﻿DATA EVALUATION RECORD
ACUTE LC50 TEST WITH AN ESTUARINE/MARINE SHRIMP
850.1035


1.  CHEMICAL:  DCPA (Dacthal)PC Code No.:  078701

2.  TEST MATERIAL: Dacthal Technical Purity: 98.6%

3.  CITATION
Authors:Claude, M.B., Martin, K.H., Gallagher S.P., Bodle, E.S. and H.O. Krueger
Title:Dacthal: A 96-Hour Static Acute Toxicity Test with the Saltwater Mysid (Americamysis bahia)
Study Completion Date:December 6, 2013
Laboratory:Wildlife International, Ltd., Easton, Maryland
Sponsor:Amvac Chemical Corporation, Los Angeles, California
Laboratory Report ID:246A-115
MRID No.:49307505
DP Barcode:420820

4.  REVIEWED BY:  Teresa Nelis, Environmental Scientist, CDM/CSS-Dynamac JV
     Signature:         Date: 10/14/2016

     APPROVED BY:  Moncie V Wright, Environmental Scientist, CDM Smith/CSS-Dynamac JV
Signature: Date: 10/31/2016

5.  APPROVED BY:  Christina M. Wendel, Biologist, OPP/EFED/ERB2

     Signature:Date: 10/19/2021
     
Michael Wagman, Senior Scientist, OPP/EFED/ERB2

     Signature:Date: 11/10/2021
     
This Data Evaluation Record may have been altered by the Environmental Fate and Effects Division subsequent to signing by CDM/CSS-Dynamac JV personnel.





6.  STUDY PARAMETERS

Age or Size of Test Organism:<24 hours
     Definitive Test Duration:96 hours
Study Method:Static
     Type of Concentrations:Time weighted average concentrations

7.  CONCLUSIONS:  

     Results Synopsis
     
     96-hour LC50:  >396 ug a.i./L (>0.396 mg a.i./L)95% C.I.:  N/A
     Probit Slope:  N/A95% C.I.:  N/A
Observational NOAEC: 24.9 ug a.i./L (0.0249 mg a.i./L)
Endpoint(s) affected: Mortality
     
8.  ADEQUACY OF THE STUDY

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

     B.  Rationale:  The dissolved oxygen (DO) was <=60% saturation in the negative and solvent controls, and the 24.9, 54.6, and 95.1 ug a.i./L treatment groups at 24 hours. The highest nominal concentration is at the solubility limit for DCPA (0.5 mg/L), and although the highest nominal concentration is at the solubility limit for DCPA, both the stock solutions and test concentrations were mixed by inversion and were clear and colorless with no undissolved test substance throughout the test. The test solutions did appear slightly cloudy and white at test termination, but this was likely due to the presence of microbial growth in all treatment and control test chambers but did not appear to have an impact on the results.

     C.  Repairability:  None

9.  GUIDELINE DEVIATIONS: This study was conducted following guidelines outlined in U.S. Environmental Protection Agency, Office of Prevention, Pesticides, and Toxic Substances (OCSPP), Ecological Effects Test Guidelines for conducting acute toxicity tests with mysids OCSPP (form. OPPTS) 850.1035 (1996), and ASTM Standard E729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians (2007). The reviewer considered these guidelines and the final OCSPP 850.1035 (2016) guideline in their evaluation. The following deviations were noted:

