Document ID: EPA-HQ-OPP-2011-0374-0028
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2022-04-28T04:00Z

Data Requirement:				PMRA DATA CODE 	{............}
                        EPA DP Barcode			460199
                        OECD Data Point		{............}
                        EPA MRID 				51499402
                        EPA Guideline			850.4500

Test material:	Dacthal W-75 [ai: chlorthal-dimethyl (Dacthal)]		Purity:	74.6%
Common name	Dacthal W-75
Chemical name:	IUPAC: dimethyl 2,3,5,6-tetrachlorobenzene-1,4-dicarboxylate
            CAS name: Not reported
            CAS No.: 1861-32-1
            Synonyms: Not reported

Primary Reviewer:	Julie Burns								Signature:  
Environmental Scientist, CDM/CSS-Dynamac JV				Date:   04/07/2021
Secondary Reviewer:   Moncie V. Wright, Ph.D.				Signature:  
Environmental Scientist, CDM/CSS-Dynamac JV				Date:   04/16/2021

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: 11/19/2021

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

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

Date Evaluation Completed: 19-11-2021

CITATION: Manson, P.S. 2004. DACTHAL W-75: Inhibition of Growth to the Alga Selenastrum capricornutum. Study conducted by Covance Laboratories Ltd., North Yorkshire, England. Laboratory report number 1708/038-D2149. Study sponsored by AMVAC Chemical UK Ltd., Surrey, England. Study initiated on September 19, 2003 and completed on April 5, 2004. 

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

EXECUTIVE SUMMARY:

In a 96-hour acute toxicity study, cultures of green algae Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) were exposed to Dacthal W-75 [ai: chlorthal-dimethyl (Dacthal); formulation of DCPA] under static conditions at nominal formulation concentrations of 0 (control), 6.3, 12.5, 25, 50, and 100 mg form/L. The corresponding nominal concentrations of the active ingredient were 4.70, 9.33, 18.7, 37.3, and 74.6 mg a.i./L. The test item was stable under the testing conditions (based on comparison to initial measured concentrations), although there were solubility issues as evidenced by the poor recoveries present at test initiation (three of the lowest test levels had recoveries <=72% of nominal) and observations made at that time. The mean-measured active ingredient concentrations of <0.003 (<LOD, control), 2.57, 6.43, 11.3, 31.3, and 61.0 mg a.i./L were used for analysis and reporting.  

The % growth inhibition in the treated algal cultures relative to the negative control ranged from 7 to 29%. The observed inhibitions after the lowest test level were considered to be dose responsive, with a general monotonic trend. The yield, growth rate, and area under the curve (AUC) were significantly affected by the test material at all test concentrations; however, the reviewer determined, that although there were effects at the lowest test level, they were not dose responsive, and could be discounted. Therefore, based on this the reviewer determined that the more reliable NOAEC was 6.43 mg a.i./L. No inhibitions reached 50% or greater and the IC50 was >61.0 mg a.i./L. The overall most sensitive endpoint was yield, growth rate, and area under the curve, based on a NOAEC of 6.43 mg a.i./L and a LOAEC of 11.3 mg a.i./L. 

There was a major change in pH over the study duration. In all treatment groups, the pH ranged from 8.0 to 8.1 at test initiation and ranged from 10.2 to 10.4 at 96 hours. In the control, pH increased from 7.9 at test initiation to 10.2 at test termination, resulting in an increase of 2.3 units in the negative control. The increase in pH was likely due to high rates of algal growth reaching the capacity of the test system. Had pH measurements been taken more frequently (i.e., daily), there would be more certainty that the similar dose-responses (and consequent NOAEC and IC50 endpoints) observed in treatment groups at 24-hours and 72-hours, compared to the treatments at 96-hours, were not a result of inhibitions in control growth due to high pH.  

