Document ID: EPA-HQ-OPP-2011-0374-0026
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2022-04-28T04:00Z

Data Requirement:				PMRA DATA CODE {............}
                        EPA DP Barcode		420906
                        OECD Data Point	{............}
                        EPA MRID 			49307509
                        EPA Guideline		850.4400

Test material:	Dacthal	Technical					Purity:	98.3%
Common name:	DCPA
Chemical name:	IUPAC:		Not reported
            CAS name:	Not reported
            CAS No.:	1861-32-1
				Synonyms:	Not reported

Primary Reviewer: Kindra Bozicevich								Signature: 
Environmental Scientist, CDM/CSS-Dynamac JV					Date: 05/16/2016
Secondary Reviewer: Adrian Graff								Signature:
Environmental Scientist, CDM/CSS-Dynamac JV					Date: 10/23/2020

Primary Reviewer:	Christina M. Wendel							Signature: 
EPA/OPP/EFED/ERB2/Biologist 									Date: 10/20/2021

Secondary Reviewer(s): Michael Wagman							Signature: 
EPA/OPP/EFED/ERB2/Senior Scientist							Date: 11/12/2021

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

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

Date Evaluation Completed: 12-11-2021

CITATION: Arnie, J.R., Martin, K.H., and J.R. Porch. 2013. Dacthal: A 7-Day Static-Renewal Toxicity Test with Duckweed (Lemna gibba G3). Study performed by Wildlife International, Ltd., Easton, Maryland. Project number 246P-104. Study sponsored by Amvac Chemical Corporation, Los Angeles, California. Study initiated August 21, 2013 and completed December 3, 2013.

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 7-day acute toxicity study, freshwater floating aquatic vascular plants (duckweed, Lemna gibba G3) were exposed to Dacthal Technical (DCPA) to nominal concentrations of 0 (negative and solvent controls, <LOQ), 31, 63, 125, 250, and 500 ug a.i./L under static-renewal conditions. Because the test item was stable, mean-measured concentrations of <10.0 (<LOQ, controls), 27, 56, 113, 230, and 470 ug a.i./L were used for analysis and reporting.

The % growth inhibition of frond number in the treated culture as compared to the control ranged from -13 to 14%. Only frond number yield and frond number growth rate were significantly affected by exposure to the test material. After seven days, the most sensitive endpoint was frond number yield, with NOAEC, LOAEC, and IC50 values of 241, 470, and >470 ug a.i./L, respectively.

After 7 days, chlorotic fronds (>=0.3%) were observed in the solvent control and 230 ug a.i./L treatment group.  Necrotic fronds (>=0.3%) were observed in the negative control and 27 and 230 ug a.i./L treatment groups. There was a notable increase in pH during the study. The pH in both controls and all exposure levels ranged from 8.0 to 8.2 at test initiation. By test termination, the pH ranged from 8.8 to 9.1. 

It should be noted that although the highest nominal concentration is at/above the solubility limit for DCPA (0.5 mg/L), both the stock solutions, and test concentrations were mixed by inversion and sonication and were clear and colorless throughout the test, indicating that the test material was in solution. The mean-measured concentrations were utilized for estimating the endpoints.

This study is scientifically sound and is classified as acceptable.

Results Synopsis

   Test Organism: Duckweed, Lemna gibba G3
   Test Type (Flow-through, Static, Static Renewal): Static-renewal
   
   Frond number yield
   IC05: 241 ug a.i./L			95% C.I.: N/A to 394 ug a.i./L
   IC50: >470 ug ai/L			95% C.I.: N/A
   NOAEC: 230 ug a.i./L
   
   Frond number growth rate
   IC05: 373 ug a.i./L			95% C.I.: 205 to 492 ug a.i./L
   IC50: >470 ug ai/L			95% C.I.: N/A
   NOAEC: 230 ug a.i./L
   
   Final biomass
   IC05: Not calculable			95% C.I.: N/A 
   IC50: >470 ug a.i./L			95% C.I.: N/A
   NOAEC: 470 ug a.i./L
   
   Biomass growth rate
   IC05: Not calculable			95% C.I.: N/A
   IC50: >470 ug a.i./L			95% C.I.: N/A
   NOAEC: 470 ug a.i./L
   
