# 349-+81 RECEiVED OPPT CBIC 

## 2012OCT 25 Af110: 

# 06
3676 Davis Road N.W. • Dover, Ohio 44622 • (330) 343-7711 • 1-800-321-8805 • Fax: (330) 364-9626


### October 18, 2012 Document Control Office (DCO) (74707M) Office ofPollution Control and Toxics (OPPT) U.S. Environmental Protection Agency (EPA) EPA East Building, Room E6428 (202-564-8930) Washington, D.C. 20460 Attn: TSCA Section4 40 CFR790 (Docket# EPA-HQ-OPPT-2007-0531) Re: TestResults Dear SirorMadam: This letter serves as DoverChemical Corporation's completion ofanalytical testing underthe above TSCA Section 4 TestRule covering CAS # 4719-04-4. Underthis rule we areprovidingthe attached testresults following theTier 1test requirements. Ifyouhave any questions, please contactme at (330-365-3610) Regards,

# ?~ TimNowicki Manager Product Compliance Dover Chemical Corporation tnowicki@doverchem.com

cc: MCD


### \IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII1111111111111 4 0 1 3 0 0 0 0 0 0 6


## CONTAINS NO CB!3676 Davis Road N.W• Dover, Ohio 44622 • (330) 343-7711 • 1-800-321-8805 • Fax: (330) 364-9626 

### July 20, 2011 Document Control Office (DCO) (74707M) Office ofPollution Control and Toxics (OPPT) U.S. Environmental Protection Agency (EPA) EPAEastBuilding, Room E6428 (202-564-8930) Washington, D.C. 20460 Attn: TSCA Section4 40 CFR 790 (Docket#EPA-HQ-OPPT-2007-0531) Re: Notice oflntentto Test Dear SirorMadam: This letter serves as DoverChemical Corporation's notice ofintention to test underthe above TSCA Section 4 Test Rule coveringCAS # 4719-04-4. Underthis rule we are providingthe following required information: 1. 

### Identification ofTestRule-76 Fed. Reg. 1067 (Jan 7, 2011) 2. Name, Address, Telephone ofSponsoring Firm3. Name, Address, Telephone ofIndividual ContactDover Chemical Corporation 3676 Davis RoadN.W. Dover, Ohio44622 Melissa Clark-Dross 3676 Davis Road N.W. Dover, Ohio44622 (330)-365-3628 4. Signatory Authority for Testing Consortium Members- N/ A 5. Testing Requirements- Tier I Testing 6. Testing ofRepresentative Substances- N/A Regards,

Melissa Clark-Dross CorporateEHS & 

### Regulatory Manager

### Commercial-in-Confidence Chemex reference: ENV9857/051201

Reportissued by: 

### Thetoxicity ofHexahydro-1,3,5,- tris(2-hydroethyl)s-triazineto the algaPseudokirchneriellasubcapitata 

### overa 72-hourexposure period Reportfor DoverChemicalCompany

Sponsor: Chemex Environmental International Limited UnitJ Dover Chemical Company 3676 Davis Road NW Dover, OHIO BroadLane Industrial Estate Cottenham Cambridge CB24 SSW 

### UK October 2012

ENV9857/051201 USA 44622

Page 1of26

### Contents Page 

### Compliance with Good Laboratory Practice standards
...........................................................3 Quality Assurance audit statement..........................................................................................4 Summary
.................................................................................................................................5 1. Introduction
.................................................................................................................6 2. Materials and methods
.................................................................................................6 2.1 Test substance
..............................................................................................................6 2.2 Reference substance
....................................................................................................6 2.3 Test organism
..............................................................................................................7 2.4 Test and culture medium
.............................................................................................7 2.5 Test methods and conditions
.......................................................................................7 3. Results
........................................................................................................................ 10 3.1 Cell density measurements......................................................................................... 1 0 3.2 Observations on cell morphology
...............................................................................11 3.3 Percent inhibition at eachtest concentration compared to the control.. .....................11 3.4 ECx and NOEC values by yield (EyCx) and growth rate (ErCx)
................................. 11 3.5 Control validity results
...............................................................................................12 4. Discussion
..................................................................................................................13 5. References
..................................................................................................................13 Graphs 

### 1. Growth curves for control and test concentrations
..................................................... 14 2. Estimation of0 to 48 hours EyCx values
.................................................................... 15 3. Estimation ofO to 72 hours EyCx values
.................................................................... 16 4. Estimation of0 to 48 hours ErCx values
..................................................................... 17 5. Estimation ofO to 72 hourErCx values
...................................................................... 18 Appendices 

