Document ID: EPA-HQ-OPP-2011-0374-0029
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 			49307508
                        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: 5/16/2016
Secondary Reviewer: Adrian Graff							Signature:   
Environmental Scientist, CDM/CSS-Dynamac JV 				Date: 10/23/2020

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 Diatom (Navicula pelliculosa). Study performed by Wildlife International, Ltd., Easton, Maryland. Project ID 246P-102. Study sponsored by Amvac Chemical Corporation, Los Angeles, CA. Study initiated August 7, 2013 and completed December 3, 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 freshwater diatom, Navicula pelliculosa (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 in the four lowest test levels with 96-hour measured recoveries ranging from 58 to 75% of the 0-hour measured concentrations. The reviewer based toxicity values on initial-measured concentrations of <10.0 (<LOQ, controls), 29.3, 59.2, 116, 258, and 514 ug a.i./L.

The % growth inhibition of cell density in the treated algal culture as compared to the control ranged from -3 to 10%. No morphological abnormalities were observed and there were no compound-related phytotoxic effects. All endpoints were significantly affected in the two highest test levels, but no effects reached 50%. All endpoints were equally sensitive with NOAEC, LOAEC, and IC50 values of 116, 258, and >514 ug a.i./L, respectively, based on the initial-measured concentrations.

The pH in both controls and all exposure levels was 7.3 at test initiation. By test termination, the pH in both controls and all test levels ranged from 9.3 to 9.4.

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, 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 to calculate risk quotients.

Results Synopsis

   Test Organism: Freshwater diatom, Navicula pelliculosa (strain not reported)
   Test Type (Flow-through, Static, Static Renewal): Static
   
   Yield
   IC05: 251 ug a.i./L			95% C.I.: 183 to 319 ug a.i./L
   IC50: >514 ug a.i./L			95% C.I.: N/A 
   NOAEC: 116 ug a.i./L
   LOAEC: 258 ug a.i./L
   
   Growth rate
   IC05: Not calculable		95% C.I.: N/A 
   IC50: >514 ug a.i./L		95% C.I.: N/A 
   NOAEC: 116 ug a.i./L
   LOAEC: 258 ug a.i./L
   
   Area under the curve (AUC)
   IC05: 176 ug a.i./L			95% C.I.: 135 to 214 ug a.i./L
   IC50: >514 ug a.i./L			95% C.I.: N/A 
   NOAEC: 116 ug a.i./L
   LOAEC: 258 ug a.i./L

   Endpoint(s) Affected: Yield, growth rate, and area under the curve (AUC)
   Most Sensitive Endpoint: Yield, growth rate, Area under the curve (AUC)

I. MATERIALS AND METHODS

   GUIDELINE FOLLOWED:		This study was designed to comply with U.S. EPA OCSPP 850.4500, OECD Guideline 201, and EU Directive 92/69/EEC, Method C.3. The following deficiency and deviations 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 in the four lowest test levels, with 96-hour measured concentrations ranging from 58 to 75% of the initial-measured concentrations. According to 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.
 Foam stoppers were used in the study, but no prior investigation was reported. 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.
 It was determined that the concentration of algal cells in the stock culture was 1.64 x10[6] cells/mL, which is two orders of magnitude higher than the guideline recommended starting density. Although it was stated in the study that in order to achieve the desired initial cell density of approx. 10,000 cells/mL; 0.61 mL of stock culture was added to each replicate test chamber at test initiation. According to OCSPP guideline the recommended initial population density is 10,000 cells/mL, although for Navicula pelliculosa higher initial inoculum concentrations may be needed. Therefore, although the concentration in the stock culture was two orders of magnitude higher than recommend, the stated starting density was within guideline recommendations, and therefore, this difference may be considered to be a minor deficiency.
 The coefficient of variation (CV) based on yield for the negative control was 2.22%, and the CV based on growth rate for the negative control was 0.37%, which meets the OCSPP guideline recommendation of yield and growth rate CV<15%.
 The light intensity used in the study (3930 to 4120 lux) was less than the OCSPP and OECD recommended level (~4300 and 4440  -  8880 lux, respectively). However, this was within the desired range of 4300 lux +- 10%.  The algae were held under continuous fluorescent lighting, as recommended in the guideline. 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. 48-49). 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 deficiency and deviations do affect the validity of the study. Although the instability of the test compound in the test system introduces uncertainty regarding the actual exposure concentrations that elicited the observed effects, the study may be used quantitatively in risk assessment.

