Data Requirement:				PMRA DATA CODE 	{............}
                        EPA DP Barcode			460199
                        OECD Data Point		{............}
                        EPA MRID 				51499401
                        EPA Guideline			850.4500


Test material:	Tetrachloroterephthalic Acid (degradate of DCPA)	Purity:	99.94%
Common name	TPA
Chemical name:	IUPAC: Not reported
            CAS name: Not reported
            CAS No.: 2136-79-0
            Synonyms: None

Primary Reviewer:	Jocelyn Marsack							Signature:  
Environmental Scientist, CDM/CSS-Dynamac JV`				Date:   04/01/2021
Secondary Reviewer:   Julie Burns								Signature:  
Environmental Scientist, CDM/CSS-Dynamac JV				Date:   04/12/2021

Primary Reviewer:	Christina M. Wendel						Signature: 
EPA/OPP/EFED/ERB2/Biologist 								Date: 10/25/2021

Secondary Reviewer(s): Michael Wagman						Signature: 
EPA/OPP/EFED/ERB2/Senior Scientist						Date: 11/19/2021

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

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

Date Evaluation Completed: 19-11-2021


CITATION: Manson, P.S. 2003. Tetrachloroterephthalic Acid (TPA): Inhibition of Growth to the Alga Selenastrum capricornutum. Study conducted by Covance Laboratories Ltd, North Yorkshire, England. Study report no. 1708/022-D2149. Study sponsored by AMVAC Chemical UK Ltd, Surrey, England. Study initiated on March 12, 2003 and completed on 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. The CDM/CSS-Dynamac Joint Venture role does not include establishing Agency policies.

EXECUTIVE SUMMARY:

In a 96-hour acute toxicity study, cultures of the green algae Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) (Chlorophyceae, CCAP 278/4) were exposed to Tetrachloroterephthalic Acid (degradate of DCPA) at nominal concentrations of 0 (negative control), 4.27, 9.39, 20.7, 45.5, and 100 mg a.i./L under static conditions. The test substance was stable under the test conditions, so the mean-measured concentrations provided by the study author and verified by the reviewer were used for analysis and reporting. The mean-measured concentrations were <0.01 (<LOD, control), 3.99, 8.90, 19.7, 46.1, and 103 mg a.i./L. 

The % growth inhibition in the treated algal culture as compared to the control ranged from 4 to 44%. All endpoints in this study were significantly affected in the two highest test levels and based on the uncertainty surrounding the pH as discussed below, the most sensitive endpoint, based on the NOAEC was all three endpoints with the NOAEC being 19.7 mg a.i./L. The IC50 for yield, growth rate, and area under the curve (AUC) was >46.1 mg a.i./L, as the highest test concentration does not appear to be reliable for use based on the uncertainties surrounding pH.

At exposure initiation, the test solutions appeared clear and colorless. After 96 hours, test vessels containing algae appeared as green suspensions in the control and four lowest treatment levels. The highest test level solutions appeared slightly hazy and paler green in color than the control.

The pH at test initiation was 7.7 in the control and ranged from 7.7 to 7.8 in the test solutions. After 96 hours, the pH increased significantly in the control (ranging from 10.0 to 10.2) and four lowest treatment levels (ranging from 8.3 to 10.1). Conversely, the pH in the highest treatment level decreased substantially by test termination (ranging from 4.9 to 5.0). Measurements on pH were only taken at 0 and 96 hours, and it is unclear if the difference in the highest test concentration was treatment related or if something else is going on within the test system. Had pH measurements been taken more frequently (i.e., daily), there would be more certainty that the similar dose-responses (and any consequently estimated NOAEC and IC50 endpoints) observed in treatment groups at 24-hour and 72-hour, compared to the treatments at 96-hour, were not a result of inhibitions in control growth due to high pH. Therefore, the highest test concentration does not appear to be reliable, and so based on these uncertainties, this test concentration should not be used to estimate endpoints. 

