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CONFIDENTIAL
DPT440/992118
ACUTE TOXICITY TO DAPHNIA MAGNA
Sponsor
DuPont Speciality Chemicals, Jackson Laboratory, Chambers Works, Deepwater, NJ 08023, USA.
Rescnrch Laboratory
Huntingdon Life Sciences Ltd., Eye, Suffolk IP237PX, ENGLAND.
Draft Report Issued 25 January 1999 Final Report Issued 4 March 1999
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DPT440/992118
CONTENTS
Page
COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS....................
4
QUALITY ASSURANCE STATEMENT............................................................................
5
CONTRIBUTING SCIENTISTS..........................................................................................
6
7
SUMMARY...........................................................................................................................
INTRODUCTION................................................................................................................. 8
TEST SUBSTANCE............................................................................................................. 9
EXPERIMENTAL PROCEDURE........................................................................................ 10
MAINTENANCE OF RECORDS......................................................................................... 13
14 RESULTS..............................................................................................................................
CONCLUSIONS................................................................................................................... 15
15 REFERENCE........................................................................................................................
FIGURE
1.
Typical sample chromatogram ...................................................................................
16
TABLES
1.
Measured concentrations............................................................................................ 17
2.
Cumulative immobilisation......................................................................................... 18
3. Environmental parameters.................................................................... 19
l)
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CONTENTS - continued APPENDICES 1. Elendt M4 medium .<....................<.............................
2. The determination ofl^^------Junaqueous media.
DPT440/992118
Page
20 21
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DPT440/992118
COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS
The study described in this report was conducted in compliance with the following Good Laboratory Practice standards and I consider the data generated to be valid.
The UK Good Laboratory Practice Regulations 1997 (Statutory Instrument No. 654). EC Council Directive, 87/18/EEC of 18 December 1986 (Official Journal No. L 15/29). OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98) 17.
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Eileen C. Daly, Nat. Ditlomaf,NCEA Eire.
Study Director, Huntingdon Life Sciences Ltd.
.^..^.<LJ.W
Date
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Company San^d. Dees.ot oo^nTSCAC.
QUALITY ASSURANCE STATEMENT
The following have been inspected or audited in relation to this study
DPT440/992118
'
Study Phases Inspected
Date of Inspection
j
Protocol Audit
06 August 1998
Process Based Inspections
j
Formulation of test media
02 October 1998
Sampling of test media
02 October 1998
Experimental set-up
15 December 1998
Daphnia observations
11 November 1998
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Report Audit
19 February 1999
Date of Reporting
06 August 1998
02 October 1998 02 October 1998 15 December 1998 11 November 1998
19 February 1999
Protocol Audit: An audit of the protocol for this study was conducted and reported to the Study Director and Company Management as indicated above.
Process based inspections: At or about the time this study was in progress inspections of routine and repetitive procedures employed on this type of study were carried out. These were conducted and
reported to appropriate Company Management as indicated above.
Report Audit: This report has been audited by the Quality Assurance Department. This audit was conducted and reported to the Study Director and Company Management as indicated above.
The methods, procedures and observations were found to be accurately described and the reported results to reflect the raw data.
Helen Comb, B.Sc.(Hons.). Principal Auditor,
Department of Quality Assurance, Huntingdon Life Sciences Ltd.
^./Vl^.raAl^^
Date
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CONTRIBUTING SCIENTISTS
STUDY MANAGEMENT
Eileen C. Daly, Nat. Diploma, NCEA Eire. Study Director
Deborah A. Blacoe, B.Sc.(Hons.), Ph.D. Study Scientist
Karen A. Firth, Higher National Certificate (Applied Biology) Study Scientist
Ben Smith, B.Sc.(Hons.), M.Sc., C.Chem., M.R.S.C. Chief Chemist.
Andrew Robertson, B.Sc.(Hons.) Senior Chemist
Richard Cubberley, B.Sc.(Hons.) Study Analyst
DPT440/992118
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SUMMARY
DPT440/992118
The acute toxicity of^^^HH--Q10 ^aP^nla tnagia was assessed under static exposure
conditions. Throughout this report, the exposure concentrations and the test results have been
expressed in terms of the active ingredient||jjjfj^f||^jf\
The study was conducted in accordance with EEC Methods for Determination of Ecotoxicity Annex to Directive 92/69/EEC (O.J. No. L383A, 29.12.92) Part C. Method 2 "Acute Toxicity for Daphnia" and the OECD Guideline for Testing of Chemicals No. 202, Part I "Daphnia Acute Immobilisation
Test".
