Document ID: EPA-HQ-OPP-2011-0374-0018
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2022-04-28T04:00Z

Data Requirement:				PMRA Data Code      	{............}
                        EPA DP Barcode		461050
                        OECD Data Point	{............}
                        EPA MRID 			51398105
                        EPA Guideline		850.1075

Test material:	DACTHAL W-75 (ai: Dacthal)		Purity:	74.6%
Common name:	DCPA
Chemical name:	IUPAC: Not reported
            CAS name: Not reported
            CAS No.: Not reported
            Synonyms: Chlorthal-dimethyl

Primary Reviewer:	Elizabeth Krupka							Signature:  
Environmental Scientist, CDM/CSS-Dynamac JV				Date:   04/02/2021
Secondary Reviewer:  Julie Burns  							Signature:  
Environmental Scientist, CDM/CSS-Dynamac JV				Date:  04/14/2021

Primary Reviewer:	Christina M. Wendel						Signature: 
EPA/OPP/EFED/ERB2/Biologist 								Date: 12/02/2021

Secondary Reviewer(s): Michael Wagman						Signature: 
EPA/OPP/EFED/ERB2/Senior Scientist						Date: 12/02/2021

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

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

Date Evaluation Completed: 02-12-2021

CITATION: Manson, P.S. 2004. DACTHAL W-75: Acute toxicity to Oncorhynchus mykiss. Study conducted by Covance Laboratories Ltd., North Yorkshire, England. Study no. 1708/036. Study sponsored by AMVAC Chemical UK Ltd., Surrey, England. Study initiated on September 19, 2003 and completed on March 19, 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, rainbow trout (Oncorhynchus mykiss) were exposed to DACTHAL W-75 (ai: Dacthal, 74.6%) at nominal concentrations of 0 (negative control), 4.66, 9.33, 18.7, 37.3, and 74.6 mg a.i./L (corresponding to formulation concentrations of 6.25, 12.5, 25, 50, and 100 mg form/L) under static conditions. The test item was unstable under the test conditions (the coefficient of variation ranged from 74 to 93% in analyzed samples, with recoveries ranging from 13 to 43% of initial measured concentrations). Poor solubility was evident as the test solutions were described as hazy to opaque white, non-homogenous liquid dispersions, with particulate material on the base of the test vessels that increased with increasing test concentration. Overall, the test material was both unstable and insoluble in all treatment levels. Therefore, the reviewer-calculated time weighted average (TWA)-measured test concentrations of <0.003 (<LOD, negative control), 1.44, 2.63. 8.64, 11.9, and 23.8 mg a.i./L were used for analysis and reporting. 

After 96 hours of exposure, no mortality was observed in the control or any treatment group. Sublethal effects were observed in the three highest test levels starting 48 hours after exposure. Sublethal behavioral effects considered mild (e.g., increased cough frequency or a swimming position different than the control) ranged from 10 to 79% in the three highest test levels at test termination and were generally dose responsive. Sustained severe sublethal behavioral effects were not observed. The 96-hour LC50 was determined to be >23.8 mg a.i./L, and an observed NOAEC, based on mild toxic sublethal effects (cough frequency, differing swimming position relative to controls) was 2.63 mg a.i./L, based on the TWA-measured test concentrations. 

All test vessels were not identical, which is a test validity requirement per OCSPP 850.1075. At test initiation, 14 fish instead of 7 fish were mistakenly added to one replicate of the TWA-measured 11.9 mg a.i./L test group. Due to the nature of the test substance forming a hazy dispersion, this error was not detected until the end of the test. While this replicate was excluded from the study author's analysis, the reviewer included this replicate as loading rate had no impact on mortality. 

Based on the results of this study, the active ingredient (Dacthal) of the formulation Dacthal W-75, would be classified as practically non-toxic to Oncorhynchus mykiss up to the highest TWA-measured concentration tested in accordance with the classification system of the U.S. EPA. 