      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 and stability of the test substance under storage conditions.

   A. MATERIALS:

   	1. Test material  				Dacthal Technical
      Description: 				Solid
      Lot No./Batch No. : 			090614-2
      Purity: 						98.3%
      Stability of compound 
      under test conditions:		The test substance was unstable in the four lowest test levels. Measured concentrations on day 0 ranged from 93 to 103% of nominal. The 96-hour measured concentrations ranged from 58 to 75% 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 diatom, Navicula pelliculosa
         Strain:					Not reported
         Source: 					In-house cultures (maintained at Wildlife International, Easton, MD since December 2006); originally obtained from the University of Texas at Austin (UTEX)
         Age of inoculum:		3 days
         Method of cultivation:	Cultured under 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 10, 100, and 1000 ug a.i./L and yielded inhibitions of 34, 6, and 11% in 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. Freshwater AAP medium (Appendix 3, pg. 47 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 24 +-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 (freshwater AAP medium)
pH at test initiation:
pH at test termination:
Chelator used:
Carbon source:
Salinity (for marine algae):

7.3 (negative control)
9.4 (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 (pg. 47 in the study report) contains the list of freshwater 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 
source/type:

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
Not reported
Not reported
Not reported
Not reported
Not reported
See Remarks
< Reporting Limits
Not reported
Water analyses were performed on samples collected December 26, 2012 (see Appendix 4 pg.48-49 of the study report) .

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

Aeration or agitation
Shaken continuously at 100 rpm

Initial cells density
Nominal 1.0x10[4] cells/mL
Concentration of algal cells in the stock culture was 1.64 x10[6] cells/mL. In order to achieve the desired initial cell density of approx. 10,000 cells/mL; 0.61 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), 24, 47, 95, 226, and 465 ug a.i./L

<10.0 (<LOQ, controls), 29.3, 59.2, 116, 258, and 514 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  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:

22.8 to 23.9ºC
Continuous
3930 to 4120 lux fluorescent light

7.3 to 9.4 
Temperature measured continuously ranged from 23.6 to 24.6ºC

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 had increased by a factor of 400 and 396, 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, inhibitions in all endpoints were low in the four lowest test levels (-3 to 8%). The maximum inhibitions were 10, 2, and 20% for yield, growth rate, and area under the curve. No morphological abnormalities were observed and there were no compound-related phytotoxic effects. The pH in both controls and all exposure levels was 7.3 at test initiation. By test termination, the pH in both controls and all test levels ranged from 9.3 to 9.4.

Table 3: Effect of Dacthal Technical on algal growth (freshwater diatom, Navicula pelliculosa).
                               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
                                     77.5
                                      323
                                      400
                                      N/A
Solvent control (<LOQ)
                                     1.00
                                     84.0
                                      316
                                      396
                                       1
29.3 (31)
                                     1.00
                                     80.3
                                      324
                                      404
                                      -1
59.2 (63)
                                     1.00
                                     87.4
                                      321
                                      411
                                      -3
116 (125)
                                     1.00
                                     77.5
                                      315
                                      402
                                       0
258 (250)
                                     1.00
                                     65.3
                                      296
                                      379
                                       5
514 (500)
                                     1.00
                                     50.8
                                      251
                                      362
                                      10
Reference chemical (if used)
Not applicable.
[a] Calculated by the reviewer relative to the negative control.
 LOQ was 10 ug a.i./L

