -  4.2
Magnesium  -  12.9
Potassium  -  6.57
Sodium  -  18.2

EPA pH: Skeletonema costatum= ~8.0 Others = ~7.5 from beginning to end of the test. EPA salinity: 30-35 ppt. EPA is against the use of dechlorinated water.

OECD: pH is measured at beginning of the test and at 72 hours, it should not normally deviate by more than one unit during the test.
Indicate how the test material is added to the medium (added directly or used stock solution)
A primary stock solution was prepared by dissolving 0.0509 g of the test substance (dacthal) in 10 mL of N,N-dimethylformamide (DMF) to achieve a nominal concentration of 5.0 mg a.i./mL (based on reported purity of 98.3%). After inversion and sonication, the resulting solution was clear and colorless.  The remaining test concentrations were prepared by serial dilution of the stock solution with DMF and freshwater algal medium. A solvent control solution was also prepared by diluting 50 uL of DMF in 500 mL of freshwater AAP medium. The negative control solution consisted of freshwater AAP medium without test substance or solvent added.
All test solutions were mixed by inversion and were clear and colorless at the time of preparation.
Aeration or agitation
Manually shaken (swirled) once each day

Initial cells density
1.0 x 10[4] cells/mL
Concentration of agal cells in the stock culture was 4.93 x10[5] cells/mL. In order to achieve the desired cell density of approx. 10,000 cells/mL; 2.0 mL of stock culture was added to each replicate test chamber at test initiation.

EPA requires an initial number of 3,000 - 10,000 cells/mL. For Anabaena flos-aquae, cell counts on day 2 are not required.

OECD recommends that the initial cell concentration be approximately 10,000 cells/ml for S. capricornutum and S. subspicatus. When other species are used  the biomass should be comparable.
Number of replicates
Control:
Solvent control:
Treatments:

4
4
4

EPA requires a negative and/or solvent control with 3 or more replicates per doses. Navicula sp.tests should be conducted with four replicate.

OECD preferably three replicates at each test concentration and ideally twice that number of controls. When a vehicle is used to solubilize the test substance, additional controls containing the vehicle at the highest concentration used in the test.
Test concentrations
Nominal:

Mean-Measured

Initial-measured:

0 (negative and solvent controls), 31, 63, 125, 250, and 500 ug a.i./L

<10.0 (<LOQ, controls), 25, 51, 103, 214, and 436 ug a.i./L

<10.0 (<LOQ, controls), 29.9, 63.5, 123, 261, and 535 ug a.i./L

Negative control = freshwater AAP medium

EPA requires at least 5 test concentrations, with each at least 60% of the next higher one. 

OECD recommends at least five concentrations arranged in a geometric series, with the lowest concentration tested should have no observed effect on the growth of the algae. The highest concentration tested should inhibit growth by at least 50% relatively to the control and, preferably, stop growth completely. 
Solvent (type, percentage, if used)
0.1 mL N,N-Dimethylformamide (DMF)

Method and interval of analytical verification
The test concentrations were measured at 0 and 96 hours using HPLC analysis with UV absorbance at 220 nm.
Samples were collected in duplicate from each experimental group.
Limit of quantitation (LOQ) was 10 ug a.i./L.
Test conditions 
Temperature:
Photoperiod:
Light intensity and quality:

24.1 to 25.1ºC
Continuous
1960 to 2230 lux fluorescent light (mean 2133 lux +- 90.1 SD)
23.8 to 24.3 ºC measured continuously

EPA temperature: Skeletonema: 20°C, Others: 24-25°C; EPA photoperiod: S. costatum 14 hr light/ 10 hr dark,  Others: Continuous; EPA light: Anabaena: 2.0 Klux (+-15%), Others: 4 - 5 Klux (+-15%)

OECD recommended the temperature in the range of 21 to25[o]C maintained at +- 2[o]C and continuous uniform illumination provided at approximately 8000 Lux measured with a spherical collector.
Reference chemical (if used)
name:
concentrations:
N/A

Other parameters, if any
N/A

      2. Observations:  

Table 2:  Observation parameters
                                  Parameters
                                    Details
                                    Remarks

                                   Criteria
Parameters measured including the growth inhibition/other toxicity symptoms
Cell density
Yield
Growth rate
Area under the growth curve (AUC)

EPA recommends the growth of the algae expressed as the cell count per mL, biomass per volume, or degree of growth as determined by spectrophotometric means.
Measurement technique for cell density and other end points
Cell counts were performed using a hemacytometer and a microscope.