### 1. Analytical confirmation oftest concentrations
..........................................................19 2. Culture media
.............................................................................................................23 3. Water quality data
......................................................................................................24 4. Reference test .............................................................................................................25 5. GLP Certificate
...........................................................................................................26
ENV9857/051201 Page 2 of26Compliancewith Good Laboratory Practice standards 

### I, the undersigned, hereby declare thatthe study described in this report was performed under my supervision, andthatthe final report fully and accurately reflects the raw data 

### generated during the conduct ofthe study, in compliance with international codes ofGood Laboratory Practice based on:
• Section II

### ofAnnex 1to the European Parliament and council Directive 2004/10/EC and Annex 1to the European Parliament and council Directive 2004/9/EC (Official Journal No. L50) and embodied within:
• The UKGoodLaboratoryPractice Regulations 1999 (The UnitedKingdom GLP Regulations 1999, Statutory/nstrument3106) 

### as amended by:
• The UKGoodLaboratoryPractice (CodificationAmendments Etc.) Regulations 2004 (Statutory InstrumentNo 994) 

### These principles are in accordance withthe OECD Principles ofGood Laboratory Practice, revised 1997 (ENVIMC/CHEM(98)17). The analytical confirmation work (page 9, analytical confirmation ofexposure concentrations and Appendix 1) was outside ofthe GLP compliant scope ofthe study and compliance is not claimed
.



# ...........

# !:\~~······················· 

# ......8.
...C:l:JclX?t
.....2.012
...................... HeatherBeattie Date StudyDirector


### Key personnel
Otherkey personnel at Chemex involved in this study were: Agnes Grabczewska Sravanthi Chitikeshi

ENV9857/051201 Page 3of26

## QualityAssurance audit statement The Quality Assurance unit inspects all operating procedures as part ofa program ofprocess and study specific based audits. Annual routine facility-based inspections are also conducted, which cover the general facilities and activities within the test facility. The inspections applicable to this study are detailed below. The dates are given as dd/mm/yy.

Audit Date ofaudit Date reported to Study Director I

### Manaaement Studyplan 18/06/12 18/06/12 Process based audits Aquatic plant oralgal test setup 23/04/12 30/04/12 Detennination ofalgal cell density 23/04112 30/04112 Equipment calibration 26/04/12 28/06/12 Inoculation with algae 23/04112 30/04112 Labelling and paperwork 23/04/12 30/04/12 Preparation ofgrowth medium 28/06/12 28/06112 Preparation ofsolutions 23/05112 29/05/12 Taking andrecording readings 26/04/12 30/04/12 Weighing outtest orreference materials 23/05/12 29/05/12
This reporthas been audited by the undersigned and, as far as 

### can be reasonably established, the methods described and the results incorporated into this report accurately reflectthe raw data generated during this study.
Finalreport auditstarted: 15/08112
Final report auditcompleted: 08/10112 Signed:

# ·····~····································· Rasa Cooke QualityAssuranceManager Date: 

## OJ

## Od~**~o/2--** 

# ...............................................................

ENV9857/051201 Page4of26

### Summary This section summarises aquatic toxicity test results obtained by Chemex Environmental International Limited on a sample as detailed below: Test commissioned by: Dover Chemical Company Substance undertest: Hexahydro-1,3,5,-tris(2-hydroethyl)s-triazine Chemex reference:
Test species: Testtype: Test conditions:

Testperiod: Test carried out at:

Results:

Period of Sample: Study: EC0051201 ENV9857 Pseudokirchneriella subcapitata, strain CCAP 278/4 Growth inhibition: 72 hour EC5o Test conditions according to SOP E203 based upon OECD Guideline for testing of chemicals, Freshwater Alga and Cyanobacteria, Growth Inhibition Test reference 201 (2011) (I) Council Regulation (EC) No 440/2008 of30 May 2008(2l. From 16 to 19 July 2012 ChemexEnvironmental International Limited Unit J BroadLane Industrial Estate Cottenham Cambridge CB24 8SW 

### UK ECx and NOEC values by yield (EyCx) and growth rate (ErCx) 

### EyCx value 

### ErCx value exposure (95% confidence limits) (95% confidencelimits)

(hours) 

