Document EvxEQBb7zya18XLygdDw68m94

o Safety &n Health Research Laboratories SPONSOR Avenue E. vAanPNMiEewenhuyse, 4 B-11B60OXBru3ssels Belgium AR226-1803 AMMONIUM PERTFELSUTORITOEOMCTANOATE (APFO) EARLY-LIFE STAGE TSOXTIUCDIYTTYITTELEST IN RAINBOW TROUT UNDER FLOW-THROUGH CONDITIONS DATA REQUIREMENT OECD Guideline No. 210, 17th July 1992 STJUacDqYueDsIL'RHEarCidToOn R [EXPERIMENTAL COMPLETIONDATE 7 August 2003 DCAaTE lOFyISS2U0E k TEST FcArCILITY BP 563 -27005 Evreus - France LABORATORY 226S39TEUCDPY NUMBER Page 10f 112, 1SSNMCmRaeCcTohaprnche 4a40?72 ena oh l gh etre 5TiR:56)_RO2072A0302R355En3rne6R3C6adox France EWoantsskCitm@cckonicocmom CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 2 of 112 STATEMENT OF CONFIDENTIALITY OR NO CONFIDENTIALITY CLAIMS (To be completed by the Sponsor) CXTStudyNo. 22659 ECP/AMMONIUM PERFLUGROOCTANOATE (APFOYAPME 3of112 STATEMENT OF THE STUDY DIRECTORAND CIT SCIENTIFIC MANAGEMENT The study was performed in compliance with the Principles of Good Laboratory Practice, except rie (a sevised in 1997), ENVMCICHEM (8) for the determination of the No Observed Effect Concentrations based on animal weight and length at the endofthe test,asdescribed in: 17. . Dcret N 98-1312 du 31 dcembre 1998 concernant les Bonnes Pratiques de Laboratoire, (Journal Officiel du 1 janvier 1999) Ministre de I'Economie. des Finances et de I'Industrie. `Commission Directive 1999/11/EC of 8 March 1999 adapting to technical progress the Principles of Good Laboratory Practice as specified in Council Directive 87/18/EEC on the htahermPorniinscaiptlieosn ooff Gloawosd, rLeagbuolraattioornsy aPnradctaidcmeinainstdratthievveerpirfoivciastiioonnsorfeltahteiinrgatpopltihceataipopnlsicfaotriotnestosf. on chemical substances (OJ No. L 77of 23.3.1999). . US Environmental Protection Agency, Federal Register, 40 CFR Part 792; Toxic Substances Control Act; Good Laboratory Practice Standards, August 17, 1989 (and subsequent Nason. 30 3 a Eo Aon. No. 030 of Nin amendments). . Requirements of the Japanese Government under the revised Chemical Substance Law of Health and Welfare and No. 1014 of Ministry of International Trade and Industry, 9th December 1986. 1 declare that this report constitutes a true and faithful record of the procedures undertaken and the results obtained during the performanceofthe study. `This study was performed at CIT, BP 563, Evreux, France, except for the determination of the N(poerOfbosremredvedbyEfJf.eWc.t GCroenecne,ntrPaht.iDo.,ns Pbha.sDe.d oPrninacniipmaallRweesiegahrtchanBdioslteantgitshtiactiant,he EenId Doufptohnettedset Nemours and Co., Newark, DE, USA). f---- = C= - Be ae J. L'Haridon Study completion date: Jw og Wek Study Director I~ Zr R. Groult Date: Doctor of Chemistry Scientific Management ox uly 1 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 4 of 112 OTHER SCIENTIST INVOLVED IN THIS STUDY For Pharmacy: S. Garapon Ing. of Chemistry Head of Pharmacy CIT/Study No. 22659 ECP/AMMONTUM PERFLUOROOCTANOATE (APFOYAPME Sof 112 STATEMENT OF QUALITY ASSURANCE UNIT `Typeof inspections Study plan Study Report N Inspections 24 July 2002 2 July 2003 2 March 2004 Dates Reported to Study Director () 26 July 2002 22 July 2003 5 April 2004 Reported to Management (*) 20 July 2002 22 July 2003 16 April 2004 Ithnisadrdeiptoirotn, t"oprtohceesasb-obvaes-emde"natnidonreodutiinnsepefcatciilointsy,iantspaebcotuitontshoefscarmietictailmepraoscetdhuersetsudreyldeevsacntritboedthiins s`TthuedyfitnydpiengwseorfethaelssoemaindsepbecytitohnesQwuearleitryepAosrstuerdatnocteheUnSittu.dy Director and to CIT Management. `The inspections were performed in compliance with CIT Quality Assurance Unit procedures and PrinciplesofGood Laboratory Practice. `The reported methods and procedures were found to describe those used and the results to constitute an accurate and complete reflectionof the study raw data. | ICn.g.GaBliloil-.Kar Date: 09 ALully Jock HeadofQuality Assurance Unit (#) The dates indicated correspond to the dates of signature of audit reports by Study Director and Management. CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 6 of 112 PAGE RESERVED FOR 40 CFR 158.34 (c) (1) CERTIFICATION (To be completed by the Sponsor) CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 7 of 112 CONTENTS STATEMENT OF CONFIDENTIALITY OR NO CONFIDENTIALITY CLAIMS 2 STATEMENT OF THE STUDY DIRECTOR AND CIT SCIENTIFIC MANAGEMENT 3 OTHER SCIENTIST INVOLVED IN THIS STUDY 4 STATEMENT OF QUALITY ASSURANCE UNIT 5 PAGE RESERVED FOR 40 CFR 158.34 (c) (1) CERTIFICATION 6 CONTENTS 7 SUMMARY 9 1. INTRODUCTION 12 2. MATERIALS AND METHODS 12 2.1 Test item 12 2.1.1 Identification 12 2.1.2 Preparation 12 2.2 TEST SYSTEM 13 2.2.1 Animals and eggs 13 2.2.2 Environmental conditions during the test 13 2.2.3 Food 14 2.3 TREATMENT 14 2.3.1 Study design 14 2.3.2 Flow-through system 15 2.3.3 Dates of treatment 15 2.3.4 Definitions of life stages 15 2.3.5 Clinical observations 15 2.4 CHEMICAL ANALYSIS 16 2.5 DATA EVALUATION 16 2.5.1 Data observed and reported 16 2.5.2 Determination of the No Observed Effect Concentration (NOEC) 16 2.6 ARCHIVING 17 2.7 STUDY PLAN ADHERENCE 17 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 8 of 112 3. 