﻿DATA EVALUATION RECORD
850.1025 -- ACUTE EC50 TEST WITH AN ESTUARINE/MARINE MOLLUSK
SHELL DEPOSITION STUDY

1.  CHEMICAL:   DCPAPC Code No.:  078701

2.  TEST MATERIAL:   Dacthal TechnicalPurity:  99.3%

3.  CITATION
Authors:Shaw, A.C.
Title:Dacthal: Acute Toxicity to the Eastern Oyster (Crassostrea virginica) Under Flow-Through Conditions, Following OCSPP Guideline 850.1025. 
Study Completion Date:December 19, 2013
Laboratory:Smithers Viscient, Wareham, Massachusetts, USA
Sponsor:AMVAC Chemical Corporation, Newport Beach, California, USA
Laboratory Report ID:11857.6108
MRID No.:49500701
DP Barcode:424913

4.  REVIEWED BY:  Teresa Nelis, Staff Scientist, CDM/CSS-Dynamac JV
     Signature:  Date:  10/12/2016

     APPROVED BY:  Moncie V. Wright, Environmental Scientist, CDM Smith/CSS-Dynamac JV
Signature: Date: 10/28/2016

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

     Signature:Date: 11/30/2021

APPROVED BY:  Michael Wagman, Senior Scientist, OPP/EFED/ERB2

     Signature:Date: 11/29/2021

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

Scientific Name of Test Organism:Crassostrea virginica
Age or Size of Test Organism:38 mm (SD +- 3.0 mm) (N = 30) at test initiation
     Definitive Test Duration:96 hours
Study Method:Flow-through
     Type of Concentrations:Mean-measured

7.  CONCLUSIONS:

     Results Synopsis
     96-hour IC50:  >0.41 mg a.i./L (>410 ug a.i./L)95% C.I.: N/A
     Probit Slope: N/A
     
     96-hour LC50:  >0.41 mg a.i./L (>410 ug a.i./L)95% C.I.: N/A
     Probit Slope: N/A 
Endpoint(s) affected: shell growth

8.  ADEQUACY OF THE STUDY

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

     B.  Rationale: Issues with dissolved oxygen (DO) occurred in the presence of gentle oil-free aeration in flow-through tanks. The DO was <=60% saturation in the solvent control, 0.23 mg/L and 0.062 mg a.i./L treatment groups at 48 hours, and in the 0.13 and 0.41 mg a.i./L treatment groups at 72 hours. Although not a validity criterion in the final OCSPP guideline, this is still an uncertainty and considered a study deficiency. Additionally, although only visual comparisons could be made due to the lack of replication, and statistical analysis could not be completed, the increase in shell growth (~9%) observed in the solvent control as compared to the negative control although growth results are highly variable, could potentially be masking effects of the test substance. Based on the available standard deviations for each level, the confidence intervals of the individual measurements would overlap for all test concentrations. However, no test concentration resulted in >=50% reduction in shell growth (maximum % effect was 27%), and overall, the results of this study support the conclusion that DCPA was not acutely toxic at the solubility limit of DCPA (0.5 mg/L) 

     C.  Repairability:  None

9.  GUIDELINE DEVIATIONS:  The procedures used in this test followed a protocol that meets the testing requirements of the U.S. Environmental Protection Agency's Ecological Effects Test Guideline OCSPP (form. OPPTS) 850.1025: Oyster Acute Toxicity Test (Shell Deposition) and OCSPP 850.1000: Special Considerations for Conducting Aquatic Laboratory Studies. The reviewer assessed the study methods and results according to OCSPP 850.1025 and 850.1000.  The following deficiencies were noted: 

