Document ID: EPA-HQ-OPP-2011-0374-0034
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2022-04-28T04:00Z

﻿Data Requirement:PMRA Data Code      {............}
                        EPA DP Barcode420869
                        OECD Data Point{............}
                        EPA MRID 49307518
                        EPA Guideline850.1075

Test material:  Tetrachloroterephthalic Acid (TPA) (Metabolite of DCPA)   Purity: 99.94%
Common name:
Chemical name:IUPAC:
            CAS name:
            CAS No. 2136-79-0
            Synonyms:
Primary Reviewer:Teresa NelisSignature:  
Environmental Scientist, CDM Smith/CSS-Dynamac JVDate:   10/11/2016
Secondary Reviewer: Moncie V. WrightSignature:  
Environmental Scientist, CDM Smith/CSS-Dynamac JV     Date:   10/28/2016

Primary Reviewer:Christina M. WendelSignature: 
EPA/OPP/EFED/ERB2/Biologist Date: 10/29/2021

Secondary Reviewer(s): Michael WagmanSignature: 
EPA/OPP/EFED/ERB2/Senior ScientistDate: 11/16/2021

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

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

Date Evaluation Completed: 16-11-2021

CITATION:  Wood, J. 2014. Tetrachloroterephthalic Acid (TPA): Acute Toxicity on Oncorhynchus Mykiss (Revised per PRN 11-03 and 86-5). Study performed by Covance Laboratories Ltd, North Yorkshire, England. Laboratory Study Number: 1708/020.  Study sponsored by AMVAC Chemical UK Ltd, Surrey, England. Study completed November 24, 2003.  

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

EXECUTIVE SUMMARY:

In a 96-hour acute toxicity study, rainbow trout (Oncorhynchus mykiss) were exposed to Tetrachloroterephthalic Acid (TPA) (a metabolite of DCPA) at nominal concentrations of 0 (negative control), 6.25, 12.5, 25, 50, and 100 mg a.i./L under static conditions. The mean-measured concentrations were <0.01 (<LOD, control), 6.72, 12.7, 23.8, 46.4, and 93.4 mg a.i./L.

Mortality and sublethal effects were observed daily, although effects were not dose responsive. After 96 hours, mortalities of 7, 7, and 21% was observed in the 6.72, 23.8, and 46.4 mg a.i./L mean-measured treatment groups, and no mortalities were observed in the control or the 12.7 and 93.4 mg a.i./L treatment groups. Sublethal effects, including swimming abnormally or lying on bottom of tank, were observed in one fish at 24 hours in the 23.8 mg a.i./L mean-measured treatment group and not in the control or at any of the other treatment levels. 

The 96-hour LC50 was >93.4 mg a.i./L, and the observed NOAEC was 23.8 mg a.i./L based on the mean-measured concentrations.  

Based on the results of this study, TPA (metabolite of DCPA) would be classified as practically non-toxic up to the concentration analytically verified to Oncorhynchus mykiss in accordance with the classification system of the U.S. EPA.  

This study is scientifically sound and is classified as acceptable.
   

   Results Synopsis

Test Organism Size/Age (mean weight or length): Juveniles (mean wet weight: 0.92 g; mean fork length: 4.6 cm)
   
   Test Type (Flow-through, Static, Static Renewal): Static
   
   96-hour LC50: >93.4 mg a.i./L95% C.I.:  N/A
   Probit Slope: N/A 
   Observational NOAEC: 23.8 mg a.i./L

   Endpoint(s) Affected: Mortality and sublethal effects
I. MATERIALS AND METHODS

   GUIDELINE FOLLOWED:This study was conducted according to a protocol that meets the testing requirements specified in the U.S. EPA's Ecological Effects Test Guideline OCSPP (form. OPPTS) 850.1075: Fish Acute Toxicity Test, Freshwater and Marine (1996), and OECD Guideline for the testing of Chemicals No 203, Fish Acute Toxicity Test (1992). The reviewer assessed the study methods and results according U.S. EPA OCSPP 850.1075 and OECD 203 and noted any differences and/or similarities between guidelines if they existed. The following deficiencies were noted by the reviewer:
   