3.1 3.2 3.3 3.4 3.4.1 3.4.2 4. RESULTS VALIDITY CRITERIA WATER QUALITY CHEMICAL ANALYSIS MORTALITY AND SUB-LETHAL EFFECTS Mortality Sub-lethal effects CONCLUSION 5. REFERENCES Table 1: Water quality data: pH Table 2: Water quality data: temperature (C) Table 3: Water quality data: dissolved oxygen concentrations (% of the air saturation value) Table 4: Water quality data: hardness (mg/L as CaCO3) Table 5a: Lethal and sub-lethal observations - Control Table 5b: Lethal and sub-lethal observations - 3.13 mg/L nominal Table 5c: Lethal and sub-lethal observations - 6.25 mg/L nominal Table 5d: Lethal and sub-lethal observations - 12.5 mg/L nominal Table 5e: Lethal and sub-lethal observations - 25.0 mg/L nominal Table 5f: Lethal and sub-lethal observations - 50.0 mg/L nominal Table 6: Cumulative mortality at embryo, larval and juvenile stages Table 7: Time to hatch and number hatched larvae in each replicate Table 8: Individual weight of surviving fish at the end of the test (mg) Table 9: Individual length of surviving fish at the end of the test (mm) Table 10: Mean weight and length of surviving fish at the end of the test APPENDICES 1. Analytical certificate and composition of the test item 2. Typical water quality characteristics 3. Chemical analysis of test solutions 4. NOEC determination based on mortality of eggs/embryons 5. NOEC determination based on mortality of larvae 6. NOEC determination based on mortality of juvenile fish 7. NOEC determination based on animal weight and length 18 18 18 19 19 19 20 21 21 22 23 24 25 26 34 42 50 58 66 74 75 76 78 80 81 82 85 87 94 97 100 104 to 112 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 9 of 112 SUMMARY At the request of APME, Brussels, Belgium, the toxicity of the test item AMMONIUM PERFLUOROOCTANOATE (APFO) (batch No. 10004) was evaluated on early-life stages (embryos, larvae and juveniles) of the rainbow trout Oncorhynchus mykiss, under flow-through conditions, according to OECD guideline (No. 210, 17th July 1992). Methods The study was started as soon as possible after fertilization (day 0). Just after fertilization, 180 eggs, divided equally between 3 replicates (60 eggs each), were exposed to test solutions at: 0 (control), 3.13, 6.25, 12.5, 25.0 and 50.0 mg/L nominal expressed as the APFO (solids) content of the test item. The test item was dissolved in water (total hardness between 150 20 mg/L as CaCO3). Test solutions were constantly replaced by continuous pumping of two stock solutions into flowing dilution water with a peristaltic pump system, using a replacement rate of 5.76 times the test vessel volume per day. Any residual food and faeces were removed from test vessels each day to avoid accumulation of waste and the risk of bacterial contamination. The number of surviving animals was reduced randomly to 30 in all test solution replicates just after the end of the hatching period in the control (day 26). The number of surviving animals was also reduced randomly to a same number in all replicates when swim-up and feeding began (day 50). Juveniles received two feeds (trout chow adapted to the life stage) per day corresponding to approximately 4% of their body weight per day. From the beginning to the end of the test, eggs, embryos, larvae and juveniles were checked for visible abnormalities and mortality each day. The study was stopped 60 days after the end of hatching in the control. Chemical analysis Chemical analysis was undertaken to measure the concentration of the test item in each test solution throughout the test. Samples were taken in all test solutions (except for the control) at the beginning of the test, at least once a week thereafter and at the end of the test. CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 10 of 112 Results Validity criteria: the hatching success was > 66% (81%) and the post-hatch success (until the end of the test) at least 70% in the control (88%), the dissolved oxygen concentration remained 60% of the air saturation value throughout the test. However: the temperature range for embryos (11.1C to 12.5C) was within the variation criterion of 1.5C, but deviated slightly from the nominal range of 8C to 12C. The temperature range for larvae and juvenile fish (11.6C to 14.4C) was within the variation criterion of 1.5C, but deviated slightly from the nominal range of 10C to 14C. This deviation was not considered to have affected the validity of the study, actual concentrations of the test item in test solutions were not maintained within 20% of the (harmonic) mean measured values. However, the means of the measured concentrations ranged from 70% to 85% of the nominals, and the coefficients of variation ranged from 18% to 33%, which were considered satisfactory. Chemical analysis Measured concentrations of the test item were not within 20% of the nominal values throughout the test. All study results were therefore based on harmonic means of measured concentrations in test solutions throughout the test (i.e. 85 days). These harmonic means were calculated as 2.18, 4.48, 10.7, 20.9 and 40.0 mg/L expressed as the APFO (solids) content of the test item, for nominal values of 3.13, 6.25, 12.5, 25.0 and 50.0 mg/L, respectively. Mortality According to analysis of variance (Dunnetts or Bonferroni test): the No Observed Effect Concentration (NOEC) based on mortality before hatching of larvae (egg and embryo stages) per replicate was 40.0 mg/L, the NOEC based on mortality of larvae per replicate was 40.0 mg/L. The NOEC based on mortality of juvenile per replicate could not be determined using analysis of variance since data failed to meet homogeneity of variance. This failure came from values obtained at 4.48 mg/L, which were the same in the three replicates. In this case, the Bartlett test can not determine homogeneity of variance. When the 4.48 mg/L group was