Document ID: EPA-HQ-OPP-2011-0374-0027
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 			49307504
                        EPA Guideline		850.4500

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: 9/28/2016
Secondary Reviewer: Adrian Graff							Signature:
Environmental Scientist, CDM/CSS-Dynamac JV				Date: 10/20/2020

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., K.H. Martin, and J.R. Porch. 2013. Dacthal: A 96-Hour Toxicity Test with the Marine Diatom (Skeletonema costatum). Study performed by Wildlife International, Ltd., Easton, Maryland. Project ID 246P-103A. Study sponsored by Amvac Chemical Corporation, Los Angeles, CA. Study initiated 21 August 2013 and completed 10 December 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:

In a 96-hour acute toxicity study, cultures of saltwater diatom, Skeletonema costatum (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 26 to 48% of 0-hour concentrations. Therefore, the reviewer based toxicity values on the initial-measured concentrations, which were <10.0 (<LOQ, controls), 33.5, 63.7, 127, 248, and 476 ug a.i./L.

The % growth inhibition of cell density in the treated algal culture as compared to the control ranged from -23 to 40%. No morphological abnormalities were observed and there were no compound-related phytotoxic effects. Cell density yield was the most sensitive endpoint (with a maximum inhibition of 40%), with NOAEC, LOAEC, and IC50 values of 127, 248 and 464 ug a.i./L, respectively, based on the initial-measured concentrations. The IC50s for growth rate and area under the curve (AUC) were greater than the highest initial-measured concentration. The results should be approached with caution as although there is some suggestion of a dose response, an increase in growth (a `positive effect') was observed in the some of the lower treatment levels. There is potential that this could be due to a positive solvent interaction, as the solvent had greater growth (3-16%) across all three endpoints, compared to the negative controls. However, there was too much variability in the study to discern a significant difference. Also notably, one of the negative control replicates had substantially less growth compared to the other three control replicates. 

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 utilizing the initial-measured concentrations for estimating the endpoints, although there is potential that this may overestimate actual exposure conditions.

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

Results Synopsis

   Test Organism: Saltwater diatom, Skeletonema costatum (strain not reported)
   Test Type (Flow-through, Static, Static Renewal): Static
   
   Yield
   IC05: 66.0 ug a.i./L			95% C.I.: N/A to 178 ug a.i./L
   IC50: 464 ug a.i./L			95% C.I.: 246 to >476 ug a.i./L
   NOAEC: 127 ug a.i./L
   LOAEC: 248 ug a.i./L
   
   Growth rate
   IC05: 156 ug a.i./L			95% C.I.: N/A to 289 ug a.i./L	
   IC50: >476 ug a.i./L			95% C.I.: N/A
   NOAEC: 127 ug a.i./L
   LOAEC: 248 ug a.i./L
   
   Area under the curve (AUC)
   IC05: 92.7 ug a.i./L			95% C.I.: N/A to 211 ug a.i./L
   IC50: >476 ug a.i./L			95% C.I.: N/A
   NOAEC: 127 ug a.i./L
   LOAEC: 248 ug a.i./L
   
   Endpoint(s) Affected: Yield, growth rate, and area under the curve (AUC)
   Most Sensitive Endpoint: Yield
I. MATERIALS AND METHODS

   GUIDELINE FOLLOWED:		This study was designed to comply with the U.S. EPA OCSPP 850.4500, OECD Guideline 201, and the Official Journal of the European Communities No. L 383 A. Method C.3 Algal Inhibition Test.  The following deficiencies from OCSPP 850.4500 (2012), OCSPP 850.4000 (2012), OCSPP 850.1000 (2016), and OECD 201 were noted:
   