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

Results Synopsis

   Test Organism: Green algae, Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum)
   Test Type (Flow-through, Static, Static Renewal): Static
   
   Yield
   IC05: 32.3 mg a.i./L				95% C.I.:  N/A to 52.7 mg a.i./L	
   IC50: >46 mg a.i./L[+]				95% C.I.:  NA	
   NOAEC: 19.7 mg a.i./L
   LOAEC: 46.1 mg a.i./L
   		
   Growth rate
   IC05: 75.1 mg a.i./L				95% C.I.:  55.8 to 92.5 mg a.i./L	
   IC50: >46.1 mg a.i./L				95% C.I.:  N/A
   NOAEC: 19.7 mg a.i./L
   LOAEC: 46.1 mg a.i./L
   
   Area under the curve (AUC)
   IC05: 0.65 mg ai/L*				95% C.I.:  N/A to 31.9 mg a.i./L	
   IC50: >46.1 mg a.i./L				95% C.I.:  N/A 
   NOAEC: 19.7 mg a.i./L	
   LOAEC: 46.1 mg a.i./L
   *Value is not bracketed by the measured concentrations and should be interpreted with caution. 
   [+] See reviewer comments regarding the IC50 endpoint in reference to the highest test concentration.
   
   Endpoint(s) Affected:  Yield, area under the curve, and growth rate
   Most Sensitive Endpoint:  Yield, area under the curve, and growth rate, based on the NOAEC
I. MATERIALS AND METHODS

   GUIDELINE FOLLOWED:		The study was conducted according to OECD 201, JMAFF 2-7-3 and OPPTS draft guideline 850.4500. The review assessed the study according to OCSPP 850.1000 (2016), OCSPP 850.4000 (2012), OCSPP 850.4500 (2012) and OECD 201 (2011) noting any similarities and/or differences where they existed. The following deviations and deficiencies from validity requirements and the listed guidelines were noted:
   
 The study author did not indicate if treatments were randomly assigned to test vessels and if test vessels were randomly assigned to positions in the growth chamber. Random treatment assignment to test vessels and test vessel location in the growth chamber are test validity requirements per OCSPP 850.4500 guidance. OECD recommends test chambers are randomly placed in the incubator. 
 The pH of the control medium increased by a minimum of 2.3 units during the test. OECD states the control medium should not increase >1.5 units during the test. OCSPP does not provide guidance for pH increases in the control. After 96 hours, the pH increased significantly in the control (ranging from 10.0 to 10.2) and four lowest treatment levels (ranging from 8.3 to 10.1). Conversely, the highest treatment level pH decreased substantially by test termination (ranging from 4.9 to 5.0). The pH measurements were only taken at 0 and 96 hours and it is unclear if the difference in the highest test concentration is chemical related or if something else is going on within the test system. The increase in the controls and lower treatment groups to extremely alkaline conditions is likely a result of the test system reaching the carrying capacity of the test vessels. Laboratory toxicity tests should be conducted in environmentally relevant conditions which may not be represented by the strongly basic water conditions present in the Day 4 samples. The alkaline conditions may have inhibited growth in the controls and lower treatment groups, resulting in less sensitive endpoints than would have occurred had the pH stayed in the environmentally relevant range. Had pH measurements been taken more frequently (i.e., daily), there would be more certainty that the similar dose-responses (and consequent NOAEC and IC50 endpoints) observed in treatment groups at 24-hours and 72-hours, compared to the treatments at 96-hours, were not a result of inhibitions in control growth due to high pH.  
 The ICx and NOAEC values for yield (cell density) were not determined by the study author. OECD requires that both average specific growth rate and yield endpoints be evaluated for the test results to be acceptable in all member countries. OCSPP requires the evaluation of yield, average specific growth rate, and area under the curve. As the reviewer was able to determine yield endpoints, this is considered a minor deviation. 
 Due to a sampling error in the mean-measured 19.7 mg a.i./L (nominal 20.7 mg a.i./L) test level at 24 hours, the data for a replicate was excluded by the study author which led to unbalanced replication across treatment levels. These data were also excluded in the MRID, so the reviewer was unable to include the replicate in the statistical analysis.
 Three replicates were used for each treatment level, except for two replicates in the 19.7 mg a.i./L concentration as described in the error listed above in #4 and six replicates were used for the negative control. OCSPP recommends a minimum of four replicates per concentration. However, OECD recommends three replicates per treatment level and preferably double the replicates for the control groups. Since the 19.7 mg a.i./L concentration was identified as the NOAEC, but only had two replicates, with inhibitions ranging from 1-5%, relative to the negative control, this introduces a degree of uncertainty into the endpoint. However, CVs for this concentration were reasonably low (2-12%), so this is not considered a major deficiency. 
 Observations of phytotoxic effects from the test material (e.g., swollen cells and aggregation) were not provided as required by OCSPP and OECD. Furthermore, OCSPP recommends daily observations on cell appearance and health. OECD recommends observations at test termination.
 The age of the inoculum at test initiation was not reported. 
 Culture conditions, acclimation, and pre-test health conditions were not reported. 
 The light intensity (7790 lux) during the test was within the range recommended by OECD (4440  -  8880 lux) but was nearly double the amount recommended by OCSPP (~4300 lux). 
 Dilution water quality characteristics were not reported. 
 Incomplete physiochemical properties of the test item were reported. This is considered to be a minor deficiency.
 The deionized water used to prepare the nutrient medium was not reagent grade quality water as recommended by OCSPP guidance. However, OECD guidance does not require that the deionized water is reagent grade. This is considered to be a minor deficiency.

