# .**~ALBEMARLE®** Albemarle Corporation 451 Florida Street Telephone: 225-388-8011 Baton Rouge, Louisiana 70801-1765 Facsimile: 225-388-7686

June 29, 2012

VIA OVERNIGHT MAIL

Document Control Office (7 407M) Office of Pollution Prevention and Toxics (OPPT) Environmental Protection Agency 1201 Constitution Ave., N.W. Washington, DC 20004-3302 

### 2 o(J 

### 7- 6 .5" 

### S J

Attn: TSCA Section 4

# P1~t:b737**~,....-,.** 

# ·-o d.:. 1 " C. I h:

.. 

### OPPT CBiC



## 2012 JUL -6 AM II: 

## 00

In compliance with the final rule, "Testing of Certain High Production Volume Chemicals; Second Group of Chemicals", 76FR 1067, January 17, 2011, Albemarle Corporation submits these Studies to meet the end points listed below for "Phenol, 4,4'-methylenebis[2,6-bis(1, 1- dimethylethyl)]-", CAS number 118-82-1.

One paper copy and one electronic copy are provided for each study. Robust summaries of submissions for each end point are also attached.

C1 -Aquatic Toxicity Test Group 2 -Chronic Toxicity to Daphnia -Toxicity to Plants (Algae)

The submission of these studies completes all the testing requirements under the test rule for this substance. Please contact me at 225-388-7776 if you have any questions.

Sincerely,

# ~':~---" Regulatory Advisor

cc: Paul Campanella (w/o attachments) Chemical Control Division (7 405M) Office of Pollution Prevention and Toxics Environmental Protection Agency 1200 Pennsylvania Ave., N.W. Washington, DC 20460-0001

1111111111111111111111111111111111111111111111111111111111111111 4 0 1 2 0 0 0 0 0 7 7



### CONTAINS NO 

## CBt"":X:w:r.Jww0.zw0.uwa:FecExNEWS®1 1SAirbi/1ExpresU•899669740992IIIII11114ExpressPat.Einpt21151005JUL12DCAA515Cl/E052/AA44Datefo/7CjjILTrackingNumber8 9 9 6 6 9 7 4 D 9 9 2NOTE:Serviceorderhaschanged.Pleaseselectcarefully.....-ExpressFnighrUSAirbi/1.Sender'sNameiE'{·'··,·.,.j:,(/-.I!,FedExFirstOvernightr""V')/0,,ft;f(·)?lt\(Phone)JSJ6-·J•..,·NextBusinessDaycompanyi\L..BEM/18L_r:.:CDF:F'em/1T I0\'.J/G.FI'·SSEf{\/:r;-,4PriorityOvernight*Fndayshlpmentsw,llbedeliven1donMondayunlessSATURDAYDel•very1sselectedAddress4lFLlJRTF'L16:[JFedExStandardOvernightNextbustnessafternoon•SaturdayDellveryNOTavallable2or3BusinessDays0SaturdayDellveryNOTava,lablenFedEx2Day'LJSecondbusmessaflernoon•ThursdayshlpmemsW1llbedeliveredonMondaylmlessSATURDAYDehvl!l"l/tsselected11FedExExpressSaverL__Th,rdbusmessday."SaturdayDehveryNOTavarlableCity·,TtJr-.1\StateLf-'.ZIP701:30l--1?OQ..•;,••'"·Envelope*FedExPak*FedExFedEx,-ther?<2YourInternalBrlhngReference.Box'Tube8:33ToRecipient'sName__··(,,;,,J:•,1.·"6.••1"1,tf1/(1I'·Phone' ).,..··{i(·1:>SATURDAYDel1very•('1,1"1+('·-·2w'LiNOTI!vailablelorFedb:Standard0vern,ght.FedEx20ayA.M.,orFedExExpressSaver)I})-Company/·fti/!')NoSignatureQ·Packagemaybelettwlthoutobtam1ngaSignaturefordel1veryc-DirectSignaturemay'!l'gn'fofde1JveryfHapplits.HOLDWeekdayFedExlocatiOnaddressDoes!hisshipmen!containdangerousg_oods?Address;'forOneboxmu"stbechecked.-----IndirectSiqnatureaddress.someoneatane1ghbonngaddressmaySIQrlfordeiiVery.ForFHapplin.Dept)Fioor/Su,te/RoomHOLDSaturdayFecl&locanonaddressil=1,YesI'Jr'iAsperattached·!IShipper's.Oe.claratlo!l)""vShipper'sDeclaratlOn'----;J·01845kgI,c;n,I·l!j()/l·'l\•,f{State!(__ZIPL111111111118996'69740992DREQUIREDAvailableONLYfotFed&Pnomy0verr11ghtandFedEx20aytoselectlocanonsDangerousgoodsl1nclud1ngdry1ceJcannotbemFedExpackagongorplacedmaFedExExpressDropBoxCargoAircraftOnly/''··'("..,-,.aymentBillto:...,L7p'-..,,-EnterFedExAcct:No..orCardNo.below.---l0CreditCard451"7;,?li.Coc:>c:>GoCoc:>c:>:,..,wwww

