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Study ID: ~ti~~~, ~y~ 

G~4-S i 0 ~ - 9 Y- 2 Page 1 of 5 

Studv Title CONTAING N 

DOWANOL* PN', GLYCOL ETHER: EVALUATION OF 

THE TOXICITY TO THE GREEN AL6li, SELENASTRUM CAPRICORNUTU ~1 PRINTZ 

A, ithors 

— 

D.C. Dill 

EPA•OTB 

D.P. Milazzo 

000611128J 

Fina i Report 96_`t9D06`k~ O 

.7Uly 13, 1988 

Performing Laboratory 

Mannalian b Environmental Toxicoiogy Research Laboratory 

Health & Environmental Sciences 

The Dow Chemical Company 

ilidland, Michigan 48674 

L aborator~v Project Study ID(S) 

irraievant, Internai COdes 

* Trademark of The Dow Chemical Company 
~ 

~ 

. . 

Pa9e 'wo is rmrved far tle ieclaion ,ei a Statamwt ef iktt 

Conf i denti a 1 i ty C 1 aim as rsqai rad Sy tlw {NN tel Statas GW rowMIttal 

Rrotectitm Assocy (Eal1) WWI&r the ProrisiaM Of PR il$tU* 04 isswd JYly 

25, 1986. 
i ne uvw 4ncmi ca i cvatpany 

Study ID: ltrotrtot, iAIOtOOCeOdN 

Page 3 of ifi 

This is to certify that this study has been carried out in compliance with 

the Good Laboratory Practice Standards. 

Irreievatat, FiIhg f?ata 

Irrelevant, Irifing 3:~~~ ''" 
• ~ rre utn~ ~na►n► ~a r V ~mNany 

Study ID: 

 Page 4 of 6 i"t9R1MCodes 

QUALITY ASSURANCE STATEMENT 

Study Titie - DOWANOL* PM GLYCOL ETHER: EVALUATION OF THE TOXICITY TO THE 

GREEN ALGA, SELENASTRUM CAPRICORNUTUM PRINTZ 

This study was examined for conformance with Good Laboratory Practices as 

published by the U.S. Environmental Protection Agency, and the U.S. Food 

and Drug Administration. The final report was determined to be an accurate 

reflection of the data obtained. The datts of Quality Assurance activities 

on this study are listed below. 

Study Start Date: 12/14/87 Date of Final Report: 07/18/88 

DATE FINDINGS REPORTED TO 

TYPE OF AUDIT: DATE OF AUDIT: STUDY DIRECTOR/MANAGEMENT: 

Preparation of 12/14/87 12/14/87 

Stock Solutions 

Draft Report 06/07/88 06/07/88 

ARCHIVING: Raw data and the final report are filed in the 

testing facility archives. 

1 ,rWevent. Fiiitag ©ata 

- DOW CONFIDENTIAL - 
141Y ✓ VI. VV.IVMYIIt 

Study ID: y . I~ COtles 

Page 5 of lb~ 

Summa ry 

The purpose of this study was to determine the growth inhibition EC50 of 

DOWANOL* PM glycol ether (100% purity), to the green alga, Selenastrum 

capricornutum Printz. The EC50 is defined as the concentration expected 

to inhibit growth by, 50 percent compared to the controls. After 7 days, 

cell counts were slightly (< 7.1%) inhibited (p<0.05) compared to the 

control at 53, 125, 250, and 1000 mg/L, but not at 500 mg/L. Total cell 

volume was not significantly different from the control after 7 days. The 

7-day EC50 values were greater than 1000 mg/L (the highest concentration 

tested), using as growth endpoints, cells/mL and total cell volume/mL, 

respectively. 
The Dow Chemical Compan.y 

Study ID : IfrpWwato lti" ~' Cbdes Page 6 of 16 

INTRODUCTION 

This study has been requested by M. Cook-King, Chemicals and Metals TS&D, 

2020 HHDC, Midland, MI. The purpose was to determine the growth 

inhibition EC50 of DOWANOL* PM glycol ether to a representative 

freshwater green alga, Selenastrum capricornutum Printz. The EC50 is 

defined as the concentration expected to inhibit growth by 50 percerit when 

compared to the controls. These data are needed for the development of 

safe handling and disposal procedures. 