      These deviations do affect the validity of the study.
   
   COMPLIANCE:				Signed and dated Quality Assurance and Good Laboratory Practice Compliance statements were provided. A No Data Confidentiality Claim statement was provided, but a signature and date were missing. The study was conducted in accordance with UK Statutory Instrument 1999 No. 3106, the OECD Principles of Good Laboratory Practice (1997), U.S. EPA 40 CFR Part 160 (1989), and the Good Laboratory Practice Standard for Agricultural Chemicals 14-Seisan-7739 (2002). 

   A. MATERIALS:

   	1. Test material  				 Tetrachloroterephthalic Acid (degradate of DCPA)

      Description: 				White powder

      Lot No./Batch No. : 			021101 (Batch no.)

      Purity: 						99.94%
      
      Stability of compound 
      under test conditions:		Stable. Analytical recoveries at 96 hours were >=101% of initial-measured 
            			concentrations. The mean-measured concentrations ranged from 93 to 103% 					of the nominal TPA exposure concentrations of nominal.
      							(OECD recommends stability in water and light)

      Storage conditions of 
      test chemicals: 				Room temperature.

		Physicochemical properties of Tetrachloroterephthalic Acid (degradate of DCPA).
Parameter
Values
Comments
Water solubility at 20°C
Not reported

Vapor pressure
Not reported

UV absorption
Not reported

pKa
Not reported

Kow
Not reported

   
   2. Test organism: 
   
         Name:					Green Alga, Chlorophyceae, Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) 
         Strain:					CCAP 278/4
         Source: 					Cultures were obtained from an axenic culture derived from the Culture Collection of Algae and Protozoa (CCAP), CEH Windermere, The Ferry House, Far Sawrey, Ambleside, Cumbria, UK.
                           EPA recommends algae be from the same source and stock culture or commercial sources.
         Age of inoculum:		Not reported
                           EPA recommends the algal inoculum should be from logarithmically growing stock cultures (typically 3- to 7-days old).
         Method of cultivation:	Not reported
   B.  STUDY DESIGN:

      1. Experimental Conditions

         a. Range-finding  -  A preliminary range finding test was conducted at nominal concentrations of 0.01, 0.1, 1, 10, and 100 mg a.i./L. After 96 hours, inhibitions for area under the curve and growth rate were only observed in the highest test group at 41% and 9%, respectively, in the highest treatment group. The % inhibitions for all other treatment levels were 0% for these endpoints. For both endpoints, the NOAEC and EC50 -  were determined to be 10 and 100 mg a.i./L, respectively. 

         b. Definitive Study
Table 1:  Experimental Parameters
                                   Parameter
                                    Details
                                    Remarks
                                       
                                       
                                   Criteria
Acclimation period:

Culturing media and conditions:  (same as test or not)


Health:  (any mortality observed)
Not reported

Liquid culture, further details provided in Appendix 1 pg 31-32 of the study report.