### Robust Summary

Study Title: "AN-2: A Flow-through Life-Cycle Toxicity Test with the Cladoceran (Daphnia magna)"

Conclusion: The cladoceran, Daphnia magna, was exposed to test article (AN-2) at

mean measured concentrations of 1.4, 2.4, 5.5, 12, 21, and 43 ng/L under flow-through conditions for 21 days. There were no statistically significant treatment-related effects

on survival, reproduction or growth at concentrations equal or less than 43 giL. In addition, the mean length of the daphnids in the 2. 4, 5. 5, 12, and 21 ng/L groups were within the ranges of the mean length of daphnids in the historical control data (3. 6 to 6. 3 nm). Consequently, the NOEC for growth (length and dry weight, reproduction and survival was 43 ng/L and the LOEC was greater than 43 ng/L.

Details:

Study Number: Project 471A-120

Study Authors: Tui Minderhout, T.S. Kendall, S.P. Gallagher, H.O. Kreuger

Study Laboratory: Wildlife International, Ltd.

Study Report Date: June 21,2012 In life Dates: March 19-April9, 2012. Dry weights collected April 11, 2012

Study Sponsor: Albemarle Corporation

Guideline: U.S. EPA OPPTS No. 850.1300 OECD Guideline 211 ASTM Standard E 1193-97

Test Substance: C 14 radio labelled test substance prepared by Quotient Bioresearch "4,4' -Methylenebis (2,6-di-tert-butyl[U-14C]phenol)" Batch CFQ41115 Specific Activity: 129 uCi/mg or 55 mCi/mmol, based on gravimetric analysis, and a radiochemical concentration of 968 uCi/mL

Study Organism: Daphnia magna
Organism Source: Wildlife International, Ltd. Easton, Maryland

Age of Test Organism: Neonates ( < 24 hours old at initiation)



### CONTAINS NO CBIDilution water: Moderately hard well water from a well on-site. Water was passed through a sand filter and pumped into 3 7,800 L storage tank. Prior to use, water

was filtered to 0.45 urn to remove fine particles and passed through an ultraviolet sterilizer. Specific conductance, hardness, alkalinity and pH of well water were monitored during the 4 week period prior to the study. Periodic analysis of the well water for selected organic and inorganic constituents was conducted.

Test Apparatus: An exposure system consisting of a continuous flow diluter was used to deliver test concentrations and controls to the test chambers. Syringe pumps delivered the test stock solutions or solvent to impartially assigned mixing chambers where mixing with dilution water occurred. Flow of dilution water was controlled with rotameters and adjusted to deliver about 9 volume additions of test water in each test chamber per day. After mixing, flow from each mixing chamber was split to deliver test solution to two replicate test chambers. Syringe
pumps were calibrated prior to the test, rotameters calibrated prior to test and verified weekly. Test flow to each replicate chamber was checked prior to test and weekly to ensure rates did not differ by +/- 10% of the mean flow for the two replicates. Delivery system and test chambers were placed in a temperature controlled environmental chamber. Test chambers were 9 L glass aquaria filled with about 8 liters of test water. Test compartments were 300 mL glass beakers, with nylon mesh screens covering two holes on opposite sides of the test chamber
to allow test solution to flow in and out of the compartment.