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

   Results Synopsis

   Test Organism Size/Age (mean weight and/or length): 1.8626 g and 5.4 cm
   Test Type (Flow-through, Static, Static Renewal): Static
   
   LC50: >23.8 mg a.i./L		95% C.I.: N/A
   Probit Slope:	N/A			95% C.I.: N/A
	Observational NOAEC:  2.63 mg a.i./L 
   
   Endpoint(s) Affected: Sublethal effects

I. MATERIALS AND METHODS

   GUIDELINE FOLLOWED:		The study was conducted according to OECD Guideline for Testing of Chemicals No. 203: Fish, Acute Toxicity Test (1992) and the US EPA Office of Prevention, Pesticides and Toxic Substances (OPPTS) draft guideline OPPTS 850.1075 (1996). The reviewer assessed the study using OCSPP 850.1075 (2016) and OECD 203 (2019), noting any similarities and/or differences where they existed, as well as OCSPP 850.1000 (2016). The following deficiencies and deviations were noted.
   
 The test 	material was both unstable and insoluble in all treatment levels. The reviewer-calculated coefficient of variation (CV) resulting from analytical test item confirmation ranged from 74 to 93%. Further, the 96-hour % of initial-measured concentrations ranged from 13 to 43%, which is well below the 80% threshold established by OCSPP 850.1000 and OECD 203. The test solutions were hazy to opaque white, non-homogenous liquid dispersions, with particulate material on the base of the test vessels. OCSPP 850.1000 recommends prior solubility trial testing if a test material has solubility issues as well as experimental design modifications. Although multiple experimental design modifications were available to the study author (e.g., saturator columns, environmental conditions, or solvent vehicles), none were utilized to improve solubility. Further, flow-through conditions may have improved stability. 
 It was not reported if test treatments and organisms were randomly assigned to test vessels, which is a validity requirement of OCSPP 850.1075. 
 All test vessels were not identical, which is another test validity requirement per OCSPP 850.1075. At test initiation, 14 fish were mistakenly added to one replicate of the 11.9 mg a.i./L test group. All other replicates only contained 7 fish. Due to the nature of the test substance forming a hazy dispersion, this error was not detected until the end of the test.
 The fish-loading rate for the 11.9 mg a.i./L test vessel containing 14 fish was calculated to be 1.04 g/fish/L. This value exceeds the OCSPP and OECD-recommended maximum fish-loading rate of 0.8 g/L.  Consequently, the biology results for this test vessel were excluded from the study author's toxicity calculations; however, the reviewer included this replicate as loading rate had no impact on mortality.
 During the test, the temperature ranged from 13.7 to 15.2 ℃ in the test solutions. OCSPP recommends a temperature of 12°C (+-2°C) for rainbow trout while OECD guidance recommends a range of 10 to 14°C.
 Each test vessel was aerated using an oil-free source of compressed air. According to OCSPP guidance, aeration of the test solutions during the test is not recommended. Gentle aeration of test vessels during the exposure period is permitted if the dissolved oxygen levels are in danger of dropping below 60% saturation. Dissolved oxygen concentrations in this study ranged from 83 to 110% saturation. According to OECD guidance, aeration can be used provided that it does not lead to a significant loss of test chemical. 
 No lighting transition period was reported. OCSPP guidance recommends that a 15- to 30-minute transition period between light and dark is used.
 The light intensity during testing was not reported. 
 The age of the fish at test initiation was not reported.  
 Incomplete reporting of the physicochemical properties. 
 Incomplete reporting of dilution water quality parameters. 
 Per OCSPP 850.1075 and 1000, dechlorinated tap water is not recommended for preparation of dilution water and should only be used as a last resort because some forms of chlorine are difficult to remove adequately. OECD guidance allows for the use of dechlorinated water, although good quality natural water or reconstituted water is preferred. However, if dechlorinated tap water is used, it should be demonstrated that test organism survival, growth, and reproduction are not affected and that test organisms do not show other signs of stress. Additionally, chlorine analysis should be performed on each batch of dilution water. The residual chlorine concentrations were 0.01-0.05 mg/mL in the dilution water during the acclimation period, and 0.00  -  0.03 mg/mL in the dilution water control during the test. Draft OPPTS 850.1075 guidance recommends residual chlorine concentrations no higher than 0.003 mg/L in dilution water. The final OCSPP 1000 guideline recommends residual chlorine concentrations no higher than 0.01 mg/L in dilution water. This is considered a minor study deficiency. 
 The statement of No Data Confidentiality Claims was missing a dated signature. 
   