### EvCIO EyC2o EyC50 ErCIO ErC2o ErC50 0.05mgll 0.146mgll 1.14mg/l 1.8lmg/l 3.13mgll 8.93mgll 0 to 48 (0.000- (0.000- (0.00- (0.139- (0.516- (4.54- 0.405mg/l) 0.749m_g/ll 3.25mgll) 3.82mg/l) 5.71mg/l) 17.2m_g/l) O.lOlmg/1 0.293mgll 2.24mg/l 6.76mg/l 8.85mgll 14.8mg/l 0 to 72 (0.000- (0.000- (0.069- (2.86- (4.84- (11.2- 0.63Imwn 1.21mgll) 6.94mgll) 9.39mg/l) I1.7mwn 20.9mg/l) NOEC <lmg/1 (0-72 hours) <lmg/1 (0-72 hours) by yield by growth rate

ENV9857 /05120I Page 5 of26

### 1. 

### Introduction This report contains a description ofthe methods used and the results obtained during a study to investigate the growth inhibition of Hexahydro-1,3,5,-tris(2-hydroethyl)s-triazine to Pseudokirchneriella subcapitata. The objective ofthis study was to determine the 72 hour EC5o, which is defined as the concentration that inhibits the growth ofthe algae by 50% after a 72 hour exposure period at 21-24°C. 

### 2. Materials and methods Unless otherwise specified all methods mentioned in this report are according to Chemex Environmental International Limited standard procedures. All records ofmeasurements and observations made during this test will be collated and held in the Chemex Environmental International Limited archives at Unit J, Broad Lane Industrial Estate, Cottenham, Cambridge CB24 SSW. 

### 2.1 Test substance Name oftest substance: Lot number: Supplied by: Chemexreference: Date ofreceipt: Active ingredients: Required storage conditions:
Actual storage conditions: Quoted stability: Quoted appearance: Observed appearance: Quoted odour: Quoted solubility in water: 

### 2.2 Reference substance Identity: Purity: Source: Hexahydro-1,3,5,-tris(2-hydroethyl)s-triazine None Dover Chemical Company ECO 051201 23/04/12 CAS#4719-04-4, 78.5% Keep containertightly closed. Store away from incompatible substances in a cool, dry, well ventilated area. Room temperature Stable under normal temperatures and pressures Water white to pale yellow liquid Water white to pale yellow liquid Mild characteristic odor Very high

Potassium dichromate (K2Cr20 7) >99.9% FisherChemicals Ltd Reference substance used in a separate in-house study in order to check test procedure.
ENV9857 /05120I Page 6 of262.3 Test organism Test species: Source:

Culture conditions: Pseudokirchneriel/a subcapitata strain: CCAP 278/4 Culture Collection ofAlgae and Protozoa SAMS Research Services Ltd Scottish Marine institute OBAN Argyll PA37 IQA Scotland, United Kingdom Temperature: Illumination: Shaking: 20.2- 21.9°C 6000

- 7000 lux continuous white light Orbital set to 200rpm 2.4 Test and culture medium The tests were performed in deionised water with added nutrients. Before use the water was sterilised by autoclaving at l20°C for I5 minutes. Sterile nutrient stock solutions were then added and the pH value adjusted to 8.0 ± 0.2 to obtain the culture medium (see Appendix 2). 2.5 Test methods and conditions The test was carried out according to the procedures given below, based on the guidelines produced by OECD 20I: Alga Growth Inhibition Test<') and Council Regulation (EC) No 440/2008 of30 May 2008 (2). Preliminary test method:

Preliminary test results:

Nominal concentration A preliminary study was conducted at concentrations of 0, O.I, I, I0 and 1 OOmg/1. The duration of the preliminary study was 72 hours. There was a single replicate at each concentration. Data from the preliminary study identified the 72 hour ECso as beingbetween I and IOmg/1 by yield and I0 and IOOmg/1 by growth rate. Percent inhibition Percent inhibition

by yield integral by growth rate (m_g/1) 0-48 hours 0-72 hours 0-48 hours 0-72 hours

0.1 22 22 II 6

I 0 -24 0 -6 10 95 62 87 25 IOO IOO 88 100 53 Note: Negative inhibition values indicate an increase in growth compared with controls.