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

Results Synopsis

   Test Organism: Green algae, Selenastrum capricornutum
   Test Type (Flow-through, Static, Static Renewal): static 

   Yield
   IC05: Not calculable				95% C.I.:  N/A
   IC50: >61.0 mg a.i./L				95% C.I.:  N/A
   NOAEC: 6.43 mg a.i./L*
   LOAEC: 11.3 mg a.i./L
   
   Growth rate
   IC05: Not calculable				95% C.I.:  N/A
   IC50: >61.0 mg a.i./L				95% C.I.:  N/A
   NOAEC: 6.43 mg a.i./L*
   LOAEC: 11.3 mg a.i./L
   
   Area under the curve (AUC)
   IC05: Not calculable				95% C.I.:  N/A 
   IC50: >61.0 mg a.i./L				95% C.I.:  N/A
   NOAEC: 6.43 mg a.i./L*
   LOAEC: 11.3 mg a.i./L
   *See reviewer's comments
   
   Endpoint(s) Affected:  Yield, growth rate, and area under the curve (AUC)
   Most Sensitive Endpoint:  Yield, growth rate, and area under the curve (AUC), based on the NOAEC
I. MATERIALS AND METHODS

   GUIDELINE FOLLOWED:		The study author conducted the test according to the requirements of Annex II to Commission Directive 91/414/EEC as amended by Directive 96/12/EC Annex I 8.2 referring to Commission Directive 92/69/EEC:C3 Algal Inhibition Test,  the OECD Guidelines for Testing Chemicals No. 201: Alga, Growth Inhibition Test, and the U.S. EPA OCSPP draft guideline 850.4500 Algal Toxicity, Tiers I and II (April 1996). The reviewer assessed the study according to OECD Guideline 201: Freshwater Alga and
                           Cyanobacteria, Growth Inhibition Test (2011) and OCSPP Number 850.4500: Algal Toxicity (2012), noting similarities and/or differences where they occurred. The following deficiencies and deviations were noted: 
   
 There were solubility issues as evidenced by the poor recoveries present at test initiation (three of the lowest test levels had recoveries <=72% of nominal) as well as the study author's visual observations of white homogeneous liquid dispersions (second and third test levels), white non-homogeneous liquid dispersions with particulate matter at the bottom of the test vessel (second to highest and highest test levels), and particulate matter at the bottom of the test vessel (highest test level only). OCSPP 850.1000 recommends pre-testing to determine the limit of solubility, to test with different solvents to aid dissolution, in addition to using sonication, centrifugation or filtration, or minor adjustments to environmental conditions in testing to improve solubility. However, the concentrations at 96-hours represented 83 to 116% of the initial-measured concentrations, which indicated that the concentrations were stable over the duration of the study, although there appears to be issues with solubility. 
 The pH in the negative control increased by 2.3 units over the course of the test (7.9 to 10.2). OECD guidance recommends that the pH of the control should not increase by more than 1.5 units during the study. OCSPP does not provide guidance for pH increases in the control. In all treatment groups, the pH ranged from 8.0 to 8.1 at test initiation and ranged from 10.2 to 10.4 at 96 hours. The increase in pH was likely due to high rates of algal growth reaching the capacity of the test system. This is supported by the 96-hour samples in test vessels without algal cells, which had measured pH values of only 7.9-8.1. Laboratory toxicity tests should be conducted in environmentally relevant conditions which may not be represented by the strongly basic water conditions present in the Day 4 algal samples. These conditions may have inhibited growth in the controls and lower treatment groups, resulting in potentially less sensitive endpoints than would have occurred had the pH stayed in environmentally-relevant ranges. Had pH measurements been taken more frequently (i.e., daily), there would be more certainty that the similar dose-responses (and consequent NOAEC and IC50 endpoints) observed in treatment groups at 24-hours and 72-hours, compared to the treatments at 96-hours, were not a result of inhibitions in control growth due to high pH.  
 The deionized water was not reagent grade quality as recommended by OCSPP. OECD does not recommend that the deionized water is of reagent grade quality.
 The age of the inoculum at test initiation was not reported. This is considered to be a minor deficiency.
 The environmental conditions during acclimation were not reported and the health of the organisms during acclimation was not reported. This is considered to be a minor deficiency.
 Several dilution water quality parameters (hardness, alkalinity, COD, TOC, conductivity, pH, etc.) were not reported. The OCSPP guideline recommends that these parameters are measured and that these water quality characteristics meet EPA specifications.
 The study author did not report if treatments were randomly assigned to test vessels, which is a test validity element under OCSPP 850.4500, and is recommended by OECD guidance.
 Each test level had three replicates per level, as accepted by OECD. OCSPP recommends a minimum of four replicates per treatment level. The control group had 6 replicates, meeting the criteria for the control. 
 Incomplete physiochemical properties of the test item were reported. 