discarded, data met homogeneity of variance and the NOEC based on mortality of juvenile per replicate was 40.0 mg/L. A non parametric method, i.e. the Wilcoxon rank sum test, confirmed this statistical result based on analysis of variance. Sub-lethal effects According to Dunn's test, using individual fish values as the unit of analysis after pooling of the replicate data: the mean weight of the fish was not significantly different from the control at any of the concentrations tested; therefore, the NOEC based on weight was 40.0 mg/L, the mean length of the fish was not significantly different from the control at the three highest test concentrations (10.7, 20.9 and 40.0 mg/L). A significant difference was identified at concentrations of 2.18 and 4.48 mg/L. However, no concentration related response trend was observed and the maximium reduction in mean weight compared to the control was less than 6%. Considering also the absence of significant effects on length at higher concentrations, and the absence of any significant differences in the weight of the fish, the apparent differences in length at the two lowest concentrations were considered to be chance occurrences. Therefore, the NOEC based on length was considered to be 40.0 mg/L. CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 11 of 112 In addition, the test item at concentrations up to and including 40.0 mg/L, did not delay significantly the hatching period of larvae. Conclusion Under our experimental conditions, the No Observed Effect Concentration (NOEC) of AMMONIUM PERFLUOROOCTANOATE (APFO) in a flow-through test system is 40.0 mg/L (highest mean measured concentration tested in this study), based on mortality and sub-lethal effects, for early-life stages of the rainbow trout Oncorhynchus mykiss. This result is expressed as the APFO (solids) content of the test item. CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 12 of 112 1. INTRODUCTION The objective of this study was to assess the lethal and sub-lethal effects of AMMONIUM PERFLUOROOCTANOATE (APFO) on hatching and early-life stages (embryos, larvae and juveniles) of Oncorhynchus mykiss, under flow-through conditions, according to OECD guideline (No. 210, 17th July 1992). The main criteria determined are the No and Lowest Observed Effect Concentrations (NOEC and LOEC) of the test item. 2. MATERIALS AND METHODS 2.1 TEST ITEM 2.1.1 Identification supplier: 3M name: - Study plan: AMMONIUM PERFLUOROOCTANOATE - labeling: FLUORAD TM Both names correspond to the same test item. batch number: 10004 description: colorless liquid containers: three drums date of receipt: 28 January 2002 storage conditions: at room temperature purity: 19.6% (expressed as solids) in purified water. (APFO) Data relating to the characterization of the test item are documented in the composition of the test item and an analytical certificate (presented in appendix 1) provided by the Sponsor. 2.1.2 Preparation Stock solutions were prepared by dissolving the test item directly in test water (see 2.2.2). The conditions of preparation of the stock solutions are reported in the following table: Days 0 to 40 Quantity of test item (as received) (mg) 1914 to 10527 Quantity of APFO (solids) mini/maxi (mg) 375.1 to 2063.3 4783 to 71745 937.5 to 14062.0 Days 41 to 85 3827 to 38270 750.1 to 7500.9 19133 to 191330 3750.1 to 37500.7 (1) expressed as the APFO (solids) content of the test item Concentration of the test item (mg/L) (1) 187.5 937.5 750 3750 Duration of the agitation (minutes) 5 5 5 5 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 13 of 112 Stock solutions were renewed every 1, 2 or 3 days for preparation of test solutions. Test solutions were constantly replaced in test vessels (aquaria) of 2.5 liters from days 0 to 40 and 20 liters from days 40 to 85 (end of the test), by means of a flow-through system. The pH of the test solutions remained within acceptable limits (> 6 and < 8.5) after preparation and there was no adjustment of pH during the test. 2.2 TEST SYSTEM 2.2.1 Animals and eggs Species: rainbow trout (Oncorhynchus mykiss). Reason for this choice: a species commonly used in Europe for aquatic toxicity testing and recommended in the OECD guideline. Origin: Brood stock: Pisciculture INRA, Gournay-sur-Aronde, France. Eggs: CIT. Unfertilized eggs and sperm of fish were fertilized in the laboratory. Approximately 2500 unfertilized eggs were placed in a rectangular plastic box (length int.: 8.5 cm; width int.: 6.0 cm; height int.: 5.0 cm). A few mL of medium for fertilization (5.52 g of sodium chloride, 3.75 g of Glycine and 2.42 g of TRIS for 1 liter of test water) were added to the box so that eggs were completely immersed in this medium. Approximately 0.5 mL of sperm were added thereafter. Eggs and sperm were left to stand for 4-5 minutes to allow fertilization. Fertilized eggs were then placed in a sieve and washed with test water to remove the medium for fertilization and sperm. Washed eggs were placed in test water and used to start the test 1 hour and 15 minutes after fertilization. Allocation of the animals: fertilized eggs were selected using a little spoon, without preference, and randomly assigned to test vessels. Number of animals: 180 fertilized eggs per concentration divided into three replicates of 60 eggs each. 2.2.2 Environmental conditions during the test Test solutions were constantly renewed over the test period. Any residual food and faeces were removed from test vessels each day to avoid accumulation of waste. The