ENV9857/05I201 Page 7 of26Definitive test method: Test period: Test volume: Test vessel Number ofreplicates Test concentrations:

Test organism:

Test conditions:

Water quality measurements:
Observations/frequency:

Calculation and expression ofresults:

ENV9857/051201 From 16 to 19 July 2012 100ml 250ml conical flask. 6 control flasks, 3 replicates at eachtest concentration. After discussion with the client, definitive test concentrations were prepared as 0 (Control), 1, 3, 10, 30, 100 and 200mgll. A stock solution of200mgil was prepared and stirred well to produce a clear colourless solution. The test concentrations were prepared by addition ofthe appropriate volume ofthe stock solution using pipettors to test media. They were then made up to the mark in volumetric flasks to give the required concentrations for the test. From a pre-culture growing in exponential phase under conditions described in 2.3 above. Inoculum level calculated to give an initial cell density of 1 x 10 4 cells/mi. Temperature: Illumination: Shaking: 20.1

- 22.0°C approximately 6,500 to 8,500 lux continuous white light Orbital set to 200 rpm The pH (to 0.1) and temperature (to 0.1 °C) were measured. Water qualities determinations on pooled replicates for each test and control solution were carried out immediately prior to initiatingthe test and at the end ofthe 72 hourtest period. The cell density was determined after 24, 48 and 72 hour exposure periods on a small volume removed from each replicate flask. The cell counts were made using a haemocytometer and microscope. In determining the cell density, cells showing morphological abnormalities were included in the count. If such abnormalities were observed, they were recorded in the observations section ofthe report. Growth curves for each test concentration were plotted as logarithm of the mean cell density against time. The inhibition of growth was calculated using both the biomass (area under the growth curve) and the growth rate method. Where possible, the ECso values were estimated graphically and 95% confidence limits calculated according to the method ofToxCalcTM Version 5.0 "Comprehensive Toxicity Data Analysis and Database Software", copyright 1994- 1996(3).
Page 8 of26Analytical confirmation of exposure concentrations:

ENV9857 /051201 A method for analytical confirmation oftest concentrations was researched, firstly using the GC/FID. Concentrations of 20, 200 and 500mgll were analysed. No peak could be identified at 20mgll and the estimated limit ofdetection was found to be approximately IOOmg/1, therefore the method of analysis was deemed to be inappropriate to the study with the EC50 expected to be <IOOmg/1 and no further attempts were made to improve the performance ofthe method. Therefore a HPLC method was looked at, although a peak could be seen without the use of a column, a column could not be found that could separate the compound peak from the mobile phase peak. As a suitably sensitive method could not be found it was decided, after communication with the study monitor, to report results as nominal concentrations.

Page 9 of26

### 3. Results Cell densities were measured microscopically by direct cell counts on each test and control replicate in triplicate. Average results ofthe cell density determinations are given in 3.1. All results in this study are calculated from the measured cell densities. 3.1 Cell density measurements Mean initial cell density: Approximately 

### 1x 104 cells/m1 based upon inoculation volume, not counted microscopically. Nominal Cell density measurements (cells/ml x 104) concentration Replicate 24 hours 48 hours 72 hours (mg/1) 1 7.7 52.0 156.7 2 10.0 46.7 152.3 3 12.7 49.0 117.3 0 4 11.3 50.7 153.7 5 11.0 51.7 146.3 6 13.0 47.7 131.3 Mean 11.0 49.6 142.9 1 8.7 23.3 41.7 1 2 8.3 21.3 67.7 3 7.0 17.7 67.7 Mean 8.0 20.8 59.0 1 9.0 23.7 102.7 3 2 10.3 28.7 107.0 3 8.3 28.7 112.3 Mean 9.2 27.0 107.3 1 2.0 6.0 33.7 10 2 1.7 9.0 40.3 3 1.3 11.3 47.3 Mean 1.7 8.8 40.4 1 3.7 0.3 2.3 30 2 1.3 1.7 2.0 3 2.3 0.3 1.3 Mean 2.4 0.8 1.9 1 0.3 0.7 1.0 100 2 1.0 2.0 1.0 3 0.7 0.0 1.3 Mean 0.7 0.9 1.1 1 0.0 0.7 1.0 200 2 0.0 0.3 1.0 3 0.0 0.0 1.3 Mean 0.0 0.3 1.1 ENV9857/051201 Page 10 of263.2 Observations on cell morphology The algal cells were examined microscopically during the determination ofthe cell density, all cells appeared normal, no cells were observed that showed any morphological abnormalities. 3.3 Percentinhibition at each test concentration compared to the control Nominal Percentinhibition Percentinhibition concentration by'ield by 2rowth rate (mg/1) 0-48 hours 0-72 hours 0-48 hours 0-72 hours 1 59 59 22 18 3 46 25 16 6 10 84 72 45 26 30 100 99 100 88 100 100 100 97 98 200 100 100 100 98 Graph 1 illustrates the growth curves for the control and all test concentrations. Graphs 2 to 5 illustrate the estimation ofthe EyCx and ErCx values. 3.4 ECx and NOECvalues byyield (EyCx) and growth rate (ErCx)