			These deficiencies and deviations do impact the acceptability of the study.
   
    COMPLIANCE:			Signed and dated Quality Assurance and GLP Standards statements were provided. An unsigned No Data Confidentiality Claims statement was included. The study was conducted in compliance with the United Kingdom Statutory Instrument 1999 No. 3106, The Good Laboratory Practice Regulations 1999, the OECD Principles on Good Laboratory Practice (revised 1997, Issued January 1998) ENV/MC/CHEM (98) 17. The following exception was noted: An expiry date for the test substance was not supplied by the Sponsor. 
    
   A. MATERIALS:

   	1. Test material  				DACTHAL W-75 (ai: Dacthal)

      Description: 				Off-white powder

      Lot No./Batch No. :			2227 

      Purity: 						74.6%

      Stability of compound 
      under test conditions:		Unstable under testing conditions. The 96-hour % of initial measured concentrations ranged from 13 to 43% and the coefficient of variation based on analyzed samples ranged from 74 to 93%. Further, the test item was insoluble with observations of solutions as hazy to opaque white, non-homogenous liquid dispersions, with particulate material on the base of the test vessels increasing in amount with concentration. 
(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: 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: 
         
      Species: 					Rainbow trout, Oncorhynchus mykiss
      							EPA recommends a cold water species (preferably rainbow trout, Oncorhynchus mykiss) and a warm water species (preferably bluegill sunfish, Lepomis macrochirus or fathead minnow, Pimephales promelas). If a marine/estuarine species is desired, sheepshead minnow, Cyprinodon variegatus, is recommended. OECD recommends choice of species at discretion of testing laboratory.
      Age at test initiation:			Not reported
                           EPA recommends juvenile fish.
      Weight at study initiation: 	1.8626 g (range 1.5628 to 2.2541 g)
                     EPA recommends: <3.0 g.
      Length at study initiation: 	5.4 cm (range 4.9 to 5.8 cm)
EPA recommends: Longest not >2x shortest; OECD recommends 2.0 +- 1.0 cm for bluegill and 5.0 +- 1.0 cm for rainbow trout.
      Source:  					Brow Well Fisheries Ltd., Phoenix, Cottage, Hebden, Skipton, North Yorkshire, BD23 5DH
      							EPA recommends that all organisms be from the same source.

   B.  STUDY DESIGN:

      1. Experimental Conditions

      a. Range-finding study  -  The nominal concentrations used in the 96-hour range-finding test were 0 (control), 0.1, 1.0, 10 and 100 mg form/L (corresponding to 0.0746, 0.746, 7.46 and 74.6 mg ai/L, based on the 74.6 % Dacthal active ingredient content of DACTHAL W-75). Each test concentration contained one replicate with 5 exposed fish. At 96 hours, two out of five fish exposed at 10 mg form/L exhibited mild toxic effects. At the 100 mg form/L exposure concentration, 3 out of 5 fish exhibited mild toxic effects and the remaining 2 fish exhibited severe toxic effects. Two fish in the 1.0 mg form/L treatment group jumped out of the aquarium at 72 hours and were found dead beside the vessel. No treatment-related mortalities were observed in this study. Based on these results, the concentrations selected for the definitive test were 6.25, 12.5, 25, 50 and 100 mg form/L. 
      
      EPA recommends that definitive testing may be waived if limit testing with at least 30 organisms shows LC50 levels to be greater than 1,000 mg/L based on 100 percent active ingredients (AI), or the limits of water solubility or dispersibility. For pesticides, a lower level of 100 mg AI/L may be tested when estimated environmental concentrations are not expected to exceed 100 mg/L (ppm).
      
      b. Definitive Study

Table 1:  Experimental Parameters
                                   Parameter
                                    Details
                                    Remarks

                                   Criteria
Acclimation 
Period:

Conditions: (same as test or not)

Feeding:

Health: (any mortality observed)

>=12 days (after a 48-hour settling period) 

Similar to test conditions (16L:8D photoperiod; pH of 7.0 to 7.4, dissolved oxygen of 78-87%, temperature of 13.2 to 14.7℃).