 The test substance was unstable under the test conditions. The 96-hour measured concentrations ranged from 26 to 48% 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). Additionally, OCSPP recommends multiple procedural modifications (e.g., use of ground glass stoppers, increasing the side of the test flasks, conducting the test at a lower temperature) if the test material is fast degrading. No procedural modifications were utilized although they were applicable in this study. 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.
 Foam stoppers were used in the study, but no prior investigation was reported as to their potential toxicity. OCSPP recommends an investigation on the acceptability of foam plugs prior to use because some brands have been found to be toxic. OECD does not have specific guidance regarding the toxicity of foam plugs. This is a minor deficiency. 
 The strain of the test organism was not reported. This is considered to be a minor deficiency.
 The light intensity used in the study (4030 to 4610 lux) was less than the OCSPP and OECD recommended level (~4300 and 4440  -  8880 lux, respectively). However, it was for a period of 14 hours light:10 hours dark under cool fluorescent lights. 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.
 Multiple dilution water characteristics (hardness, alkalinity, pH, conductivity, TOC, COD, particulate matter, and chlorine) were not reported. The OCSPP guideline recommends that these parameters are measured and that these water quality characteristics meet EPA specifications. The most recent analyses performed to measure concentrations of selected organic and inorganic constituents in well water that was used to prepare the algal medium are presented in Appendix 4 of the study report (pgs. 51-52). 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 29% and 19%, respectively, which exceeds the OCSPP guideline recommendation of yield CV<15%. The PMSD for AUC was 28-36% and cell density was 36-49%, indicating there is too much variability in the power of the study to discern significant differences. This is considered a major study deficiency.
         
   	The deficiencies do affect the validity of the study. Specifically, the high control CVs make interpretation
   	of potential statistical differences difficult. 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 not be used quantitatively in risk assessment, it may be used for risk 
   	characterization.

    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 characterization and stability of the test substance in storage. 
   
   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:		The test substance was unstable under the test conditions. Measured concentrations on day 0 ranged from 95 to 108% of nominal. The 96-hour measured concentrations ranged from 26 to 48% 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

      (OECD recommends water solubility, stability in water and light, pKa, Pow, and vapor pressure of test compound)

   2. Test organism: 
   
         Name:					Marine diatom, Skeletonema costatum
         Strain:					Not reported
         Source: 					In-house cultures (maintained at Wildlife International, Easton, MD since September 2008); originally obtained from the Provasoli-Guillard Nation Center for Culture of Marine Phytoplankton (CCMP)
         Age of inoculum:		3 days
         Method of cultivation:	Cultured under the same conditions as the definitive test

   B.  STUDY DESIGN:

      1. Experimental Conditions

      a. Range-finding study:  A non-GLP, preliminary, range-finding toxicity test was conducted at nominal concentrations of 0 (negative control), 10, 100, and 1000 ug a.i./L and yielded -10, 92, and 74% inhibition of mean cell density relative to the negative control, respectively, after 96 hours of exposure.

         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)
At least 2 weeks prior to test initiation.

Same as test. Saltwater algal medium (Appendix 3, pg. 50 in the study report)

Exponential growth phase. No further details regarding health or appearance were reported.
Transferred to fresh medium 3 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 20 +-2°C equipped with a mechanical shaker, continuously shaken at 100 rpm.

Duration of the test
96 hours

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

Glass Erlenmeyer flask
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 (saltwater AAP medium)
pH at test initiation:
pH at test termination:
Chelator used:
Carbon source:
Salinity (for marine algae):

7.9 (negative control)
8.3 (negative control)
Na2EDTA::2H2O
N/A
30 ppt

The pH of the medium was adjusted to 8.0+-0.1 using 10% HCl.

The medium was sterilized by filtration (0.22 um) prior to use.
Appendix 3 (pg. 50 in the study report) contains the list of saltwater algal medium constituents. Stock nutrient solutions were prepared by adding reagent-grade chemicals to NANOpure(R) water (purified Wildlife International well water).