Period of EyCx value ErCx value exposure (95% confidence limits) (95% confidence limits)

(hours) Ey_Cto EyC2o Ey_Cso ErCto ErC2o ErCso 0.05mgll 0.146mg!l 1.14mg/l 1.81mg/l 3.13mg!l 8.93mg!l 0 to 48 (0.000- (0.000- (0.00- (0.139- (0.516- (4.54- 0.405mg!l) 0.749mg/l) 3.25mg!l) 3.82mg/l) 5.71mg/l) 17.2mg/l) 0.101mg!l 0.293mg!l 2.24mg/l 6.76mg/l 8.85mg!l 14.8mg!l 0 to 72 (0.000- (0.000- (0.069- (2.86- (4.84- (11.2- 0.631mg/l) 1.21mgll) 6.94mg!l) 9.39mgill_ 11.7mg/1)_ 20.9mg/l) NOEC <1mg/l (0-72 hours) <1mg!l (0-72 hours) by yield by growth rate

ENV9857/051201 Page 11 of26

### 3.5 Control validity results Control growth rate data coefficient ofvariation calculations

Control Celldensity (1.0 x 104 cells/ml) replicate no. 0 hr 24 hr 48 hr 72 hr

I 1.0 7.7 52.0 I56.7 2 1.0 IO.O 46.7 I52.3 3 1.0 I2.7 49.0 II7.3 4 1.0 Il.3 50.7 I53.7 5 1.0 Il.O 51.7 I46.3 6 1.0 13.0 47.7 131.3 Mean: Standard deviation: Coefficient ofvariation: 0-72 hr average specific growth rates 1.68 1.68 1.59 1.68 1.66 1.63 1.65 0.0367

2.22 Note: 0 hr cell density based upon inoculation volume, not counted microscopically.

Control Day-by-day specific growth rates Coefficient of replicate variation no. 0-24 hr 24-48 hr 48-72 hr

I 2.04 1.9I 1.10 30.26 2 2.30 1.54 1.18 34.17 3 2.54 1.35 0.87 54.19 4 2.42 1.50 1.11 40.I2 5 2.40 1.55 1.04 41.31 6 2.56 1.30 1.01 50.76 Coefficient ofvariation: 22.I4

ENV9857/051201 Page 12 of26

### 4. Discussion The EC50 values (by yield) of Hexahydro-1,3,5,-tris(2-hydroethyl)s-triazine to Pseudokirchneriella subcapitata were estimated as 1.14mgll at 48 hours and 2.24mg/l at 72 hours. The EC50 value (by growth rate) was estimated as 8.93mgll from 0 to 48 hours and 14.8mg/l from 0 to 72 hours. The NOECr (0-72h) (growth rate) was estimated as <lmg/1. The NOECy (0-72h) (yield) was estimated as <lmg/1. Both were determined by the Bonferroni T test. The algal cells were examined microscopically during the determination ofthe cell concentration, all cells appeared normal, no cells were observed that showed any morphological abnormalities. The full water quality measurements are given in the summary tables in Appendix 3 and were within acceptable limits. The cell density ofthe control replicates increased by a factor of 143, the coefficient ofvariation for the control replicates was less than 35% for each ofthe 24 hour time periods and less than 7% for the whole time period, 0- 72 hours. This fulfils the validity criteria ofthe study and it is therefore compliant with the OECD Guideline for Testing of Chemicals reference 201 Alga, Freshwater Alga and Cyanobacteria, Growth Inhibition Test 2011 guideline. There were no deviations to the protocol that affected the results ofthe study. There were a number of deviations from the protocol; the temperature during the culturing period was low, the temperature during the test period was low and the light was high. As the control validity criteria were met it does not appearthat these deviations were significant.


### 5. References 1. OECD Guideline for Testing of Chemicals reference 201 Alga, Freshwater Alga and Cyanobacteria, Growth Inhibition Testreference 201(2011). 2. Council Regulation (EC) No 440/2008 of30 May 2008. 3. ToxCalcTM Version 5.0 "Comprehensive Toxicity Data Analysis and Database Software", copyright 1994-1996.