METHODS AND MATERI ALS 

This study included a 7-day algal toxicity test. Methodology for algal 

[1] growth inhibition tests have been described in the Standard Operating 

Procedures (SOPs) for Mammalian & Environmental Toxicology. The algal SOP 

is based on procedures recommended by the U.S. EPA [2]. The algal test 

specifications are listed in Table I. A11 calculations are based on 

nominal test concentrations of the test material on an as-received basis. 

The primary purpose of these tests is to determine the acute toxicity of 

the test substance to algae. This is achieved by exposing algae to a 

series of test substance concentrations in algal growth medium. 

TEST MATERIAL 

A sample of the test material has been received from G.J. Reese, 

Alkanolamines QAC, Louisiana Division, Plaquemine, LA. Information on the 

material is summarized below. 

Sample Name: Dowanol* PM glycol ether 

Synonym: 1-methoxy-2-propanol 

Purity: 100% 

Sampl e I D: Lot # Irrelevant, Filktg E)gft 

CAS No: 000107-98-2 

Dow Registry No.: DR-0005-1945 

Molecular Weight (g/mol): 90.1 

Boiling Point ( oC @ 760 mm Hg): 120.1 

Vapor Pressure (mm Hg @ 20 0

C): 10.9 

Water Solubility (mg/L): miscible 
lig6 vVA V11cirl11~ ti1 4v111Fla11J 

Study ID: Irrelevant.IniernwMr,p ~!~ 

Page 7 of 16 

Sased on its high water soltibility, volatilization was not expected to be 

a significant inter-media transfer mechanism. It was expected to be 

stable in the test system due to the lack of readily or easily 

hydrolyzable functional groups. 

TEST ORGANISM. The alga used in this test was Selenastrum capricornutum 

Printz, obtained from laboratory stock ctiltures. The original culture was 

obtained from the U.S. EPA, Corvallis, Oregon. Stock cultures are 

maintained in algal assay medium (AAM) [2] przpared using double distilled 

water. The final concentrations of nutrients in AAM are given in Table II. 

The AAM used for toxicity testing was identical except that it did not 

contain the chelant, ha 2EDTA . 2H20. 

EXPERIMENTAL. A range-finding test indicated that the EC50 was greater 

than 1000 mg/L. The definitive test consisted of a series of five test 

concentrations set with three replicates each, and six replicate coni.rois. 

A counting blank for each test and control concentration was set 

containing AAM and the test material but no algal inoculum. The pH of each 

t.est and control treatment was recorded at the start and end of the test. 

A stock solution of 6.67 mg/mL in sterile-filtered AAM was prepared by 

weighing 0.6669 g of the test substance and diluting to 100 mL. 

Test concentrations were prepared by adding appropriate volumes of the 

stock soiution to AAM in 250 mL volumetric flasks to yield nominal 

concentrations of 63, 125, 250, 500, and 1000 mg/L. After mixing, 50 mL 

of each concentration was added to each of four replicate 125 mL Pyrex 

Erlenmeyer, flasks. The algal inoculum was prepared from a 6-day old stock 

culture. The stock culture was diluted with AAM to contain approximateiy 

500,000 cells/mL. One mL of the inoculum was added to each flask, except 

the counting blank, giving a nominal initial concentration of about 10,000 

cells/mL. Flasks were placed in a temperature-controlled shaker-incubator 

set to maintain 24 ± 2°C. The temperature was recorded continuously 

during exposure. Flasks were continuousiy shaken at 100 oscillations/min, 

and continuous illumination of 4304 t 430 lux was provided by cool-white 

fluorescent lights. 
The Dow Ghenical Company 

Study I D: irnetevant. intsnnsl Cpda 

Page 8 of 16 

Cell growth was assessed by measuring both the number (cells/mL) and site 

(total cell volume (TCV)/mL) of the algal population on days 2, 3, 4, and 

7 of exposure using a Coulter Counter Model TA-II electronic particle 

ccunter equipped with the TA-II Population Accessory. Two counts per 

replicate were made on each sampling day. A11 counts were corrected for 

background count and dilution factor to give cells/mL and TCV/mL. Samples 

were collected aseptically using autornatic micropipettes and sterile tips. 