Test Concentrations:

Nominal Mean Measured Negative control <LOQ Solvent Control <LOQ 1.6 ng/1 1.4 ng/1 3.1 ng/1 2.4 ng/1 6.3 ng/1 5.5 ng/1 13 ng/1 12 ng/1 25 ng/1 21 ng/1 50 ng/1 43 ng/1

Preparation of Test Concentrations: Individual stock solutions for each of the six concentrations were prepared once during the study. Each stock solution was prepared by mixing a calculated amount of radio labelled test article into HPLCgrade dimethylformamide (DMF) at a nominal concentration of 0.064, 0.124, 0.252, 0.52, 1.0, and 2.0 ug/mL. Solutions were inverted to mix. Stock solutions
up to 1.0 ug/mL were clear, and the 2.0 ug/mL solution had a slight purple tint. Stock solutions were stored refrigerated in amber bottles, and aliquots placed in the syringe pump every 4 to 7 days as needed during the study. The stock solutions were injected into the diluter mixing chambers at a rate of2.5 uL/minute, where they were mixed with dilution water delivered at a rate of 100mL/minute to achieve the desired test concentrations. Negative control was dilution water only. Solvent control was prepared by delivering HPLC-grade DMF to the appropriate mixing chamber. The concentration ofDMF in the solvent control and all treatment groups was 0.025 mLIL.

Analytical Sampling and Method: Samples were collected from each treatment group and controls five days prior to the start of the test. Water samples were also collected from alternating replicate test chambers in each group at the beginning of the test, at weekly intervals during the test and at the end of the test to measure test substance concentration. Samples ofthe stock solutions being delivered to the diluters were collected on Day -11 to confirm stock concentrations. Test solutions were also collected on Day 22 to confirm the concentration of the 1.6 ng/L treatment level when the analytical data on Day 21 for this group was suspect. Radioactive stock solutions were transferred to scintillation vials and analyzed by liquid scintillation counting (LSC), using a Packard Liquid Scintillation Analyzer. Test waters were extracted twice with dichloromethane, evaporated to dryness, and reconstituted with acetonitrile. Extracts were then analyzed by LSC.

Environmental Conditions: Ambient light was supplied by fluorescent light bulbs that emitted wavelengths similar to sunlight for a photoperiod of 16 hours light and 8 hours darkness. Light intensity was measured at the water surface of one representative chamber at test initiation. Target test temperature was 20 +/-degrees C. Temperature was measured in each test chamber at beginning oftest, weekly during test, and at end oftest using liquid-in-glass thermometer. Temperature was also monitored continuously in one negative control test chamber using a Fulscope ERIC recorder. The recorder was verified prior to test initiation and weekly during the test using a liquid-in-glass thermometer. Dissolved oxygen was measured in one replicate of each treatment and control at beginning oftest, about 3 times weekly during the test and at end. Measurement of pH was made in one replicate of each group at beginning of test, about weekly during the test and at the end. Hardness, alkalinity and specific conductance were measured in alternating replicates of the negative control and the highest test concentration at beginning oftest, weekly during test and at end of
test. Total organic carbon was measured in dilution water at beginning and end of
test.

Length of Study: 21 days

Experimental Design: Daphnids were exposed to a geometric series of 6 test concentrations, a negative control (dilution water) or a solvent control (0.025 ml/L HPLC-grade dimethyl formamide). Two replicate test chambers were tested for each treatment and controlgroup. Each replicate contained two compartments with five daphnids for a total of 20 daphnids in each treatment and control group. Nominal concentrations were selected based on exploratory range finding data and solubility limit in the test system. Nominal concentrations were 1.6, 3.1, 6.3, 13, 25, and 50 ng test article per liter. Mean measured concentrations were determined from samples of test water collected from each treatment and control group at study initiation, at approximately weekly intervals, and at test termination. Test substance delivery to the test chambers was initiated seven days prior to introduction of the daphnids in order to achieve equilibrium of the test substance in the chambers. At test initiation, neonate daphnids were randomly assigned to test chambers. First-generation daphnids were observed daily for mortality, onset of reproduction, and clinical signs of toxicity. Following onset of reproduction, numbers of second-generation daphnids were counted three times weekly and at test termination. Body lengths and dry weights of surviving first-generation daphnids were measured at the end of the exposure period. Test article effects on survival, reproduction and growth were used to determine no-observed-effect-concentration (NOEC), the lowest-observed-effect concentration (LOEC), and the maximum acceptable toxicant concentration. EC50 values were based