Table 4: Effect of Dacthal Technical on algal growth (freshwater diatom, Navicula pelliculosa).
                        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[a]
                                  0-96 hours
                                % inhibition[a]
                                  0-96 hours
                                % inhibition[a]
Negative control (<LOQ)
                                     1.00
                                    0.0624
                                      N/A
                                  145,723,350
                                      N/A
                                      399
                                      N/A
Solvent control (<LOQ)
                                     1.00
                                    0.0623
                                       0
                                  145,188,873
                                       0
                                      395
                                       1
29.3 (31)
                                     1.00
                                    0.0625
                                       0
                                  146,956,914
                                      -1
                                      403
                                      -1
59.2 (63)
                                     1.00
                                    0.0627
                                       0
                                  148,924,842
                                      -2
                                      410
                                      -3
116 (125)
                                     1.00
                                    0.0624
                                       0
                                  143,788,734
                                       1
                                      401
                                       0
258 (250)
                                     1.00
                                    0.0619
                                       1
                                  133,501,266
                                       8
                                      378
                                       5
514 (500)
                                     1.00
                                    0.0614
                                       2
                                  116,778,969
                                      20
                                      361
                                      10
[a] Calculated by the reviewer relative to the negative control.
Data were obtained from Table 6 on page 26 of the study report.
LOQ was 10 ug a.i./L

Table 5: Statistical endpoint values calculated by the study author based on mean-measured (nominal) concentrations.
Statistical endpoint
Cell density
Yield
Growth rate
Area under the curve (AUC)
NOAEC (ug a.i./L)
LOAEC (ug a.i./L)
Not calculated
95 (125)
226 (226)
95 (125)
226 (250)
95 (125)
226 (250)
IC50 or EC50 (ug a.i./L) (95% C.I.)
Not calculated
>465 (>500) (N/A)
>465 (>500) (N/A)
>465 (>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 The 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).
   
   Due to maximum effects of 20, 10, and 2% for area under the curve, yield, and growth rate, respectively, and unreasonably large confidence limits based on linear regression, the reviewer chose to visually estimate the IC50 values as greater than the highest initial-measured concentration tested. The IC05 values for yield and area under the curve were calculated using non-linear regression.

   Yield
   IC05: 251 ug a.i./L			95% C.I.: 183 to 319 ug a.i./L
   IC50: >514 ug a.i./L			95% C.I.: N/A 
   NOAEC: 116 ug a.i./L
   LOAEC: 258 ug a.i./L 
   
   Growth rate
   IC05: Not calculable		95% C.I.: N/A 
   IC50: >514 ug a.i./L		95% C.I.: N/A 
   NOAEC: 116 ug a.i./L
   LOAEC: 258 ug a.i./L
   
   Area under the curve (AUC)
   IC05: 176 ug a.i./L			95% C.I.: 135 to 214 ug a.i./L
   IC50: >514 ug a.i./L			95% C.I.: N/A 
   NOAEC: 116 ug a.i./L
   LOAEC: 258 ug a.i./L

   Endpoint(s) Affected: Yield, growth rate, and area under the curve (AUC)
   Most Sensitive Endpoint: Area under the curve (AUC)

   D.  STUDY DEFICIENCIES: 

   The test substance was unstable under the test conditions in the four lowest test levels, with 96-hour measured concentrations ranging from 58 to 75% 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).
   
   E.  REVIEWER'S COMMENTS: 

   The reviewer's and study author's determinations of the level that constituted the NOAEC were in agreement. The IC50 values were not comparable because the reviewer used the initial-measured concentrations while the study author used the mean-measured concentrations. Although not statistically analyzed, after 72-hours, the effect appears greater than at 96-hours in the two highest dose levels, the maximum inhibition of cell density was 10 and 20%, respectively compared to 5 and 10% inhibition at 96 hours in the same two dose levels, when both are compared to the negative control. 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 12 to 16, 2013.

   The coefficient of variation (CV) based on yield for the negative control was 2.22%, which meets the guideline recommendation of yield CV<15%. The CV based on growth rate for the negative control was 0.37%, which meets the guideline recommendation of growth rate CV<15%. Cell counts in the controls exhibited logarithmic growth with an increase of 400x after 96 hours.  

   F. CONCLUSIONS:  

   This study is scientifically sound and is classified as supplemental and may be used to calculate risk quotients. No morphological abnormalities were observed and there were no compound-related phytotoxic effects. All endpoints were significantly affected in the highest test level, but effects never reached 50%. All endpoints were equally sensitive with NOAEC, LOAEC, and IC50 values of 116, 258, and >514 ug a.i./L, respectively, based on the initial-measured concentrations.

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.