Dissolved oxygen (DO) was below the recommended test parameter of >=60% of saturation in one replicate of the negative control and both replicates of the solvent control, 24.9, 54.6, and 95.1 ug a.i./L treatment groups at 24 hours. The test chambers were gently aerated to address the low DO and maintain DO levels at >=60% of saturation. Although the study authors determined the drop in DO at 24 hours did not affect the overall findings because there were no sufficient negative effects to create 50% mortality in the control or treatment groups. This is considered a study deficiency for both the draft OPPTS and final OCSPP 850.1035 guideline.
  The photoperiod was 16L:8D; which is within the range for the final OCSPP 850.1035 guideline, although the draft OPPTS 850.1035 guideline suggests 14L:10D. However, based on the final OCSPP 850.1035 guideline, this is not considered a deficiency, as a photoperiod should be selected from regimes of 12 hours light:12 hours dark to 16 hours light:8 hours dark. For any given test, the light regime should be constant. 
The TOC of the dilution water was not reported.  The lack of measurement in dilution water is only considered a deficiency based on the final OCSPP 850.1035 guideline. In the draft OPPTS 850.1035 guideline, the measurement of TOC was only recommended when deionized water was used to prepare artificial seawater. 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 likely not result in an underestimation of toxicity.
       Pre-test mortality was not reported, this is only considered a deficiency based on the final OCSPP 850.1035 guideline, and not the draft OPPTS 850.1035 guideline. However, the even with the lack of this information, the mortality in the controls was acceptable (<=10%) during this test.  

      These deficiencies do impact the acceptability of the study.

10.  SUBMISSION PURPOSE:  This study was submitted to provide data on the effects of Dacthal (DCPA) on juvenile mysids (Americamysis bahia) following acute exposure for the purpose of registration review.

11.  MATERIALS AND METHODS

     A.  Test Organisms
Guideline Criteria
Reported Information
Species
Preferred species are Mysidopsis bahia, Penaeus setiferus, P. duorarun, P. aztecus and Palaemonetes sp.

Saltwater mysid; Americamysis bahia
(form. Mysidopsis bahia)
Age
Juvenile, mysids should be 
< 24 hours old

Juveniles; <24 hours old
Supplier
Laboratory in-house cultures; identification of the species was verified by the supplier of the original stock culture.
All shrimp are from same source?
Yes
All shrimp are from the same year class?
Yes

     B.  Source/Acclimation
Guideline Criteria
Reported Information
Acclimation Period

Acclimation period was continuous. Mysids held in water from the same source and at approx. same temperature as used in test. During the two-week period immediately preceding the test, temperatures ranged from 24.7 to 26.1ºC; pH ranged from 8.0 to 8.3; DO was >=6.7 mg/L (>=91% saturation); and salinity of the filtered seawater ranged from 20 to 21 ppt (%.). 
Wild caught organisms were quarantined for 7 days?
N/A
Were there signs of disease or injury?
No signs of disease or stress were observed in the adults in the holding period.
If treated for disease, was there no sign of the disease remaining during the 48 hours prior to testing?
N/A
Feeding
No feeding during the study and no feeding for 24 hour before the beginning of the test if organisms are over 0.5 g each. Mysids should be fed throughout the study.
Culture mysids were fed live brine shrimp (Artemia sp.) nauplii (INVE Aquaculture, Salt Lake City, Utah) daily, which were periodically enriched with a nutrient enrichment (Al DHA Selco from INVE Thailand, Ltd., Phichit, Thailand). During the test, mysids were fed live brine shrimp nauplii (Artemia sp.) daily. 
Pretest Mortality
<3% mortality 48 hours prior to testing
Pre-test mortality was not reported.

     C.  Test System
Guideline Criteria
Reported Information
Source of dilution water 
Soft reconstituted water or water from a natural source, not dechlorinated tap water
Natural seawater collected at Indian River Inlet, Delaware. Water was pumped into a 5000 gallon holding tank and was ozonated and passed through a sand filter (~25 micron), it was then pumped into a 37,800 L storage tank and diluted with well freshwater (on the Wildlife International site) to a salinity of ~20%., and aerated with spray nozzles. Diluted water was then filtered to 0.45 um to remove fine particles and UV-sterilized prior to use.
Does water support test animals without observable signs of stress?
Yes
Salinity
30-34 %. (ppt, parts per thousand) for marine (stenohaline) shrimp and 10-17 ppt for estuarine (euryhaline) shrimp, weekly range < 6 ppt

20-21 %.
Salinity was measured in the dilution water at test initiation and termination.
Water Temperature
Approx. 22 + 1 °C

24.3-27.2°C
pH
8.0-8.3 for marine (stenohaline) shrimp, 7.7-8.0 for estuarine (euryhaline) shrimp, monthly range < 0.8

7.9-8.5
Dissolved Oxygen
Static: >= 60% during 1st 48 hrs and >= 40% during 2nd 48 hrs, Flow-through: >= 60%
At 0, 48, 72 and 96 hours: 6.0 to 7.4 mg/L (>=81% of saturation)

At 24 hours: 3.4 to 6.0 mg/L (>=46% of saturation); below 60% of saturation. Test vessels were gently aerated after low DO found at 24 hours, to maintain adequate levels of DO.