on reproduction, and on first-generation immobility at the test termination.

Experimental conditions: Adult daphnids were cultured in water from same source and temperature as during the test. The six adult daphnids used to supply the neonates were held at least 17 days prior to the test, and had each produced at least one previous brood. Over the 7 day period just prior to the test, the adults had produced an average of at least 3 young per adult per day. No signs of disease, stress, or ephippia were observed during the holding period. At test initiation, juvenile daphnids were collected from the cultures and randomly transferred one or two at a time to test chambers using wide-bore pipettes. During culture and testing, daphnids were fed a mixture of yeast, cereal grass media, and trout chow (YCT), as well as a suspension of the freshwater green algae (Pseudokirchneriella subcapitata). Daphnids were fed two or three times per day through Day 7 of test and then four times daily until the end of the test. At each feeding, each test chamber was fed 0.75 mL ofYCT and 1.5 mL of algae, amounting to about 0.7 mg C/daphnid/day. This exceeds the OECD guideline, but was fed in order to maintain sufficient feed in the flow-through system to support acceptable reproduction rates. Biological observations of each first-generation daphnid were made daily during these tests. Numbers of immobile (lack of movement except for minor spontaneous random movement of appendages) daphnids and signs of clinical toxicity (inability to maintain body position in water column, uncoordinated swimming, cessation of feeding)

were recorded. Presence of eggs in the brood pouch, aborted eggs, males or epihippia

were recorded daily. With onset of reproduction, number of neonates produced were counted and then discarded every Monday, Wednesday and Friday of the test. At the end of the test, body length and dry weight of each surviving first-generation daphnid were measured.

Statistical Analysis: Test endpoints analyzed statistically for first-generation daphnids

were survival, reproduction and growth (length and dry weight). Reproduction was basedon number of live young produced per reproductive day. Negative control and solvent control data for each parameter were compared with appropriate statistical tests. No significant differences were seen between the control groups; therefore the control groups data were combined.

Survival data was considered a discrete variable, whereas reproduction and growth were considered continuous. Discrete variable data were analyzed using Chi-Square and Fisher's Exact test to identify treatment groups that showed a statistically significant (p</= 0.05) difference from control. All continuous data were evaluated for normality using Shapiro-Wilk's and for homogeneity of variance using Levene's (a= 0.01). When data passed the assumptions of normality and homogeneity, those treatments that were significantly different from control means were identified using Dunnett's test (a= 0.05).

Results of the statistical analysis were used to aid in determine the NOEC, LOEC, and MATC. Scientific judgement was also used to determine if statistical differences were biologically meaningful, and if the data showed a concentration-dependent relationship.Results: Based on mean measured concentrations

No-Observed- Effect-Concentration 43 ng/1 Lowest -0 bserved Effect-Concentration 

### > 43 ng/1 Maximum Acceptable Toxicant Concentration 

### > 43 ng/1 21-Day EC50 (Immobility) 

### > 43 ng/1 95% Confidence Level Not calculated 21-Day EC50 (Reproduction) 

### > 43 ng/1 95% Confidence Level Not Calculated

Sumary of Survival, Reproduction, and Growth of Daphnia magna exposed to R d' 1 b 11 dAN 2 fi 21 d

awaee 

# - or ays Mean Measured %Adult Mean Neonates Mean Length Mean Dry Weight

Concentration 

## s . 112 +/- STD +/- STD 1 urv1va · per (ng/L) Reproductive Day+/- STD 1

' 3 (mm) (mg)