Proprietary food (Nutrafry AB 02 (TROUW UK)). Fish were not fed in the 48-hours preceding the definitive test.

No mortality during the 7-day period preceding the test. The stock of fish would not be used for testing if mortality exceeded 5% or if disease was apparent. 

The recommended acclimation period is 14 days (at a minimum 12 days), and a minimum of 7 days acclimation in test dilution water at test temperature. OECD guideline recommends a minimum of 12 days. Wild caught fish should be quarantined 7 days before acclimation begins.  

Acclimation temperature change should be <3°C per day.
No feeding or disease treatments permitted within 48 hrs of test initiation or during test.

Pretest mortality must be <5% 48 hrs prior to testing. If >10%, batch must be rejected.  OECD pretest mortality criteria: >10%= rejection of entire batch; 5-10%= continued acclimation for 7 days; <5%= acceptable.
Duration of the test
96 hours

The recommended test duration is a minimum 96 hours.
Test condition

Static/static renewal/flow-through

Type of dilution system - for flow- through method

Renewal rate for static renewal

Static

N/A

N/A

Static renewal or flow-through is necessary when test concentrations are expected to decline during the study period to <70% of the day-0 measured concentrations.  A reproducible supply of toxicant is recommended.  Consistent flow rate should be 6-10 vol/24 hours for flow-through systems; diluter systems should be monitored for proper calibration before and every 24 hours during study. Flow rate to each replicate should be measured at the beginning and end of each test, and not vary by more than a factor of 10 between replicates.
Aeration, if any
Each test vessel was aerated using an oil-free source of compressed air.

Aeration is not recommended for flow-through tests, but gentle aeration is permitted in static systems only to keep oxygen levels at 60%. OECD guideline recommends aeration.  If aeration is necessary, test solutions must be analyzed periodically to verify exposure.
Test vessel
Material: (glass/stainless steel)

Size:

Fill volume:

Glass aquariums 

30 L

25 L 

Loading
Static or static-renewal tests: <=0.8 g wet weight per liter;
Flow-through test: <=0.5 g/L per 24 hours and <5 g/L at any time
Source of dilution water

Quality of dilution water
Particulate matter:
COD:
TOC:
Boron and fluoride:
Residual chlorine:
Un-ionized ammonia:
Aluminum, arsenic, chromium, cobalt, copper, iron, lead, nickel and zinc:

Cadmium, mercury and silver:

Total organophosphorus pesticides:
Total organochlorine pesticides +
    PCBs or organic chlorine:

Specific conductivity:	
Hardness as CaCO3:
Dechlorinated municipal mains water*

Not reported
Not reported
Not reported
Not reported
<=0.05 mg/mL 
Not reported 

Copper: 65 ug/L; Zinc: 73 ug/L; Aluminum, Iron, Lead: <reporting limit; Values for other metals not reported
Cadmium: <reporting limit; Values for other metals not reported
Not reported

<Reporting limit

187 uS/cm
27 mg/L Ca
* Pumped to the laboratory via a
Purite AC9 activated carbon filter and a Purite ROPF 20 particulate filter, then chilled to approximately 14 °C. 

Recommended source of dilution water is clean surface or ground water, seawater (for estuarine or marine species), and reconstituted water. EPA does not recommend the use of dechlorinated tap water; however, if used, daily chlorine analysis is recommended.  Maximum allowable concentrations for water quality parameters are provided in EPA's 850.1075 guideline (http://www.epa.gov/ocspp/pubs/frs/home/draftguidelines.htm).  OECD permits dechlorinated tap water.
Water quality during testing
Hardness:

pH:

Dissolved oxygen:

Temperature:

Other measurements
Photoperiod:

Light intensity:

Intervals of water quality measurement

74 to 80 mg/L as CaCO3

7.3 to 7.6

83 to 110% saturation

13.7 to 15.2 ℃

16-hr L:8-hr D

Not reported

Dissolved oxygen, temperature, and pH were measured daily in each test item vessel. The temperature range (min and max) of the control test media was determined every 24 hours. 

Total hardness was determined at the start and end of the test in the control test solution.