ENV9857 /051201 Page 13 of26

### Graph 1 Growth curves for control and test concentrations

16.00 

### ::- 

# e 14.00**~** 

# -**~** CJ 

### 12.00 

# -**~** 

# ·-**~** 

# = 10.00**~** "0 

# - 

# -**~** CJ 

### 8.00 

# = 

# =**~** 

# = 6.00 

## ""' Q CIJ 

# .s 4.00 

### -; a. 

# = .... 2.00 

# = z 0.00

Statistical data \ ,£3".

# .- I \ I \ I \ I \ I \ I 

### 0 24 48 Exposure period (hours)

Control



### _ ... _ 10.0

- {3- 

### 200.0 Concentration in mg/1
• 1.0 

### --30.0 --3.0 • 100.0

NOECy (yield)= <lmg/1 NOECr (growth rate) = <lmg/l As determined by the Bonferroni T test, based on the 0-72 hour values.

ENV9857 /05120I Page 14 of26 72

### Graph 2 Estimation of0 to 48 hours 

### EyCx values
1.0 0.9 0.8 0.7 

# = .s 0.6 

### .... 

## :c := 

# = 

# ... '; 

### 0.5 

# = Q 

## t Q 

### 0.4 c. Q 

## ...**~** 

### 0.3 0.2 0.1 0.0 0.000001 0.0001 0.01 100 

### 10000 Concentration mg/L

Indicates 95% confidence limits +-t

### --+• Indicates averaged dataplot Statistical data 

### EyC 10 value 

### EyC2o value 

### EyCso value

ENV9857/051201 0.050mgll (0.00 to 0.405) 0.146mgll (0.00 to 0.749) 1.14mg/l (0.00 to 3.25) (as determined by the Maximum Likelihood-Probit Method)

Page 15 of26

### Graph 3 Estimation of0 to 72 hours 

### EyCx values
1.0 0.9 0.8 0.7 

## = .s 

# ... 0.6 

## :c :a 

## = .... -; 0.5 

## = .s t: 0.4 

# = c. 

# = 

# .. =- 0.3 0.2 0.1 0.0

0.0001 0.001 

### O.ot 0.1 10 100 1000 10000 Concentration mg/L
Indicates 95% confidence limits +-•

## --+• Indicates averaged data plot Statistical data EyCIO value EyC2o value EyCso value

ENV9857/051201 0.10Img/1 (0.00 to 0.631) 0.293mgll (0.00 to 1.21) 2.24mg/l (0.069 to 6.94) (as determined by the Maximum Likelihood-ProbitMethod)

Page 16 of26

### Graph 4 Estimation of0 to 48 hours ErCx values
1.0 

# • 0.9 0.8 0.7 

## = .s 

# .. 0.6 

## :c 

# := = .... -; 0.5 

## = Q 

## t 0.4 Q c. Q 

## "'**~** 

### 0.3 0.2 0.1 0.0 0.01 0.1 

### 10 100 1000 Concentration mg/L
Indicates 95% confidence limits +-•--+• Indicates averaged data plot Statistical data 

### ErCJO value 

### ErC2o value 

### ErCso value

ENV9857/051201 1.81mg/l (0.139 to 3.82) 3.13mg/l (0.516 to 5.71) 8.93mg/l (4.54 to 17.2) (as determined by the Maximum Likelihood-Probit Method)

Page 17 of26

### Graph 5 Estimation of0 to 72 hourErCx values
1.0 

# • • 0.9 0.8 0.7 

## = .5! 0.6 

### .... 

## :c 

# := = 

# ... -; 0.5 

## = Q 

## t: Q 0.4 c. Q 

### ""'**~** 0.3 0.2 0.1 0.00.1 10 100 1000 Concentration mg/L
Indicates 95% confidence limits 

# ••---+• Indicates averaged data plot Statistical data ErCIO value ErC2o value ErCso value

ENV9857/051201 6.76mg/l (2.86 to 9.39) 8.85mgll (4.84 to 11.7) 14.8mgll (11.2 to 20.9) (as determined by the Maximum Likelihood-Probit Method)

