Document RpoLLn8EoJ1YmBQEvYkeQDbLV
AR226-2955
Du Pont HLO 235-91
BU PORT USE OSLY
Study Title Static Acute Toxicity ofi4------f to the
Freshwater Alga, Seleaastrua capricornutua
Authors Timothy J. Bard Robert L. Boeri
Study Coippleted
April 15, 1991
Performing Laboratory
EnTiroSystems Division Resource Analysts, Incorporated
P.O. Box 2130 One Lafayette Road Hampton, New Hampshire 03842
Submitted To
naskell
E. I. du Pont de Hemours and Company
Laboratory for Toxicology end Industrial Ellttcn Road, Post Office Box 50 Newark, Delaware 19714
Hedicine
Haskell Laboratory Project ID Haskell Laboratory Outside Report No. 235-91
Page 1 of 20
Resource AnaWs .Inc.. Subsidiary olMUllFORE
Company SanWzed. Does ro cnnn?n T<;r*fl /pri
^hpany Sahifize'd. Does noi contain TSCA CBf
Du Font HLO 235-91
I. GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
This study was performed in compliance with Good Laboratory Practices as defined in 40 CFli part 792, and presented in the Federal Register, Thursday, August 17, 1989 (Toxic Substances Control Act [TSCA]; Good Laboratory Practice Standards; Final Rule). Light intensity was not measured daily during the study. Neither the Study Director nor the sponsor are aware of any circumstances that would affect the integrity of this study.
9fA^----y/^ 'i,
Tim/thy'3J1/ Wa/d Study Director
En'.'iroSystems Study Number , Page 2 of 20
Reso-jice Anolvsts. Inc. Suteiaicuv ot MILLIPQRE
.Company Sanitized. Does not contain T0'
Ou Font HLO 235-91
II. QUALITY ASSURANCE STATEMBff
Submitted by:
EnviroSiiifcess Division
Resource Analysts, Incorporated P.O. Box 2130 Oie Lafayette Road Hanpton, Hew Hampshire 03820
Certification:
This study meets requirements of 40 CFR part 792. Data
presented in this report were derived by aethods and with materials identified in the section of the report entitled "Methods and Materials." The test was performed in accordance with EnviroSysteas Protocol
Kand the Product Registration Aquatic _y Laboratory Standard Operating Procedures
Manual. The toxicity test was performed by Jeanne
Magazu, Robert Boeri, and Timothy Ward.
All data transcribed from the raw data to the report were checked for accuracy, and all data were verified by Quality Assurance Auditors. Quality assurance
audits were performed (and reported to management) on:
Aspect Audited
Audit Reported to Reported to Date Study Director Hanagenent
Protocol
In-life
Draft report Final report
7/ 5/90
9/25/90 2/ 4/91 4/18/91
7/ 5/90 9/25/90
11 4/91 4/18/91
10/ 2/90 4/18/91 4/18/91
& ^ c^Ag^4ux^^
Quality Assurance Representative
^ .1 ^ ^
EnviroSystems Study number, Page 3 of 20
Resource Anotvsts. inc. Subsidiary ol M1111PORE
^ompan.Y'Sanlfi2sa. Doss notconiain TSC^. ^s
Du Font HLO 235-91
III. TABLE OF COKTEKTS
SECTION:
I.
II. III.
IV.
V.
VI.
VII. VIII.
IXX. XI.
XII.
Good Laboratory Practice Compliance Statement Quality Assurance Statement Table of Contents Index of Tables
Summary
Records General Information
Methods and Materials Results Protocol Deviations Signature Page References
APPENDIX: Appendix A.