Seven fish were tested per replicate. In the draft and final OPPTS and OCSPP 850.1075 guideline seven fish per replicate (at a minimum) is required but prefers 10 fish per replicate (1 replicate minimum and 2 are preferred) to obtain a more statistically accurate representation of the dose-response curve, to allow for mortality which may occur, yet be unrelated to chemical effect, and to avoid necessary repetitions of the test due to excessive control mortality. OECD guidance only requires seven fish per replicate. Given that no mortality occurred in the controls, this is considered to be a minor deficiency. 
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. 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 0.03-0.05 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. 
Zinc and copper concentrations in dilution water during the acclimation period were 0.073 and 0.065 mg/L, respectively, which is higher than the 0.001 mg/L concentration recommended by OPPTS guidance. OECD guidance and the final OCSPP 850.1075 do not address specific requirements for these specific water quality parameters. However, OCSPP 850.100 does provide specifications for dilution water quality. This is considered a minor deficiency. 
Dilution water TOC content was not provided. Both draft and final OPPTS and OCSPP 850.1010 guidelines recommends specific maximum concentrations. However, this is considered a minor deficiency a minor deficiency as the solubility (175.4 mg/L) of TPA in water would not result in an underestimation of toxicity; and details of the holding conditions and quality of the dilution water were presented in Appendix 1 of the study report (pg. 25-26).
Light intensity measurements were not reported, nor was the use of a transition period. It was only reported that a 16L:8D hour photoperiod was utilized. All  guidelines (draft OPPTS and final 850.1075 and OECD) suggest that light intensity be reported. The draft OPPTS and final OCSPP 850.1705 guidelines recommends 30-minute transition periods between light and dark conditions, while OECD guidance does not address the use of transition period. This is considered a minor deficiency.
            
These deficiencies do not have an impact on the scientific integrity of this experiment.
   
   COMPLIANCE:Signed and dated GLP, Quality Assurance and Data Confidentiality statements were provided.  This study was conducted in compliance with U.S. EPA Good Laboratory Practice regulations (40 CFR, Part 160), UK Statutory Instrument 1999 No. 3106, the OECD Principles on GLP ENV/MC/CHEM (98)17, and GLP Standards for Agricultural Chemicals 14-Seisan-7739, with no exceptions indicated.

   A. MATERIALS:
   1. Test material:  Tetrachloroterephthalic Acid (TPA) (metabolite of DCPA) 

      Description: White powder

      Lot No./Batch No.:021101

      Purity: 99.94%

      Stability of compound 
      under test conditions:Analytical verification of the test material at time 0 yielded recoveries ranging from 91 to 108% of the nominal concentrations. At test termination (96 hours), recoveries ranged from 92 to 107% of the nominal concentrations, indicating the test material was stable under the experimental conditions.  
(OECD recommends water solubility, stability in water and light, pKa, 
Pow, and vapor pressure of test compound)

      Storage conditions of 
      test chemicals: The test material was stored at room temperature. 

Physicochemical properties of TPA.
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). OECD recommends choice of species at discretion of testing laboratory.
      Age at test initiation:Juveniles
      Weight at study initiation: Mean wet weight: 0.9241 (range 0.6065  -  1.1953) g (N = 10)   
      EPA recommends: mean 0.5 - 5 g
      Length at study initiation: Mean fork length: 4.6 (range 4.1  -  5.0) cm (N = 10) 
      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, North Yorkshire, UK; Batch No. used in definitive test was P03/02.
      EPA recommends that all organisms be from the same source

   B.  STUDY DESIGN:

      1. Experimental Conditions

         a. Range-finding study:  A 96-hour flow-through range-finding study was conducted with a negative control and concentrations of 0.1, 1.0, 10 and 100 mg a.i./L. Five fish (one replicate, five fish per replicate), were exposed in each control and treatment group. There was 60% mortality in the 100 mg ai/L treatment group, and no mortality in the control or any of the other treatment groups tested. Sublethal effects (i.e., mild severe toxic effects) were observed after 48 and 72 hours in the fish in the 100 mg a.i./L treatment.  The range of concentrations selected for use in the definitive test were selected based on the results of the range-finding test. Results of the range finding test are found in Appendix 3 of the study report, pg. 33-37.
         