Negative Control 75 8.1 +/-0.53 4.6 +/- 0.06 0.63 +/- 0.11

Solvent Control 95 8.7 +/- 1.3 4.5 +/- 0.05 0.69 +/- 0.033 Pooled Control 85 8.4 +/- 1.0 4.5 +/- 0.05 0.66 +/- 0.074 1.4 ng/1 90 8.9 +/- 1.2 4.6 +/- 0.06 0.76 +/- 0.10 2.4 ng/1 90 6.7+/-1.1 4.3 +/- 0.10 ' 4 0.60 +/- 0.013 5.5 ng/1 100 8.1 +/- 0.74 4.4 +/- 0.08 ' 4 0.74 +/- 0.046 12 ng/1 95 6.9 +/- 1.1 4.4 +/- 0.00 ' 4 0.76 +/- 0.095 21 ng/1 90 7.7 +/- 1.7 4.4 +/- 0.05 ' 4 0.63 +/- 0.055 43 ng/1 90 8.3 +/- 1.6 4.5 +/- 0.13 0.60 +/- 0.039 

### * Statistically significant difference in mean total from pooled control (Dunnett's one-tailed test, p</=0.05)
1 No significant difference in survival (Fisher's Exact Test, p > 0.05), reproduction or mean dry weight (Dunnett's one-tailed test, p > 0.05)
2 21-day EC50 survival:> 43 ng/L
3 21-day EC50 for reproduction: > 43 ng/L
4 Statistically significant differences in mean total length noted at 2.4, 5.5, 12 and 21 ng!L not considered biologically significant

### Robust Summary

Study Title: "AN-2: A 96-Hour Toxicity Test with the Freshwater Alga (Pseudokirchneriella subcapitata)"

Conclusion: The freshwater alga (Pseudokirchneriella subcapitata), was exposed to a geometric series of five concentrations of test article (AN-2) ranging/rom 1.50 to 26.5 ng/L (based on day 0 measured test concentration). There were no statistically significant treatment-related effects on cell density, yield and growth rate at concentrations equal or less than 26.5 ng/L. Consequently, the 72 and 96 hour NOECs for cell density, yield and growth rate were 26.5 ng/1 (the highest concentration tested). EC50 values at 72 hours for the same effects were all greater than 26.5 ng!L.

Details:

Study Number: Project 471A-119

Study Authors: J.R. Arnie, J.R. Porch, T.Z. Kendall, H.O. Kreuger Study Laboratory: Wildlife International, Ltd.

Study Report Date: June 26, 2012

In life Dates: February 20-24, 2012. Cell counts completed February 27, 2012

Study Sponsor: Albemarle Corporation

Guideline: U.S. EPA OPPTS No. 850.5400 OECD Guideline 201 ASTM Standard E 1218-04

Test Substance: C 14 radiolabelled test substance prepared by Quotient Bioresearch "4,4 '-Methylenebis (2,6-di-tert-butyl[U-14C]phenol) Batch CFQ41115 Specific Activity: 129 uCi/mg or 55 mCi/mmol, based on gravimetric analysis, and a radiochemical concentration of 968 uCi/mL

Study Organism: Pseudokirchneriella subcapitaa

Organism Source: Originally University of Toronto Culture collection, and maintained in culture medium at Wildlife International, Ltd. Easton, MarylandAge of Test Organism: Cells had been growing in culture medium at least 2 weeks prior
to test. Cells for this study were taken from culture that had been transferred to fresh media 3 days prior to test initiation.

Culture medium water: Freshwater AAP medium was used. Stock nutrient solutions

were prepared by adding reagent grade or better components to purified Wildlife International Laboratory well water. This was moderately hard well water from a well on site. Water was passed through a sand filter and pumped into 37,800 L storage tank. Prior to use, water was filtered to 0.45 urn to remove fine particles and passed through an ultraviolet sterilizer. Periodic analysis of the well water for selected organic and inorganic constituents was conducted. pH ofthe medium was 7.5 

### +/- 0.1; adjustment where necessary was with 10% hydrochloric acid. Medium was sterilized by filtration (0.22 urn).

Test Apparatus: Test chambers were sterile 250 mL Erlenmeyer flasks, with foam stoppers and contained 100 ml of solution. Test chambers were randomly placed in position on one or more mechanical shaking tables in an environmental chamber and

were shaken continuously at about 100 rpm.