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:

Purified Wildlife International, Ltd. well water

Not reported
Not reported
Not reported
Not reported
30 ppt
Not reported
Not reported
Not reported
Not reported
Calcium  -  33.5 mg/L
Chloride  -  4.2 mg/L
Magnesium  -  12.9 mg/L
Potassium  -  6.57 mg/L
Sodium  -  18.2 mg/L

< Reporting Limit
Not reported
Analyses performed by Lancaster Laboratories on samples collected December 26, 2012 (see Appendix 4 pg.51-52 of the study report).

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 saltwater algal medium. A solvent control solution was also prepared by diluting 50 uL of DMF in 500 mL of saltwater algal medium. The negative control solution consisted of saltwater algal medium without test substance or solvent added.

Aeration or agitation
Shaken continuously at 100 rpm

Initial cells density
1.0x10[4] cells/mL
Concentration of agal cells in the stock culture was 7.45 x10[5] cells/mL. In order to achieve the desired cell density of approx. 10,000 cells/mL; 1.34 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:

Geometric mean-measured:

Initial measured:

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

<10.0 (<LOQ, negative and solvent controls), 23, 44, 87, 158, and 245 ug a.i./L

<10.0 (<LOQ, negative and solvent controls), 33.5, 63.7, 127, 248, and 476 ug a.i./L
Negative control = culture 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/L (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.
Limit of quantitation (LOQ) was 10 ug a.i./L.
Test conditions 
Temperature:
Photoperiod:
Light intensity and quality:

pH:

19.1 to 20.1ºC
14 hours light:10 hours dark
4030 to 4610 lux cool-white fluorescent light
7.9-8.3
18.1 to 19.9ºC 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 density was determined using an electronic particle counter (Coulter Electronics, Inc.). 

Yield was calculated as final minus initial cell density.  

Growth rate:
u= ln Nn  -  ln No
           tn  -  to
where:
μ = average specific growth rate 
No = Nominal cell density (cells/mL) at to
Nn = Measured cell density (cells/mL) at tn
to = Time of 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-ti)+ ((Nn-1+Nn-2N0)/2)(tn-tn-1) 
where:
A = Area under the growth curve
N0 = Mean nominal number of cells/mL at t0
N1,2 = Mean measured number of cells/mL at t1,2 etc.
Nn = Mean measured number of cells/mL at tn
t0 = test initiation
t1,2 = time of first/second measurement (hours)
tn = time of n[th] measurement after beginning of test

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 121 and 137 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, cell density % inhibitions in the two highest treatment levels were 36 and 40% compared to the remaining treatment levels with % inhibitions of -23 to -1%. The increase in % inhibition appeared to be dose responsive. A similar trend (appearing dose responsive) was observed for growth rate and area under the curve data, although growth rate and area under the curve only achieved maximum inhibitions of 13 and 33%, respectively.  
   
   No morphological abnormalities were observed and there were no compound-related phytotoxic effects. 
   There were slight increases in pH during the test for the controls and three lowest test levels where pH increased from 7.9/8.0 to 8.1-8.3. In the two highest test levels, pH decreased from 8.0 to 7.9. 

Table 3: Effect of Dacthal on algal growth of the saltwater diatom Skeletonema costatum.
                   Initial-measured Concentration (Nominal)
                                  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
                                     18.0
                                     68.0
                                      121
                                      N/A
Solvent control (<LOQ)
                                     1.00
                                     20.8
                                     84.4
                                      137
                                      -13
33.5 (31)
                                     1.00
                                     19.3
                                     79.1
                                      149
                                      -23
63.7 (63)
                                     1.00
                                     17.6
                                     61.7
                                      123
                                      -1
127 (125)
                                     1.00
                                     22.0
                                     73.7
                                      133
                                      -10
248 (250)
                                     1.00
                                     17.7
                                     51.8
                                     77.5
                                      36
476 (500)
                                     1.00
                                     18.7
                                     40.7
                                     72.7
                                      40
Reference chemical 
(if used)
Not applicable.
[a] Calculated by the reviewer relative to the negative control.
Data were obtained from Appendix 6 on pages 65-66 of the study report.
LOQ was 10 ug a.i./L