Sample dilutions were made using HEMATALL isotonic diluent (Fisher 

Diagnostics). The instrument settings for the Coulter Counter are given 

in Table III. A11 particles having volumes from approximately 10 to 320 

cubic microns were counted. 

STATISTICAL ANALYSIS. The algal data were processed to give mean values 

for cells/mL and TCV/mL measurements of the repiicate flasks. Percent 

inhibition of growth compared to the control were then calculated for cell 

count and TCV using the following equation. 

I = C-X x 100 

C 

Mlhere I= percent inhibition (A negative I indicates stimulation) 

X= growth of test treatment. 

C= growth of the control 

Cells/mL and TCV/mL means for each test concentration were compared to the 

control using Dunnett's t-test [3]. 
The Dow Chemical Company 

Study I D: tttebymf, ktawrg CdKlw 

Page 9 of 16 

RESULTS 

Algal Growth Test - 158 h 

The sumnary data for this test (started on 12/14/87) are given below: 

Initial and final pH values 

Nominal Initial Final 

Conc. (mg/L) pH pH 

control 7.4 7.5 

63 7.5 7.6 

125 7.5 7.6 

250 7.5 7.6 

500 7.6 7.6 

1000 7.5 7.6 

Percent inhibition values * , relative to the controls, based upon mean 

cells/mL and total cell volume/mL (TCV) values, on days 4 and 7. 

Nominal Day 4 Percent Day 4 Percent 

Conc. (mq/L) cells/mL Inhibition TCV/mL (X 104 ) Inhibition 

control 2,471,517 -- 9,300 -- 

63 2,699,633 # -9.2 10,333 # -11.1 

125 2,749,000 # -11.2 10,400 # -11.8 

250 2,694,333 # -9.0 10,200 # -9.7 

500 2,571,733 -4.1 9,500 -2.2 

1000 2,268,300 8.2 8,933 3.9 

Nominal Day 7 Percent Day 7 Percent 

Conc. (mg/L) cells/mL Inhibition TCV/mL (X 104 ) Inhibition 

controi 4,151,367 -- 17,067 -- 

63 3,953,133 # 4.8 16,667 2.3 

125 3,855,467 # 7.1 16,467 3.5 

250 ' 3,964,533 # 4.5 16,733 2.0 

500 4,045,333 2.6 16,867 1.2 

1000 3,909,533 # 5.8 16,667 2.3 

7-day EC50a >1000 mg/L >1000 mg/L 

*= a negative value indicates growth stimulation. 

#= Differs significantly from control, p< 0.05, two-tailed Dunnett's T 

on raw data, except for day 7 TCV/mL data where the Dunnett's T-test 

was performed on log-transformed data. 

a EC50 by inspection of data. 
The Dow Chemical Companv 

Study ID: ImM1ew8W' :~ rnsrr~i ~ 

Pagc 10 of 16 

OISCUSSION AND SUMMARY 

The daily replicate cell count and total cell volume values for the algal 

test are presented in Tables IV and V, respectively. Although, after 7 

days there was a statistical difference (ps0.05) between the control cell 

counts and four of the five test concentrations, there was a distinct lack 

of a concentration-response relationship. The observed differences lack 

biological significance and may reflect the normal variability of this 

parameter. This interpretation is supported by the lack of any 

significant differences noted for the total cell volume (TCV) parameter. 

The 7-day EC50 values were greater than 1000 mg/L (the highest 

concentration tested), using as growth endpoints, cells/mL and total cell 

volume/mL, respectively. 