Residual chlorine was measured every 24 hours in one of the control test vessels. 
Residual chlorine: 0.00 to 0.03 mg/L

EPA Recommendations:
Hardness: 40-180 mg/L as CaCO3 (OECD recommends 10 - 250 mg/L)
pH:  > 6.0 and < 8.0 for freshwater testing, and > 7.5 and < 8.5 for marine testing. (OECD recommends  pH 6.0 - 8.5)
Dissolved Oxygen: Static: > 60% saturation;  flow-through: > 75% saturation; (OECD recommends at least 80% saturation value).
Temperature:10-12 +-2 °C for coldwater species and 22-23+-2 °C for warmwater species, (OECD recommends 21-25°C for bluegill and 13-17[o]C for rainbow trout). Temperature should not vary >1°C in any 24-hour period.
Salinity:  20+-5 %. for estuarine species.  

Photoperiod: 12 hours light and 12 hours dark, to 16 hours light and 8 hours dark, with a 15 to 30 minute transition period. (OECD recommends a photoperiod of 12-16 hours light).
Light intensity should range from 30-100 lumens at the water surface;

DO, pH and temperature should be measured in each replicate at test initiation and every 24 hours. Temperature should also be measured in one replicate at least hourly. Water hardness or salinity, as appropriate, should be measured at test initiation; in marine flow-through tests, salinity should be recorded at test initiation, on day 4, and if extended, on days 7 and 14.
Number of replicates/groups:
Negative control:
Solvent control:
Treatments:

2
N/A
2/level

Recommended number of replicates is 2 per test concentration and control.
Number of organisms per replicate /groups:
Negative control:
Solvent control:
Treatments:

7
N/A
7/level*
*At test initiation 14 fish instead of 7 fish were added to one replicate of the TWA-measured 11.9 mg ai/L test group. Due to the nature of the test substance forming a hazy dispersion, this error was not detected until the end of the test. 

Number of organisms per replicate per test concentration and control/solvent control should be a minimum 7, >= 10 is recommended; OECD guideline recommends at least 7 fish/concentration.
Biomass loading rate
0.51 g fish/L/day*

* The fish-loading rate of the TWA-measured 11.9 mg ai/L test group was calculated to be 1.04 g/fish/L. This value exceeds the required maximum fish-loading rate for the test (0.8 g/L). Consequently, the biology results for this test vessel were excluded from the study author's toxicity calculations.

Recommended static conditions are <= 0.8 g FWF/L per replicate and for flow-through are <= 0.5 g FWF/L per replicate within 24 hours. OECD recommends a maximum of 1 g fish/L for static and semi-static, while higher rates are recommended for flow-through.
Test concentrations:

Nominal (formulation):

Nominal (active ingredient):

TWA-measured:

0 (negative control), 6.25, 12.5, 25. 50, and 100 mg form/L

0 (negative control), 4.66, 9.33, 18.7, 37.3, and 74.6 mg a.i./L 

<0.003 mg a.i./L (<LOD, control), 1.44, 2.63, 8.64, 11.9, and 23.8 mg a.i./L*

*Calculated by the reviewer (refer to copy of Excel worksheet in Appendix I). 
EPA recommends a minimum of 5 test concentrations, in geometric series with a ratio of 1.5 to 2.2, plus a control/solvent control. Test concentrations should be selected to produce a no-observable-effect concentration (NOEC) and, preferably, at least two partial mortalities, i.e. one greater than and the other less than 50% after 96 hours.

Test concentration must be measured at test initiation and every 48 hours in each replicate until 96 hours, and after every malfunction in the appropriate chamber, and for static tests after every renewal. After 96 hours, test concentration measurements are recommended only every 96 hours.

No more than 25 percent variation is allowed between test concentrations within the same treatment during the test.
Solvent (type, percentage, if used)
N/A

The solvent should not exceed 0.5 ml/L for static tests or 0.1 ml/L for flow-through tests; OECD recommends that the solvent not exceed 100 mg/L. Preferred solvents are dimethyl formamide, triethylene glycol, methanol, acetone, or ethanol.
Feeding
The fish were not fed in the 48-hour period preceding the test or during testing. 