Dissolved oxygen (DO) was 3.9, 4.1 and 4.3 mg/L at 48 hours in the solvent control, 0.23 mg/L and 0.062 mg/L treatment groups, respectively, which correspond to DO saturation of <60% (DO saturation of 49, 52 and 54%, respectively). At 72 hours the DO saturation was 4.7 and 4.6 mg/L in the 0.13 and 0.41 mg/L treatment groups, respectively (corresponding to a DO saturation of 59, 58%, respectively). The draft OPPTS 850.1025 guideline lists DO as a validation criterion and specifies that the DO concentration should be at least 60% of air saturation throughout the test. In the final OCSPP 850.1025 guideline, DO >=60% of saturation is a recommended test parameter, and not a validity criterion. Additionally, issues with DO occurred despite the study using gentle oil-free aeration in flow-through tanks in an attempt to increase oxygenation. Therefore, this is an uncertainty and considered a study deficiency.
There was no holding period before acclimation was initiated. The draft and final OPPTS and OCSPP 850.1025 guideline suggests that oysters be held for a minimum of 10 to 15 days before testing. The oysters used in the study arrived 6 days prior to exposure initiation, and the holding and acclimation conditions were monitored for the 6 days prior to exposure initiation. No mortalities were observed during the acclimation period. This is considered a minor study deficiency.
The number of replicate test chambers (aquaria) that were used meets the draft OPPTs 850.1025 guideline, which does not explicitly recommend replication, and just uses 20 individuals per control/treatment level (each treatment level in a single aquarium). However, according to the final OCSPP 850.1025 guideline test conditions, the number of replicate test chambers per treatment level for a `multiple-concentration definitive test' is recommended to be a minimum of 2, with a minimum of 20 individuals between the 2 chambers. As the test was conducted according to the draft OCSPP 850.1025 guideline, this is considered a minor deficiency. However, the lack of replication prevents most statistical analyses, including identifying whether there are statistically significant differences in shell deposition between the solvent and negative controls.
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 not result in an underestimation of toxicity. 

     These deficiencies do have an impact on the acceptability of this study.
     
10.  SUBMISSION PURPOSE: This study was submitted to provide data on the effects of Dacthal (DCPA) on oyster (Crassostrea virginica) shell deposition following acute exposure for the purpose of registration review.

11.  MATERIALS AND METHODS

     A.  Test Organisms
Guideline Criteria
Reported Information
Species
Preferred species are the Pacific oyster (Crassostrea gigas) and the Eastern oyster (Crassostrea virginica)
Eastern Oyster (Crassostrea virginica)
Mean valve height
25 - 50 mm along the long axis
Mean: 38 mm (SD +- 3.0 mm) (N = 30)
Supplier
Northside Shellfish, Barnstable, Massachusetts, USA
Are all oysters from same source?
Yes
Are all oysters from the same year class?
Yes

     B.  Source/Acclimation
Guideline Criteria
Reported Information
Acclimation Period
Minimum 10 days
6 days
The seawater oysters were held in had a temperature range of 18-20ºC; a pH range of 7.8-8.1; a DO concentration range of 83 to 96% saturation; and a salinity range of 20 to 23 ppt.
Wild caught organisms were quarantined for 7 days?
N/A
Were there signs of disease or injury?
No mortality was observed among the oyster population during the six days before exposure initiation. Oysters that appeared less than optimal were discarded prior to testing.
If treated for disease, was there no sign of the disease remaining during the 48 hours prior to testing?
No treatments for disease were described.
Amount of peripheral shell growth removed prior to testing
ca. 3-5 mm
Feeding during the acclimation
Must be fed to avoid stress.
Oysters were fed a supplementary diet of algae (Shellfish Diet 1800), an algal mixture prepared in seawater from a concentrate at least once a day. 
Pretest Mortality
<3% mortality 48 hours prior to testing
0%, no mortality was observed prior to testing.

     C.  Test System
Guideline Criteria
Reported Information
Source of dilution water Natural unfiltered seawater from an uncontaminated source.
Natural, filtered seawater was pumped from the Cape Cod Canal, Bourne, Massachusetts (1 to 4 meters offshore at a depth of 0.5 meters). In the laboratory, the seawater was recirculated within an epoxy-coated concrete reservoir prior to use. Routine analyses of the water by GeoLabs, Braintree, Massachusetts, did not detect pesticides, PCBs, or toxic metals at concentrations that are considered toxic in agreement with ASTM (2002) standard practice. The results of these analyses were not provided in the study report for reference.
Does water support test animals without observable signs of stress?
Yes
Salinity
Artificial or natural seawater diluted with freshwater: 20%. (parts per thousand) (range +-2%. during test); Natural seawater not diluted with freshwater: >12%. (range <5%. during test)
20 - 21 ppt
Water Temperature
15°-30°C, consistent in all test vessels
20 - 22ºC
pH
7.4  -  7.9
Dissolved Oxygen
>=60% throughout
Range:  3.9  -  8.4 mg/L 
3.9, 4.1 and 4.3 mg/L at 48 hours in the solvent control, 0.25 mg/L and 0.063 mg/L nominal test solutions, respectively, were <60% saturation. 4.7 and 4.6 mg/L at 72 hours in the 0.13 and 0.50 mg/L nominal test solutions, respectively, were <60% saturation.
[4.7  -  4.8 mg/L at salinities of 20-21 ppt and temperatures of 20-22ºC represent 60% of dissolved oxygen saturation]
Aeration 
Gentle, oil-free aeration was initiated at test initiation in all test vessels to maintain dissolved oxygen levels at >=60% of saturation.
Total Organic Carbon (TOC)
Not reported
Test Aquaria
Should be constructed of glass or stainless steel.
Glass aquaria measuring 49.5 x 25.5 x 29 cm (L x W x H) that were equipped with an overflow side drain positioned at a height of 14 cm which maintained a test solution volume of ca. 18 L.
Type of Dilution System
Must provide reproducible supply of toxicant
Constant-flow serial diluter with a dilution factor of 50% between concentrations, a temperature-controlled water bath with seven exposure aquaria. A Harvard Syringe pump was calibrated to deliver 0.015 mL/minute of the 5.0 mg/mL diluter stock solution into a mixing chamber that also received 150 mL/minute of dilution water. Mixing chamber was positioned over a magnetic stir plate and partially submerged in an ultrasonic water bath. 