      These deficiencies and deviations do affect the validity of the study.
   
   COMPLIANCE:				Signed and dated Quality Assurance and GLP compliance statements were provided. A statement of No Data Confidentiality Claims was provided, but a signature was missing from the statement. This study was performed in compliance with the UK Statutory Instrument 1999 No. 3106, the GLP regulations 1999, and the OECD principle of GLP (revised 1997) ENV/MC/CHEM(98)17. 
   A. MATERIALS:

   	1. Test material:				Dacthal W-75 [ai: chlorthal-dimethyl (Dacthal)] 

      Description: 				Off-white powder
      Lot No./Batch No.: 			2227 (batch no.)

      Purity: 						74.6%
      
      Stability of compound 
      under test conditions:		Stable. The concentrations at 96-hours represented 83 to 116% of the initial-measured concentrations. There were solubility issues as evidenced by the poor recoveries present at test initiation (three of the lowest test levels had recoveries <=72% of nominal) as well as the study author's visual observations of precipitates in the culture media at test termination. 
      							(OECD recommends stability in water and light)

      Storage conditions of 
      test chemicals: 				All precautions required in the handling, storage, and disposal of the test substance normally taken for a new chemical undergoing safety evaluation was outlined by the Sponsor. Further details were not provided. 

		Physicochemical properties of Dacthal W-75 [ai: chlorthal-dimethyl (Dacthal)].
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:					Green algae, Selenastrum capricornutum
                           This test is conducted with a nonvascular species, including at a minimum the freshwater alga P. subcapitata (formerly S. capricornutum), freshwater diatom N. pelliculosa, and the marine diatom S. costatum. Other test species may need modification of the test method. For Tier I studies, only the freshwater alga P. subcapitata is recommended. The cyanobacterium A. flos-aquae test is found in EPA guideline 850.4550.
                           OECD suggests the following species are considered suitable: S. capricornutum, S. subspicatus, and C. vulgaris. If other species are used, the strain should be reported.
         Strain:					CCAP 278/4
         Source: 					Sourced from an axenic culture derived from the Culture Collection of Algae and Protozoa (CCAP), CEH Windermere, The Ferry House, Far Sawrey, Ambleside, Cumbria, UK.
                           EPA recommends algae be from the same source and stock culture or commercial sources.
         Age of inoculum:		Not reported
                           EPA recommends the algal inoculum should be from logarithmically growing stock cultures (typically 3- to 7-days old).
         Method of cultivation:	Maintained in liquid culture. Further details provided in Appendix 1 pages										31-32 of the study report.

   B.  STUDY DESIGN:

      1. Experimental Conditions

         a. Range-finding study: A range finding study was conducted at nominal active ingredient concentrations of 0.00746, 0.0746, 0.746, 7.46, and 74.6 mg/L based on the 74.6% purity of the active ingredient. After 96 hours, treatment group inhibitions in area under the curve relative to the control were 0, 24, 0, 21, and 0%, respectively. Average specific growth rate inhibitions were 0, 5, 0, 5, and 0%, respectively. The EC50 for area under the curve and average specific growth rate was determined to be >74.6 mg a.i./L. 

         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)
Green algae cultures were maintained in liquid culture which were used to inoculate the test media in the test vessels. 

Further details provided in Appendix 1 pg 31-32 of the study report.

Not reported.

EPA recommends the algal inoculum used to initiate toxicity testing is from a liquid culture shown to be actively growing (i.e. capable of logarithmic growth within the test period) in at least two subcultures lasting 7 days each prior to the start of the definitive test. A culture should not be used if it is contaminated by fungi/other algae or if test algae were used in a previous test.

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 recommends a static exposure technique. Although semi-continuous algal culturing techniques are available, they have not been commonly employed in algal toxicity testing and their use is not recommended.
Incubation facility
Orbital incubator 

Duration of the test
96 hours

EPA recommends 96 hours at a minimum.
OECD:  72 hours.  
Test vessel
Material: (glass/stainless steel)
Size:
Fill volume:

Erlenmeyer conical glass flasks
250-mL
100-mL

EPA recommends 125-500 mL Erlenmeyer flasks and test solution volume <=50% of flask volume. Flasks may be covered with foam plugs (that are proven non-toxic), stainless steel caps, Shimadzu enclosures, glass caps or screw caps.