Water Quality Data from Toxicity Tesr
PAGE
2 3 4 5 6 7 8
10 13 15 16 17
18
EnviroSystems Study Number 1 Page 4 of 20
Resource AnaWs. lrc. Subsidiary of WU-PORE
Du Font HLO 235-91
IV. INDEX OF TABLES
PAGE
Table 1. Chemi-al characterization of a representative
11
sample of deionized water used to prepare sterile enriched media used in the toxicity test with ^----MB and the freshwater alga
Seleaastruis capricoraatus
Table 2. Cell growth data from the toxicity test with
14
--------^^and the freshwater alga
Seleaastrua capricornutum
Table A.I. Temperature measured during toxicity test
19
with ----^^^and the freshwater alga
Seleaastrum capricornuturn
Table A.2. pH values measured during toxicity test with
20
^BBBPRid the freshwater alga
Selenastrua capricornutum
EnviroSystems Study Number
Resource Analysis, inc. Subsidiary 01 MILUPORE
|
w
Page 5 of 20
Company Sanitized. Does nol contain T8&A ABf
Du Font HLO 235-91
V. STOHm
Jim^ An acute toxicity test was conducted with
and the
freshwater alga. Selenastrua capricorautua, to deterffiine the effective
concentrations (EC10, EC50, EC90). The test was conducted for E.I. duPont
de Nemours and Company for 96 hours during September 21 to 25, 1990, at
It the EnviroSystens Division of Resource Analysts, Inc. in Baapton, Hew
Hampshire,
was conaucteJ according to the protocol developed for
EnviroSystems Study Number I^BHR) The test substance was supplied by
the sponsor.
The test was performed under static conditions with five concentrations of test substance and a dilution water control with 3
replicates per level. The test was conducted at a temperature of 24 t
2C.
The dilution water was sterile enriched media adjusted to a pH of
7.5.
and
Nominal concentrations were: 0 (control), 315, 625, 1,250, 2,500,
5,000 rog/L ------I----
Cell counts were made daily with a
haemocytometer.
Nominal concentrations were used for all calculations.
Bater quality parameters were within acceptable limits throughout the
test.
The test substance was not toxic to Seleaastrua capricorautua
at any tested concentration. Exposure of algae to the test substance for 96 hours resulted in a median effective concentration (EC50) of '>5,000 mg/L----^^IR the highest tested concentration.
EnviroSystens Study Number Page 6 of 20
Resource Analysts, inc. Subsidw of MIU.1PCWE
"SSMpahy'Sanhized. Does noi contain rsCA CtJi
VI. RECORDS
Du Font HLO 235-91
All records from those portions of this study conducted at Resource
Analysts, Inc. and EnviroSystems Division
Hampton, New Hampshire.
the final report will be maintained at the of Resource Analysts, Inc., 1 Lafayette Road,
EnviroSysteiss Study Number Page 7 of 20
Resource Anolysts. Inc. SubsOioiv of M1U.1PORE
VII. GENERAL INTOMUTiaf Du Font HLO 235-91
Haterial Tested:
Medical Research Number:
Haskell No.:
18,267
Synonyms:
Submitter's
Notebook Number:
Purity;
Contaminants:
Composition:
Sponsor: Submitted by:
Du Font Chemicals and Pigments
E.I. du Font de Honours and Company
vilmington, Delaware 19898
Du Font CheBicals and Pigments
E.I du Pont de Kemours and Company
Jackson Laboratory Deepwater, Wew Jersey 08023
EnviroSys terns Study Number ' Page 8 of 20
Resource AnaWs. inc SuEiaaiaiv of MmiPORE
j
Company SaniHzed. Does hot contain TSftA Cw
Du Font HLO 235-91
Study Schedule:
Haskell Laboratory Test Code: Distribution:
Study Initiation Date: August 6, 1990 Experimental Start Date: September 21, 1990
Experimental 1990
Conpletion
Date: SepteiBber 25,
Study Coapletion Date: April 15, 1991
EnviroSystems Study Number Page 9 of 20
Resource Analysts, me. Subsidiary of MIUJOOBE
JSQ^pafiSyanitizes!, Does not contain YSCA CB<
TEST SUBSTANCE:
Till. METHODS AND KATERIALS Du Font HLO 235-91
EnviroSystems on June 18, 1990. It was contained in a glass bottle
that was labelled with the following information: "H 18,267, 5-22-90,
412 grans gross".