Not reported





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

OECD recommends an amount of algae suitable for the inoculation of test cultures and incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about 3 days. When the algal cultures contain deformed or abnormal cells, they must be discarded.
Test system
Static/static renewal

Renewal rate for static renewal

Static

N/A



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

Duration of the test
96 hours



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

Glass Erlenmeyer flask
250 mL
100 mL



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

EPA recommends all test vessels and closures to be identical.
OECD recommends 250 ml conical flasks are suitable when the volume of the test solution is 100 ml or use a culturing apparatus.
Details of growth medium 
Name:
pH at test initiation:
pH at test termination:
Chelator used:
Carbon source:

Salinity (for marine algae):

Algal Culture Medium (EC/AM) 
7.7 (negative control)
10.0 to 10.2 (negative control)
Na2EDTA ● 2H2O
NaHCO3  (added after filter sterilization (0.2um)
N/A
Additional details found in Appendix 1 pg 31-32 of the study report.


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

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

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

Dilution water used to prepare media 
Source of dilution water:

Quality of dilution water
Hardness:
Alkalinity:
pH:
Specific conductivity:
Salinity (for marine algae):
Water pretreatment (if any):
TOC:
COD:
Particulate matter:
Metals:
Pesticides/PCBs:
Chlorine:


Deionized water 


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



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

Marine algal nutrient medium is prepared by adding reagent grade chemicals to synthetic salt water or filtered natural salt water, or by preparing a complete saltwater medium. Salinity for saltwater medium should be 30 +- 5 %..


Indicate how the test material is added to the medium (added directly or used stock solution)
A nominal 100 mg a.i./L stock solution was made by dissolving 201.8 mg of the test material to a final volume of 2 L with EC media. The stock solution was stirred for 1 hour using a magnetic stirrer. Secondary stock solutions at nominal concentrations of 9.39, 20.7, and 45.5 mg a.i./L were made by serial dilution of the 100 mg ai/L stock solution in EC media. A nominal 4.27 mg a.i./L stock solution was made from dilution of the 9.39 mg a.i./L stock solution in EC media. A control solution was prepared using EC media only.

Aeration or agitation


Oscillation rate:
Agitated in a cooled orbital incubator

100 cycles/min under constant illumination



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



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

OECD recommends that the initial cell concentration be approximately 10,000 cells/ml for S. capricornutum and S. subspicatus. When other species are used the biomass should be comparable.
Number of replicates
Negative control:
Solvent control:
Treatments:

6
N/A
3/level* 

*Due to a sampling error at 24 hours in the nominal 20.7 mg a.i./L test level, only two of the replicate test vessels were used for cell counts and growth inhibitions, causing unbalanced replication.



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

OECD preferably three replicates at each test concentration and ideally twice that number of controls. When a vehicle is used to solubilize the test substance, additional controls containing the vehicle at the highest concentration used in the test.

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


Initial


Mean-Measured:

0 (negative control), 4.27, 9.39, 20.7, 45.5, and 100 mg a.i./L

<0.01 (<LOD, control), 3.98, 8.82, 19.3, 44.7, and 101 mg a.i./L

<0.01 (<LOD, control), 3.99, 8.90, 19.7, 46.1, and 103 mg a.i./L
Mean-measured concentration is a mean of 0 and 96-hour (with algae) values. 
Limit of determination (LOD) = 0.01 mg/L


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

OECD recommends at least five concentrations arranged in a geometric series, with the lowest concentration tested should have no observed effect on the growth of the algae. The highest concentration tested should inhibit growth by at least 50% relatively to the control and, preferably, stop growth completely. 
Solvent (type, percentage, if used)
N/A 
EPA recommends the solvent N,N-dimethyl-formamide. The concentration of solvent should be the same in all test treatments and should not exceed 0.1 mL/L.
Method and interval of analytical verification
Test solutions were analytically verified at 0 and 96 hours.

Each test solution was analyzed to determine the test substance concentration using high performance liquid chromatographic analysis with UV detection (HPLC-UV) at 220 nm. 
EPA recommends confirmation of dissolved test concentrations at a minimum at test initiation and at test termination for static tests. 
Test conditions 
pH:
Temperature (media):
Temperature (air):
Photoperiod:
Light intensity and quality:

7.7  -  10.2
22.9  -  25.1 ℃
Not reported
Constant illumination
7790 lux 



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

OECD recommended the temperature in the range of 21 to 25[o]C maintained at +- 2[o]C and continuous uniform illumination provided at approximately 8000 Lux measured with a spherical collector. OECD: pH is measured at beginning of the test and at 72 hours, it should not normally deviate by more than one unit during the test.