Test Concentrations:

Nominal Mean Measured Negative control <LOQ Solvent Control <LOQ 1.99 ng/L 1.50 ng/L 3.99 ng/L 3.35 ng/L 7.98 ng/L 6.84 ng/L 16.0 ng/L 12.9 ng/L 31.9 ng/L 26.5 ng/L

Preparation of Test Concentrations: A primary stock solution was prepared by mixing 0.213 mL of radio labelled test article into 50 ml HPLC-grade dimethylformamide (DMF)
to achieve a nominal concentration of 32.0 ug/mL. The solution was inverted to mix and the resulting solution had a clear purple tint. A secondary stock solution was made by diluting 1.0 mL of the primary stock solution into 100 mL DMF to achieve a nominal concentration of 0.319 ug/mL. Additional stock solutions were prepared at nominal concentrations of0.16, 0.08, 0.04, and 0.02 ug/mL in DMF by serial dilution. The nominal test solutions of2.0, 4.0, 8.0, 16.0 and 32.0 ng/L were prepared by diluting 100 ul of each respective stock solution in 1000 mL freshwater AAP medium. Solvent control solution was prepared by diluting 100 ul DMF in 1000 mL freshwater AAP medium to achieve a nominal concentration of 0.1 mL DMF per liter. The negative control solution was freshwater AAP algal medium without solvent or test article added.

Analytical Sampling and Method: Samples were collected from each treatment group and controls at the beginning of the test and at 96 hours. Samples at initiation

were from individual batches of test solution prepared for each treatment andcontrol group after distribution to test chambers. At termination, samples were collected from pooled replicates of each treatment and control. Samples were processed immediately for liquid scintillation counting. Radioactive stock solutions were transferred to scintillation vials and analyzed by liquid scintillation counting (LSC), using a Packard Liquid Scintillation Analyzer. Test waters were extracted twice with dichloromethane, evaporated to dryness, and reconstituted with acetonitrile. Extracts were then analyzed by LSC.

Environmental Conditions: Ambient light was supplied by continuous fluorescent lighting (cool white light tubes). Light intensity was measured at five sites on the shaker table at test initiation. Target light intensity was 6,000 lux+/- 10%. Target test temperature was 24 +/- 2 degrees C. Temperature was measured twice daily in a container of water placed adjacent to the test chambers using a hand-held liquid in glass thermometer. Temperature was also monitored continuously in the environmental chamber using a minimum/maximum thermometer. Measurement of pH was made in each group and control at beginning of test, and from samples taken from each of the treatment and control groups at termination, and pooled by treatment at the end of test.

Length of Study: 96 hours

Conditions for a valid study: 1) Mean Cell Density in control replicates must have increased by a factor of at least 16 within 3 days and at least 1 00 within 4 days 2) Coefficient of variation of the average specific growth rate in the control replicates during the first 72 hours should not exceed 7%. 3) Mean percent coefficient of variation for the section-by-section specific growth rats (days 0-1, 1-2, and 2-3) in the control replicates must not exceed 35%

Experimental Design: Algal cells were exposed to a geometric series of five test concentrations, a negative control (dilution water) or a solvent control (0.1 ml/L HPLC-grade dimethylformamide ). Three replicate test chambers were tested for each treatment group and six replicates for the negative control group. At test initiation, an inoculum of algal cells was added to each test chamber to achieve a nominal concentration of about 10,000 cells/mL. Prior to test initiation, concentration of algal cells in the stock culture was determined using a hemacytometer and microscope. Density of stock culture was 7.35 x 10 5 cells/mL. To achieve the desired initial cell density in test chambers, 1.36 mL of culture was added to each chamber.