REFERENCES 

1. Test Method ET-11-1987-1. Algal toxicity test. Mammalian and 

Environmental Toxicology Research Laboratory, The Dow Chemical 

Company, Midland, MI. 

2. U.S. Environmental Protection Agency. The Selenastrum capricornutum 

printz Algal Assay: Bottle Test. EPA-600/9-78-018. Corvallis, OR 

(1978). 

3. Winer, B. J. Statistical Methods in Experimental Design (;econd 

edition) McGraw-Hill, NY. 1971. 

4. Draper, N., and H. Smith. Applied Regression Analysis . John Wiley 

and Sons, Inc., NY. 1966. 
The Qow Chemical Companv 

Study ID : irrolwant. InWAti CetlBer 

Page 11 of 16 

Table I. Summary of Algal Growth Test Parameters. 

Alga l Test 

Species Name Selenastrum capricornutum Printz 

Common Name green alga (family Chlorophaceae, order Chlorococcales) 

Source of Organisms laboratory cultures; originally obtained fran 

the U.S. EPA, Corvallis, Oregon 

Length of Test 7 days 

Test Temperature 24 t 2`C 

Lighting continuous, cool-white r'luorescent, 4304 ± 430 lux 

Diet Algal Assay Medium without Na 2 EDTA (Table '') 

Nominal concentrations Control, 63 mg/L, 125 mg/L, 250 mg/L, 500 mg/L and 

1000 mg/L 

Test Vessels sterile, 125 mL Pyrex Erlenmeyer flasks 

with foam plug closures 

Test Volume 50 mL 

Test Chamber LAB-LINE environmental shaker-incubator; continuous 

shaking at 100 oscillations/min 

Observation Schedule Day 2, 3, 4, and 7 

Observations initial and final pH; continuous temperature; 

cell numbers and total cellular volume (Coulter 

Counter Model TA II with the Population Accessory) 

Effect Criteria cell growth 

Statistical Analysesa Dunnett's T-test comparing cells/mL and 

TCV/mL from each test concentration with the 

control 

a not used for range-finding test 
The Dow Chemical Comanv 

Study ID : lrreWvant, *** COd9S 

Page 12 of 16 

Tabie II. The nutrient components of the algal assay medium (AAi9) use for 

algal maintenance and testing. 

Macronutrients 

NaNO3 

MgC1 2 .6H2 O 

CaC1 2 .2H2O 

MgSO4 .7H2 O 

NaHCO3 

K

2

HPO4 

Micronutrients 

H3 B03 

MnC1 2 .4H2O 

ZnC1 2 

Na2Mo0.2H2 0 

CoC1 2 .2H2O 

CuC1 2 .2H2O 

Na 2 E0TA.2H 2O 

FeC1 3 .6H 2O 

Final AAM 

Concentration 

mg/L 

25.50 

12.16 

4.41 

14.70 

15.00 

1.04 

ug/L 

185.60 

417.20 

3.27 

7.26 

1.43 

0.011 

300.00 (a) 

160.0 

a0mitted from AAM used for toxicity testirg. 
S tu dy I D: ltselevant, &tbmef C0dqq 

Page 13 of 16 

Table iII. Operating parameters for the Coulter Counter Model TA-II with 

the Population Accessory. 

Aperture Tube 

Volume 

Cisplay Gain 

Readout 

Channel Select 

Sampling 

Active Channels 

Population Accessory 

cdit Switch 

100 um 

500 uL 

X10 Manual 

Percent Total Volume or Population 

4or9 

Manometer Mode 

13-4 

On 

Off 
• - c vam YIIYIII . YM . vv...*rv..,~ 

Study ID

of 

: ~r~Ant, In~ ~ 1 Co 

• ~ Page 14 

Table IV. Cell counts (cells/mL)a during an algal test with DOWANOL* PM 

glycol ether. 