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

N/A 
N/A

Other parameters, if any
None


      2. Observations:  

Table 2:  Observation parameters
                                  Parameters
                                    Details
                                    Remarks


                                   Criteria
Parameters measured including the growth inhibition/other toxicity symptoms
Cell density, growth rate, and area under the curve



Recommended parameters measured per replicate include:
-Algal cell density (cell count/mL)
-yield (final population density)
-average specific growth rate
-mean area under growth curve (AUC)
Measurement technique for cell density and other end points
Cell counts were performed using a Z2 Coulter Counter (Coulter Electronics Ltd, Luton, UK). Corrections for cell counts were made by subtraction of the background cell counts from the test flasks without algae. Initial cell concentration of the starter culture was measured using a hemacytometer and confirmed using the Z2 Coulter Counter. 

Growth rate:
u= logeNn  -  logeN0
                tn 
where:
N0 = initial measured cell concentration at time t0 (cells/mL)
N1 = measured cell concentration at time t1 (cells/mL)
Nn = measured cell concentration at time tn (cells/mL)
N2 = measured cell concentration at time t2 (cells/mL)
t1 = time of first measurement after the beginning of the test (h)
t2 = time of second measurement after the beginning of the test (h)
tn = time of n[th] measurement after the beginning of the test (h)

Area under the curve:
A = ((N1-N0)/2)(t1)+((N1+N2-2N0)/2)(t2-t1)+ ((Nn-1+Nn-2 N0)/2)(tn-tn-1) 

where:
N0 = initial measured cell concentration at time t0 (cells/mL)
N1 = measured cell concentration at time t1 (cells/mL)
Nn = measured cell concentration at time tn (cells/mL)
N2 = measured cell concentration at time t2 (cells/mL)
t1 = time of first measurement after the beginning of the test (h)
t2 = time of second measurement after the beginning of the test (h)
tn = time of n[th] measurement after the beginning of the test (h)
The reviewer calculated cell density, yield, growth rate, and area under the curve according to the formulas provided by OCSPP 850.4500 guidance.


EPA recommends the measurement of cell counts by microscopic observation or electronic particle counter, with alternative option of measuring chlorophyll a. 

OECD recommends the electronic particle counter, microscope with counting chamber, fluorimeter, spectrophotometer, and colorimeter. (note: in order to provide useful measurements at low cell concentrations when using a spectrophotometer, it may be necessary to use cuvettes with a light path of at least 4 cm).
Observation intervals 
Observations were conducted at 0, 24, 48, 72, and 96 hours.



EPA and OECD: every 24 hours.
Other observations, if any
Test solutions were clear and colorless at exposure initiation (0 hours). After 96 hours, test vessels containing algae appeared as green suspensions in the control and four lowest treatment levels. The highest test level solutions appeared slightly hazy and paler green in color than the control. 
Limit of determination (LOD) = 0.01 mg/L
Indicate whether there was an exponential growth in the control
Yes, cell density in the negative control increased by a factor of 462X during the test.  



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

OECD: cell concentration in control cultures should have increased by a factor of at least 16 within three days.
Were raw data included?
Yes, except for phytotoxic effects. 


II. RESULTS and DISCUSSION:
   
	A. INHIBITORY EFFECTS:

   At 96 hours, yield inhibitions relative to the control were low (<=17%) in the four lowest test groups and increased to 44% in the highest test level. Inhibitions for area under the curve and growth rate were low in every test level, reaching a maximum inhibition of 17% and 10%, respectively. For all endpoints, effects were highest in the mean-measured 103 mg a.i./L treatment group. 

   At exposure initiation, the test solutions appeared clear and colorless. After 96 hours, test vessels containing algae appeared as green suspensions in the control and four lowest treatment levels. The highest test level solutions appeared slightly hazy and paler green in color than the control. Further details on phytotoxic effects were not provided. 
   