Table 4: Effect of Dacthal on algal growth of the saltwater diatom, Skeletonema costaum,
                   Initial-measured Concentration (Nominal)
                                  ug a.i./L
                                 Initial cell
                                    density
                               (x10[4] cells/mL)
                               Mean growth rate
                                  (hour[-1])
                 Mean area under the curve (AUC) (x 10[4])[a]

                                  0-96 hours
                                % inhibition[a]
                                  0-96 hours
                                % inhibition[a]
Negative control (<LOQ)
                                     1.00
                                    0.0496
                                      N/A
                                     3546
                                      N/A
Solvent control (<LOQ)
                                     1.00
                                    0.0512
                                      -3
                                     4219
                                      -19
33.5 (31)
                                     1.00
                                    0.0521
                                      -5
                                     4199
                                      -18
63.7 (63)
                                     1.00
                                    0.0498
                                       0
                                     3412
                                       4
127 (125)
                                     1.00
                                    0.0509
                                      -3
                                     3986
                                      -12
248 (250)
                                     1.00
                                    0.0445
                                      10
                                     2656
                                      25
476 (500)
                                     1.00
                                    0.0429
                                      13
                                     2369
                                      33
[a] Calculated by the reviewer relative to the negative control.
Data were obtained from Table 6 on page 27 of the study report.
LOQ was 10 ug a.i./L

Table 5: Statistical endpoint values calculated by the study author in terms of geometric mean-measured (nominal) concentrations.
Statistical endpoint
Cell density
Yield
Growth rate
Area under the curve (AUC)
NOAEC (ug a.i./L)
Not calculated
87 (125)
87 (125)
158 (250)
IC50 or EC50 (ug a.i./L) [95% C.I.]
Not calculated
242 [144 to >245]
>245(500) (N/A)
>245 (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 endpoints for area under the curve, yield, and growth rate using SAS System for Windows (Version 8.2). ICx values were calculated using nonlinear regression. The endpoint data were evaluated for normality and homogeneity of variance using Shapiro-Wilk's and Levene's tests, respectively. Negative and solvent control data were compared using a t-test and were not significantly different. Treatment data were compared to the negative control data using Dunnett's test.  Non-parametric analyses (Jonckheere-Terpstra Trend Test) was also conducted to evaluate the results of the Dunnett's test, and these results were reported as they provided the most conservative estimate of toxicity. Toxicity values were based on geometric mean-measured exposure concentrations.

	C. VERIFICATION OF STATISTICAL RESULTS:

   Statistical Method: The reviewer statistically analyzed the 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. 
   
   The 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. All treatment data were normally distributed and homoscedastic based on Shapiro-Wilk's (α = 0.01) and Bartlett's (α = 0.01) tests, respectively. These data were monotonically decreasing and therefore analyzed using a Williams Multiple Comparison test (α = 0.05).
   
   ICx values were determined using non-linear regression, however, estimates for area under the curve and growth rate were above the highest initial-measured concentration tested. 

   Yield
   IC05: 66.0 ug a.i./L			95% C.I.: N/A to 178 ug a.i./L
   IC50: 464 ug a.i./L			95% C.I.: 246 to >476 ug a.i./L
   NOAEC: 127 ug a.i./L
   LOAEC: 248 ug a.i./L
   
   Growth rate
   IC05: 156 ug a.i./L			95% C.I.: N/A to 289 ug a.i./L	
   IC50: >476 ug a.i./L			95% C.I.: N/A
   NOAEC: 127 ug a.i./L
   LOAEC: 248 ug a.i./L
   
   Area under the curve (AUC)
   IC05: 92.7 ug a.i./L			95% C.I.: N/A to 211 ug a.i./L
   IC50: >476 ug a.i./L			95% C.I.: N/A
   NOAEC: 127 ug a.i./L
   LOAEC: 248 ug a.i./L
   
   Endpoint(s) Affected: Yield, growth rate, and area under the curve (AUC)
   Most Sensitive Endpoint: Yield