A similar system was configured to deliver 0.015 mL/minute of 500 uL/mL solvent control stock solution into the diluters solvent control mixing chamber which received 0.074 L/minute of dilution water, to achieve 0.10 mL/L solvent. 

The dilution water control contained dilution water only and was maintained under the same conditions as the treatment level containers but contained no treatment substance or solvent. 
Flow rate
Consistent flow rate
75 mL/minute provided ca. 6 solution volume replacements/24 hours. The contents of each aquarium were continuously circulated using an impeller pump and a Teflon tube (1.75 L/minute flow rate in each test aquarium or 5.25 L/oyster/hour). The circulating system aided in evenly distributing the algae fed to the oysters and in mixing the flow of the test solution throughout each aquarium (as applicable). 
Was the loading of organism such that each individual sits on the bottom with water flowing freely around it?
Yes
Photoperiod
16 hours light, 8 hours dark
16 hours light, 8 hours dark with sudden transitions between light and dark avoided. Lighting provided by Sylvania Octron(R) fluorescent bulbs with an intensity of 200 to 2200 lux. 
Test conditions: light intensity ranged from 30 to 82 footcandles (320 to 880 lux).
Solvents
Not to exceed 0.5 ml/L
Solvent:  DMF at 0.10 mL/L

     D.  Test Design
Guideline Criteria
Reported Information
Range Finding Test
If EC50 >100 mg/L with 30 or more oysters, then no definitive test is required.
A 96-hour static-renewal acute exposure was conducted with a dilution water control, a solvent (DMF) control and nominal concentrations of 0.000050, 0.00050, 0.0050, 0.050, and 0.50 mg a.i./L. All test solutions were observed to be clear and colorless with no visible undissolved test substance present. One replicate aquarium containing ten oysters was established for the controls and each test group. Following 96-hours of exposure, shell growth reductions were 12, 27, 16, 19 and 50% in the 0.000050, 0.00050, 0.0050, 0.050, and 0.50 mg a.i./L treatment groups, respectively, compared to the dilution water control. Mortality of 10% occurred in the dilution water control. No mortality or adverse effects were observed among the oysters in the solvent control or any of the treatment levels tested. Based on these results, and in consultation with the Study Sponsor, the nominal concentrations were selected for the definitive test. 
Nominal Concentrations of Definitive Test
Control & 5 treatment levels;
each conc. should be 60% of the next highest conc.; concentrations should be in a geometric series
0 (negative dilution water control and solvent control), 0.031, 0.063, 0.13, 0.25, and 0.50 mg a.i./L
Number of Test Organisms
Minimum 20 individuals per test level and in each control

20 per treatment level and controls; no replication was used (all organisms per a treatment level/control were in single aquaria)
Test organisms randomly or impartially assigned to test vessels?
Yes
Biological observations made every 24 hours?
Yes, noting visible abnormalities (excessive mucus production or failure to siphon and feed as evidenced by a lack of fecal and pseudofecal production) and other sublethal effects. 
Water Parameter Measurements
1. Temperature
     Measured hourly in at least one chamber
2. DO and pH
     Measured at beginning of test and every 48 h in the high, medium, and low doses and in the control

Temperature was measured daily in each aquarium, and also monitored continuously in the nominal 0.13 mg a.i./L aquarium. 
Dissolved oxygen and pH were measured daily in each aquarium.
Was chemical analysis performed to determine the concentration of the test material at the beginning and end of the test?  (Optional)
Yes; samples were collected from the controls and treated aquaria at test initiation and termination. Samples were collected from the approximate midpoint of the test vessel using a pipet. The samples were analyzed using HPLC with UV detection. 
The method validation was conducted prior to test initiation (24 June 2013) and established an average recovery of 97.9 +- 4.86% for dacthal from 20 ppt filtered seawater (Appendix 2; pg. 54-66 in the study report).