Nominal concentrations were selected based on exploratory range finding data and solubility limit in the test system. Nominal concentrations were 2.0, 4.0, 8.0, 16.0 and 32 nanograms per liter (ng/L) oftest article. Mean measured concentrations were determined from samples of test water collected from each treatment and control group at study initiation, and at test termination (96 hours).Samples were collected from each replicate test chamber at about 24 hour intervals to determine cell densities, yield and growth rates. These endpoints were used to calculate percent inhibition values relative to control over the 96 hour exposure period. Samples were held for a maximum of three days under refrigerated conditions sufficient
to inhibit growth until cell counts could be performed. At the end of the test, samples of
test solution were collected from each replicate, and pooled treatment samples were made. Subsamples were removed and examined microscopically for atypical cell morphology. Cells in the test chambers were also observed for aggregation or flocculation of cells, or adherence to test chamber.

Data analysis: Calculation of cell densities, yield, growth rates and percent inhibition values, as well as all statistical analyses were conducted using the "SAS System for Windows, Version 8.2".

Growth Rates were calculated for each treatment and control replicate at 24, 48, 72 and 96 hours using the formula:

where: u= (ln Nn -ln No) 

### I (tn- to)

u = Average specific growth rate No= Nominal cell density (cellslmL) at to Nn =Measured cell density (cellslmL) attn tn =Time after beginning oftest (hours)
to = Time of beginning of test (hours)

Yield was calculated for each treatment and control replicate at 72 and 96 hours as the final biomass (cell density) in the respective exposure period minus the initial biomass (cell density)

Percent inhibition of cell density, growth rate and yield was calculated as: Percent inhibition = (Mean control response

- Mean treatment response) 

### I (Mean control response) x 1 00

EC50 and ErC50 values were calculated for all time periods when possible and EyC50

was calculated for 72 and 96 hours using non-linear regression or other suitable method with the treatment response (cell density, growth rate, yield) and exposure concentration data (eg mean measured concentrations).

NOEC (No observed effect concentration) for 72 and 96 hours was based on evaluation ofthe concentration-effect pattern and results of the statistical analyses ofthe cell growth, density, growth rate, and yield data at 72 and 96 hours.

Statistical Analysis: Test endpoints analyzed statistically were cell density, growth, growth rate and yield. Negative control and solvent control data for each parameter werecompared with appropriate statistical tests. No significant differences were seen between the control groups (Student's t-test, a= 0.05); therefore the control groups data were combined.

The 72 and 96 hour cell density, yield, and growth rate data were evaluated for normality and homogeneity of variances (a= 0.01) using Shapiro-Wilk's and Levene's tests respectively. Statistically significant differences between the control and treatment groups were identified using a test to compare treatment mean responses to the control
response (Dunnett's one-tailed t-test, a= 0.05)

Results of the statistical analysis were used to aid in determine the NOEC for each endpoint at 72 and 96 hours. Scientific judgment was also used to determine if statistical differences were biologically meaningful, and if the data showed a concentrationdependent relationship.

Results: Based on mean measured concentrations Cell Density Growth Rate Yield Exposure EC50 95% EC50 95% EC50 95% Duration (ng/L) 1 Confidence (ng/L) 1 Confidence (ng/L) 1 Confidence (hours) Interval Interval Interval (ng/L) 1 (ng/L) 1 (ng/L) 1
24 

### > 26.5 n/aj 

### > 26.5 n/aJ

__ L

__ 1.

48 

### > 26.5 n/aj 

### > 26.5 n/aJ __ L

__ L
72 

### > 26.5 n/aj 

### > 26.5 n/aj 

### > 26.5 n/aj
96 

### > 26.5 n/aj 

### > 26.5 n/aj 

### > 26.5 n/aj

NOEC (ng/L) NOEC (ng/L) NOEC (ng/L) 72 26.5 26.5 26.5 96 26.5 26.5 26.5

1

..

EC50, Ey50 and Er50 values and confidence hmtts, where posstble, were calculated by non-lmear regression, with replicate data (cell density, yield or growth rate) and day 0 measured test concentrations 2 EyC50 values were only determined at 72 and 96 hours as per protocol 3 95% confidence intervals could not be calculated with the data obtained