^H^BBlO^ Groups of twenty Daphnia, less than 24 hours old, were exposed for 48 hours
nominal concentrations of 4.27, 9.39, 20.7, 45.5 and 100 mg/1 (as active ingredient). The test media were prepared in Elendt M4 medium and to aid dissolution ultrasound treatment was employed.
The measured concentrations o^HMIHiB|in samples unfiltered of media indicated that the
intended exposure concentrations were adequately achieved (between 85 and 97% of their nominal values) and maintained (between 93 and 100% of their starting values). The overall mean measured levels were 4.01, 8.92, 18.6, 40.2 and 85.9 mg a.i./l.
Observations of the Daphnia in each control and test vessel were made after 24 and 48 hours. After 48 hours, 20% immobility had occurred at the highest concentration (85.9 mg a.i./l); the highest
concentration at which no immobility had occurred was 40.2 mg a.i./l.
The 48-hour EC could not be calculated but must be >85.9 mg a.i./l and the "no-observed-effect concentration" (NOEC) was 40.2 mg a.i./l.
Under the EC^General Classification and Labelling Requirements for Dangerous Substances and
^ H B U U n i s Preparations,
not considered to require classification as the 48-hour EC,o is
considered to be greater than the highest nominal concentration, 100 mg a.i./l (mean measured level
of 85.9 mg a.i./l),
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INTRODUCTION
DPT440/992118
The objective of the study was to determine the acute toxicity (48 hour median effect concentration
- ECfo) "dBB^lBK0 )fl'/7/'"'a magnet at 20C.
The study was conducted in accordance with EEC Methods for Determination of Ecotoxicity Annex to Directive 92/69/EEC (O.J. No. L383A. 29.12.92) Part C, Method 2 "Acute Toxicity for Daphnia" and the OECD Guideline for Testing of Chemicals No. 202, Part I "Daphnia Acute Immobilisation
Test".
The protocol was approved by Huntingdon Life Sciences Management on 7 July 1998, by the Sponsor on 17 July 1998 and by the Study Director on 3 August 1998.
The experimental phase of the study was conducted between 1 September and 20 November 1998 and the results of chemical analysis were issued by 8 December 1998.
o^jHHiB^11 Information provided by the Sponsor indicated that the solubility
water was 25%
by weight at 35 - 40C and that its^^l^--------U Throughout this report, the exposure
concentrations and test results have been expressed in terms of the active ingredient. Also, the
Sponsor indicated that at room temperature the test substance was a suspension in water and upon
standing it would separate out into its component phases; accordingly, at the recommendation of the
Sponsor, the suspension was warmed to 35 - 40C (in a water bath) with gentle stirring to obtain an
homogenous composition before use.
The sensitivity of juvenile Daphnia cultured in this laboratory is periodically assessed using the
reference substance potassium dichromate. The results for the most recent test performed prior to this study indicated that its 48-hour ECso to Daphnia magna was 0.57 mg/I; this was within the range typically obtained in this laboratory (0.3 to 0.8 mg/1).
:8: TSCACBl
Company San-.d. edH. Dooees. nr ot contain
Identity: Chemical name:
Appearance: Storage conditions: Lot number: Expiry date: Purity: Sample received:
TEST SUBSTANCE
DPT440/992118
Pale-yellow slurry Room temperature in the dark
2 years from date of receipt
c
23 June 1998
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^s------00--------""50"31
EXPERIMENTAL PROCEDURE
DPT440/992118
TEST ORGANISM
Daphnia magna (Straus) used in this study were cultured in-house and were obtained from a strain originating from the Institute National de Recherche Chimique Applique (IRChA), France.
Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 1.5 litres ofElendt M4 culture medium in a temperature-controlled laboratory at nominally 20 2C. A photoperiod of 16 hours light: 8 hours dark was maintained, with periods of subdued lighting at the beginning and end of each light phase. The culture medium was renewed at least twice each week.