   The pH at test initiation was 7.7 in the control and ranged from 7.7 to 7.8 in the test solutions. After 96 hours, the pH increased significantly in the control (ranging from 10.0 to 10.2) and four lowest treatment levels (ranging from 8.3 to 10.1). Conversely, the highest treatment level pH decreased substantially by test termination (ranging from 4.9 to 5.0). 
   For a satisfactory test, cell counts in the controls should increase by a factor of at least 100X for P. subcapitata and a factor of at least 30X for S. costatum by test termination (i.e., logarithmic growth in the controls). At test termination the coefficient of variation (CV) for mean control yield should be < 15% and the CV for average specific growth rate should be < 15%, which is a logarithmically-transformed variable.

Table 3:  Effect of Tetrachloroterephthalic Acid (degradate of DCPA) on Algal Growth Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum).
            Mean-Measured 
(and Nominal) Concentrations,
 mg a.i./L
                                 Initial Cell
                                    Density
                              (x 10[4] cells/mL)
                      Cell density (x 10[4] cells/mL)[a]


                                   24 hours
                                   72 hours
                                   96 hours


                                       
                                       
                                 Cell Density
                                 % Inhibition
Negative control (<LOD)
                                     1.00
                                     6.38
                                      185
                                      462
                                      N/A
3.99 (4.27) 
                                     1.00
                                     6.15
                                      171
                                      389
                                      16
8.90 (9.39)
                                     1.00
                                     6.29
                                      175
                                      421
                                       9
19.7 (20.7)
                                     1.00
                                     5.96
                                      175
                                      442
                                       4
46.1 (45.5)
                                     1.00
                                     5.82
                                      168
                                      384
                                      17
103 (100)
                                     1.00
                                     6.22
                                      214
                                      258
                                      44
  a Calculated by the reviewer. 
  N/A - Not Applicable. 
  Data were obtained from Table 3 on page 25 of the study report.
  LOD was 0.01 mg a.i./L

Table 4:  Effect of Tetrachloroterephthalic Acid (degradate of DCPA) on Algal Growth Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum).
                 Mean-Measured 
(and Nominal) Concentrations 
                                  (mg a.i./L)
                                 Initial cell
                                    density
                              (x 10[4] cells/mL)
                               Mean growth rate
                                 (day[-1]) [a]
         Mean area under the curve (AUC; x 10[4] cells x days/mL) [a]
                                       
                                       
                                  0-96 Hours
                                 % Inhibition
                                  0-96 Hours
                                 % Inhibition
Negative control (<LOD)
                                     1.00
                                     1.53
                                      N/A
                                      460
                                      N/A
3.99 (4.27) 
                                     1.00
                                     1.49
                                       3
                                      406
                                      12
8.90 (9.39)
                                     1.00
                                     1.51
                                       2
                                      427
                                       7
19.7 (20.7)
                                     1.00
                                     1.52
                                       1
                                      437
                                       5
46.1 (45.5)
                                     1.00
                                     1.49
                                       3
                                      399
                                      13
103 (100)
                                     1.00
                                     1.39
                                      10
                                      384
                                      17
  [a]     Calculated by the reviewer.
  N/A - Not applicable
  Data were obtained from Table 3 on page 25 of the study report.
  LOD was 0.01 mg a.i./L
  
Table 5:  Statistical endpoint values calculated by the study author based on nominal concentrations
                             Statistical Endpoint
                                     Yield
                                  Growth Rate
                             Area Under 
the Curve
NOAEC (mg a.i./L) (95% C.I.)
                                 Not reported
                                     45.5
                                   <4.27
IC50/EC50 (mg a.i./L) (95% C.I.)
                                 Not reported
                                    >100
                                    >100

   B. REPORTED STATISTICS: 

   The NOAEC was determined using ANOVA followed by Dunnett's test (α=0.05). Due to the lack of inhibitory effects, the IC50 values were empirically to be greater than the highest nominal concentration tested. The study author's results are based on the nominal concentrations. 

	C. VERIFICATION OF STATISTICAL RESULTS:

   The data were analyzed using CETIS Version 1.9.6.12 with database backend settings implemented by EFED on 7/25/17. The mean-measured concentrations provided by the study author were used for analysis.
   