12.  REPORTED RESULTS

     A.  General Results
Guideline Criteria
Reported Information
Quality assurance and GLP
compliance statements were
included in the report?
Yes; this study was conducted in compliance with all pertinent U.S. EPA (40 CFR, Part 160) Good Laboratory Practice Regulations with the following exception: routine food and water contaminant screening analyses were conducted using standard U.S. EPA procedures.
Control Mortality
Not more than 10% of control organisms may die or show abnormal behavior.
0% (dilution water and solvent controls)
Control Shell Deposition
Must be at least 2 mm.
3.3 mm dilution water control; 3.6 mm solvent control
Recovery of Chemical
Analytical recoveries at time 0 ranged from 92-106%, and from 70-116% at 96 hours. The coefficient of variation ranged from 6-22% (refer to copy of Excel worksheet in Appendix I). The highest nominal concentration is at the solubility limit for DCPA (0.5 mg/L), but both the stock solutions and test solutions were clear and colorless with no visible undissolved test substance throughout the test. 
Limit of quantitation (LOQ) = 0.0024 mg/L
Raw data included?
Yes
Signs of toxicity (if any) were described?
Yes

 Table 1. Effect of DCPA on Shell Growtha
Concentration (mg/L)
Number Per Level
Number Dead
Mean Shell Deposition +-  SDb
(mm)
Mean % Reduction Compared to Pooled Control (Comparison to Negative Control)
[Comparison to solvent control]
Nominal
Mean-Measured




Dilution Water
Negative Control
<LOQc
20
0
3.3 +- 1.6
-- [8]
Solvent Control
<LOQc 
20
0
3.6 +- 1.1
--
0.031
0.035
20
0
3.5 +- 1.5
-3 (-6) [3]
0.063
0.062
20
0
3.7 +- 1.2
-7 (-12) [-3]
0.13
0.13
20
0
3.3 +- 1.3
4 (0) [8]
0.25
0.23
20
0
3.1 +- 0.9
11 (6) [14]
0.50
0.41
20
0
2.4 +- 1.1
31 (27) [33]
  a Data were obtained from Table 2 to Table 5 on pages 25 to 28 of the study report.
  b SD = Standard Deviation
  c LOQ = 0.0024 mg/L
     B.  Statistical Results

     Method: The IC50 value was visually determined due to <50% reductions in shell deposition compared to the pooled control. Geometric mean-measured concentrations were used for the analysis.

     96-hour IC50:  >0.40 mg a.i./L 95% C.I.: N/A
     Probit Slope: N/A

13.  VERIFICATION OF STATISTICAL RESULTS 

The reviewer entered shell deposition and mortality data and the corresponding mean-measured concentrations into CETIS statistical software version 1.8.7.12 with database backend settings implemented by EFED on 10/20/15.  The toxicity values were visually determined due to a lack of effects >=50%.

Parameter
Result
Statistical Method for LC/IC50
N/A - reductions were <=27% in this study compared to negative control (max inhibition of 33% compared to solvent control)
LC/IC50 (95% C.I.)
>0.41 mg a.i./L 
(based on mean-measured concentrations)
Probit Slope
N/A

14.  REVIEWER'S COMMENTS:  

The reviewer's and the study author's results were in agreement, when accounting for the slight differences in the reported toxicity value due to the use of geometric mean-measured versus mean-measured concentrations. Although only visually determined, as statistical analysis could not be completed due to the lack of replication, the increase in shell growth observed in the solvent control as compared to the negative control could potentially be masking effects of the test substance. However, the individual measurements of shell growth are highly variable in both the controls and treatment groups and not dose-responsive, except at the higher treatment levels; the mean and standard deviation (SD) for shell growth are presented Table 1 above. Using the standard deviations, the confidence intervals for the mean individual measurements for all test concentrations would overlap. For the negative control the individual measurements of shell growth range from 1.5  -  7.3 mm (mean 3.3 +- 1.6 mm (SD), the solvent control measurements range from 2.2  -  6.1 mm (mean 3.6 +- 1.1 mm (SD), the 0.035 mg a.i./L (lowest) treatment level range from 1.0 to 7.2 mm (mean 3.5 +- 1.5 mm (SD), 0.062 mg a.i./L  treatment level range from 1.8 to 6.7 mm (mean 3.7 +- 1.5 mm (SD), 0.13 mg a.i./L treatment level range from 1.7 to 6.0 mm (mean 3.3 +- 1.3 mm (SD), 0.23 mg a.i./L treatment level range from 1.3 to 5.5 mm (mean 3.1 +- 0.9 mm (SD), 0.41 mg a.i./L mg a.i./L (highest) treatment level range from 0.3 to 5.1 mm (mean 2.4 +- 1.1 mm (SD). No test substance concentration resulted in >=50% reduction in shell growth, but the possible solvent effect presents a potential uncertainty of the test, however the individual growth results are highly variable (as described above), even though effects on shell growth were observed in all treatment groups, effects were not dose-responsive, except at the higher treatment levels, and these could not be statistically analyzed due to the lack of replication. There were no mortalities in the controls or treatment groups and overall the results of this study support the conclusion that DCPA was not acutely toxic at the solubility limit of DCPA. The reviewer's results are reported in the Conclusions section of this DER.