Cultures were fed daily with a suspension of the unicellular green alga, Chlorella vulgaris, to provide 0.1 to 0.2 mg carbon per daphnid, per day. Culture conditions ensure that the stock animals reproduce by parthenogenesis.
The day before the start of the study, all juvenile Daphnia were removed from the laboratory cultures. The following morning, juveniles produced by the gravid (egg-bearing) adult Daphnia were removed from the culture vessels and held in a separate holding vessel; these animals, which were less than 24 hours old, were used in the test.
DILUTION MEDIUM
The test organisms were maintained and the tests conducted in Elendt M4 medium (Appendix 1).
The medium was prepared in reverse osmosis water.
TEST SUBSTANCE PREPARATION
Method of preparation
Based on information provided by the Sponsor, the test substance was warmed (to c.38C) in a water bath and gently swirled to provide a homogenous mixture before weighing.
At all concentrations, the test media were individually prepared by adding the appropriate weights of substance (17.1, 37.6, 82.8, 182 and 400 mg) to dilution medium in a volumetric flask (1 1); to aid dissolution, the contents of the flasks were treated with ultrasound for thirty minutes before being made up to volume with dilution medium.
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DPT440/992118
Stability of test concentrations
The test concentrations of^B--------Rvermeeasured using an HPLC method of chemical analysis
(see Appendix 2). The stability of the test substance in dilution medium under refrigerated storage conditions was determined before the start of the study (Table 2, Appendix 2).
At the start of the definitive test, four samples (100 ml) were taken from the freshly-prepared control and test media; after 48 hours, the contents of the test vessels from each group were pooled and further samples were taken for analysis. They were stored in a refrigerator until two samples from each set were transferred to Huntingdon Research Centre, Cambridgeshire; one sample per concentration was analysed and all of the other samples remained in storage at Eye Research Centre
in case further analysis was required.
EXPOSURE CONDITIONS
Experimental design
A rangefinding test was conducted, followed by a definitive (limit) test with one test concentration (100 mg a.i./l) plus a control (dilution medium). Because 60% immobility occurred in the limit test it was repeated and 50% immobility was observed. Another test was then undertaken employing five test concentrations (4.27, 9.39, 20.7, 45.5 and 100 mg a.i./l) but this gave variable results. It was suspected that repeated warming (to 38C) of the small volume of test substance in the sub-sample
container used for the aquatic studies may have affected the chemical characteristics of the test
substance. Therefore, a new sub-sample of the test substance was obtained and a definitive test was conducted with five test concentrations and the results are reported here.
Twenty Daphnia, four replicates of five animals per vessel, were exposed in each control and test
group.
The first instar Daphnia were placed in groups of five, at random into glass dishes containing 100 ml of medium to give a loading of 20 ml medium per organism. The dishes were loosely covered.
i
Test concentrations
j
The preliminary tests employed test concentrations within the range 1 to 100 mg/1 (as active
I
ingredient). The definitive test concentrations, which were selected based on the results of the
preliminary tests, were:
Nominal concentrations:
4.27, 9.39, 20.7,45.5 and 100 mg active ingredient/I.
(Expressed in terms of the test substance as received: 17.1, 37.6, 82.8, 182 and 400 mg/1)
Medium renewal
Daphnia were exposed to the test or control conditions for a period of 48 hours without renewal of
test media.
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DPT440/992118
Environmental conditions
The temperature of the test area was maintained at 20 2C and constant to within PC during the
study. Temperature was continuously monitored in an additional vessel containing the same volume
of dilution medium. A photoperiod of 16 hours light: 8 hours dark was maintained, with periods of subdued lighting at the beginning and end of each light phase. No supplementary aeration was
employed and no feed was given during the exposure period.
The temperature, pH and dissolved oxygen levels of control and test media were recorded at the start and at the end of the study.
CRITERION OF EFFECT Daphnia were considered to be immobile if they were unable to swim within approximately 15 seconds following gentle agitation of the test vessel. The numbers of mobile, immobile and floating Daphnia were counted 24 and 48 hours after the start of the study.
EVALUATION OF DATA The "no-observed-effect concentration" (NOEC) was derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% is considered to be significant.