Growth Rate

u = average specific growth rate
No = nominal cell density (cells/mL) at to
Nn = measured cell density at tn
to = time at beginning of test (hours)
tn = time after beginning of test (hours)

Area under the curve
A = ((N1-N0)/2/)(t1)+((N1+N2-2N0)/2/(t2-t1) + ... ((Nn-1+Nn-2N0)/2)(tn-tn-1)
A = area under the growth curve
N0 = Mean nominal number of cells/mL at t0
N1 = Mean measured number of cells/mL at t1
N2 = Mean measured number of cells/mL at t2
Nn = Mean measured number of cells/mL at t0
t0 = test initiation
t1 = time of first measurement (hours)
t2 = time of second measurement (hours)
tn = Time of n[th] measurement (hours)

Yield was calculated as final biomass minus initial biomass.

EPA recommends the measurement technique of cell counts or chlorophyll a

OECD recommends the electronic particle counter, microscope with counting chamber, fluorimeter, spectrophotometer, and colorimeter. (note: in order to provide useful measurements at low cell concentrations when using a spectrophotometer, it may be necessary to use cuvettes with a light path of at least 4 cm).
Observation intervals 
Every 24 hours

EPA and OECD: every 24 hours.
Other observations, if any
Cells were observed for morphological effects at test termination.

Indicate whether there was an exponential growth in the control
Yes, after 96 hours, the mean cell densities of the negative and solvent controls were 144 and 101 x 10[4] cells/mL, respectively.

EPA requires control cell count at termination to be >2X initial count or by a factor of at least 16 during the test.

OECD: cell concentration in control cultures should have increased by a factor of at least 16 within three days.
Were raw data included?
Yes.

II. RESULTS and DISCUSSION:
   
	A. INHIBITORY EFFECTS:

   After 96 hours, mean cell density inhibition in the solvent control was 30% compared to the negative control. In the initial-measured 261 ug a.i./L treatment group, mean cell density inhibition was 25% compared to the negative control. This is the only treatment group where positive inhibitions were observed, as all other treatment groups exhibited a promotion effect in growth (% inhibitions ranging from -79 to -41%). This same trend was observed for cell density yield, growth rate, and area under the curve, although growth rate and area under the curve did not exhibit the same level of growth promotion with % inhibition values ranging from -7 to 12% and -7 to 37% in the treatment groups, respectively. Mean inhibitions were 11 and 36% for growth rate and area under the curve in the solvent control group, respectively. The data were not dose responsive. 
   
   No morphological abnormalities were observed and there were no compound-related phytotoxic effects. The pH on Day 0 was 7.4 and 7.5 in the negative and solvent control groups, respectively, increasing to 8.3 and 8.9, respectively, by Day 4. The pH of the treatment groups on Day 0 ranged from 7.3 to 7.5, increasing to a range of 8.3 to 8.7 on Day 4. The greatest pH increase from Day 0 to 4 was observed in the solvent control, from 7.5 to 8.9.

Table 3: Effect of Dacthal Technical on algal growth (freshwater blue-green alga, Anabaena flos-aquae).
                               Initial-Measured 
                          (and Nominal) Concentration
                                  ug a.i./L
                                 Initial cell
                                   density 
                               (x10[4] cells/mL)
                        Cell Density (x10[4] cells/mL)
                                       
                                       
                                   48 hours
                                   72 hours
                                   96 hours
                                       
                                       
                                       