Fish should not feed during the study.

Recovery of chemical 
Frequency of determination
Level of quantization
Level of detection

0, 48, and 96 hours 
Not reported
0.003 mg a.i./L (0.004 mg form/L)

Positive control:
Nominal concentrations:
N/A 

Other parameters, if any
None

   2. Observations:

Table 2:  Observations
                                   Parameter
                                    Details
                                    Remarks
                                       
                                       
                                   Criteria
Parameters measured including the sublethal effects/toxicity symptoms
Mortality and behavioral abnormalities (sublethal effects)

Recommended parameters measured include:
- mortality;
- immobility;
- incidence and description of morphological
     abnormalities and behavioral effects;
- observations of other effects or clinical signs.
Observation intervals 
Observations were made at 17.42, 24, 48, 72, and 96 hours. 

Mortality observations should be recorded at 6, 24, 48, 72 and 96 hours, and if continued, every 24 hours until test termination.
Were raw data included?
Yes.

Other observations, if any
Stock solutions appeared as white hazy emulsions with foaming on the surface at the two highest concentrations. 

The test media generally appeared as hazy to opaque white, non-homogenous liquid dispersions, with particulate material on the base of the test vessels increasing in amount with concentration. At 24, 48, and 72 hours, foaming was present on the surface of the test media at the highest test concentration.

II. RESULTS AND DISCUSSION:
   
   A. MORTALITY:

   By 96 hours, there was a complete absence of mortality in the control and all treatment groups. 
   
   EPA/OECD recommend pretreatment control mortality <10%. EPA recommends control or solvent control mortality be <10 % (or 1 mortality if 7 to 10 control fish are used) for a 96 - h period of testing. If the test is continued past 96 h, the maximum-allowable additional mortality is 10%.  OECD also recommends that maximum-allowable control or solvent control mortality is 10% (or 1 mortality if 7 to 10 control fish are used) for a 96-h period of testing. 
   
Table 3:  Effect of DACTHAL W-75 (ai: Dacthal) on Mortality of Oncorhynchus mykiss[1].
                                 TWA-Measured 
                   (and Nominal) Concentrations, 
mg a.i./L
                         No. of fish at start of study
                              Observation period

                                   24 Hours
                                   72 Hours
                                   96 Hours

                                   No. Dead
                                  % Mortality
                                   No. Dead
                                  % Mortality
                                   No. Dead
                                  % Mortality
Control (<LOD)
                                      14
                                       0
                                       0
                                       0
                                       0
                                       0
                                       0
1.44 (4.66)
                                      14
                                       0
                                       0
                                       0
                                       0
                                       0
                                       0
2.63 (9.33)
                                      14
                                       0
                                       0
                                       0
                                       0
                                       0
                                       0
8.64 (18.7)
                                      14
                                       0
                                       0
                                       0
                                       0
                                       0
                                       0
11.9 (37.3)
                                      21*
                                       0
                                       0
                                       0
                                       0
                                       0
                                       0
23.8 (74.6)
                                      14
                                       0
                                       0
                                       0
                                       0
                                       0
                                       0
LC50
>100 mg form/L (based on study author's results)
Positive control, if used
mortality:
LC50:

N/A
     [1] Data obtained from Table 3, p. 23 of the study report. 
    * At test initiation 14 fish, instead of 7, were added to one replicate of the 11.9 mg a.i./L test group. Due to the nature of the test substance forming a hazy dispersion, this error was not detected until the end of the test.
  LOD = 0.003 mg/L

   B. SUB-LETHAL TOXICITY ENDPOINTS:

   All fish appeared normal after 24 hours of exposure. Mild toxic effects, such as increased cough frequency or a swimming position different than the control, were observed in the three highest test concentrations from 48 hours through the end of the test. The number of fish exhibiting mild effects generally increased with increasing test concentration. Severe toxic effects (e.g., swimming abnormally or lying on bottom of tank) were only observed in one fish in the highest test concentration after 72 hours of exposure. 