PROTOCOL DEVIATIONS
None.
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DPT440/992118 MAINTENANCE OF RECORDS All specimens, raw data and study related documents generated during the course of the study at Huntingdon Life Sciences, together with a copy of the final report will be lodged in the Huntingdon Life Sciences Archive. Such specimens and records will be retained for a minimum period of five years from the date of issue of the final report. At the end of the five year retention period the Sponsor will be contacted and advice sought on the future requirements. Under no circumstances will any item be discarded without the Sponsor's knowledge.
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RESULTS
DPT440/992118
Chemical analysis
The results of chemical analysis are given in Table 1 and an example chromatogram is illustrated in Figure 1. Concentrations are expressed in terms of the measured levels of the active ingredient.
Results for unfiltered samples ofi------Hindicated that the intended exposure concentrations
were adequately achieved (between 85 and 97% of nominal values) and were maintained (between 93 and 100% of starting values). The overall mean measured levels were 4.01,8.92, 18.6,40.2 and 85.9 mg a.i./l.
Immobility
Observations of the Daphnia in each control and test vessel, made after 24 and 48 hours, are listed in Table 2.
After 48 hours, 20% immobility occurred at the highest exposure concentration (85.9 mg a.i./l) and the highest concentration at which no immobilisation had occurred was 40.2 mg/1.
The 48-hour EC could not be calculated but must be >85.9 mg a.i./l.
Under the EC General Classification and Labelling Requirements for Dangerous Substances and
Preparations,!^^--------Uisnot considered to require-classification as the 48-hour ECy, is
considered to be greater than the highest nominal concentration, 100 mg a.i./l (mean measured level of 85.9 mg a.i./l).
Environmental parameters
The measurements of water quality (temperature, pH, dissolved oxygen and total hardness) are summarised in Table 3; they remained within acceptable limits during the study.
The test media were clear and colourless.
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CONCLUSIONS
DPT440/992118
The 48-hour EC% onBi^--------vbr the immobilisation ofDaphnia magna could not be calculated
but must be >85.9 mg active ingredient/I.
of||fBI|with The 'no-observed-effect concentration'
ingredient/I.
^^
Daphnia magna was 40.2 mg active
^
REFERENCE
Official Journal of the European Communities Commission Directive (1 March 1991). Annex VI
General classification and labelling requirements for dangerous substances and preparations. Part II "Classification on the Basis of Environmental Effect" p.62 - 64.
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DPT440/992118 FIGURE 1 Typical sample chromatogram 37.6 ing/I (9.39 mg a.i./l) taken from Day 2 of the study
CHANNEL A___INJECT 01-12-98 19:52:24 STORED TO BIN tt 57 6.76
DATA SAVED TO BIN 57
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TABLE 1
Measured concentrations
DPT440/992118
Nominal cone.,* (ing/I)
0 4.27
9.39
20.7
45.5
100
MeasuredpH^HH iq-oncentrations (mg/1)
0 hours
48 hours
%ti
Overall mean
nd
nd
-
-
4.14
[97]
3.87
93
[91]
4.01 (94)
8.93
8.90
100
8.92 (95)
[95]
[95]
K.7
18.5
99
18.6(90)
[90]
[89]
41.5
38.9
94
40.2 (88)
[91]
[85]
86.2
85.5
99
85.9 (86)
[86]
[86]
$
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nd
:
% ti :
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in terms of the active ingredient] none detected (< 0.5 mg active ingredient/I).
measured concentration after 48 hours expressed as a percentage
of the starting concentration. measured concentration expressed as a percentage of the nominal concentration. overall mean measured concentration expressed as a percentage of the
nominal concentration.