4.4 mg/L DO represents 60% saturation at a salinity of 20%. and 25°C.
Total Organic Carbon
Should be <5 mg/L in reconstituted seawater

Not Reported
Test Aquaria
1. Material:
   Glass or stainless steel
2. Size:
     19.6 L is acceptable for organisms >= 0.5 g (e.g. pink shrimp, white shrimp, and brown shrimp), 3.9 L is acceptable for smaller organisms (e.g. mysids and grass shrimp).
3. Fill volume:
     15 L is acceptable for organisms >= 0.5 g, 2-3 L is acceptable for smaller organisms.

1. Glass beakers


2. 2 L





3. 1.5 L

The depth of the test water was 12.0 cm.
Type of Dilution System
Must provide reproducible supply of toxicant
N/A; test was conducted under static conditions
Flow Rate
Consistent flow rate of 5-10 vol/24 hours, meter systems calibrated before study and checked twice daily during test period 
N/A; test was conducted under static conditions
Biomass Loading Rate
Static: Pound 0.8 g/L at # 17°C, Pound 0.5 g/L at > 17°C; flow-through: Pound 1 g/L/day
(N/A for mysids)
 
N/A
Photoperiod
16 hours light, 8 hours dark
16 h light, 8 h dark with 30-minute transitional light periods to avoid sudden changes. Light intensity at test initiation was 379 lux at surface of one representative test chamber.
Solvents
Not to exceed 0.5 ml/L for static tests or 0.1 ml/L for flow-through tests

Solvent: DMF
Maximum conc.: 0.06 mL/L (0.006%)
     
     D.  Test Design
Guideline Criteria
Reported Information
Range Finding Test
If LC50 >100 mg/L with 30 shrimp, then no definitive test is required.
The range finding study (results summarized in Table 1, pp. 18 of study report) indicated that DCPA was completely soluble in the 24 to 900 ug a.i./L solutions (solutions were clear and colorless at test initiation and test termination) containing 0.06 mg/L (0.006%)DMF but not in the 3000 ug a.i./L solution (fine white precipitate was on the surface of the solution at test initiation and test termination). No information was provided if the test solutions were measured.

Mysid mortality was 0% in the negative control, solvent control and 81 ug a.i./L test solution, and was 10, 30, 80 and 100% in the 24, 270, 900 and 3000 ug a.i./L test solutions, respectively. Additional observations were recorded every 24 hours beginning at test initiation and included the following: appeared normal, surfacing, lethargic or erratic swimming. Mysids appeared normal in in the negative control, solvent control 24, 81 and ug a.i./L test solutions. In the in the 270, 900 and 3000 ug a.i./L test solutions mysids observations included appearing normal, surfacing, lethargy, and erratic swimming.
Nominal Concentrations of Definitive Test
Control & 5 treatment levels;
a geometric series in which each concentration is at least 60% of the next higher one.
0 (negative control), 0 (solvent control), 28, 56, 113, 225, and 450 ug a.i./L

Number of Test Organisms
Minimum 20/level, may be divided among containers
20/level, equally divided among two replicates
Test organisms randomly or impartially assigned to test vessels?
Yes
Biological observations made every 24 hours?
Yes. Observations were made approx. 3, 24, 48, 72, and 96 hours after test initiation. All organisms were observed to determine the number of mortalities, signs of toxicity and abnormal behavior in each treatment group.
Water Parameter Measurements
1. Temperature
     Measured constantly or, if water baths are used, every 6 hrs, may not vary > 1°C
2. DO and pH
     Measured at beginning of test and ever 48 h in the high, medium, and low doses and in the control

1. Temperature was measured at test initiation and termination and daily at approx. 24 hr intervals in each replicate test vessel, and continuously in a surrogate test chamber.