EPA recommends all test vessels and closures to be identical.

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 
Name:
pH at test initiation:
pH at test termination:
Chelator used:
Carbon source:
Salinity (for marine algae):

Algal culture medium (EC/AM)
7.9 (in the negative control)
10.2 (in the negative control) *
Na2EDTA·2H2O
NaHCO3
N/A
*The 96-hour pH in the negative control replicate without algae was 7.9.  Additional details found in Appendix 1 pg 31-32 of the study report.

EPA recommends an AAP medium with chelating agents (e.g. EDTA) prepared according to EPA's 850.4500 guideline (http://www2.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-850-ecological-effects-test-guidelines).  Lower concentrations of chelating agents (down to one-third of the normal concentration recommended for AAP medium) may be used in the nutrient medium for test solution preparation if it is suspected that the chelator will interact with the test material. 

EPA recommends adjustment of pH before adding inoculum, if pH of test solution is <5 or highly basic. 

OECD recommends the medium pH after equilibration with air is ~8 with less than 0.001 mmol/L of chelator if used.
If non-standard nutrient medium was used, detailed composition provided (Yes/No)
N/A  -  standard medium used. 

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:

Deionized water

Not reported
Not reported
Not reported
Not reported
N/A
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported
Not reported

Water used for preparation of nutrient medium should be of reagent quality (e.g., ASTM Type I water).

Marine algal nutrient medium is prepared by adding reagent grade chemicals to synthetic salt water or filtered natural salt water, or by preparing a complete saltwater medium. Salinity for saltwater medium should be 30 +- 5 %..
Indicate how the test material is added to the medium (added directly or used stock solution)
A primary stock solution was prepared at a nominal formulation concentration of 100 mg form/L by bringing 0.1002 g of test material to a final volume of 1 L with EC media. The stock solution was stirred for 30 minutes, and the remaining test media were prepared by serial dilution.  

Aeration or agitation

Oscillation rate:
None

N/A

EPA recommends rotary shaking apparatus to oscillate vessels at approximately 100 cycles/min during the test. The rate of oscillation should be determined at test initiation or at least once daily during testing if the shaking rate changes. S. costatum should be shaken by hand 1-2X daily or shaken at 60 cycles/min.
Initial cell density
1.0 x 10[4] cells/mL

EPA recommends an initial population density of 10,000 cells/mL for P. subcapitata, S. costatum and at a minimum 10,000 cells/mL for all other test species. Other species may need a higher initial inoculum density and should be determined on a case-by case basis.

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
Negative control:
Solvent control:
Treatments:

6
N/A
3

EPA recommends a minimum number of 4 replicates per treatment and control/ solvent control. 

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.

EPA recommends treatments be randomly assigned to test vessels, and test vessels randomly assigned to positions in the growth chamber.
Test concentrations
Nominal:

Geometric Mean-Measured:

Mean-Measured:

Initial 

Formulation: 0 (control), 6.3, 12.5, 25, 50, and 100 mg form/L

Active ingredient: 0 (control), 4.70, 9.33, 18.7, 37.3, and 74.6 mg a.i./L

<0.003 (<LOD, control), 2.56, 6.42, 11.2, 31.2, and 60.7 mg a.i./L

<0.003 (<LOD, control), 2.57, 6.43, 11.3, 31.3, and 61.0 mg a.i./L

<0.003 (<LOD, control), 2.38, 6.73, 11.8, 29.7, and 66.6 mg a.i./L
Geometric mean-measured concentrations based on study author report. Mean-measured concentrations are reviewer calculated, refer to copy of Excel worksheet in Appendix I. 
Both are based on the 0 and 96 hour measured concentrations. 

EPA recommends at least 5 test concentrations, in geometric series with a ratio of 2 to 4, and insure bracketing the NOAEC or IC05 and the IC50, plus a control/solvent control.

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)
N/A
EPA recommends the solvent N,N-dimethyl-formamide. The concentration of solvent should be the same in all test treatments and should not exceed 0.1 mL/L.
Method and interval of analytical verification
Samples were collected at 0 and 96* hours and analyzed using high performance liquid chromatography analysis with UV detection (HPLC-UV) at 230 nm. 

LOD: 0.003 mg/L
*Analysis at 96 hours included samples from replicates containing algae and replicates without algae. 