         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

Fish were held in holding tanks at a density appropriate to their size in a temperature controlled room under a 16L:8D photoperiod; filtered mains dechlorinated water supplied at 14°C was replaced continually. The pH ranged from 6.9 to 7.6, total hardness as CaCO3 ranged from 57 to 65 mg/L, water temperature ranged from 14.3 to 14.6ºC, and dissolved oxygen (% air saturation) ranged from 89 to 95%. 

Fish were fed a proprietary food (Nutrafry AB 02, TROUW UK) in quantities based on the size of the fish. The food was not considered to contain contaminants likely to affect the study. Fish were not fed in the 48 hours preceding the test.

Fish in the batch were used for testing because mortality was less than 5% and no disease was observed in the 7-day period prior to testing. 
After a 48-hour settling period the fish were acclimated to test conditions for a minimum of 12 days before tests began. Acclimation period was from April 9-22, 2003 (water quality during holding provided in Appendix 1, pg 26;latest value April 16, 2003). 

Uneaten food and debris were cleaned from tanks as required.

Fish mortalities were recorded daily. The stock of fish would not be used for testing if mortality exceeded 5% or if disease was apparent. 

Test fish from one holding tank were allocated to the test vessels at the start of the test. 

The recommended acclimation period is a minimum of 14 days; OECD guideline recommends a minimum of 12 days.  Pretest mortality should be < 3% 48 h. prior to testing.  OECD pretest mortality criteria: >10% = rejection of entire batch; > 5 and < 10% = continued acclimation for 7 days; <5% = acceptable.
Duration of the test
96 hours

The recommended test duration is 96 hours.
Test condition

Static/flow-through

Type of dilution system - for flow- through method.

Renewal rate for static renewal

Static

N/A

N/A

A reproducible supply of toxicant is recommended.   Consistent flow rate is usually 5-10 vol/24 hours; meter systems should be calibrated before and after study and checked twice daily during test period. 
Aeration, if any
Oil-free aeration during test.  
Compressed air

Aeration is not recommended; 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 aquaria; covered

30 L

20 L

Test vessel size is usually 19 L (5 gal) or  30 x 60 x 30 cm.
Fill volume is usually 15-30 L of solution.
Source of dilution water
Quality:
Dechlorinated municipal mains tap water filtered by a Purite AC9 activated carbon filter and a Purite ROPF 20 particulate filter, then chilled to approximately 14°C prior to use.

Recommended source of dilution water is soft, reconstituted water or water from a natural source.  EPA does not recommend the use of dechlorinated tap water; however, its use may be supportable if the biological responses for the organisms and  chemical analyses of residual chlorine meet conditions in the Agency=s 850.1010 guidelines for dilution water (http://www.epa.gov/opptsfrs/OPPTS_Harmonized/850_Ecological_Effects_Test_Guidelines/Draft/850.1010.pdf)  Dilution water should be intensely aerated before the study.  OECD permits dechlorinated tap water.
Water parameters:
Total Hardness

pH

Dissolved oxygen

Total Organic carbon

Particulate Matter

Metals

Pesticides

Chlorine (residual chlorine)

Temperature

{Salinity for marine or estuarine species}

Intervals of water quality measurement

65 mg/L as CaCO3

6.9  -  7.4

88  -  104% (% air saturation)

Not reported

Turbidity: <0.15 FTU

Aluminium, iron, lead, and manganese were not detected (<13 ug/L, <7 ug/L, <0.1 ug/L, and <1.5 ug/L, respectively).
Chloride: 17.0 mg/L
Calcium: 20.9 mg/L
Copper: 65 ug/L
Magnesium: 3.76 mg/L
Potassium: 1.58 mg/L
Sodium: 10.1 mg/L
Zinc: 73 ug/L

None detected

0.00  -  0.03 mg/L 
(during acclimation 0.03-0.05 mg/L)

13.8 - 14.7 ºC (Continuous monitoring to determine minimum and maximum temperature every 24 hours in control vessel 1: 14.3 - 14.9ºC)

N/A

Dissolved oxygen, temperature, and pH were measured once daily in both replicate vessels of each treatment level aquaria and the control throughout the exposure period. The temperature was monitored continuously in vessel 1 of the control. Other measurements were taken in dilution (holding) water during acclimation period with latest value taken 16 April 2003.
Conductivity (20ºC): 187 uS/cm
Alkalinity Total: 33.4 mg/L as CaCO3
Nitrate: 6.962 mg/L NO3