2. DO and pH were measured at test initiation and termination and daily (at approx. 24 hr intervals) in each replicate test vessel.
Chemical Analysis
needed if solutions were aerated, if chemical was volatile, insoluble, or known to absorb, if precipitate formed, if containers were not steel or glass, or if flow-through system was used
Samples were collected for analysis at 0, 48, and 96 hours (+- 1 hour) and analyzed by HPLC with UV detection (220 nm).


12.  REPORTED RESULTS

     A.  General Results
Guideline Criteria
Reported Information
Quality assurance and GLP
compliance statements were
included in the report?
Yes. Signed and dated No Data Confidentiality, GLP, and Quality Assurance statements were provided. This study was conducted in compliance with US EPA Good Laboratory Practice Standards (40 CFR Parts 160 and 792), that are compatible with: OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98)17), and Japan MAFF (11 NohSan Notification Number 6283, Agricultural Production Bureau, 1 October 1999), with the following exception: periodic analyses of saltwater for potential contaminants were not performed according to Good Laboratory Practice Standards but were performed using a certified laboratory and standard U.S. EPA analytical methods.
Recovery of Chemical
Recovery of DCPA in test solutions for Day 0 and Day 2 were 82 to 105% of nominal based on individual samples; recovery of DCPA for Day 4 was much lower and was 58 to 83% based on individual samples. Average recovery data for 0, 2 and 4 days combined was 81 to 95%. TWA was reviewer calculated from the measured concentrations (refer to copy of Excel worksheet in Appendix I).
Recovery of QC fortification spikes ranged from 98 to 104% of nominal.

Test solutions appeared clear and colorless at test initiation, with no evidence of precipitation observed. The test solutions did appear slightly cloudy and white at test termination, but this was due to the presence of microbial growth in all treatment and control test chambers.
Control Mortality
Not more than 10% of control organisms may die or show abnormal behavior.
After 96 hours, there was 5% mortality in the negative control, and 10% mortality in the solvent control.
Raw data included?
Yes
Signs of toxicity (if any) were described?
Yes
     
     Mortality
Concentration (ug a.i./L)
Number of Mysids
Cumulative Number Dead
Nominal
TWA1
(Mean-Measured)

Hour of Study



24
48
72
96
96
(%)
Negative Control
<10.0 (<LOQ)a
20
0
0
0
1b
5
Solvent Control
<10.0 (<LOQ)a
20
0
2
2
2
10
28
24.9 (24)
20
0
0
0
0
0
56
54.6 (53)
20
0
2
2
2
10
113
95.1 (92)
20
0
2b
2b
2b
10
225
201 (197)
20
0
0
0
0
0
450
396 (391)
20
1
2
2
2
10
  1 Time weighted average (TWA) concentrations were reviewer calculated
  a LOQ was 10 ug a.i./L.
  b One mysid was missing and assumed dead.

Two mysids were missing and assumed dead in the 95.1 ug a.i./L Time weighted average (TWA)-measured treatment group (equivalent to 92 ug a.i./L mean-measured concentrations) at 48 hours, the mysids were never found. Additionally, one mysid was missing and assumed dead in the negative control at 96 hours. After 96 hours, there was no mortality in the 24.9 and 201 ug a.i./L TWA-measured treatment groups (equivalent to 24 and 197 ug a.i./L mean-measured concentrations, respectively), 5% mortality in the negative control (the missing/assumed dead mysid), and 10% mortality in the solvent control and the 54.6, 95.1 (the missing/assumed dead mysids), and 396 ug a.i./L treatment groups (equivalent to 53, 92, and 391 ug a.i./L mean-measured concentrations, respectively). The study author reported a 96-hour LC50 value of >391 ug a.i./L, based on the mean-measured concentrations. Which correspond to a 96-hour LC50 value of >396 ug a.i./L, based on the TWA-measured concentrations.

Other Significant Results:  No sublethal effects were observed in the controls or in any of the test solutions. 