test conditions were as follows: Water: filtered and dechlorinated potable water which is then softened by a Permo water softening system. Half of the water passes directly into a holding tank attached to the flow-through system while the remaining half passes through a deionizer producing ultra-pure water which is then fed into the holding tank. This results in a 1:1 dilution of potable dechlorinated:deionized water giving a supply of the desirable hardness. The pH of the solution is between 6.0 and 8.5 and the total hardness is 150 20 mg/L as CaCO3. The typical quality characteristics of this water are presented in appendix 2. Temperature: between 11.1C and 12.5C for embryos and between 11.6C and 14.4C for larvae and juvenile fish. CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 14 of 112 Light/dark cycle: Duration of test: Loading: pH: Forced aeration: Oxygen concentrations: Test vessels: darkness for larvae until 7 days after hatching (except for observation periods), then 16/8 hours. 85 days (60 days after the end of hatching in the control). maximum 0.5 g of biomass (eggs, embryos, larvae or juvenile fish) per liter of test solution per 24 hours and not exceeding 5 g/L of test solution at any time. The loading was 1.82 g/L of test solution at the beginning of the test, based on weighing of 50 unfertilized eggs, and 0.0432 g/L maximum per replicate at the end of the test, based on weighing of juvenile fish on day 85. not adjusted during the test. was not used during the test. were maintained at > 60% of the air saturation value at that temperature. glass aquaria of 2.5 liters until day 41 (when larvae began to swim) and then glass aquaria of 20 liters from days 41 to the end of the test. Eggs and embryos were placed in the bottom of nest boxes (one nest box per aquarium). The bottom of each nest box, pierced with many holes (diameter lower than that of eggs/embryos), was located at circa 3 cm under the surface of the test solution and 7 cm above the bottom of the aquarium. Temperature, dissolved oxygen and pH were measured in all aquaria at the beginning of the test, once a week thereafter and at the end of the test. Temperature was also monitored continuously in a vessel placed near the test vessels. Total hardness was measured in one vessel of the control and the highest concentration at the start of the test, once a week thereafter and at the end of the test. 2.2.3 Food Juveniles were fed with two to four feeds (trout chow adapted to the life stage) per day (corresponding to approximately 4% of their body weight per day) from day 50 to the end of the test. 2.3 TREATMENT 2.3.1 Study design The study was started as soon as possible after fertilization of trout eggs (day 0). The concentrations used in this study were: 0 (control), 3.13, 6.25, 12.5, 25.0 and 50.0 mg/L nominal, expressed as the APFO (solids) content of the test item. Just after fertilization, 180 eggs, divided equally between 3 replicates (60 eggs each), were exposed to test solutions at these concentrations. The number of surviving animals was reduced randomly to 30 in all replicates 1 day (day 27) after the end of the hatching period in the control (day 26). This reduction was not carried out sooner since it is not recommended to move eggs/embryos and some unfertilized eggs could not be distinguished from fertilized ones without moving eggs/embryos. The number of surviving animals was also reduced randomly to 20 in all replicates when swim-up and feeding began (day 50) to make observations easier and also to avoid the risk of bacterial contamination. CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 15 of 112 2.3.2 Flow-through system Test solutions were constantly replaced by continuous pumping of the stock solutions (see 2.1.2) into flowing dilution water with a peristaltic pump system, using a replacement rate of 5.76 times the test vessel volume per day for the entire study. Each test solution was prepared in a mixing (by magnetic stirring) chamber receiving, by two peristaltic pumps, the appropriate flows of stock solution and dilution water. The out-flow of the mixing chamber was dispatched in three equivalent flows supplying the three replicate test vessels per concentration. Flow rates delivered to each replicate were calculated to be 10 mL/min for aquaria of 2.5 liters (until day 41) and 80 mL/min for aquaria of 20 liters (from day 41). Prior to the introduction of the test animals, the test system was operated for 2 days (pre-conditioning period) and the correct operation of the system was verified by the analysis of samples from the test vessels for the test item. Peristaltic pumps were calibrated at least once a week and tubes of the system were changed every week. Flow rates of stock solutions and dilution water used to prepare the test solutions, were also checked, and adjusted when necessary, on days -2 (beginning of the pre-conditioning period), 0 (beginning of the test), 1, 6, 13 and 41 (change of test vessels) thereafter. Flow rates of test solution delivered in each test vessels were also controlled on day -1. 2.3.3 Dates of treatment The test started on 14 May 2003 and terminated on 7 August 2003. 2.3.4 Definitions of life stages Unfertilized/fertilized eggs: unfertilized eggs can surely be distinguished from fertilized ones when eyes of the embryo are visible (through the membrane). Eggs/Embryos: from fertilization of eggs to hatching. Larvae: from hatching of embryo to complete resorption of the vitellus. Juvenile fish: from complete resorption of the vitellus, when animals swim correctly and can eat alone. The duration of these stages depends mainly of the temperature of rearing water. 