EPA recommends confirmation of dissolved test concentrations at a minimum at test initiation and at test termination for static tests. 
Test conditions 
pH:
Temperature (media):
Temperature (air):
Photoperiod:
Light intensity and quality:

7.9 - 10.4 
21.5 - 22.7°C
Not reported
Constant illumination 
8030 - 8180 lux

EPA Recommendations
pH at test initiation: 7.5+-0.1 for freshwater and 8.0+-0.1 for marine.
Temperature for P. subcapita and N. pelliculosa is 24+-2 °C, and for S. costatum is 20+-2 [o]C
Photoperiod for P. subcapita and N. pelliculosa is continuous, and for S. costatum is 14 hr light/ 10 hr dark.
Light intensity: 60 μmol/m2/s or 4300 lux.

OECD recommended the temperature in the range of 21 to 25[o]C maintained at +- 2[o]C and continuous uniform illumination provided at approximately 8000 Lux measured with a spherical collector. 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.

EPA recommends measuring pH at test initiation and at end of the test (or daily if pH adjustment was necessary); temperature on a separate vessel or hourly/daily on the air; and light intensity at test initiation (or daily if intensity changed by >15%);
Reference chemical (if used)
name:
concentrations:

N/A

Other parameters, if any
None

      2. Observations:  

Table 2:  Observation parameters
                                  Parameters
                                    Details
                                    Remarks

                                   Criteria
Parameters measured including the growth inhibition/other toxicity symptoms
Cell density
Area Under the Curve
Average Specific Growth Rate

Recommended parameters measured per replicate include:
-Algal cell density (cell count/mL)
-yield (final population density)
-average specific growth rate
-mean area under growth curve (AUC)
Measurement technique for cell density and other end points
Cell concentrations were measured using a Z2 Coulter Counter (Coulter Electronics Ltd., Luton, UK). The starting cell concentration of the starter culture was determined using the Z2 Coulter Counter and confirmed using a haemocytometer.

Area under the curve:
A = ((N1-N0)/2)(t1)+((N1+N2-2N0)/2)(t2-t1)+((Nn-1+Nn-2N0)/2)(tn-tn-1) 
Where:
A = Area under the Curve
N0 = initial measured cell concentration at time t0 (cells/mL)
N1 = measured cell concentration at time t1 (cells/mL)
Nn = measured cell concentration at time tn (cells/mL)
N2 = measured cell concentration at time t2 (cells/mL)
t1 = time of first measurement after the beginning of the test (h)
t2 = time of second measurement after the beginning of the test (h)
tn = time of nth measurement after the beginning of the test (h)

Growth Rate:
μ = (LogeNn - LogeN0) / tn
Where: 
μ = average specific growth rate
N0 = initial measured cell concentration at time t0 (cells/mL)
Nn = measured cell concentration at time tn (cells/mL)
tn = time of nth measurement after the beginning of the test (h)

EPA recommends the measurement of cell counts by microscopic observation or electronic particle counter, with alternative option of measuring 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
At the end of the exposure period, the control test media appeared as green cultures. In the mean-measured 2.57, 6.43, 11.3, and 31.3 mg a.i./L test solutions, the general appearance of the test media was as green cultures with small amounts of white particulate material on the base of the vessels, increasing in amount with concentration. In the 61.0 mg a.i./L test solutions, cultures appeared green with white particulate material on the base of the vessels. In flasks not inoculated with algae, the test media were unchanged relative to the appearance at the start of the test. 

Indicate whether there was an exponential growth in the control
Cell density increased by a factor of 353X in the negative control. 

During the 96 hour test period, cell counts in the controls did not increase by a factor of at least 100X for P. subcapitata and a factor of at least 30X for S. costatum (i.e., logarithmic growth in the controls was not reached 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:

   For mean growth rate, the % inhibition in the exposure groups compared to the control were low (<= 6%) in all treatment groups. For cell density, the % inhibitions fluctuated across test concentrations but did demonstrate a general dose response. The largest inhibition was determined for the highest test level (29%). Area under the curve followed the same pattern as cell density, and the largest inhibition was determined in the highest test level (34%). Effects were dose responsive.
   
   At the start of the exposure period the control and lowest test concentration solutions appeared as clear solutions. The mean-measured 6.43 and 11.3 mg a.i./L test solutions were white homogeneous liquid dispersions, while the 31.3 and 61.0 mg a.i./L test solutions were white non-homogeneous liquid dispersions with particulate matter at the bottom of the test vessel in the highest test level.
   
