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Studv Title 

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## DOWANOL* PN', GLYCOL ETHER: EVALUATION OF THE TOXICITY TO THE GREEN AL6li, SELENASTRUM 

## CAPRICORNUTU

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### — D.C. Dill 

### EPA•OTB
D.P. Milazzo 000611128J Fina i Report 

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.7Uly 13, 1988



### Performing Laboratory Mannalian b Environmental Toxicoiogy Research Laboratory

Health & Environmental Sciences The Dow Chemical Company ilidland, Michigan 48674



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### v Project Study ID(S) irraievant, Internai COdes

* Trademark of The Dow Chemical Company~

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## 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.



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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.



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## 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 : IfrpW

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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 ( o C @ 760 mm Hg): 120.1 Vapor Pressure (mm Hg @ 20 0 C): 10.9 Water Solubility (mg/L): misciblelig6 vVA V11cirl11

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### 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 2 EDTA . 2H 2 0.
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 10 4 ) 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 10 4 ) 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. 

### Alg a 

### 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 

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### Page 12 of 16
Tabie II. The nutrient components of the algal assay medium (AAi9) use for

algal maintenance and testing.

Macronutrients 

### NaNO 3 MgC1 2 .6H 2 O CaC1 2 .2H 2 O MgSO 4 .7H 2 O NaHCO 3 K 2 HPO 4

Micronutrients 

### H 3 B0 3 MnC1 2 .4H 2 O ZnC1 2 Na 2 Mo0.2H 2 0 CoC1 2 .2H 2 O CuC1 2 .2H 2 O Na 2 E0TA.2H 2 O FeC1 3 .6H 2 O 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



### a 0mitted from AAM used for toxicity testirg.

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### 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.., ~ 

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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 )] a during

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 STD b 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.

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### APPROVED BY:

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### 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 prod uced__!



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