The test substance (a viscous, beige liquid) was
shipped from E. Research Center.
I. duPont de Reaours and Conpany, Stine-Haskell
P.O. Box 30, Elkton Road, Newark, Delaware. Prior to
use the test aaterial was stored in the dark at 2-<C. & reserve
sample (approximately 1.2 grams) will be archived at EnviroSystens for a minimum of 10 years, and unused test substance is returned to the
sponsor^
A_ 5,000 mg/L stock solution_was. prepared by combining 5.0-.g
of test substance and dilution water and adjusting the mixture to 1,000
ml. Thetfis^substance was tested as received and assumed to have a
purity of^^^^^^ ^^^^^^^^^^^^^^r
DILUTION ffATER:
Water used for acclimation of test organisms and for all
toxicity testing was sterile enriched media (U.S. EPA, 1978; Resource Analysts, Inc. Standard Operating Procedure 01-02-1101) adjusted to a pH of 7.3. Results of chemical analysis of a representative sample of
deionized water used to prepare the media are presented in Table 1.
TEST ORGANISM.
Algae used for the test was from a culture originally procured from the Culture Collection of Algae at the University of Texas at Austin and delivered to EnviroSystems on October 31, 1989. The culture was transferred to sterile enriched media identical to media used for this test and maintained for at least 14 days.
TOXICITY TESTING:
A screening test was conducted during August 9 to 13, 1990.
'J----------fr Nominal concentrations of tke^s^vt s9uuub^sLtaaun*wc^e were: 1. 10, 100, and
1,0 the
0
0 c
o
mg/L ntrol
.
v
Aft alue
e
r a
t
9
6
1
,
hou
000
r
s
m
gtht/iLej^jmm^j
^nnh^hf^aii^^i^A^^^wcfe
l
l
s
per
ml
was
greater
than
The definitive toxicity test was performed during Septeaber 21
to 25, 1990, according to the protocol developed for EnviroSystems Study 4------BB^ The protocol is based on procedures of the U.S. Environmental" Protection Agency (1985, 1987). The test was conducted at a target temperature of 24 2C with five concentrations of test material and a dilution water control. Nominal concentrations of
|were: 0 (control), 315, 625, 1,250, 2,500, and 5,000 mg/L.
EnviroSystems Study Number Page 10 of 20
Resource Anotysts. me. Subsdioiv of kSliroCE
Table 1.
Du Foot BLO 235-91
Chemical eb ^erization of representative 9--pie of
deionized water used to prepare sterile enriched aedia used in the f-oxicity test with|^----^teind the freshwater alga
Seleasstma caprieorawtUB
Parameter
Unit of
Measurement
Detection Limit
Measured Value
Total organic carbon 0 hour
96 hour Total suspended
solids
0 hour 96 hour
Ammonia
Aluminum
mg/L mg/L
mg/L as N mg/L
Arsenic
Boron
mg/L mg/L
Cadmium
mg/L
Chromium
ng/L
Copper Cobalt
Fluoride Iron
Lead
mg/L ng/L ng/L mg/L mg/L
Mercury Nickel
Silver
Zinc
Organochlorine and
-mg/L
mg/L ng/L mg/L ug/L
Organophosphorus
Pesticides and PCBs
1
10
0.2 0.025 0.01 0.1 0.001 0.002 0.005 0.008 0.05 0.01 0.005 0.0003 0.008 0.005 0.005 5.0
HD 11
ND
21 HO
NO ND HD HD HP HD
'- HD
ND
0.01
HD HD HD KD HD HD
Notes: 1. 2.
MB = Not detected at or above the detection limit. Total organic carbon and total suspended
solids
determination was conducted on a sample of dilution water
collected from a control test vessel during this toxicity test (96 hour values represent analysis of the test organism
in the dilution water). All other chemical data are the
result of analysis of a representative sample of deionized
water collected during March, 1990 as part of routine
bi-annual water quality analysis.