2.3.5 Clinical observations In all test vessels, eggs, embryos, larvae and juveniles were checked for visible abnormalities (abnormal appearance and behavior) and mortality each day. Criteria for death were as follows: for eggs: particularly in the early stages, marked loss of translucency and change in coloration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance, for embryos: absence of body movement and/or absence of heart-beat, for larvae and juvenile fish: immobility and/or absence of respiratory movement and/or absence of heart-beat and/or white opaque coloration of central nervous system and/or lack of reactions to mechanical stimulus. Dead embryos, larvae and juvenile fish were removed as soon as observed. The other criteria were: the time to hatch and number hatched, the length of all surviving fish to the nearest 0.1 mm at the end of the test, the individual weight of these fish (blotted wet weight) to the nearest 0.1 mg. CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 16 of 112 2.4 CHEMICAL ANALYSIS Chemical analysis was undertaken to measure the concentration of the test item in each test solution, except for the control, throughout the test. Each sample contained 5mL. Samples were taken in all test test vessels (except for the control vessels) and pooled per concentration (total of 5 mL for each pooled sample), at the beginning of the test, at least once a week thereafter and at the end of the test. The analytical procedure is presented in appendix 3. 2.5 DATA EVALUATION 2.5.1 Data observed and reported The following data are presented for each concentration: cumulative mortality at embryo, larval and juvenile stages, time of start of hatching and end of hatching, number of larvae hatching each day, incidence and description of morphological abnormalities, if any, incidence and description of behavioral effects, if any, length and weight of surviving fish at the end of the study, numbers of healthy fish at the end of the test. 2.5.2 Determination of the No Observed Effect Concentration (NOEC) Lethal effects and some sub-lethal effects (weight and length of surviving fish at the end of the study) were compared with control values to determine the NOEC. The data were checked for normality (Chi-square and Shapiro-Wilks tests) and homogeneity of variance (Bartlett's test or Levene's test). For data for that were found to be normal and homogeneous (mortality), the NOEC was determined by ANOVA followed by Dunnett's test or Bonferroni-T test. For data that were found to be not normally distributed (length and weight), a Kruskal-Wallis test and Dunn's test were employed. Further details are given in appendices 4, 5, 6 and 7. CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 17 of 112 2.6 ARCHIVING The following study materials are archived by CIT (BP 563, 27005 Evreux, France) for 5 years after the end of the in vivo phase of the study: Study plan and possible amendments, raw data, correspondence, final report and possible amendments. On completion of this period, the archived study materials will be returned to the Sponsor, or may be archived at CIT for a further period (at additional cost). The total duration of archiving (depending on regulations) will be the responsibility of the Sponsor. In addition, raw data not specific to the study including, but not limited to, records of environmental data and equipment calibration, will also be archived by CIT and retained for at least 30 years. 2.7 STUDY PLAN ADHERENCE The study was performed in accordance with the Study plan No. 22659 ECP and subsequent amendments, with the following deviations from the agreed Study plan: temperature in test solutions exceeded slightly 12C for embryos (maximum of 12.5C) and 14C for larvae and juveniles fish (maximum of 14.4C), test item concentrations in test solutions were not maintained within 20% of the (harmonic) mean measured values (see 3.3 for further information). These minor deviations were not considered to have compromised the validity or integrity of the study. CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 18 of 112 3. RESULTS 3.1 VALIDITY CRITERIA Two validity criteria were fulfilled: the hatching success was > 66% (81%) and the post-hatch success (until the end of the test) at least 70% in the control (88%), the dissolved oxygen concentration remained 60% of the air saturation value throughout the test, However: the temperature range for embryos (11.1C to 12.5C) was within the variation criterion of 1.5C, but deviated slightly from the nominal range of 8C to 12C. The temperature range for larvae and juvenile fish (11.6C to 14.4C) was within the variation criterion of 1.5C, but deviated slightly from the nominal range of 10C to 14C. This deviation was not considered to have affected the validity of the study, actual concentrations of the test item in test solutions were not maintained within 20% of the (harmonic) mean measured values. However, the means of the measured concentrations ranged from 70% to 85% of the nominals, and the coefficients of variation ranged from 18% to 33%, which were considered satisfactory. 