Nominal Day 2 Day 3 Day 4 Day 7 

Conc.(mg/L) 12-16-87 12-17-87 12-18-87 12-21-87 

1000 A 78870 465700 2170000 3933000 

B 83630 508160 2398200 3925600 

C 79930 495420 2236700 3870000 

MEAN 80810 489760 2268300 3909533 

STDb 2499 21789 117336 34436 

500 A 95050 588700 2570900 4009400 

B 95830 617260 2726600 4041200 

C 88580 573300 2417700 4035400 

MEAN 93153 593087 2571733 4045333 

STD 3980 22306 154452 38158 

250 A 105030 610660 2706100 4080400 

B 98530 584880 2650100 3944600 

C 96690 590820 2726800 3868600 

MEAN 100083 595453 2694333 3964533 

STD 4382 13500 39681 107298 

125 A 109240 628240 2713600 3863000 

B 113040 630780 2911900 3850200 

C 93640 596380 2621500 3853200 

MEAN 105307 618467 2749000 3855467 

STD 10281 19170 148401 6694 

63 A 107930 632620 2709900 3928000 

B 100070 587600 2657300 3865200 

C 103030 578220 2731700 4066200 

MEAN 103677 599480 2699633 3953133 

STD 3970 29081 38248 102830 

0 A 105770 588820 2554100 4064600 

B 94880 561800 2390800 4202600 

C 103980 565020 2387700 4079200 

D 95930 561340 2427500 4075800 

, E 102780 566400 2499700 4200600 

F 97060 572140 2569300 4285400 

MEAN 100067 559253 2471517 4151367 

STD 4653 10348 80813 91061 

°Each value represents the average of two sample readings. 

b Std = standard deviation. 
The Dow chemical Gompany .,. 

• • . Study I D : }h*&M4, InWW ~ 

• Page 15 of 16 

Table V. Total cell volumes [TCV/mL (cubic microns X 10 4 )] aduring 

an algal test with DOWANOL* PM glycol ether. 

Nominal Day 2 Day 3 Day 4 Day 7 

Conc.(m /L) 12-16-87 12-17-87 12-18-87 12-21-87 

1000 A 620 2420 8700 1 

B 680 2740 9300 16600 

C 660 2580 8800 16400 

MEAN 653 2580 8933 16667 

STDb 31 160 321 306 

B 760 3000 10000 16600 

C 660 2680 9100 17000 

MEAN 720 2847 9500 16867 

STD 53 160 458 231 

N 830 3260 10300 ---= 

B 770 2920 10000 16600 

C 770 2980 10300 16400 

MEAN 790 3053 10200 16733 

STD 35 181 173 416 

B 890 3480 11000 16200 

C 730 3080 9900 16600 

MEAN 833 3313 10400 16467 

STD 90 208 557 231 

1 

B 780 3060 10300 16400 

C 790 3060 10300 17000 

MEAN 810 3173 10333 16667 

STD 44 196 58 306 

1 

B 710 2620 9100 17600 

C 800 2780 8900 16800 

D 710 2620 9100 16600 

E 800 2700 9200 17000 

F 760 2900 9700 17400 

MEAN 763 2777 9300 17067 

STD 44 167 363 372 

b Each value represents the average of two sample readings. 

Std = standard deviation. 
The Dow Chemical Company 

Study ID:1,~t.l~~ 

Page 16 0 

This report, ES -1065 , has been written and approved by the following 

personnel. 

. . Di ll 

APPROVED BY: 

lttelevant. Fiiing Daca 

E~ . iP /I't• ~~l.o~~ o G/rs~/,~c

~ 

'b. P-MiaT zzo 

- DOW CONFIDENTIAL - 
CERTIFICATE OF AUTHENTICITY 

T1iIS IS TO CERTIFY that the microimages appearing on Ws microttche are accuraie 

and complete reproductions ot the records of U.S. Environmental Protection Agency 

documents at delivered In the regular couree o( buainess for mtcrofllming. 

Data produced__! 

(Month) (Day) (Year) Camera Operator 

~ 

Place Syracuse N=w York 

(City) (State) 

A 

AMTW 

corp