EnviroSysterns Study Number] Page 11 of 20
Resource AnoWs i-ic. Su&sidiaiy ol MiLlPOOt
.Company Sanitized. Doas not contain TSCA CRI\
Du Font HLO 235-91
Algae was randonly and equally distributed aaong three
replicates of each treatment at the rate of 10,000 cells/al. The test
was performed in 250 ol glass erienneyer flasks that contained 100 !
of test solution. Test vessels were randomly arranged on a rotary
shaker adjusted to 100 rpa.
The test vessels were placed in an
incubator during the test (a random numbers table was used to select the location of each vessel). A 24 hour light and 0 hour dark
photoperiod was automatically maintained with cool-white fluorescent
lights that provided a light intensity of 24 uEs-'n2.
The number of algal cells/mi in each test vessel and the
occurreice of relative size differences, unusual cell shapes, colors,
flocculations, adherence of cells to test containers, or aggregation of cells was determined visually by means of direct microscopic
examination with a haeBocytoneter. Cell counts were made and recorded
daily during the 96 hour test.
Temperature of the incubator was
measured and recorded daily (thermometer number 2211), and pB (Becfcman
model pHI 12 meter; instrument number PRL-4) was determined in each
test vessel at the beginning and end of the test.
STATISTICAL METHODS:
Results of the toxicity test could not be interpreted by
D standard statistical techniques because cell growth at the highest tested concentration was greater than 50% of control growth. Determination of EC10, EC50, and EC90 values were performed by the author using percent of control growth data and the nominal concentrations of the test substance.
EnviroSystems Study Numbec Page 12 of 20
Resomce Analysts. Inc.. Subsidiary dt MU11PORE
T?oWpnv''g'a^^stl;<Kdr:Dnoo?ec^o' ntain TSCA C3f
IX. RESULTS
Du Font HIiO 235-91
presented acceptable the test
9.0.
Water quality data generated by the acute toxicity test are in Appendix A. ffater quality throughout the test was within limits. The range of values measured in test vessels during
were: incubator temperature - 23.2 to 24.21>C, and pH - 7.5 to
Biological and water quality data generated by the acute toxicity test are presented in Table 2 and Appendix A, respectively. The 96 hour EC10, EC50, and EC90 values are >5,000 ag/L. the highest
tested concentration. Ho effects (size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells) were noted.
EnviroSysteros Study Number] Page 13 of 20
Resource Anolyrt. Inc. Subsictofy 01 MILUPORE
jpompany Sanitized. Poas hot contain TSCA CBi
1)
i
/
Table 2.
Du Font BLO 235-91
Cell growth data from the acute toxicity test with --------Band the freshwater dga, Seleaastrua capricfrautua.
Nominal
Concentrat ion (Bff/L)
rep.
Hiumber of Cella/nl 3i 10 (hi)ur)
0
24
48
72
96
0
1
10
5
(control)
2
3
10
61
10
54
mean
10
57
% control 100
100
238
945 1,878
212 1,070 1,929
246 1,325 1,842
232
1,113
1,883
100
fob
100
315
1
10
60
209 1,212 1.750
2
10
49
227
938 1,825
3
10
52
230 1,110 1,803
mean
10
54
222 1,087 1,793
% control 100
95
96
98
95
625
1
10
52
198 1,070 1,821
2
1C
48
212 1,083 1,800
3
10
54
226
962 1,862
mean
10
51
212 1,038 1,828
t control 100
89
91
93
97
.250
1
10
53
204
924 1,760
2
10
51
221
981
1.740
3
10
49
240 1.042 1,826
mean
10
51
222
982 1,775
% control 100
89
96
88
94
,500
1
10
46
218 1.221 1,912
2
10
57
212
989 1.716
3
10
53
227 1.014 1,844
mean
10
52
219
1,075
1,824
% control 100
91
94
97
97
5,000
1
10
58
222
845
1.861
2
10
47
241
910 1,840
3
10
52
210
885
1,962
mean
10
52
224
880 1,888
% control 100
91
97
79
100
EnviroSystems Page 14 of 20
Study
Number
JH^f
Resomce Analysts, me. Suteiaoiv d MUJPORE
L'Offipariy.Sanitized. Doeano? contain TSCACSS
S. PBOTOCOL DCTHiTIOHS
Du Pont HLO 235-91
Ho deviations Mere made from the protocol.