3.2 WATER QUALITY The test item was completely soluble in water. A translucent colorless solution was obtained at all concentrations tested. The minimum and maximum values of parameters measured during the test were: pH: 7.36 and 8.10, temperature in test vessels: eggs/embryos: 11.1C and 12.5C; larvae/juveniles fish: 11.6C and 14.4C, dissolved oxygen concentration: 83% and 100% (> 60% of the air saturation value), water hardness: 140 and 168 mg/L as CaCO3. The water quality parameters are presented in tables 1, 2, 3 and 4. CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 19 of 112 3.3 CHEMICAL ANALYSIS Results are presented in appendix 3, table A. Measured concentrations of the test item were not within 20% of the nominal or mean measured values throughout the test. However, actual concentrations were considered satisfactory in this study for the following reasons: accuracy of harmonic means obtained from these measured concentrations ranged from 70% to 85%, relative to nominal values. This variation compared to nominal values could be explained, at least partly and especially for the two lowest concentrations (3.13 and 6.25 mg/L nominal), by an interference between test water and the test item peak on chromatograms. This interference, probably related to natural variations in the ionic composition of test water, induced a more or less important underestimation of actual concentrations, depending on the test item level and the sampling time, coefficients of variation (CVs) of harmonic means were relatively high (18% to 33%). These CVs could partly be explained by the lower accuracy of measured values coming from the interference mentioned above. This decrease in accuracy was generally more pronounced as the measured concentration approached the limit of quantification obtained during validation of the analytical method (1 mg/L). Nevertheless, the ratios between harmonic means of measured concentrations (1.91 to 2.39) were close to that between nominal values (2). Under these conditions, although this could not explain completely some marked variations between nominal and measured values, the study results were based on these harmonic means of measured concentrations in test solutions throughout the test (i.e. 85 days). Harmonic means were calculated as 2.18, 4.48, 10.7, 20.9 and 40.0 mg/L expressed as the APFO (solids) content of the test item, for nominal values of 3.13, 6.25, 12.5, 25.0 and 50.0 mg/L, respectively. 3.4 MORTALITY AND SUB-LETHAL EFFECTS 3.4.1 Mortality Mortality noted each day is recorded in tables 5a to 5f and cumulative mortality for each stage of life is presented in table 6. The test item did not induce significant mortality in eggs/embryos at concentrations tested. At the end of the hatching period, the cumulative mortality (mean per replicate) was 19% in the control, 23% at 2.18 mg/L, 19% at 4.48 mg/L, 22% at 10.7 mg/L, 18% at 20.9 mg/L and 19% at 40.0 mg/L. According to analysis of variance (Dunnetts test; see appendix 4), the No Observed Effect Concentration (NOEC) based on mortality before hatching of larvae (egg and embryo stages) per replicate was 40.0 mg/L (50.0 mg/L nominal). After hatching, the cumulative mortality in larvae (mean per replicate) increased slightly with the test item concentration: 2% in the control, 3% at 2.18 mg/L, 4% at 4.48 mg/L, 5% at 10.7 and 20.9 mg/L and 7% at 40.0 mg/L. However, this effect was not significant. According to analysis of variance (Dunnetts test; see appendix 5) the NOEC based on mortality of larvae per replicate was 40.0 mg/L (50.0 mg/L nominal). CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 20 of 112 On the other hand, the cumulative mortality in juvenile fish (mean per replicate) was not concentration related: 10% in the control, 3% at 2.18 mg/L, 5% at 4.48 mg/L, 20% at 10.7 mg/L, 12% at 20.9 mg/L and 13% at 40.0 mg/L. The NOEC based on mortality of juvenile per replicate could not be determined using analysis of variance since data failed to meet homogeneity of variance. This failure came from values obtained at 4.48 mg/L (6.25 mg/L nominal), which were the same in the three replicates. In this case, the Bartlett test can not determine homogeneity of variance. When the 4.48 mg/L group was discarded, data met homogeneity of variance and the NOEC based on mortality of juvenile per replicate was 40.0 mg/L (50.0 mg/L nominal) (see appendix 6). A non parametric method, i.e. the Wilcoxon rank sum test, confirmed this statistical result based on analysis of variance (see appendix 6). Consequently, the test item at 40.0 mg/L had no statistically significant effect on mortality of early-life stages (eggs/embryos, larvae and juvenile fish) of the rainbow trout. 3.4.2 Sub-lethal effects Sub-lethal effects such as deformation of the body, immobile animal or abnormal respiratory rate were observed in all test solutions except for the control (tables 5a to 5f), but these effects concerned only a few animals per test solution and were not concentration related. These were generally transient effects, except for one juvenile exposed to 40.0 mg/L (replicate No. 3) showing deformation of the body (comma-shaped) that died on day 60, 10 days after the initial observation of this effect (day 50). Therefore, none of these effects were attributed to the test item. The time of hatching for each test vessel (replicate) is reported in table 7. This time corresponded to 1 or 2 day(s), depending on the replicate, and was observed between days 24 and 27 for all test vessels. According to these data, the test item up to 40.0 mg/L, did not delay significantly the hatching period of larvae. The weight and length data of surviving animals at the end of the test (day 85) are presented in tables 8, 9 and 10. The