                                       
                                 Cell Density
                               % Inhibition [a]
Negative control (<LOQ) 
                                     1.00
                                     13.7
                                     62.0
                                      144
                                      N/A
Solvent control (<LOQ) 
                                     1.00
                                     11.3
                                     36.8
                                      101
                                      30
29.9 (31)
                                     1.00
                                     15.6
                                     41.0
                                      218
                                      -51
63.5 (63)
                                     1.00
                                     12.0
                                     30.4
                                      206
                                      -42
123 (125)
                                     1.00
                                     12.5
                                     47.0
                                      203
                                      -41
261 (250)
                                     1.00
                                     12.0
                                     30.9
                                      108
                                      25
535 (500)
                                     1.00
                                     13.5
                                     22.6
                                      259
                                      -79
Reference chemical 
(if used)
N/A
[a] Calculated by the reviewer relative to the negative control.
Data were obtained from Appendix 6 on pgs. 61-62 of the study report.
LOQ was 10 ug a.i./L
Table 4: Effect of Dacthal Technical on algal growth (freshwater blue-green alga, Anabaena flos-aquae).
                        Initial-Measured (and Nominal)
                                 Concentration
                                  ug a.i./L
                                 Initial cell
                                    density
                               (x10[4] cells/mL)
                               Mean Growth Rate
                                  (hour[-1])
                      Mean Area Under the Curve (AUC)[a]
                                  Mean Yield 
                   (based on cell density; x10[4] cells/mL)

                                  0-96 hours
                                % Inhibition[b]
                                  0-96 hours
                                % Inhibition[b]
                                  0-96 hours
                                % Inhibition[b]
Negative control (<LOQ)
                                     1.00
                                    0.0509
                                      N/A
                                  36,975,000
                                      N/A
                                      143
                                      N/A
Solvent control (<LOQ)
                                     1.00
                                    0.0453
                                      11
                                  23,571,000
                                      36
                                     99.9
                                      30
29.9 (31)
                                     1.00
                                    0.0526
                                      -3
                                  39,642,000
                                      -7
                                      217
                                      -52
63.5 (63)
                                     1.00
                                    0.0528
                                      -4
                                  34,368,000
                                       7
                                      205
                                      -43
123 (125)
                                     1.00
                                    0.0512
                                      -1
                                  39,330,000
                                      -6
                                      202
                                      -41
261 (250)
                                     1.00
                                    0.0446
                                      12
                                  23,451,000
                                      37
                                      107
                                      25
535 (500)
                                     1.00
                                    0.0543
                                      -7
                                  39,462,000
                                      -7
                                      258
                                      -80
[a] Calculated by the reviewer as the area under the cell density growth curve. 
[b] Calculated by the reviewer relative to the negative control.
Data were obtained from Table 6 on page 25 of the study report.
LOQ was 10 ug a.i./L
	
Table 5: Statistical endpoint values calculated by the study author in terms of mean-measured (nominal) concentrations.
Statistical endpoint
Cell density
Yield
Growth rate
Area under the curve (AUC)
NOEC (ug a.i./L)
Not calculated
436 (500)
436 (500)
436 (500)
IC50 or EC50 (ug a.i./L) 
(95% C.I.)
Not calculated
>436 (500) (N/A)
>436 (500) (N/A)
>436 (500) (N/A)
Reference chemical, if used
NOAEC
IC50/EC50
N/A
N/A = Not applicable.

   B. REPORTED STATISTICS: 

   The study author statistically analyzed the yield, growth rate, and area under the curve data using The SAS System for Windows (Version 8.2).  ICx values were calculated using non-linear regression. Negative and solvent control data were compared using a t-test (α = 0.05) and no significant differences were observed. The treatment data were evaluated for normality and homogeneity of variance using Shapiro-Wilk's and Levene's tests, respectively. Because all assumptions were met, the treatment group data were compared to the negative control data using Dunnett's test. Toxicity values were based on mean-measured exposure concentrations.
   C. VERIFICATION OF STATISTICAL RESULTS:

   Statistical Method: The reviewer statistically analyzed 96-hour yield, growth rate, and area under the curve data using CETIS version 1.8.7.12 statistical software with database backend settings implemented by EFED on 10/20/15. The initial-measured concentrations were used for analysis and reporting. 
   
   Negative and solvent control data were compared using an Equal Variance t Two-Sample test (α = 0.05) and no significant differences were noted. All further hypothesis testing was conducted comparing treatment data to negative control data only. Based on Bartlett's test (α = 0.01) and Shapiro-Wilk's test (α = 0.01), assumptions of homoscedasticity and normal distribution were met. Because the data were not monotonically decreasing, the reviewer analyzed the endpoint data using Dunnett's Multiple Comparison test (α = 0.05). Due to an absence of statistically significant differences, the reviewer visually determined the IC50 values as greater than the highest initial-measured concentration tested.  