Table 4:  Sub-lethal Effect of DACTHAL W-75 (ai: Dacthal) on Oncorhynchus mykiss[1].
                                 TWA-Measured 
                   (and Nominal) Concentrations, 
mg a.i./L
                              Observation period

                                   24 Hours
                                   48 Hours
                                   72 Hours
                                   96 hours

                              % with mild effects
                          % with severe toxic effects
                              % with mild effects
                          % with severe toxic effects
                              % with mild effects
                          % with severe toxic effects
                              % with mild effects
                          % with severe toxic effects
Control (<LOD)
                                       0
                                       0
                                       0
                                       0
                                       0
                                       0
                                       0
                                       0
1.44 (4.66)
                                       0
                                       0
                                       0
                                       0
                                       0
                                       0
                                       0
                                       0
2.63 (9.33)
                                       0
                                       0
                                       0
                                       0
                                       0
                                       0
                                       0
                                       0
8.64 (18.7)
                                       0
                                       0
                                       7
                                       0
                                      21
                                       0
                                      21
                                       0
11.9 (37.3)*
                                       0
                                       0
                                      36
                                       0
                                      29
                                       0
                                      10
                                       0
23.8 (74.6)
                                       0
                                       0
                                      86
                                       0
                                      93
                                       7
                                      79
                                       0
[1] The % effects were calculated by the reviewer in Excel using the data provided in Table 3, p. 23 of the study report. Mild toxic effects: swimming normally but exhibiting mild effects e.g., increased cough frequency, swimming position in test vessel different to controls. Severe toxic effects: e.g., swimming abnormally or lying on bottom of tank.
*At test initiation 14 fish, instead of 7, were mistakenly added to one replicate of the 11.9 mg a.i./L test group. Due to the nature of the test substance forming a hazy dispersion, this error was not detected until the end of the test. The % with sublethal effects is based on 21 fish in this treatment group for the 96-hour results only.
LOD = 0.003 mg/L

   C. REPORTED STATISTICS: 
   
   No significant mortality was recorded during the definitive test. Therefore, the 96-hour LC50 value could not be calculated and was visually estimated to be greater than the highest test concentration. The NOAEC was established by observation of the mortality and sublethal effect raw data. The study author's results were based nominal concentrations of the formulation. 
   
   LC50: >100 mg form/L		95% C.I.:  N/A
   Probit Slope:	N/A				95% C.I.: N/A
   Observational NOAEC:  12.5 mg form/L
   
   Endpoint(s) Affected: Sublethal effects

   D. VERIFICATION OF STATISTICAL RESULTS:

   The mortality data were analyzed using CETIS Version 1.9.6.12 with database backend settings implemented by EFED on 7/25/17. Since no mortality was observed in any of the treatment levels, the LC50 was empirically determined to be greater than the highest TWA-measured test concentration. A No Comparisons report was generated.   

   LC50: >23.8 mg a.i./L		95% C.I.: N/A
   Probit Slope:	N/A			95% C.I.: N/A
	Observational NOAEC:  2.63 mg a.i./L 
   
   Endpoint(s) Affected: Sublethal effects

   
   