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DPT440/992118
TABLE 2 Cumulative immobilisation
Nominal cone.,* mg/1 (measured)
Cumulative numbers of immobile Daphnia
24 hours
48 hours
R, (t, R3 R< total % R, R; Ri R, total %
Control (nd) 4.27(4.01) 9.39 (8.92) 20.7(18.6) 45.5 (40.2) 100(85.9)
000000000000 000000000000 000000000000 000000000000 000000000000 02 02 00 00 04 020
nd
: none detected (< 0.5 mg active ingredient/I)
R
:
replicate number
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TABLE 3
Environmental parameters -
temperature, pH and dissolved oxygen
DPT440/992118
Nominal conc.,^ mg/1
(measured)
Control (nd) 4.27(4.01) 9.39 (8.92) 20.7(18.2) 45.5 (40.2) 100(85.9)
Temperature C
Oh
48 h
19.7 20.0 20.0 19.9 20.2 19.8
19.8 19.9 20.0 19.9 19.9 20.0
pH
Oh
48 h
7.8
7.3
7.8
7.8
7.7
7.6
7.6
7.8
7.5
7.8
7.2
7.8
Dissolved oxygen
%ASV
Oh
48 h
95
92
93
91
92
92
92
92
91
91
89
89
$
nd
ASV
none detected (<0.5 mg active ingredient/I). air saturation value.
Tlie total hardness and alkalinity of the batch ofElendt M4 medium used were 240 mg/1 and 65 mg/1 as CaCO,
respectively.
Continuous monitoring of an additional vessel containing diluent = 19.9 to 20.4C.
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APPENDIX 1 EbLLIELNINDUTl M1V144 MiVIEILDI IUM
1. Trace elements
H,BO,
MnCL4H,0 Lid
RbCl SrCl,.6H;0 NaBr Na3MoO<.2H,0 CuC1.2H,0 ZnCI, CoCl,.6H,0
KI
Na;.SeO,
NH',VO, Fe-EDTA solution
2. Macro nutrients
CaCL2H,0
i^04-7"20
NaHCO,
3. Buffer nutrients
Na;.SiO,.9H;0 NaNO, K;H,PO, K;HPO,
4. Vitamins
Thiamine hydrochloride Cyanocobalamine (B12) Biotin
mg/1
2.86 0.36 0.31 0.071 0.152 0.016 0.063 0.0165 0.013 0.010 0.0033 0.0022 0.00058 3.50
mg/1
294
123 5.80
64.8
mg/1
10 0.274 0.143
0.184
mg/1
0.075 0.0010 0.00075
The above analytical grade reagents are dissolved in revers
DPT440/992118
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DPT440/992118 APPENDIX 2
THE DETERMINATION OF^ta--------AQftUNEOUS MEDIA
SAMPLE ANALYSIS
The aqueous samples were diluted with sodium hydroxide and acetonitrile to bring the expected
^1------IJconccntrationwisthin the calibration range. Determination ofgP^B----jwasby
high performance liquid chromatography (HPLC) using a conductivity detector'.
CHROMATOGRAPHY INSTRUMENTATION AND CONDITIONS
A high performance liquid chromatography system comprising autosampler, pump, conductivity detector, anion suppressor and data collection system was used.
Column Type: Dimensions (1 x id):
Temperature: Mobile phase
Composition: Flow rate: Suppressor type: Regenerant composition: Flow rate: Injection volume:
PLRP-S supplied by Polymer Laboratories 250 x 4.6 mm Ambient
Acetonitrile : aqueous buffer solution (25 : 75% v/v) l.Oml/rnin
50 mN sulphuric acid 2.5 ml/min 100 ul
Aqueous buffer solution: 2mM ammonium hydroxide/I mM sodium carbonate (made up in ultra high purity water) and filtered through 0.2 micron cellulose nitrate filter paper.
Under the above conditions
:hromatographed as a single peak (see Figure 3).
^ ^
^
' A method contained in a fax dated 23 July 1998 from Kavsy D Dastur, Dupont Specialty Chemicals was modified to comply with Huntingdon Life Sciences standard operating procedures and instrumentation.
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DPT440/992118
CALIBRATION SOLUTIONS
Calibration solutions were prepared with the same batch of||B|^^----jused in the preparation of
the toxicity test solutions.
Trout and Algae Studies
Working calibration solutions in the nominal range 40 to 500 mg/1 (equal to 10 to 125 mg a.i./l) were prepared by volumetric dilution with acetonitrile: 100 mM sodium hydroxide (25: 75% v/v) of a primary standard prepared in ultra high purity water.
Daphnia Study
Working calibration solutions in the nominal range 4.0 to 50.0 mg/1 (equal to 1.0 to 12.5 mg a.i./l) were prepared by volumetric dilution with acetonitrile : 100 mM sodium hydroxide (25: 75% v/v) of a primary standard prepared in ultra high purity water.