   At the end of the exposure period, the control test media appeared as green cultures. In the mean-measured 2.57, 6.43, 11.3, and 31.3 mg a.i./L test solutions, the general appearance of the test media was as green cultures with small amounts of white particulate material on the base of the vessels, increasing in amount with concentration. In the 61.0 mg a.i./L test solutions, cultures appeared green with white particulate material on the base of the vessels. In flasks not inoculated with algae, the test media were unchanged relative to the appearance at the start of the test. 
   
   There was a major change in pH over the study duration. In the control, pH increased from 7.9 at test initiation to 10.2 at test termination. In all treatment groups, the pH ranged from 8.0 to 8.1 at test initiation and ranged from 10.2 to 10.4 at 96 hours. 

Table 3:  Effect of Dacthal W-75 [ai: chlorthal-dimethyl (Dacthal)] on growth of Green Algae, Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum)
Treatment
Mean-measured 
(and nominal), 
mg a.i./L 
                                 Initial cell
                          Density (x 10[4] cells/mL)
                              Cell density at[1]

                                       
                                   24 hours
                                   72 Hours
                                   96 hours

                                       
                                       
                                       
                                 cell density
                                % inhibition[1]
Negative control (<LOD)
                                      1.0
                                     3.57
                                      125
                                      353
                                      N/A
2.57 (4.70)
                                      1.0
                                     3.09
                                      109
                                      259
                                      27
6.43 (9.33)
                                      1.0
                                     3.12
                                      117
                                      327
                                       7
11.3 (18.7)
                                      1.0
                                     3.19
                                      109
                                      283
                                      20
31.3 (37.3)
                                      1.0
                                     3.07
                                     99.5
                                      297
                                      16
61.0 (74.6)
                                      1.0
                                     -6.12
                                     86.5
                                      251
                                      29
 1 Calculated by the reviewer. 
  Data were obtained from Table 3 on page 25 of the study report.
  LOD was 0.003 mg a.i./L 

Table 4:  Effect of Dacthal W-75 [ai: chlorthal-dimethyl (Dacthal)] on growth of Green Algae, Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum)
Treatment
Mean-measured 
(and nominal), 
mg a.i./L
                                 Initial cell
                                    density
                              (x 10[4] cells/mL)
                               Mean growth rate
                                 (day[-1]) [1]
            Mean area under the curve (x 10[4] cells x days/mL) [1]

                                  0-96 Hours
                                % Inhibition[1]
                                  0-96 Hours
                                % Inhibition[1]
Negative Control (<LOD)
                                      1.0
                                     1.47
                                      N/A
                                      336
                                      N/A
2.57 (4.70)
                                      1.0
                                     1.39
                                       5
                                      268
                                      20
6.43 (9.33)
                                      1.0
                                     1.45
                                       1
                                      310
                                       8
11.3 (18.7)
                                      1.0
                                     1.41
                                       4
                                      280
                                      17
31.3 (37.3)
                                      1.0
                                     1.42
                                       3
                                      273
                                      19
61.0 (74.6)
                                      1.0
                                     1.38
                                       6
                                      222
                                      34
 1 Calculated by the reviewer.
  Data were obtained from Tables 3 & 4 on pages 25 & 26 of the study report.
  LOD was 0.003 mg a.i./L 
Table 5:  Statistical endpoint values. {calculated by the study author based on geometric mean-measured (nominal) concentrations}
                             Statistical Endpoint
                                 Cell density
                                     Yield
                                  Growth Rate
Area under the curve
NOAEC 
(mg a.i./L)
                                Not determined
                                Not determined
                              <2.56 (<4.70)
                             <2.56  (<4.70)
IC50/EC50 (mg a.i./L) (95% C.I.)
                                Not determined
                                Not determined
                              >60.7 (>74.6)
                              >60.7 (>74.6)
Reference chemical, if used
NOAEC
IC50/EC50
N/A

   B. REPORTED STATISTICS: 

   The NOAEC values for growth rate and area under the curve were determined using ANOVA and Dunnett's test (α = 0.05). The 96-hour EC50 values could not be calculated due to a lack of significant (>= 50%) inhibition compared to the control. The toxicity values were therefore visually estimated to be greater than the highest test level. The study author's results are based on the geometric mean-measured concentrations. 
	