   Endpoint(s) Affected: Frond number yield and frond number growth rate
   Most Sensitive Endpoint: Frond number yield and frond number growth rateI. MATERIALS AND METHODS

   GUIDELINE FOLLOWED:		This study was designed to comply with OECD 221 (2006) and OCSPP 850.4400 (2012). The reviewer assessed the study according to these same guidelines and OCSPP 850.4000 (2012), and OCSPP 850.1000 (2016), noting several deviations:
   
 Use of aeration or agitation was not reported. This is considered to be a minor deficiency.
 The hardness, alkalinity, specific conductivity, TOC, COD, particulate matter, and chlorine content of the dilution water were not reported as recommended by OCSPP Guidance. These water quality components are not specifically addressed by OECD. The OCSPP guideline recommends that these parameters are measured and that these water quality characteristics meet EPA specifications. The most recent analyses performed to measure concentrations of selected organic and inorganic constituents in well water that was used to prepare the algal medium are presented in Appendix 4 of the study report (pgs. 51-52). 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.
 The physicochemical properties of the test substance were not reported as recommended by both OCSPP and OECD Guidance. This is considered to be a minor deficiency.
 Test solutions were not measured immediately before and after renewal as recommended by OCSPP Guidance (850.1000). However, the study author did provide sufficient analytical to determine that the static-renewal test design was sufficient to maintain the test concentrations at >=80% of the initial-measured concentrations of the previous interval. This is considered to be a minor deficiency.
 The light intensity (4930 to 5990 lux) was within the OCSPP recommended range of 4200 to 6700 lux but below the OECD recommended range of 6500 to 10000 lux. The Lemna were held under continuous fluorescent lighting, as recommended in the guideline. This is considered to be a minor deficiency.
   
   	These deviations do not affect the validity of the study.

   COMPLIANCE:			Signed and dated GLP, Quality Assurance, and No Data Confidentiality statements were provided.  The study was conducted in compliance with the GLP standards of the U.S. EPA (40 CFR parts 160 and 792), OECD Principles of GLP ENV/MC/CHEM (98)17, and Japan MAFF 11 NohSan Notification No. 6283 with the following exceptions: periodic analyses of water for potential contaminants and characterization of the test substance and stability of the test substance under storage conditions.

   A. MATERIALS:

   	1. Test material  				Dacthal Technical

      Description: 				Solid

      Lot No./Batch No. : 			090614-2
   
      Purity: 						98.3%
      
      Stability of compound 
      under test conditions:		The test substance was stable under test conditions. The reviewer-calculated %CV values ranged from 5 to 10%. Measured concentrations ranged from 87 to 94% of nominal.

      Storage conditions of 
      test chemicals: 				Stored at ambient temperature in the dark.
		Physicochemical properties of Dacthal Technical.
Parameter
Values
Comments
Water solubility at 20C
Not reported

Vapor pressure
Not reported

UV absorption
Not reported

pKa
Not reported

Kow
Not reported

   2. Test organism: 
         
         Name:  Duckweed, Lemna gibba
         Strain, if provided: G3
         Source:  In-house cultures originally (maintained at Wildlife International, Easton, MD since June 			1995) obtained from the United States Department of Agriculture (USDA).
         Age of inoculum:  7 days
         Method of cultivation: Cultured and tested in 20X AAP medium under continuous warm-white fluorescent light (5450 +- 1250 lux) at 25  2C.
         
   B.  STUDY DESIGN:

      1. Experimental Conditions

         a. Range-finding study: A non-GLP range-finding test was conducted at nominal concentrations of 5.0, 50, and 500 ug a.i./L, and yielded 5, 2, and 15% inhibition of mean frond number relative to the mean negative control response, respectively, after 7 days of exposure. Nominal test concentrations were selected in consultation with the Sponsor and were based upon the results of the exploratory range-finding test.
         
         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)
Continuous, 7-day old batch culture 

Same as test; 20X AAP medium (Appendix 3, pg 50 in the study report).