statistical analysis of the data is described in appendix 7. For both weight and length, the individual fish value was used as the unit of analysis after pooling of the replicate data. For both weight and length, the data were not normally distributed, and no normalising transformation was found. Consequently, a non-parametric analysis was performed (Kruskal-Wallis and Dunn's test). No monotonic concentration-response trend was detected statistically or visually for either weight or length. The results of the statistical analysis were as follows: the mean weight of the fish was not significantly different from the control at any of the concentrations tested; therefore, the NOEC based on weight was 40.0 mg/L, the mean length of the fish was not significantly different from the control at the three highest test concentrations (10.7, 20.9 and 40.0 mg/L). A significant difference was identified at concentrations of 2.18 and 4.48 mg/L. However, no concentration related response trend was observed and the maximium reduction in mean length compared to the control was less than 6%. Considering also the absence of significant effects on length at higher concentrations, and the absence of any significant differences in the weight of the fish, the apparent differences in length at the two lowest concentrations were considered to be spurious chance occurrences. Therefore, the NOEC based on length was considered to be 40.0 mg/L. Consequently, the test item at 40.0 mg/L did not induce statistically significant sub-lethal effects on early-life stages (eggs/embryos, larvae and juvenile fish) of the rainbow trout. CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 21 of 112 4. CONCLUSION Under our experimental conditions, the No Observed Effect Concentration (NOEC) of AMMONIUM PERFLUOROOCTANOATE (APFO) in a flow-through test system is 40.0 mg/L (highest mean measured concentration tested in this study), based on mortality and sub-lethal effects, for early-life stages of the rainbow trout Oncorhynchus mykiss. This result is expressed as the APFO (solids) content of the test item. 5. REFERENCES Bliss, C.I.: The determination of dosage-mortality curves from small number. Quart. J. Pharm. 11, 192-216 (1938). Fieller: A fundamental formula in the statistics of biological assay and some applications. Quarterly Journal of Pharmacy and Pharmacology, 117-123 (1944). Weber, E: Grunden der biologischen Statistik, Gustav Fisher Verlag, Stuttgart, 1972. CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 22 of 112 Table 1: Water quality data: pH Day Replicate 0 2 7 14 20 28 34 44 48 55 62 69 76 83 85 0 mg/L 123 7.68 7.68 7.66 7.91 7.90 7.89 7.77 7.78 7.66 7.86 7.97 7.96 7.76 7.92 7.90 7.88 7.84 7.69 7.96 7.92 7.86 7.73 7.79 7.75 7.88 7.78 7.59 7.62 7.58 7.59 7.78 7.75 7.74 7.83 7.76 7.58 7.66 7.74 7.55 7.44 7.57 7.37 7.46 7.55 7.37 3.13 mg/L 123 7.70 7.69 7.68 7.91 7.91 7.93 7.69 7.59 7.71 7.93 7.66 7.84 7.92 7.86 7.94 7.71 7.70 7.72 7.86 7.80 7.78 7.63 7.62 7.59 7.59 7.66 7.64 7.49 7.49 7.46 7.68 7.64 7.59 7.61 7.66 7.65 7.66 7.72 7.71 7.43 7.52 7.53 7.36 7.39 7.37 Nominal concentration 6.25 mg/L 12.5 mg/L 25.0 mg/L 50.0 mg/L 123 7.64 7.61 7.63 7.90 7.90 7.93 7.69 7.68 7.68 7.86 7.83 7.87 7.88 7.97 8.10 7.76 7.74 7.74 7.83 7.78 7.81 7.53 7.54 7.53 7.61 7.60 7.60 7.44 7.40 7.39 7.54 7.54 7.52 7.61 7.60 7.58 7.58 7.59 7.58 7.45 7.41 7.45 7.37 7.39 7.41 123 7.76 7.77 7.75 7.88 7.91 7.96 7.71 7.71 7.75 7.90 7.92 7.95 8.01 8.02 8.04 7.76 7.75 7.79 7.78 7.80 7.79 7.53 7.55 7.55 7.60 7.58 7.59 7.42 7.41 7.43 7.53 7.48 7.49 7.59 7.59 7.61 7.63 7.57 7.57 7.44 7.50 7.50 7.39 7.37 7.41 123 7.78 7.78 7.76 7.94 7.92 7.90 7.55 7.71 7.54 7.97 7.96 7.94 7.96 7.92 7.92 7.66 7.69 7.71 7.79 7.74 7.79 7.52 7.54 7.55 7.54 7.56 7.55 7.40 7.39 7.39 7.50 7.47 7.48 7.61 7.57 7.59 7.61 7.58 7.56 7.56 7.55 7.55 7.40 7.41 7.43 123 7.70 7.64 7.66 7.88 7.86 7.90 7.76 7.77 7.79 7.89 7.90 7.94 7.94 7.96 7.98 7.71 7.74 7.74 7.80 7.78 7.79 7.59 7.60 7.62 7.66 7.76 7.78 7.43 7.45 7.45 7.48 7.52 7.52 7.62 7.66 7.68 7.58 7.62 7.59 7.59 7.60 7.57 7.44 7.47 7.50 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 23 of 112 Table 2: Water quality data: temperature (C) Day Replicate 0 2 7 14 20 28 34 44 48 55 62 69 76 83 85 0 mg/L 3.13 mg/L Nominal concentration 6.25 mg/L 12.5 mg/L 25.0 mg/L 50.0 mg/L 1 23 11.8 11.8 11.7 11.5 11.4 11.4 11.8 11.8 11.9 12.2 12.2 12.1 12.5 12.3 12.5 12.2 11.9 11.8 12.4 12.2 12.3 12.2 12.1 12.4 12.8 13.2 13.2 13.3 13.3 13.2 13.0 12.9 13.2 12.2 12.6 12.6 13.3 13.3 13.2 13.7 13.8 14.1 14.4 14.4 14.4 123 11.7 11.7 11.8 11.1 11.1 11.1 11.8 11.8 11.8 11.9 11.9 12.1 11.9 11.8 11.9 11.8 12.1 12.2 12.2 12.0 12.1 12.4 12.8 12.8 12.9 13.0 13.2 13.2 13.2 13.1 13.2 13.0 13.1 12.9 12.8 12.6 13.5 13.0 12.4 14.1 14.1 13.8 14.4 14.4 14.4 123 11.5 11.4 11.2 11.1 11.1 11.2 11.5 11.4 11.5 11.6 11.7 11.7 11.8 11.9 11.9 11.7 11.6 11.7 11.9 11.8 11.8 12.8 12.8 12.8 13.2 13.1 13.2 13.0 13.0 13.0 13.3 13.2 13.0 12.6 12.8 12.7 13.2 13.2 13.0 14.1 14.1 14.1 14.4 14.4 14.4 123 11.3 11.3 11.3 11.1 11.1 11.1 11.5 11.5 11.5 11.7 11.7 11.7 11.8 11.8 11.8 11.7 11.7 11.8 11.8 11.7 11.8 12.8 12.8 12.8 13.2 13.1 13.1 13.0 13.0 13.0 13.0 13.1 12.8 12.8 12.7 12.6 13.4 13.3 12.9 14.1 14.1 13.9 14.4 14.4 14.3 123 11.3 11.3 11.3 11.2 11.2 11.3 11.5 11.4 11.4 11.9 11.8 12.1 11.9 11.8 11.9 11.6 11.6 11.6 11.8 11.8 11.9 12.8 12.8 12.8 12.8 12.7 12.7 13.0 13.0 13.0 13.0 13.0 12.9 12.7 12.7 12.6 13.4 13.4 13.3 13.8 13.5 13.5 14.4 14.4 14.4 123 11.4 11.4 11.4 11.4 11.4 11.4 11.6 11.6 11.8 11.9 11.9 11.9 12.0 11.9 