   The data were tested for normality and equality of variance using Shapiro-Wilk's and Bartlett's tests (α = 0.01), respectively. The data for all endpoints were normally distributed and homoscedastic. Area under the curve data were monotonically decreasing and analyzed using William's Multiple Comparison test. Growth rate and cell density data responses straddled monotonicity and non-monotonicity. Further, hypothesis testing results were the same for both William's and Dunnett's test, so the shared NOAEC results are reported. 
   
   The IC05 values were calculated using non-linear regression. The yield IC50 was slightly extrapolated using non-linear regression results, and results should be interpreted with caution. The growth rate IC50 was highly extrapolated to a value that was approximately 5X greater than the highest measured test concentration and should be interpreted with great caution. The reviewer attempted non-linear regression to determine the area under the curve IC50, but the value was highly extrapolated and confidence intervals could not be determined. Additionally, the effects were low, with a maximum effect of 17%. Therefore, the IC50 value was visually determined to be greater than the highest mean-measured concentration tested. 

   Yield
   IC05: 32.3 mg a.i./L				95% C.I.:  N/A to 52.7 mg a.i./L	
   IC50: >46 mg a.i./L[+]				95% C.I.:  NA	
   NOAEC: 19.7 mg a.i./L 
   LOAEC: 46 mg a.i./L
   		
   Growth rate
   IC05: 75.1 mg a.i./L				95% C.I.:  55.8 to 92.5 mg a.i./L	
   IC50: >46.1 mg a.i./L[+]				95% C.I.:  N/A
   NOAEC: 19.7 mg a.i./L
   LOAEC: 46 mg a.i./L
   
   Area under the curve (AUC)
   IC05: 0.65 mg a.i./L*				95% C.I.:  N/A to 31.9 mg a.i./L	
   IC50: >46.1 mg a.i./L[+]				95% C.I.:  N/A 
   NOAEC: 19.7 mg a.i./L	
   LOAEC: 46 mg a.i./L
   *Value is not bracketed by the measured concentrations and should be interpreted with caution.
   [+] See reviewer comments below regarding the IC50 endpoint in reference to the highest test concentration.
   
   Endpoint(s) Affected:  Yield, area under the curve, and growth rate
   Most Sensitive Endpoint:  Yield, area under the curve, and growth rate, based on the NOAEC


   D.  STUDY DEFICIENCIES: 
	
   The pH of the control medium increased by a minimum of 2.3 units during the test, and OECD guidance states that the pH of the control medium should not increase by more than 1.5 units during the test. Whereas OCSPP does not provide guidance for pH increases in the control. Also, the highest test level solutions appeared slightly hazy and paler green in color than the control. After 96 hours, the pH increased significantly in the control (ranging from 10.0 to 10.2) and four lowest treatment levels (ranging from 8.3 to 10.1). Conversely, the highest treatment level pH decreased substantially by test termination (ranging from 4.9 to 5.0). Measurements were only taken at 0 and 96 hours and it is unclear if the effect in the highest test concentration are treatment related or if something else is going on within the test system. 
   
   Three replicates were used for each treatment level, except the 19.7 mg a.i./L (nominal 20.7 mg a.i./L) test level (where a sampling error occurred in one replicate), and six replicates were used for the negative control. OCSPP recommends a minimum of four replicates per concentration. However, OECD recommends three replicates per treatment level and preferably double the replicates for the control groups. Due to the noted sampling error, a replicate was excluded by the study author which led to unbalanced replication across treatment levels. These data were also excluded in the MRID, so the reviewer was unable to include the replicate in the statistical analysis. This is not considered a major deficiency as the two remaining replicates had fairly similar responses (CVs of 2-12%) and variation throughout the test system was low (maximum CV of 15% across all concentrations and endpoints).  
   