Actively growing. Appeared healthy and uniform in size.
Obtained from cultures that had been actively growing in culture medium (20X AAP; same as test) for at least 2 weeks prior to test initiation. Plants used in this test were transferred to fresh medium 7 days prior to start of test.
Test system
Static/static renewal

Renewal rate for static renewal

Static-renewal

Renewed on days 3 and 5

EPA expects the test concentrations to be renewed every 3 to 4 days (one renewal for the 7 day test, 3-4 renewals for the 14 day test).  
Incubation facility
Temperature-controlled environmental chamber at a temperature of 25 +-2°C

Duration of the test
7 days

EPA requires a duration of 14 days.  Seven day studies will be accepted for review by the Agency.  
Test vessel
Material: (glass/stainless steel)
Size:
Fill volume:

Glass beakers
250 mL
100 mL
Beakers covered with sterile, polystyrene Petri dishes
Details of 20X AAP medium
pH in new solutions:
pH in old solutions:
Chelator used:
Carbon source:

8.0 (negative control)
8.6 to 9.0 (negative control)
Na2EDTA::2H2O
NaHCO3

The pH of the medium was adjusted to 7.5+-0.1 using 10% HCl and 0.1N sodium hydroxide.

The medium was sterilized by filtration (0.22 um) prior to use.
Appendix 3 (pg. 50 in the study report) contains the list of the medium constituents. Stock nutrient solutions were prepared by adding reagent-grade chemicals to NANOpure(R) water (purified Wildlife International well water).
Appendix 4 contains the results of the most recent analyses (December 26, 2012) performed to measure concentrations of contaminants in well water used to prepare the 20X AAP medium.

EPA recommends the following culture media:  Modified Hoagland's E+ or 20X-AAP. Chelating agents (e.g. EDTA) are recommended in the nutrient medium for optimum cell growth. Lower concentrations of chelating agents (down to one-third of the normal concentration recommended for AAP medium) may be used in the nutrient medium used for test solution preparation if it is
suspected that the chelator will interact with the test material. ASTM reference, E1415-91and D 3978-80 (reapproved 1987).
If non-standard nutrient medium was used, detailed composition provided (Yes/No)
A standard medium was used and a detailed composition was provided.

Dilution water used to prepare media
Source of dilution water:

Quality of dilution water
Hardness:
Alkalinity:
pH:
Specific conductivity:
Water pretreatment (if any):
TOC:
COD:
Particulate matter:
Metals:

Pesticides/PCBs:

Chlorine:

Purified Wildlife International, Ltd. well water

Not reported
Not reported
Not reported
Not reported
None
Not reported
Not reported
Not reported
Calcium  -  33.5 mg/L, Chloride  -  4.2 mg/L, Magnesium  -  12.9 mg/L, Potassium  -  6.57 mg/L, Sodium  -  18.2 mg/L

All others < Reporting Limit

< Reporting Limit

Not reported
Water analyses were performed on samples collected on December 26, 2012 (see Appendix 4 pg. 51-52 of the study report).

EPA recommends a pH of ~5.0. A solution pH of 7.5 is acceptable if type 20X-AAP nutrient media is used.
Indicate how the test material is added to the medium (added directly or used stock solution)
Primary stock solutions were prepared on days 0, 3, and 5 by adding 0.0509 g of Dacthal to 10 mL of N,N-Dimethylformamide (DMF) to achieve a nominal test concentration of 5.0 mg a.i./mL (based on reported purity of 98.3%). After inversion and sonication, the resulting solution was clear and colorless.  Aliquots of the primary stock solution were diluted with DMF and 20X AAP medium to prepare the remaining nominal test concentrations. The solvent control contained 0.1 mL DMF/L whereas the negative control consisted of 20X AAP medium only.

Aeration or agitation
Not reported

Sediment used (for rooted aquatic vascular plants)
Origin:
Textural classification (%sand, silt, and clay):
Organic carbon (%):
Geographic location:
N/A

Number of replicates
Negative control:
Solvent control:
Treatments:

4
4
4
One recovery replicate was also maintained in each experimental group.
Number of plants/replicate
4 plants/replicate

EPA requires 5 plants.
Number of fronds/plant
3 (12 fronds total per replicate)

EPA requires 3 fronds per plant.  
Test concentrations
Nominal:

Mean-measured:

0 (negative and solvent controls), 31, 63, 125, 250, and 500 ug a.i./L 

<10.0 (<LOQ, controls), 27, 56, 113, 230, and 470 ug a.i./L 
Negative control = 20X AAP medium

EPA requires at least 5 test concentrations with a dose range of 2X or 3X progression.  
Solvent (type, percentage, if used)
0.1 mL/L N,N-Dimethylformamide (DMF)

Method and interval of analytical verification
The test concentrations were measured on Day 0 (new), Day 3 (new), Day 3 (old), Day 5 (new), Day 5 (old), and Day 7 (old) using HPLC analysis with UV absorbance at 220 nm.
Samples were collected in duplicate from each experimental group (refer to copy of Excel worksheet in Appendix I). 
Limit of quantitation (LOQ) was 10 ug a.i./L.
Test conditions 
Temperature:
Photoperiod:
Light intensity and quality:

pH:

23.2 to 24.2ºC
Continuous
4930 to 5990 lux fluorescent light
(mean 5460 lux +- 379.6 SD)

8.0 to 9.1
Temperature measured continuously ranged from 22.6 to 24.9ºC
Reference chemical (if used)
name:
concentrations:

N/A

Other parameters, if any
N/A

      2. Observations:  

Table 2: Observation parameters
                                  Parameters
                                    Details
                               Remarks/Criteria
Parameters measured (e.g., number of fronds, plant dry weight or other toxicity symptoms)
Frond number
Frond number growth rate
Frond number yield
Biomass (dry weight)
Biomass growth rate
Biomass yield

Measurement technique for frond number and other end points
Frond number was determined by direct counting.  

Frond number yield and biomass yield were determined by subtracting the final values for each respective parameter from the initial values.

Growth Rates were calculated using a logarithmic growth equation.

Plants used in biomass determinations were dried at approximately 60°C for 48 hours prior to final weighing.

Observation intervals 
Fronds were counted on days 0, 3, 5, and 7. 

Other observations, if any
Fronds were observed for abnormalities (chlorotic and necrotic fronds).

Indicate whether there was an exponential growth in the control
Yes, the doubling time in the negative control was 1.9 days.

Were raw data included?
Yes

II. RESULTS and DISCUSSION:
   
	A. INHIBITORY EFFECTS:

Effects ranged from potential promotion of growth to at most low inhibitions in every test level for all endpoints tested. The maximum inhibition observed was for frond number and frond yield (14 and 15%, respectively). The detailed results are presented in Tables 3 to 5 below.

After 7 days, chlorotic fronds (>=0.3%) were observed in the solvent control and 230 ug a.i./L treatment group.  Necrotic fronds (>=0.3%) were observed in the negative control and 27 and 230 ug ai/L treatment groups. There was a notable increase in pH during the study. The pH in both controls and all exposure levels ranged from 8.0 to 8.2 at test initiation. By test termination, the pH ranged from 8.8 to 9.1. 

Table 3: Effect of Dacthal Technical on frond number of duckweed, Lemna gibba.
                          Mean-Measured (and Nominal)
                                 Concentration
                                  ug a.i./L
                      Initial frond number/test solution
                                frond number at
                                       
                                       
                                    3 days
                                    5 days
                                    7 days
                                       
                                       
                                       
                                       
                                 frond number
                               % inhibition [a]
Negative control
                                      12
                                      30
                                      61
                                      160
                                      N/A
Solvent control
                                      12
                                      27
                                      56
                                      149
                                       7
27 (31)
                                      12
                                      31
                                      62
                                      167
                                      -4
56 (63)
                                      12
                                      28
                                      67
                                      180
                                      -13
113 (125)
                                      12
                                      31
                                      66
                                      173
                                      -9
230 (250)
                                      12
                                      29
                                      61
                                      161
                                      -1
470 (500)
                                      12
                                      29
                                      59
                                      137
                                      14
Reference chemical (if used)
N/A
[a] Calculated by the reviewer relative to the negative control group.
Data were obtained from Table 6 on page 27 of the study report.
LOQ was 10 ug a.i./L

Table 4: Effect of Dacthal Technical on frond number of duckweed, Lemna gibba.
                                Mean-Measured 
                                 (and Nominal)
                                 Concentration
                                  ug a.i./L
            Initial frond number/test solution (or other endpoint)
                           Frond number growth rate
                                   (day[-1])
                              Frond number yield