CALCULATIONS
iXfHIUkfoncentrations were determined using mean bracketing standards. The mean peak height responses jyere calculated for ||p9MBtt^Jin bracketing standard chromatograms. The1)|^BBBK|concentrationin each sample was then calculated using the
following equation:
Where
Y
Z
A
F
Detector response tolWj^------^
Mean detector response to bracketing standard. Concentration of bracketing standard (mg/1). Factor to take into account sample processing.
SheeMf( The purityullBBHBIBofthe test substance isj;iven in the Tfcst Substance Data
as^JUBUps Nominal and fortified concentrations are reported
supplied and in terms oTthe
active ingredient.
VALIDATION OF THE ANALYTICAL PROCEDURE
The analytical procedure was validated by determining the linearity of response of the analytical system, specificity ofchromatographic analysis, the limit of detection, and the method's accuracy and
precision.
During the course of the study the performance of the method was monitored by the analysis of
quality control samples.
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DPT440/992118
Validation recoveries o;
TABLE 1
rom fortified samples of dilution media
Medium Dechlorinated tap water
Algal
Elendt M4
Overall mean (RSD)
Fortification level (mg/1)
As supplied Control 387.9 387.9 Control 535.5 530.0 214.2 212.0 Control 9.93 9.93 496.5 496.5
As active ingredient Control 96.98 96.98 Control 133.9 132.5 53.55 53.00 Control 2.483 2.483 124.1 124.1
Recovery as a % of
fortification level
ND
95.8 104 ND 92.0 101 92.6 95.5
ND
98.5 95.9 103 103 98.1 (4.2)
RSD: relative standard deviation. ND: none detected; less than the limit of detection (trout and algal studies : 2.5 mg a.i./l; Daphnia study: 0.5 mg
a.i./l).
The limit of detection is defined as the analyte concentration in a processed sample which would give a peak
equal to 3 x local base-line noise.
;osnpa^"y s.^."-"0"0-'"73"'81
Stability o
TABLE 2
n dilution medium
DPT440/992118
Storage conditions
Procedural recovery ' Procedural recovery ' Light, sealed, room temperature ' Light, sealed, room temperature '
Dark, sealed, 4C'
Dark, sealed, 4C ' Procedural recovery2 Procedural recovery 2
' Fortification level: 11.20 mg/1 as
2 Fortification level: 11.65 mg/1 as|
Results are given as percentage recoveries ol
Time-point
0 hours
20 hours
110
-
112
-
-
103
- 106
-
109
-
93.1
-
109
-
98.9
ifter storage for the indicated time.
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FIGURE 1
Standard Calibration fora (cff 40 500 mg/1)
DPT440/992118
100
200
300
400
500
600
Concentration (mgj
Runno.:DPT/439/013
Standard concentration (mg/1) 0.0
43.57 108.9 217.9 435.7 544.7 NOP: no observable peak
Peak height NOP 4075 10668 20734 38644 47864
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FIGURE 2
Standard Calibration forJ (ca 4 to 50 mg7I)
DPT440/992118
Run no.:DPT/440/002
Standard concentration (mg/1) 0.0
4.720 11.80 23.60 47.20 59.00 NOP: no observable peak
Peak height NOP 3295 8657 18669 38224 48699
Conteins TSCA CB&,
DPT440/992118
FIGURE 3 Typical calibration chromatography - 217.9 mg/I (= 54.47 ing/I as a.i.)
CHANNEL A
INJECT 09-09-98 14:45:47 STORED TO BIN tt 84
DATA SAVED TO BIN It 84
FIGURE 4 Typical chromatography - Unfortified algal medium
CHANNEL A'
FNJECT" 09-09-78' 14:55:17' STORED TU^TR B 85
DATA SAVED TO BIN 85
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DPT440/992118
FIGURE 5 Typical chromatography Sample of algal medium fortified at 535.5 mg/1 (= 133.9 mg/1 as a.l.)
CHANNEL A
INJECT 09-09-98 13:19:59 STORED TO BIN It 75
6.36
DATA SAUED TO BIN 75
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