   E.  REVIEWER'S COMMENTS: 

   The reviewer's and study author's NOAEC results for area under the curve (AUC) and growth rate were not in agreement. The study author reported the NOAEC for AUC to be undefined based on Dunnett's test (<4.27 mg a.i./L, the lowest test concentration, which was "the highest concentration that did not produce a statistically significant adverse effect when compared to the control"), whereas the reviewer determined a higher toxicity value using William's test, effects were not dose-responsive. Conversely, the study author determined a higher NOAEC value for growth rate using Dunnett's test then was determined by the reviewer. The reason for this discrepancy is unclear as the reviewer's results based on Dunnett's and William's test produced a higher NOAEC value, but one potential fact is that effects in the three lowest test concentrations were not dose-responsive. The reviewer's and study author's IC50 results for area under the curve and growth rate were in general agreement, both were visually determined to be greater than the highest test level. Variations in all results can be attributed to the study author using nominal concentrations for analysis, compared to the reviewer using the mean-measured concentrations for analysis. Additionally, the reviewer's `highest concentration' was the second highest test concentration, as the highest test concentration does not appear to be reliable, due to issues with pH (as discussed in the study deficiency section above), and so based on these uncertainties, this test concentration should not be used to estimate endpoints. The study author did not assess yield, which was an affected endpoint in this study. The reviewer's results are presented in the Executive Summary and Conclusions section of this DER.
   
   All endpoints in this study were significantly affected in the two highest test levels, and based on the uncertainty surrounding the pH as discussed below, the most sensitive endpoint, based on the NOAEC was all three endpoints, with the NOAEC being 19.7 mg a.i./L.  
   
   With pH measurements taken at 0 and 96 hours, it is unclear if the difference (increased inhibition, relative to control plants) in the highest test concentration is treatment related or if something else was going on within the test system. Therefore, the highest test concentration does not appear to be reliable, based on these uncertainties, and was not used to estimate endpoints. Consequently, the reviewer concluded that the IC50 for all endpoints was greater than the 2[nd] highest tested concentration (46.1 mg a.i./L).

   All OCSPP 850.4500 (2012) validity criteria were met except: The study author did not indicate whether treatments were randomly assigned to test vessels. Although based on the description of the study summary this was likely completed. All OECD 201 (2011) validity criteria were met. 
   
   The highest test level solutions appeared slightly hazy and paler green in color than the control. After 96 hours, the pH increased significantly in the control (ranging from 10.0 to 10.2) and four lowest treatment levels (ranging from 8.3 to 10.1). Conversely, the highest treatment level pH decreased substantially by test termination (ranging from 4.9 to 5.0). Measurements on pH were only taken at 0 and 96 hours, and it is unclear if the difference in the highest test concentration was treatment related or if something else is going on within the test system. The increase in the controls and other treatment groups to extremely alkaline conditions are likely a result of the test system reaching the carrying capacity of the test vessels. Laboratory toxicity tests should be conducted in environmentally relevant conditions which may not be represented by the strongly basic water conditions present in the Day 4 samples. The alkaline conditions may have inhibited growth in the controls and lower treatment groups, resulting in less sensitive endpoints than would have occurred had the pH stayed in the environmentally relevant range. Had pH measurements been taken more frequently (i.e., daily), there would be more certainty that the similar dose-responses (and any consequently estimated NOAEC and IC50 endpoints) observed in treatment groups at 24-hours and 72-hours, compared to the treatments at 96-hours, were not a result of inhibitions in control growth due to high pH.  
   
   After 96 hours, the reviewer-calculated %CV for control yield and growth rate were 8 and 1%, respectively, which meets the OCSPP 850.4500 Guideline requirement of <15% (yield and growth rate) and OECD 201 Guideline requirement of <10% (growth rate).
   
   The in-life phase of the study was conducted sometime between March 12, 2003 and April 11, 2003.
	
   F. CONCLUSIONS:  
   
   This study is scientifically sound and is classified as supplemental and may be used for risk characterization. All endpoints in this study were significantly affected in the two highest test levels and based on the uncertainty surrounding the pH, the most sensitive endpoint, based on the NOAEC was all three endpoints with the NOAEC being 19.7 mg a.i./L. With pH measurements taken at 0 and 96 hours, it is unclear if the difference (increased inhibition, relative to control plants) in the highest test concentration is treatment related or if something else was impacting the test system. Therefore, the highest test concentration does not appear to be reliable, and so based on these uncertainties, this test concentration should not be used to estimate endpoints. The IC50 for yield, growth rate and area under the curve (AUC) was >46.1 mg a.i./L, as the highest test concentration does not appear to be reliable for use based on the uncertainties surrounding pH. 

III.  REFERENCES:  

No references were provided in the MRID. 