	C. VERIFICATION OF STATISTICAL RESULTS:

   Statistical Method:  The reviewer analyzed yield, growth rate, and area under the curve using CETIS version 1.9.6.12 with database backend settings updated by EFED on 7/25/17. The mean-measured concentrations were used for analysis and reporting. 
   
   The data were tested for normality and homogeneity of variance using Shapiro-Wilk's and Bartlett's test, respectively (α = 0.01). For all endpoints, the ANOVA assumptions were met. The growth rate data were determined to be non-monotonic and were analyzed using Dunnett's test. Area under the curve and yield were generally monotonic, so were analyzed using Williams' Multiple Comparison test. The IC05 values were calculated, where possible, using non-linear regression results. The IC05 values for yield, growth rate, and area under the curve were far lower than the lowest test level or were unreliable and the confidence interval was unreasonably large. Therefore, this endpoint was not considered a reliable regression endpoint for yield, growth rate, and area under the curve. The IC50 values were visually estimated to be greater than the highest test level due to highly extrapolated non-linear regression results and/or unreliable or undetermined confidence intervals.      

   Yield
   IC05: Not calculable				95% C.I.:  N/A
   IC50: >61.0 mg a.i./L				95% C.I.:  N/A
   NOAEC: 6.43 mg a.i./L*
   LOAEC: 11.3 mg a.i./L
   
   Growth rate
   IC05: Not calculable				95% C.I.:  N/A
   IC50: >61.0 mg a.i./L				95% C.I.:  N/A
   NOAEC: 6.43 mg a.i./L*
   LOAEC: 11.3 mg a.i./L
   
   Area under the curve (AUC)
   IC05: Not calculable				95% C.I.:  N/A 
   IC50: >61.0 mg a.i./L				95% C.I.:  N/A
   NOAEC: 6.43 mg a.i./L*
   LOAEC: 11.3 mg a.i./L
   *See reviewer's comments below
   
   Endpoint(s) Affected:  Yield, growth rate, and area under the curve
   Most Sensitive Endpoint:  Yield, growth rate, and area under the curve (AUC) (based on the NOAEC)
   
   D.  STUDY DEFICIENCIES: 

   There were solubility issues as evidenced by the poor recoveries present at test initiation (three of the lowest test levels had recoveries <=72% of nominal) as well as the study author's visual observations of white homogeneous liquid dispersions (second and third test levels), white non-homogeneous liquid dispersions with particulate matter at the bottom of the test vessel (second to highest and highest test levels), and particulate matter at the bottom of the test vessel (highest test level only). OCSPP 850.1000 recommends pre-testing to determine the limit of solubility, to test with different solvents to aid dissolution, in addition to using sonication, centrifugation or filtration, or minor adjustments to environmental conditions in testing to improve solubility. However, the concentrations at 96-hours represented 83 to 116% of the initial-measured concentrations, which indicated that the concentrations were stable over the duration of the study, although there appears to be issues with solubility.
   
   The pH in the negative control increased by 2.3 units over the course of the test (7.9 to 10.2), and OECD guidance recommends that the pH of the control should not increase by more than 1.5 units during the study. Whereas OCSPP does not provide guidance for pH increases in the control. In all treatment groups, the pH ranged from 8.0 to 8.1 at test initiation and ranged from 10.2 to 10.4 at 96 hours. The increase in pH was likely due to high rates of algal growth reaching the capacity of the test system. This is supported by the 96-hour samples in test vessels without algal cells, which had measured pH values of only 7.9-8.1. Measurements were only taken at 0 and 96 hours and it is unclear if the effect in the highest test concentration are treatment related or if something else is going on within the test system.

   E.  REVIEWER'S COMMENTS: 

   The reviewer's and the study author's determination of the level that constituted the NOAEC for growth rate and area under the curve (AUC) were not in agreement. The study author reported the NOAEC for growth rate and area under the curve (AUC) to be less than the lowest test concentrations.  However, the reviewer determined, that although there were effects at the lowest test level, they were not dose responsive, and could be discounted. Although for all test concentrations that were greater than the lowest test concentration, they were considered to be dose responsive, with a general monotonic trend. Therefore, the reviewer determined that a more reliable NOAEC would be 6.43 mg a.i./L for yield, growth rate, and area under the curve (AUC). The IC50 values were in agreement when considering the differences between the mean-measured concentrations (used by the reviewer, refer to copy of Excel worksheet in Appendix I) and the geometric mean-measured concentrations (used by the study author). However, the study author did not analyze yield, which was a significantly affected endpoint. The reviewer's results are presented in the Executive Summary and Conclusions sections of this DER.