Hardness:
EPA recommends  40 - 48 mg/L as CaCO3 (OECD recommends 10 - 250 mg/L)
pH:
EPA recommends  7.2 - 7.6; 8.0-8.3 for marine-stenohaline fishes, 7.7-8.0 for estuarine-euryhaline fishes, monthly range < 0.8); (OECD recommends  pH 6.0 - 8.5)
Dissolved Oxygen:
EPA recommends:  Static: $ 60% during  first 48 hrs and $ 40% during second 48 hrs; flow-through: $ 60%; (OECD guideline recommends at least 80% saturation value).
Temperature:  
EPA recommends 12 EC  for coldwater species, 17 or 22 EC  for warmwater species, and  22 + 1 EC  for  estuarine/marine organisms. (OECD recommends 21 - 25oC for bluegill and 13 - 17oC for rainbow trout).
Salinity:  
EPA recommends 30-34%. (parts per thousand) for marine, 10-17%. for estuarine fish, weekly range < 6%..  

Water quality should be measured at beginning of test and every 48 hours.
Number of replicates/groups:
control:
solvent control:
treated ones:

2
N/A
2 per level

Recommended number of replicates include a control and five treatment levels. Each concentration should be 60% of the next highest concentration; concentrations should be in a geometric series.
Number of organisms per replicate / groups:
control:
solvent control:
treated ones:

7
N/A
7 per level
14 fish per control and each treatment level

Number of organisms per replicate should be   $ 10/concentration; OECD guideline recommends at least 7 fish/concentration.
Biomass loading rate
0.32 g of fish biomass per liter of solution per day

Recommended static conditions are # 0.8 g/L at # 17EC and # 0.5 g/L at > 17EC.  Recommended flow-through conditions are # 1 g/L/day.  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:

mean-measured:

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

<0.01 (<LOD, control), 6.72, 12.7, 23.8, 46.4, and 93.4 mg a.i./L
Both stock solutions as well as the test concentrations were clear and colorless throughout 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.
Lighting
Artificial light and a photoperiod of 16L:8D; transition period and light intensity measurements not reported.

The recommended photo period is 16 hours of light and 8 hours of dark with a 15-30 minute transition period.  OECD recommends a photo period of 12 -16 hours.
Feeding
Fish were not fed during the 48-hour period prior to exposure initiation or during the exposure period. 

Fish should not feed during the study.
Recovery of chemical 
Frequency of determination

Level of quantization (LOQ)
Level of detection (LOD)

Test solutions were sampled in every treatment vessel at 0, 48 and 96 hours. Test solutions were analyzed for TPA using HPLC with UV detection (220 nm).

Not reported
0.01 mg a.i./L
Duplicate samples (20 mL volumes) of each test solution were taken; one sample was analyzed by the Residue Chemistry department at Covance Laboratories Ltd and the other was stored as a reserve. 
Both the stock and test solutions appeared as clear and colorless throughout the test. The mean-measured concentrations of TPA ranged from 92 to 107% of nominal concentrations indicating the test material was stable under the experimental conditions (refer to copy of Excel worksheet in Appendix I). The method of validation was completed on 20 March 2003 (Appendix 2, pg. 27-32 of study report).
Positive control {if used, indicate the chemical and 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 sublethal effects (toxic symptoms)

Observation intervals 
1, 3, 24, 48, 72, and 96 hours

Observation intervals should be a minimum of every 24 hours.
Were raw data included?
Yes

Other observations, if any
No

II. RESULTS AND DISCUSSION:
   
   A. MORTALITY:

   Mortality was 7 and 14% at 24 hours, and 7 and 21% at 48 hours, in the nominal 25 and 50 mg a.i./L treatment groups, respectively. At 96 hours, mortality of 7, 7, and 21% was observed in the 6.5, 25 and 50 mg a.i./L treatment groups, and no mortalities were observed in the control or the 12.5 and 100 mg a.i./L treatment groups.  The resulting LC50 value was reported to be >100 mg a.i./L, based on the nominal test concentrations. 