Issues with DO occurred in the presence of gentle oil-free aeration in flow-through tanks. The dissolved oxygen (DO) was <=60% saturation (49, 52 and 54% saturation, respectively) in the solvent control, 0.23 mg/L and 0.062 mg a.i./L treatment groups at 48 hours, and (59, 58% saturation, respectively) in the 0.13 and 0.41 mg a.i./L treatment groups at 72 hours. Although not a validity criterion in the final OCSPP guideline, this is still an uncertainty and considered a study deficiency.

The coefficient of variation of the measured test concentrations ranged from 0 to 22% (refer to copy of Excel worksheet in Appendix I).

The study was conducted following the draft OPPTS 850.1025 (1996), and OCSPP 850.1000 (1996) guidelines, and the reviewer considered both the draft and the final OCSPP 850.1025 (2016) guideline and OCSPP 850.1000 in their evaluation of the data.

An initial definitive static-renewal acute test was initiated on August 19, 2013, exposing Eastern oysters to DCPA at nominal concentrations of 0.031, 0.063, 0.13, 0.25 and 0.50 mg/L; however, due to the low analytical results, this study was terminated. As a result, the study design was changed to a flow-through exposure in an effort to maintain the desired concentrations. 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. This in combination with flow-through exposure aided in the ability of keeping the test material in solution and deeming the analytical measurements to be reliable.

The experimental phase of the test was conducted from September 23 to September 27, 2013.

 CONCLUSIONS:

This study is scientifically sound and is classified as supplemental and may be used for risk characterization. Mortality was 0% in the dilution water negative control, the solvent control and treatment groups. Inhibitions in new shell growth ranged from -12 to 27% relative to the negative control and -3 to 33% relative to the solvent control and exhibited a dose-response at the higher treatment levels; however the individual growth results are highly variable. As there was overlap of the confidence intervals based on the standard deviations for all test concentrations. Although only visual comparisons could be made due to the lack of replication, and statistical analysis could not be completed, the apparent increase in shell growth observed in the solvent control (~9%) as compared to the negative control, although results are highly variable, could potentially be masking effects of the test substance. However, no test substance concentration resulted in >=50% reduction in shell growth (maximum % effect was 27%). Therefore, overall, the results of this study support the conclusion that DCPA was not acutely toxic at the solubility limit of DCPA (0.5 mg/L). The 96-hour LC50 value was > 0.410 mg a.i./L (>410 ug a.i./L).

     96-hour LC50:  > 0.410 mg a.i./L (>410 ug a.i./L)95% C.I.:  N/A 
     Probit Slope: N/A
Endpoint(s) affected: shell growth

16.  REFERENCES:

ASTM, 2002. Standard practice for conducting acute toxicity tests with fishes, macroinvertebrates and amphibians. Standard E729-96. American Society for Testing and Substances, 100 Barr Harbor Road, West Conshohocken, Pennsylvania 19428.

Benoit, D.A., V.R. Mattson, and D.C. Olson, 1982. A continuous flow mini-diluter system for toxicity testing. Water Research. 16:457-464.

Ives, M., 2013. CETIS, Comprehensive Environmental Toxicity Information System(TM). User's Guide. Tidepool Scientific Software, McKinleyville, California.

U.S. EPA, 1996a. Office of Chemical Safety and Pollution Prevention. Ecological Effects Test Guideline, OCSPP 850.1025. Oyster Acute Toxicity Test (Shell Deposition). "Public Draft". EPA712-C-96-115. April 1996. U.S. Environmental Protection Agency, Washington, D.C. 

U.S. EPA, 1996b. Office of Chemical Safety and Pollution Prevention. Ecological Effects Guideline, OCSPP 850.1000. Special Consideration for Conducting Aquatic Laboratory Studies. "Public Draft". EPA 712-C-96-113. April 1996. U.S. Environmental Protection Agency, Washington, D.C.


APPENDIX I. Copy of Excel Worksheet with Measured Concentrations