                                   0-7 days
                               % inhibition [a]
                                   0-7 days
                               % inhibition [a]
Negative control (<LOQ)
                                      12
                                     0.369
                                      N/A
                                      148
                                      N/A
Solvent control (<LOQ)
                                      12
                                     0.359
                                       3
                                      137
                                       7
27 (31)
                                      12
                                     0.375
                                      -2
                                      155
                                      -5
56 (63)
                                      12
                                     0.386
                                      -5
                                      168
                                      -14
113 (125)
                                      12
                                     0.381
                                      -3
                                      161
                                      -9
230 (250)
                                      12
                                     0.370
                                       0
                                      149
                                      -1
470 (500)
                                      12
                                     0.347
                                       6
                                      125
                                      15
[a] Calculated by the reviewer relative to the negative control group.
Data were obtained from Table 7 on page 28 and Appendix 7 on pages 28 and 67, respectively, in the study report.
LOQ was 10 ug a.i./L
 
Table 5: Effect of Dacthal Technical on biomass of duckweed, Lemna gibba.
                          Mean-Measured (and Nominal)
                                 Concentration
                                  ug a.i./L
                    Initial frond weight/test solution (mg)
                              Final biomass (mg)
                              Biomass Yield (mg)
                        Biomass growth rate (days[-1])

                                     Day 7
                               % inhibition [a]
                                   0-7 days
                               % inhibition [a]
                                   0-7 days
                               % inhibition [a]
Negative control (<LOQ)
                                      1.2
                                     25.5
                                      N/A
                                     24.3
                                      N/A
                                     0.436
                                      N/A
Solvent control (<LOQ)
                                      1.2
                                     24.5
                                       4
                                     23.3
                                       4
                                     0.430
                                       1
27 (31)
                                      1.2
                                     26.3
                                      -3
                                     25.1
                                      -3
                                     0.441
                                      -1
56 (63)
                                      1.2
                                     27.0
                                      -6
                                     25.8
                                      -6
                                     0.445
                                      -2
113 (125)
                                      1.2
                                     27.7
                                      -9
                                     26.5
                                      -9
                                     0.448
                                      -3
230 (250)
                                      1.2
                                     25.3
                                       1
                                     24.1
                                       1
                                     0.435
                                       0
470 (500)
                                      1.2
                                     24.1
                                       5
                                     22.9
                                       6
                                     0.427
                                       2
[a] Calculated by the reviewer relative to the negative control group.
Data were obtained from Table 8 and Appendix 8 on pages 29 and 68, respectively, in the study report.
LOQ was 10 ug a.i./L

Table 6: Statistical endpoint values calculated by the study author based on mean-measured concentrations.
Statistical Endpoint
Frond number (final) 
Frond number growth rate 
Frond yield
Biomass yield
Biomass growth rate
NOAEC (ug a.i./L)
Not calculated
470
470
470
470
LOAEC (ug a.i./L)
Not calculated
>470
>470
>470
>470
IC50 or EC50 
(ug a.i./L) (95% C.I.)
Not calculated
>470 (N/A)
>470 (N/A)
>470 (N/A)
>470 (N/A)
Reference chemical
NOAEC
IC50/EC50
N/A
N/A - Not applicable.
   
   B. REPORTED STATISTICS: 

   The study author statistically analyzed mean frond number, growth rate, biomass, and biomass yield using The SAS System for Windows, Version 8.2. Negative and solvent control data were compared using a t-test and no statistically significant differences were noted. Endpoint data were evaluated for normality and homogeneity of variance using Shapiro-Wilk's and Levene's tests, respectively.  Treatment data were compared to the negative control data using Dunnett's t-test. The ICx values were determined using nonlinear regression. Toxicity values were based on mean-measured exposure concentrations.
   
C. VERIFICATION OF STATISTICAL RESULTS:

   Statistical Method: The reviewer assessed frond number yield, frond number growth rate, biomass, and biomass growth rate data using CETIS version 1.8.7.12 statistical software with database backend settings implemented by EFED on 10/20/15. The reviewer compared the negative and solvent control data using an Equal Variance t-Two Sample test (α = 0.05) and no statistically significant differences were observed. All further hypothesis testing was conducted comparing treatment data to negative control data only. 
   