   Yield
   IC05: Not calculable			95% C.I.: N/A
   IC50: >535 ug a.i./L			95% C.I.: N/A
   NOEC: 535 ug a.i./L
   
   Growth rate
   IC05: Not calculable			95% C.I.: N/A
   IC50: >535 ug a.i./L			95% C.I.: N/A
   NOEC: 535 ug a.i./L
   
   Area under the curve (AUC)
   IC05: Not calculable			95% C.I.: N/A
   IC50: >535 ug a.i./L			95% C.I.: N/A
   NOEC: 535 ug a.i./L
   
   Endpoint(s) Effected: None
   Most Sensitive Endpoint: N/A

   D.  STUDY DEFICIENCIES: 

	The test substance was unstable under the test conditions, with 96-hour measured concentrations ranging from 
   62 to 68% of the initial-measured concentrations. According to OCSPP 850.1000, a chemical is unstable if it declines to <80% of the initial-measured test concentrations. According to the current OCSPP/EFED policy with regards to measured concentrations for algae studies, if the chemical is stable throughout the test period, then mean-measured concentrations are used for evaluation of endpoints. If the chemical degrades rapidly then the initial (Day 0) test concentrations are used for evaluation of endpoints. The use of the initial or Day 0 test concentrations are more appropriate for current EFED models. This is the guidance set forth in the EPA Rejection Rate Analysis Ecological Effects handbook (EPA 738-R-94-035, 1994). The coefficient of variation (CV) based on yield and area under the curve (AUC) for the negative control was 52% and 38%, respectively, which exceeds the guideline recommendation of yield CV<15%. The PMSD for AUC was 56-85% and cell density were 89-179%, indicating there is substantial variability in the study and limiting the study's power to discern significant differences. These are considered major study deficiencies that limit the ability to use this study quantitatively in risk assessment for these two endpoints (cell density (yield) and AUC).   

   E.  REVIEWER'S COMMENTS: 

   The reviewer's results were in general agreement with the study author's results (no biological effects) with differences were attributed to the reviewer's use of initial-measured concentrations for analysis and reporting compared to the study author's use of mean-measured concentrations. The reviewer's results are reported in the Executive Summary and Conclusions sections of this report.

   The in-life phase of the definitive test was conducted from August 26 to 30, 2013, with cell counts completed on September 4, 2013.
   
   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 sonication and were clear and colorless throughout the test, indicating that the test material was in solution, even though there was low recovery of the test material. Overall this study can still be deemed to be reliable and utilize the initial measured concentrations for estimating the endpoints.

   The coefficient of variation (CV) based on yield and area under the curve (AUC) for the negative control was 52% and 38%, respectively, which exceeds the guideline recommendation of yield CV<15%.  The CV based on growth rate for the negative control was 9%, which meets the guideline recommendation of growth rate CV<15%.  The PMSD for AUC was 56-85% and cell density were 89-179%, indicating there is too much variability in the power of the study to discern significant differences. This is considered a major study deficiency.

   F. CONCLUSIONS:  

   This study is scientifically sound and is classified as supplemental and the results for the growth endpoint may be used to calculate risk quotients, while the results for the yield and area under the curve endpoints may be used for risk characterization. No morphological abnormalities were observed and there were no compound-related phytotoxic effects. No endpoints were significantly affected and therefore the NOEC and IC50s for all endpoints (yield, growth rate, and area under the growth curve) were 535 and >535 ug a.i./L, respectively, based on the initial-measured concentrations. Although, the results should be considered with caution given the high variability in the test system, including in control replicates, there were no effects observed at the solubility limit of DCPA.

III.  REFERENCES:

American Society for Testing and Materials. 2004. ASTM Standard Guide E1218-04. Standard Guide for Conducting Static Toxicity Tests with Microalgae.

Bruce, Robert D. and Donald J. Versteeg. 1992. A Statistical Procedure for Modeling Continuous Toxicity Data. Environmental Toxicology and Chemistry. 11: 1485-1494.

The SAS System for Windows. 1999-2001. Version 8.2. SAS Institute, Inc. Cary, North Carolina.

Organization for Economic Cooperation and Development. 2006. OECD Guidelines for Testing of Chemicals, Guideline 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test. Adopted 23 March 2006.

U.S. Environmental Protection Agency. 2012. Series 850-Ecological Effects Test Guidelines, OCSPP Number 850.4550: Cyanobacteria (Anabaena flos-aquae) Toxicity.