     B.  Statistical Results

     Method:  The absence of mortality in any of the DCPA treatment groups during the test precluded the statistical calculation of LC50 values. Therefore, the LC50 values were estimated to be greater than the highest concentration tested. The no-mortality concentration and the no-observed-effect concentration (NOEC) were determined by visual interpretation of the mortality and observation data. The study author used mean-measured concentrations for the analyses.
     
     96-hr LC50:  >391 ug a.i./L (>0.396 mg a.i./L)95% C.I.:  N/A 
     Probit Slope: N/A 
     
13.  VERIFICATION OF STATISTICAL RESULTS

   The mortality data and TWA-measured concentrations of the test material were estimated by the reviewer and were entered into the program CETIS, (Version 1.8.7.12) with backend settings implemented by EFED on 10/20/15. The reviewer visually determined the LC50 due to a lack of effects >=50%. 

Parameter
Result
Probit LC50 (95% C.I.)
>396 ug a.i./L (>0.396 mg a.i./L)
Probit Slope (95% C.I.)
N/A
Statistical Method for NOAEC
Visual estimation
Observational NOAEC
24.9 ug a.i./L (0.0249 mg a.i./L)
   

REVIEWER'S COMMENTS:

     The reviewer's and study author's results were in agreement when accounting for the differences between the mean-measured and TWA-measured concentrations.
     
     The study was conducted following the draft OPPTS 850.1035 (1996) guideline, ASTM Standard E729-96 (2007), and the reviewer considered both the draft and the final OCSPP 850.1035 (2016) guideline and ASTM E729-96 in their evaluation of the data.
     
     The in-life portion of the definitive toxicity test was conducted from August 5, 2013 to August 9, 2013.
     
     The measured test concentrations had a coefficient of variation (CV) of >=20%. The CV for the time-weighted average concentrations ranged from 12 to 20%.  
     
     Time-weighted average (TWA) concentrations were reviewer-calculated (refer to copy of Excel worksheet in Appendix I) and are reported in the Conclusions sections of the DER.  TWA concentrations were calculated by the reviewer using the following equation:


     where:
     C TWA  is the time-weighted average concentration,
     C j  is the concentration measured at time interval j (j = 0, 1, 2,...n)
     t j  is the number of hours (or days or weeks, units used just need to be consistent in the equation) of the test at time interval j
     (e.g., t 0  = 0 hours (test initiation), t 1 =48 hours, t 2 =96 hours)
     
     Results from the periodic screening analysis of the saltwater dilution water indicated the presence of the following analytes: calcium (239 mg/L), chloride (11,400 mg/L), magnesium (498 mg/L), potassium (238 mg/L), and sodium (6,050 mg/L). Pesticides and PCBs were not detected in the saltwater dilution water.
     
     The dissolved oxygen (DO) was <=60% saturation in the negative and solvent controls, and the 24.9, 54.6, and 95.1 ug a.i./L treatment groups at 24 hours.  
     
CONCLUSIONS

This study is scientifically sound and classified as supplemental and may be used for risk characterization. Mortality was 5% in the dilution water negative control, and <=10% in the solvent and treatment groups. No sublethal effects were observed in the controls or in any treatment groups. The 96-hour LC50 value was >396 ug a.i./L (>0.396 mg a.i./L), and the observed NOAEC was 24.9 ug a.i./L (0.0249mg a.i./L).

     96-hour LC50:  >396 ug a.i./L (>0.396 mg a.i./L)95% C.I.:  N/A 
     Probit Slope: N/A 95% C.I.:  N/A 
     Observational NOAEC: 24.9 ug a.i./L (0.0249 mg a.i./L)
Endpoint(s) affected: Mortality 

REFERENCES:  
       
American Society for Testing and Materials. 2007. ASTM Standard E729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians.

U.S. Environmental Protection Agency.  1996.  Ecological Effects Test Guidelines, OPPTS 850.1035(draft), Mysid Acute Toxicity Test, EPA 712-C-96-136, 8pp.


APPENDIX I. Copy of Excel Worksheet with TWA Concentrations