   All data were confirmed to be normally distributed using Shapiro-Wilk's test (α = 0.01) and exhibit equal variance according to Bartlett's test (α = 0.01). The data were monotonically decreasing and were therefore analyzed using Williams' test (α = 0.05). The final biomass and biomass growth rate data did not violate assumptions of monotonicity although the means of the four lowest test levels were higher than or similar to the negative control mean. 
   
   Despite significant effects observed for frond number yield and frond number growth rate, the reviewer chose to estimate the IC50 values as greater than the highest concentration tested because the results were highly extrapolated for both endpoints and the 95% confidence limits weren't calculable for frond number growth rate. Final biomass and biomass growth rate IC50 values were estimated as greater than the highest concentration tested due to a lack of significant effects. The IC05 values were calculated using non-linear regression when possible.

   Frond number yield
   IC05: 241 ug a.i./L			95% C.I.: N/A to 394 ug a.i./L
   IC50: >470 ug a.i./L			95% C.I.: N/A
   NOAEC: 230 ug a.i./L
   
   Frond number growth rate
   IC05: 373 ug a.i./L			95% C.I.: 205 to 492 ug a.i./L
   IC50: >470 ug a.i./L			95% C.I.: N/A
   NOAEC: 230 ug a.i./L
   
   Final biomass
   IC05: Not calculable			95% C.I.: N/A 
   IC50: >470 ug a.i./L			95% C.I.: N/A
   NOAEC: 470 ug a.i./L
   
   Biomass growth rate
   IC05: Not calculable			95% C.I.: N/A
   IC50: >470 ug a.i./L			95% C.I.: N/A
   NOAEC: 470 ug a.i./L
   
   Endpoint(s) Affected: Frond number yield and frond number growth rate
   Most Sensitive Endpoint: Frond number yield and frond number growth rate

   D.  STUDY DEFICIENCIES: 

   No deficiencies were noted. 

   E.  REVIEWER'S COMMENTS: 

   The reviewer's and study author's results were in complete agreement for biomass and biomass growth rate. Complete agreement was also noted over IC50 values for frond number yield and frond number growth rate. However, the reviewer determined more toxicologically sensitive NOAECs for these endpoints. The reason for this is likely attributed to the reviewer's use of Williams' test compared to the study author's use of Dunnett's test for analysis and reporting. The reviewer's results are presented in the Executive Summary and Conclusions sections of this report.

   The laboratory portion of the definitive test was conducted from August 23 to 30, 2013, with dry weight 	measurements completed on September 1, 2013.
   
   The coefficient of variation (CV) based for frond number yield, biomass yield and growth rates based on both frond number and biomass were all <20%, which meets the guideline requirement of CV<20%.

   F. CONCLUSIONS:  

   This study is scientifically sound and is classified as acceptable. Only frond number yield and frond number growth rate were significantly affected by exposure to the test material. After seven days, the most sensitive endpoint was frond number yield, with NOAEC, LOAEC and IC50 values of 230, 470, and >470 ug a.i./L, respectively.

III.  REFERENCES:

American Society for Testing and Materials. 1991. ASTM Standard Guide 1415-91E. Standard Guide for Conducting Static Toxicity Tests with Lemna gibba G3.

Bruce, Robert, D. and Donald J. Versteeg. 1992. A Statistical Procedure for Modeling Continuous Toxicity Data. Environmental Toxicology and Chemistry. 11: 1485-1494.

Microsoft Corporation, Microsoft Excel 2010. Copyright 1985-2010.

Organization for Economic Cooperation and Development. 2006. OECD Guidelines for Testing of Chemicals, Guideline 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test. Adopted 23 March 2006.

The SAS System for Windows. 1999-2001. Version 8.2. SAS Institute, Inc. Cary, North Carolina.
U.S. Environmental Protection Agency. 2012. Series 850-Ecological Effects Test Guidelines, OCSPP Number 850.4400: Aquatic Plant Toxicity Test Using Lemna spp.

APPENDIX I. Copy of Excel Worksheet with Measured Concentrations