Table 3:  Effect of TPA on Mortality of Rainbow Trout (Oncorhynchus mykiss).
Treatment
Nominal               [Mean-Measured]
(mg a.i./L)
No. of Fish at Start of Study
Observation Period

Day 1
Day 2
Day 3
Day 4

No. Dead
% Mortality
No. Dead
% Mortality
No. Dead
% Mortality
No. Dead
% Mortality
<LOD (Control; dilution water only)
14
0
0
0
0
0
0
0
0
6.25 [6.72]
14
0
0
0
0
0
0
1
7
12.5 [12.7]
14
0
0
0
0
0
0
0
0
25 [23.8]
14
0
0
1
7
1
7
1
7
50 [46.4]
14
0
0
2
14
3
21
3
21
100 [93.4]
14
0
0
0
0
0
0
0
0
LC50
>100 mg a.i./L
Positive control, if used. mortality: LC50:
N/A
  Data were obtained from Table 3 on page 23 of the study report.
  LOD = 0.01 mg a.i./L
   
   B. NON-LETHAL TOXICITY ENDPOINTS:

   Sublethal effects, including swimming abnormally or lying on bottom of tank, were observed in one fish at 24 hours in the nominal 25 mg a.i./L treatment group and not in the control or at any of the other treatment levels. 

Table 4:  Sub-lethal Effect of TPA on Rainbow Trout (Oncorhynchus mykiss). 
Treatment
Nominal 
[Mean-Measured]
(mg a.i./L)
Observation Period

24 hours
48 hours
72 hours
96 hours

% Affected
% Affected
% Affected
% Affected
<LOD (Control; dilution water only)
0
0
0
0
6.25 [6.72]
0
0
0
0
12.5 [12.7]
0
0
0
0
25 [23.8]
0 a
7
0
0
50 [46.4]
0
0
0
0
100 [93.4]
0
0
0
0
EC50
Not determined
Positive control, if used. % sublethal effect: EC50:
N/A
  a One fish was swimming abnormally or lying on bottom of tank. 
  Data were obtained from Table 3 on page 23 of the study report.
  LOD = 0.01 mg a.i./L
C. REPORTED STATISTICS: 

   The 24, 48, 72 and 96 hour LC50 values were calculated using probit analysis after log10 transformation of the nominal exposure concentrations.

   D. VERIFICATION OF STATISTICAL RESULTS:

   Statistical Method: The mortality data and mean-measured concentrations of the test material 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 mortalities of <50% in this study.

   96-hour LC50: >93.4 mg a.i./L95% C.I.:  N/A
   Probit Slope: N/A

   E.  STUDY DEFICIENCIES: 

   There were no major study deficiencies. However, 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. 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 0.03-0.05 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. Given the adequate control performance, this is considered a minor study deficiency. 
   
   
   F.  REVIEWER'S COMMENTS:  

   The reviewer's and the study author's results were in general agreement, with the differences attributable to the study author using nominal concentrations. The reviewer's results were based on the mean-measured concentrations (refer to copy of Excel worksheet in Appendix I). No test substance concentration resulted in >=50% effect, the maximum was 21% mortality, and effects were not dose responsive. The reviewer's results are reported in the Executive Summary and Conclusions sections of this DER.

   The study was conducted following the draft OPPTS 850.1075 (1996) guideline, OECD 203 guideline, and the reviewer considered both the draft and the final OCSPP 850.1075 (2016) guideline and OECD 203 guideline (1992) in their evaluation of the data.

   The range-finding test was initiated on 24 March 2003; and the definitive test was completed on 26 April 2003. The analytical phase of the study was completed on 28 April 2003. 
   
   G. CONCLUSIONS:

   This study is scientifically sound and is classified as acceptable.  After 96 hours, mortality was 7, 7 and 21% in the 6.72, 23.8 and 46.4 mg a.i./L treatment groups, and effects were not dose-responsive. Sublethal effects was observed in one fish at 24 hours in the 23.8 mg a.i./L treatment group, but not in any other treatment groups at any time. The 96-hour LC50 value was >93.4 mg a.i./L, and the observed NOAEC was 23.8 mg a.i./L, based on mean-measured concentrations.

III.  REFERENCES:

None.

APPENDIX I. Copy of Excel Worksheet with Measured Concentrations