The following OCSPP 850.4500 validity criterion may not have been met:

 It was not reported if treatments were randomly assigned to test vessels or if vessels were randomly assigned to positions in the growth chamber. Although based on the description of the study summary this was likely completed.

   There were solubility issues as evidenced by the poor recoveries present at test initiation (three of the lowest test levels had recoveries <=72% of nominal) as well as the study author's visual observations of white homogeneous liquid dispersions (second and third test levels), white non-homogeneous liquid dispersions with particulate matter at the bottom of the test vessel (second to highest and highest test levels), and particulate matter at the bottom of the test vessel (highest test level only). OCSPP 850.1000 recommends pre-testing to determine the limit of solubility, to test with different solvents to aid dissolution, in addition to using sonication, centrifugation or filtration, or minor adjustments to environmental conditions in testing to improve solubility. Measurements on pH were only taken at 0 and 96 hours, and over the course of the test, the pH in the negative control increased by 2.3 units (from 7.9 at test initiation to 10.2 at test termination). In all treatment groups, the pH ranged from 8.0 to 8.1 at test initiation and ranged from 10.2 to 10.4 at 96 hours. The increase in pH was likely due to high rates of algal growth reaching the capacity of the test system. This is supported by the 96-hour samples in test vessels without algal cells, which had measured pH values of only 7.9-8.1. Laboratory toxicity tests should be conducted in environmentally relevant conditions which may not be represented by the strongly basic water conditions present in the Day 4 algal samples. These conditions may have inhibited growth in the controls and lower treatment groups, resulting in potentially less sensitive endpoints than would have occurred had the pH stayed in environmentally-relevant ranges. Had pH measurements been taken more frequently (i.e., daily), there would be more certainty that the similar dose-responses (and consequent NOAEC and IC50 endpoints) observed in treatment groups at 24-hours and 72-hours, compared to the treatments at 96-hours, were not a result of inhibitions in control growth due to high pH.  
   
The 96-hour %CV values in the control replicate for mean growth rate and yield were 1.58 and 8.88%, respectively, meeting the OCSPP guideline requirement of <15%, respectively, and the OECD guideline requirement for average specific growth rate of <=10%. The guideline requirement of 100x logarithmic growth in controls was reached during the tests (336 factor increase).

The in-life phase of the definitive test was conducted sometime between September 19, 2003 and October 28, 2003. 

   F. CONCLUSIONS:  
   
   This study is scientifically sound and is classified as supplemental and may be used for risk characterization. The observed inhibitions after the lowest test level were considered to be dose responsive, with a general monotonic trend. The yield, growth rate, and area under the curve (AUC) were significantly affected by the test material at all concentrations; however, the reviewer determined, that although there were effects at the lowest test level, they were not dose responsive, and could be discounted. Therefore, based on this the reviewer determined that the more reliable NOAEC was 6.43 mg a.i./L. There was a major change in pH over the study duration. In all treatment groups, the pH ranged from 8.0 to 8.1 at test initiation and ranged from 10.2 to 10.4 at 96 hours. In the control, pH increased from 7.9 at test initiation to 10.2 at test termination, resulting in an increase of 2.3 units in the negative control. The increase in pH was likely due to high rates of algal growth reaching the capacity of the test system. Had pH measurements been taken more frequently (i.e., daily), there would be more certainty that the similar dose-responses (and consequent NOAEC and IC50 endpoints) observed in treatment groups at 24-hours and 72-hours, compared to the treatments at 96-hours, were not a result of inhibitions in control growth due to high pH. No inhibitions reached 50% or greater and the IC50 was >61.0 mg a.i./L, and the most sensitive endpoints were yield, growth rate, and area under the curve (AUC) based on a NOAEC of 6.43 mg a.i./L and a LOAEC of 11.3 mg a.i./L.

III.  REFERENCES:  

All references were standard guidelines or statistical methods. 

APPENDIX I. Copy of Excel Worksheet with Measured Concentrations