Page 18 of26

### Appendix 1 Analytical confirmation ofexposure concentrations GC/FID method development: On 22 May 2012 a calibration standard of200mg/l was prepared by adding 0.0200g ofHexahydro-1,3,5,-tris(2-hydroethyl)s-triazine to 100ml ofwater. This was analysed used on the following dates and conditions using the GC/FID 23rd and 241hMay 2012 Phenomenex Chromatography Capillary Column: Zebron ZB5 (column phase: 5%-phenyl-95%-dimethylpolysiloxane) PartNo: 7HM-C002-22 30m x 1.0um film thickness x 0.32mm Internal Diameter. Various different temperature programs were attempted to see where the compound eluted from the system. The peak was not observed. 25th May 2012 J & W Chromatography Megabore Capillary Column: DB-624 (column phase: 6% Cyanopropylphenyl-94%-dimethylpolysiloxane) PartNo: 125-1334 30m x 3.0um film thickness 0.53mm internal Diameter. Carriergas: Nitrogen Detector: Flame Ionisation Detector (FID) Injectionporttemperature: 250 C Detector temperature: 300 C Column temperature: JOOC isothermal Total analysis time: 6 minutes Sample volume: lui (DirectAqueousInjection) A peak believed to be Hexahydro-1,3,5,-tris(2-hydroethyl)s-triazine was observed and successfully resolved from the solvent front. Peak eluted after approximately 1.1 minutes. From this point it was decided that this was to be the best capillary column for validation purposes. The peak appeared to be reproducible in terms ofretentiontime and peak intensity.
In order to determine that the peak identified was the sample, two more calibration standards were prepared on the 28 May 2012, a 503mg/l standard was prepared by 0.0503g ofHexahydro1,3,5,-tris(2-hydroethyl)s-triazine being added to 1 OOml ofdeionised water, a 20mg/l standard was prepared by adding 4ml ofthe 503mg/l stock to 1 OOml ofdeionised water. 11th June 2012 Using the conditions above (25th May 2012). The calibration standard (@20mg/l) was analysed to determine the approximate limit of detection. No peak was observed. The two further
ENV9857/051201 Page 19 of26standards of200mg/l and 500mgll were analysed. On each occasion peaks were observed. (with peak areas consistent withthe concentration ofthe standard). With an estimated limit of detection at approximately IOOmgll, the method of analysis was deemed to be inappropriate to the study and no further attempts were made to improve the performance ofthe method. HPLC method development: On I2 June 20I2 a stock solution of IOOmg/l was prepared by 0.0IOOg of Hexahydro-I,3,5,-tris(2-hydroethyi)s-triazine being added to IOOml of deionised water. 25 June 2012 Used a cyano column in normal phase with a mobile phase of 45:4:I (hexane:DCM:H20) at 225nm isocratic 1 OOmgll Triazine identical to water result (one small peak at about 3 mins) 26 June 2012 Used short CIS column (150mm) with 3:I MeCN:H2

### 0 at I95nm IOOmgll Triazine similar to water result with some small additional unresolved peaks at around 1.5 mins 27 June I2 I95nm 3:I MeCN:H2

### 0 I) Chiral column Some small peaks co-eluting with water peaks. Retention times less than 2 mins 2) SAX column Some small chromatography at around 5 minutes 3) No column Sample and solvent both eluted very quickly with sample peak about 5 times bigger in height (decent sized peak) 4) NH2 column Some small peaks with extra small sample peak at about 3.5 mins 5) Phenyl column

ENV9857/05I20I Page 20 of26No difference between sample and solvent chromatography with two small peaks noted at around 1.2 and 2.5 mins On 28 June 2012 a fresh stock solution was prepared by adding 0.0105g ofHexahydro-1,3,5,- tris(2-hydroethyl)s-triazine to 1 OOml ofdeionised water. 28 June 2012 195nm 3:1 MeCN:H20 1) Different Cyano column Some small peaks in both acetonitrile and sample- small peaks at 2.6 and 3.7 mins 2) Nocolumn Peak area of 100mg/l triazine is about 11 times bigger than acetonitrile (1188297 and 105435). Eluted straight away 3) Anothercyano column Small sample peak at about 0.3 mins 4) Anotherp}1enyl column Sample and acetonitrile both hardly retained (0.25 mins)- no separation 5) Another C18 column Very small peaks for both sample and acetonitrile- no difference 29 June 2012
1) Gemini-NX C18 195nm Reasonably busy chromatograms with some small unresolved peaks eluting around 1.2 to 1.6 minutes. 2) Gemini-NX C18 225nm Not as busy at this wavelength with larger unresolved sample peaks at 1.3 and 1.6 mins (still too small) 3) Hypersil C12 195nm Some small number ofpeaks with a small additional resolved sample peak at 0.6mins
ENV9857/051201 Page 21 of264) Gemini-NX Cl8 225nm Similarto 195nm with a small resolved sample peak at 0.6 mins 3 July 2012 Gradient method with 15min gradient from 100% H2 0 to 100% MeCN, return to 100% H20 over a minute and equilibration for a further 4 minutes. 225nm with a very short C18 column. Busy baseline with additional, random, peaks. Sorted out random peaks but no peaks noted for sample which weren't in acetonitrile. In all this work, whilst some small peaks were noted for the various column/mobile combinations, they were too small as this was for a IOOmg/1 standard. When the column was removed, a much larger peak was noted (even after removing the solvent area) but the peak or peaks noted with all the various attempts were not as large suggesting that the sample was not being eluted (ie retained by the various columns).