t)
Eir.-iroSystenis Study Number , Page IS of 70
Resource Anolvsll me.. Sutisiaiory of MIUJPORE
XX. SIGNATURE PAGE
Du Font RLO 235-91
Mq^~--~^, TiiBs(i?ththyy 3Jf. Mxi
V f T d " Study Director
1^L^./^^ v^i Robert L. Boeri
Coauthor
u ^ ^ ^ Snianne P. Magazu \ Aquatic Toxicol-bgist
w^
EnviroSystems Study Number Page 16 of 20
Resouice Anolysts. me. SutMidioiv ol MRiiPORt
(Company Sanitized. Does not contain TSCA CBI
XII. REFERENCES
Du Font HLO 235-91
O.S. EPA. 1978. The Seleaastrwa capricorautwa Printz Algal Assay
Bottle Test. EPA-600/9-78-018.
Environmental Research Laboratory,
Corvallis, Oregon.
O.S. EPA. 1985. Guidelines; Final
Section 797.1050.
40 CFR Rules.
Part 797. Toxic Substances Control Act Test Federal Register, Friday, September 27, 1985.
D.S. EPA. 1987.
40 CFR Part 797. Toxic Substances Control Act Test
Guidelines; Amendments.
Federal Register, Wedaesday, Hay 20, 1987.
Section 797.1050.
U.S. EPA. 1989.
40 CFR Part 792. Toxic Substances Control Act (TSCA);
Good Laboratory Practice Standards; Final Rule.
Thursday, August 17, 1989
Federal Register,
EnviroSystems Study Number^ Page 17 of 20
Resource Analysts. Inc. Subsidiary of MIU.IPORE
^Wparw'Sarutbsd- D^es fici contain TOCA C^i
Dti Font HLO 235-91 Appendix A. VATER QgALITY DflTA FROM TOXTCITY TEST
D
EnviroSystens Study Humbert Page 18 of 20
Resource Anaiysl. inc.. SubsBioiv ol M1UIPOBE
Du Font BLO 235-91
U^f^^ Table R.I. Temperature aeasured during toidcity test with and the freshvater alga, Seleaastrua capricornutaa.
Hour of Exposure
Tenperature of Incubator,C
0
24.2
24
23.6
48
23.7
72
23.7
96
23.2
EnviroSystems Study Number^ Page 19 of 20
Resouice Analysts. Ire. Subsidioly of MILUPORE
Company Ssaannn'i.l-'zteid"-. Does not contain T8CA cr
Du Font HLO 235-91
Table A.2. pH values measured during toxicity test withJ
freshwater alga, Seleaastrua capricorautua.
land the
Nominal
concentration
(ing/L)
0 (control)
315
625
1,250
2,500
5,000
Replicate
1 2 3
1 2 3
1 2 3
1 2 3
1 2 3
1 2 3
pH
Initial
Final
7.5
8.3
7.5
8.7
7.5
8.7
7.5
8.9
7.5
9.0
7.5
8.9
7.5
9.0
7.5
9.0
7.5
9.0
7.5
9.0
7.5
9.0
7.5
9.0
7.5
9.0
7.5
9.0
7.5
9.0
7.5
9.0
7.5
9.0
7.5
9.0
EnviroSystems Study Number Page 20 of 20
Resource Analysis. Inc.. Subsidiary of MILUPORE
^^nv "owpa'"iyyS5n^Kfe..-et-d.- D"o- es iw co^n TSCA ^