   D.  STUDY DEFICIENCIES: 

   The test substance was unstable under the test conditions. The 96-hour measured concentrations ranged from 26 to 48% 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). Additionally, OCSPP recommends multiple procedural modifications (e.g., use of ground glass stoppers, increasing the side of the test flasks, conducting the test at a lower temperature) if the test material is fast degrading. No procedural modifications were utilized although they were applicable in this study. The coefficient of variation (CV) based on yield and area under the curve (AUC) for the negative control was 29% and 19%, respectively, which exceeds the OCSPP guideline recommendation of yield CV<15%. The PMSD for AUC was 28-36% and cell density were 36-49%, 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.

   E.  REVIEWER'S COMMENTS: 

   The reviewer's and study author's determination of the level that constituted the NOAEC were in agreement. The reviewer observationally determined a lower NOAEC than was determined through the statistical analysis; this was done for a number of reasons: 1) there was 36% and 40% mortality at 248 and 476 ug a.i./L, respectively; 2) In the negative control, one low preforming replicate brought the average down; 3) in both the 248 and 476 ug a.i./L groups, one high performing replicate is bringing up the average; 4) the PMSD for Williams is 38%, given the variability, the statistical test does not have that much power. However the overall results, NOAEC, do align with the study author's results, when accounting for the difference in geometric mean vs initial-measured concentrations. The ICx values were not comparable because the reviewer used the initial-measured concentrations while the study author used the geometric mean-measured concentrations. The reviewer's results are reported in the Executive Summary and Conclusions sections of this DER.
   
   According to OCSPP guidance, the algal toxicity test is considered valid if the following criteria were met:
   
 Test vessels were identical.
 Treatments were randomly assigned to individual test vessel and test vessels were randomly assigned to positions in the growth chamber.
 An untreated medium control (and solvent control, if used) was included in the test.
 The solvent concentration used did not affect the growth of the test species.
 Cell counts in the controls increased by a factor of >=100 for P. subcapitata and a factor of >=30 for S. costatum. 
 Five test concentrations were used in the definitive test.
 Controls were not contaminated with the test substance.
 The lowest test concentration was less than the 96-hour yield, growth rate, and area under the curve IC50 values.
 There was no use of surfactants or dispersants during the preparation of the stock and test solutions.
 Temperature and light intensity were measured as specified in the guideline during the test.
   
   	These ten criteria were met.
   
   The in-life phase of the definitive test was conducted from September 5 to 9, 2013. With cell counts completed on September 9, 2013. A recovery test was conducted from September 9 to 13, 2013 following the four day exposure period. 

   The coefficient of variation (CV) based on yield and area under the curve for the negative control was 29% and 19%, respectively, which exceeds the guideline recommendation of yield CV<15%.  The CV based on growth rate for the negative control was 7%, which meets the guideline recommendation of growth rate CV<15%. Based on high variability for yield and AUC, the study had low power to discern significant differences. This is considered a major study deficiency.

   F. CONCLUSIONS:  

   This study is scientifically sound and is classified as supplemental and may be used for risk characterization. No morphological abnormalities were observed and there were no compound-related phytotoxic effects. Yield was the most sensitive endpoint (maximum inhibition of 40%), with NOAEC, LOAEC, and IC50 values of 127, 248, and 464 ug a.i./L, respectively, based on the initial-measured concentrations. The IC50 values for area under the curve and growth rate were above the highest initial-measured concentration. The results should be considered with caution given the high variability in the test system, including in control replicates, the instability of the test compound and subsequent use of initial-measured concentrations, the observed increased (but not statistically significant) growth observed in the solvent treatment (compared to the negative control) and slight increase in growth observed in the lower treatment levels, which may have been due to the potential solvent interaction.

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. 

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.

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

U.S. Environmental Protection Agency. 2012. Series 850-Ecological Effects Test Guidelines, OCSPP Number 850.4500: Algal Toxicity.