ENV9857/051201 Page 22 of26Culture medium: Dilution water:
Method ofpreparation: Composition ofculture medium:

ENV9857/051201 

### Appendix 2 Culture media According to OECD guidelines. De-ionised, filtered and sterilised by autoclaving at l20°C for 15 minutes. Sterile nutrient stock solutions were prepared and added to the dilution waterto obtain the culture medium. Nutrient Final concentration in culture medium (mWJ) NRtCl 15 MgCh.6H20 12 CaCh.2H20 18 MgS04.?H20 15 KH2P04 1.6 FeCh.6H20 0.064 Na2EDTA.2H20 0.1 H3B03 0.185 MnChAH20 0.415 ZnCh 0.003 CoCh.6H20 0.0015 CuCh2H20 

### 10-5 Na2Mo04.2H20 0.007 NaHC03 50

Page 23 of26

### Appendix 3 Waterquality data
Nominal pH Temperature concentration 

### (OC) (mg/1) Startoftest End oftest 0 Hours 24 48 72 

### Hours Hours Hours 

### 0 7.8 7.3 I 

### 7.8 7.3 3 8.1 7.2 10 8.7 7.3 21.1 20.3 20.3 20.4 30 9.2 7.4 100 9.7 7.7 200 10.0 7.8 Note: Water qualities determinations on pooled replicates for each test and control solution were carried out immediately priorto initiating the test and at the end ofthe 72 hourtest period.



### ENV9857/051201 Page 24 of26

### Appendix4 Reference test This section summarises aquatic toxicity test results obtained by Chemex International Environmental Ltd. on a sample as detailed below: Test commissioned by: Chemex Environmental International Ltd Substance undertest: Potassium Dichromate Chemex reference: Test species: Test type: Test conditions:

Testperiod: Test carried out at:

Results:

Period of exposure

(hours) Oto48
0 to 72 NOEC Historical Data: Mean:
Standard deviation

ENV9857 /051201 Sample: Study: EC0110706 ENV9820 Pseudokirchneriellasubcapitata, strain CCAP 278/4 Growth inhibition: 72 hour EC50 Test conditions according to SOP E203 based upon OECD Guideline for testing of chemicals, Alga Growth Inhibition Test reference 201 (2006)Ol. Council Regulation (EC) No 440/2008 of30May 2008(2). From 5 to 8 March 2012 Chemex Environmental International Limited UnitJ Broad Lane Industrial Estate Cottenham Cambridge CB24 8SW UK ECx and NOEC values by yield (EyCx) and growthrate (ErCx) 

### ErCx value 95% confidence limits

EyCso (0 to 72 hours) 0.53mg/l ErCso (0 to 72 hours) 0.86mg/l EyCso (0 to 72 hours) 0.11 ErCso (0 to 72 hours) 0.17 Page 25 of26

### Appendix 5 GLP Certificate


## f

# o-;;, Department 

## C ofHealth

THEDEPARTMENTOFREALmOFTHEGOVERNMENT OFTHEUNITED KINGDOM GOOD LABORATORYPRACI'ICE STATEMENTOFCOMPLIANCE INACCOitDANCEWITH DIRI'.CTIVE**~** TESTFACILITY**a.-.~**

### ............Ltd UllltJ

..... ._...........l:lale ()!tteettam Culbl. CBJ48SW

DATEOFINSI'I!C110N 25N.-ber2t10

A general iaspection for compliance with the Principles ofGood Labol'lllory Practice was carried out atthe above test facility aspanoftheUK GLPCompliance Programme. AI the time ofinspection no deviations were found ofsufficient magnitude to affect the validity ofIJOD.Ciinical studies performedat these facilities
.

Dr.AlldrewJ,c..., H.d,UK GLPMeaitoriJI&Alldlorily 

# •

ENV9857/051201 

### Page 26 of26

# Chemical Corporation A Subsidi•ry of ICC lndustrlfl Inc West Fifteenth &Davis •

### _e; ' ! 19 • Dover, Ohio 44622-0040