   E.  STUDY DEFICIENCIES: 
   
 The test 	material was unstable and insoluble in all treatment levels. The reviewer-calculated coefficient of variation (CV) resulting from analytical test item confirmation ranged from 74 to 93% (refer to copy of Excel worksheet in Appendix I). Further, the 96-hour % of initial-measured concentrations ranged from 13 to 43%, which is well below the 80% threshold established by OCSPP 850.1000 and OECD 203. The test solutions were hazy to opaque white, non-homogenous liquid dispersions, with particulate material on the base of the test vessels. OCSPP 850.1000 recommends prior solubility trial testing if a test material has solubility issues as well as experimental design modifications. Although multiple experimental design modifications were available to the study author (e.g., saturator columns, environmental conditions, or solvent vehicles), none were utilized to improve solubility. Further, flow-through conditions may have improved stability.   
 It was not reported if test treatments and organisms were randomly assigned to test vessels, which is a validity requirement of OCSPP 850.1075. 
 At test initiation, 14 fish were mistakenly added to one replicate of the 11.9 mg a.i./L test group. All other replicates only contained 7 fish. Due to the nature of the test substance forming a hazy dispersion, this error was not detected until the end of the test. It is a test validity requirement for OCSPP 850.1075 that all test vessels are identical.
 Per OCSPP 850.1075 and 1000, dechlorinated tap water is not recommended for preparation of dilution water and should only be used as a last resort because some forms of chlorine are difficult to remove adequately. OECD guidance allows for the use of dechlorinated water, although good quality natural water or reconstituted water is preferred. However, if dechlorinated tap water is used, it should be demonstrated that test organism survival, growth, and reproduction are not affected and that test organisms do not show other signs of stress. Additionally, chlorine analysis should be performed on each batch of dilution water. The residual chlorine concentrations were 0.01-0.05 mg/mL in the dilution water during the acclimation period, and 0.00  -  0.03 mg/mL in the dilution water control during the test. Draft OPPTS 850.1075 guidance recommends residual chlorine concentrations no higher than 0.003 mg/L in dilution water. Draft OPPTS 850.1075 guidance recommends residual chlorine concentrations no higher than 0.003 mg/L in dilution water. The final OCSPP 1000 guideline recommends residual chlorine concentrations no higher than 0.01 mg/L in dilution water. Given the adequate control performance, this is considered a minor study deficiency. 
   
   F.  REVIEWER'S COMMENTS:  

   The study author's and the reviewer's LC50 values were in agreement after taking into consideration the difference in concentrations used for analysis. The study author's results were based on the nominal formulation concentrations, whereas the reviewer's results were based on the TWA-measured test concentrations (refer to copy of Excel worksheet in Appendix I). The reviewer's results are presented in the Executive Summary and Conclusions sections of this DER because the test material was unstable and TWA-measured concentrations are preferred when the coefficient of variation of the analytical data is >=20%. 
   
   At test initiation, 14 fish instead of 7 fish were mistakenly added to one replicate of the TWA-measured 11.9 mg a.i./L test group. Due to the nature of the test substance forming a hazy dispersion, this error was not detected until the end of the test. The fish-loading rate for this test vessel was calculated to be 1.04 g/fish/L. This value exceeds the OCSPP and OECD-recommended maximum fish-loading rate for the test (0.8 g/L). While this replicate was excluded from the study author's analysis, the reviewer included this replicate as loading rate had no impact on mortality. 
   
	The following OCSPP 850.1075 (2016) validity criteria were NOT met:
 All test vessels were not identical. (In this study, one replicate in the 11.9 mg a.i./L treatment group contained 14 fish. All other replicates contained 7 fish.)
 Treatments were not reported to be randomly or indiscriminately assigned to individual test vessel locations, or individual test organisms were not reported to be randomly or indiscriminately assigned to test vessels. (It was not reported if treatments or organisms were randomly assigned in this study.)

   All OECD 203 (2019) validity criteria were met. 

   The study was conducted following the draft OPPTS 850.1075 (1996) guideline, OECD 203 guideline (1992), and the reviewer considered both the draft and the final OCSPP 850.1075 (2016) guideline and updated OECD 203 guideline (2019) and OCSPP 850.1000 guideline in their evaluation of the data.
   
   The in-life phase of the study was conducted sometime between October 7 and November 16, 2003. 

   G. CONCLUSIONS:

   This study is scientifically sound and is classified as supplemental and may be used for risk characterization.  No mortality was observed in the control or any treatment groups throughout the 96-hour exposure. Sublethal effects (mild effects, e.g., increased cough frequency, swimming position in test vessel different to controls) were observed in the >=8.64 mg a.i./L treatment groups starting at 48 hours. Effects were generally dose responsive. The 96-hour LC50 is >23.8 mg a.i./L, and an observed NOAEC based on mild sublethal behavioral effects (cough frequency, differing swimming position relative to controls) was 2.63 mg a.i./L, based on the TWA-measured test concentrations. 

III.  REFERENCES:

Stephan, C.E., K.A. Busch, R. Smith, J. Burk and R.W. Andrews. 1978. A computer program for calculating an LC50. U.S. Environmental Protection Agency, Duluth, Minnesota, pre-publication manuscript, August, 1978.

All other references listed referred to standard guidelines.  
APPENDIX I. Copy of Excel Worksheet with TWA Concentrations