12.1 11.7 11.6 11.8 11.9 12.1 12.2 12.8 12.7 12.8 12.7 12.3 12.5 13.0 12.6 12.6 12.9 12.6 12.8 12.5 12.1 12.4 13.5 12.6 13.4 13.9 12.7 13.1 14.1 14.4 13.9 Time period for eggs/embryos: between days 0 and 27 Minimum temperature: 11.1C Maximum temperature: 12.5C Time period for larvae and juvenile fish: between days 27 and 85 Minimum temperature: 11.6C Maximum temperature: 14.4C CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 24 of 112 Table 3: Water quality data: dissolved oxygen concentrations (% of the air saturation value) Day Replicate 0 2 7 14 20 28 34 44 48 55 62 69 76 83 85 0 mg/L 123 99 98 100 100 100 100 99 99 100 99 97 97 100 100 100 100 95 94 97 97 99 100 100 97 98 96 92 95 94 89 100 95 94 95 94 88 94 97 91 97 96 89 100 98 90 3.13 mg/L 123 99 100 100 100 100 100 100 100 100 98 99 100 100 100 100 89 93 91 98 86 77 95 95 94 94 93 92 91 88 85 94 91 89 94 89 84 96 89 80 92 91 80 94 87 79 Nominal concentration 6.25 mg/L 12.5 mg/L 123 98 99 100 100 100 100 100 100 99 100 100 94 100 100 99 90 93 93 71 96 98 93 94 94 93 93 94 88 89 91 91 93 92 91 91 90 90 92 89 92 92 88 89 90 87 123 99 100 100 100 100 100 100 100 100 100 100 100 100 100 100 94 95 94 95 97 97 93 94 94 97 99 95 90 89 88 93 93 89 93 93 88 92 92 88 91 92 87 91 90 88 25.0 mg/L 123 98 100 100 100 100 100 100 100 100 100 100 100 100 100 100 96 95 100 97 95 98 92 95 95 94 94 100 91 89 89 93 91 93 91 91 91 91 91 91 86 89 88 88 90 89 50.0 mg/L 123 98 100 100 100 100 100 100 100 100 100 100 100 100 100 100 98 97 97 97 98 100 96 97 97 97 96 96 100 98 98 95 93 96 95 85 92 94 84 85 97 83 92 86 92 86 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 25 of 112 Table 4: Water quality data: hardness (mg/L as CaCO3) Nominal concentration Day 0 mg/L 50.0 mg/L Replicate 1 1 0 152 159 2 155 158 7 158 157 14 155 158 20 154 168 28 144 153 34 148 153 44 143 141 48 140 142 55 140 145 62 142 145 69 141 146 76 143 156 83 151 153 85 148 145 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 26 of 112 Table 5a: Lethal and sub-lethal observations - Control Days Eggs/Embryos Larvae Juveniles Alive NumberNormal Dead Unfert NumberNormal Immob Deform Dead NumberNormal Immob RR Deform Dead out of 60 0 Rep. 1 60 60 60 14/05/2003 Rep. 2 60 60 60 Rep. 3 60 60 60 1 Rep. 1 60 60 60 15/05/2003 Rep. 2 60 60 60 Rep. 3 60 60 60 2 Rep. 1 60 60 60 16/05/2003 Rep. 2 60 60 60 Rep. 3 60 60 60 3 Rep. 1 60 60 60 17/05/2003 Rep. 2 60 60 60 Rep. 3 60 60 60 4 Rep. 1 60 60 60 18/05/2003 Rep. 2 60 60 60 Rep. 3 60 59 1 59 5 Rep. 1 60 60 60 19/05/2003 Rep. 2 60 60 60 Rep. 3 59 59 59 6 Rep. 1 60 60 60 20/05/2003 Rep. 2 60 60 60 Rep. 3 59 59 59 7 Rep. 1 60 60 60 21/05/2003 Rep. 2 60 60 60 Rep. 3 59 56 3 56 8 Rep. 1 60 60 60 22/05/2003 Rep. 2 60 59 1 59 Rep. 3 56 55 1 55 9 Rep. 1 60 59 1 59 23/05/2003 Rep. 2 59 59 59 Rep. 3 55 55 55 10 Rep. 1 59 59 59 24/05/2003 Rep. 2 59 59 59 Rep. 3 55 55 55 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 27 of 112 Table 5a (continued): Lethal and sub-lethal observations - Control Days Eggs/Embryos Larvae Juveniles Alive Number Normal Dead Unfert Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 60 11 Rep. 1 59 59 59 25/05/2003 Rep. 2 59 59 59 Rep. 3 55 55 55 12 Rep. 1 59 59 59 26/05/2003 Rep. 2 59 59 59 Rep. 3 55 55 55 13 Rep. 1 59 58 1 58 27/05/2003 Rep. 2 59 59 59 Rep. 3 55 55 55 14 Rep. 1 58 58 58 28/05/2003 Rep. 2 59 58 1 58 Rep. 3 55 55 55 15 Rep. 1 58 58 58 29/05/2003 Rep. 2 58 56 2 56 Rep. 3 55 55 55 16 Rep. 1 58 58 58 30/05/2003 Rep. 2 56 56 56 Rep. 3 55 55 55 17 Rep. 1 58 58 58 31/05/2003 Rep. 2 56 56 56 Rep. 3 55 55 55 18 Rep. 1 58 56 2 56 01/06/2003 Rep. 2 56 55 1 55 Rep. 3 55 54 1 54 19 Rep. 1 56 56 56 02/06/2003 Rep. 2 55 54 1 54 Rep. 3 54 54 54 20 Rep. 1 56 56 56 03/06/2003 Rep. 2 54 54 54 Rep. 3 54 54 54 21 Rep. 1 56 56 56 04/06/2003 Rep. 2 54 54 54 Rep. 3 54 54 54 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 28 of 112 Table 5a (continued): Lethal and sub-lethal observations - Control Days Eggs/Embryos Larvae Juveniles Alive Number Normal Dead Unfert Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 60 22 Rep. 1 56 56 56 05/06/2003 Rep. 2 54 53 1 53 Rep. 3 54 54 54 23 Rep. 1 56 56 56 06/06/2003 Rep. 2 53 53 53 Rep. 3 54 53 1 53 24 Rep. 1 50 49 1 6 6 55 07/06/2003 Rep. 2 50 49 1 3 3 52 Rep. 3 49 49 4 4 53 25 Rep. 1 8 6 2 47 47 53 08/06/2003 Rep. 2 19 18 1 33 33 51 Rep. 3 27 24 3 26 26 50 26 Rep. 1 3 2 1 50 50 52 09/06/2003 Rep. 2 1 1 50 50 50 Rep. 3 6 4 2 44 44 48 27 Rep. 1 2 10/06/2003 Rep. 2 Rep. 3 4 27 10/06/2003 Rep. 1 Rep. 2 Rep. 3 2 1 3 50 50 50 50 44 44 30 30 30 30 30 30 50 50 44 Alive out of 30 30 30 30 28 Rep. 1 30 30 30 11/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 29 Rep. 1 30 30 30 12/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 30 Rep. 1 30 30 30 13/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 31 Rep. 1 30 30 30 14/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 29 of 112 Table 5a (continued): Lethal and sub-lethal observations - Control Mortality and sub-lethal effects in the control Days Eggs/Embryos Larvae Juveniles Alive Number Normal Dead Unfert Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 30 32 Rep. 1 30 30 30 15/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 33 Rep. 1 30 29 1 29 16/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 34 Rep. 1 29 29 29 17/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 35 Rep. 1 29 29 29 18/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 36 Rep. 1 29 29 29 19/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 37 Rep. 1 29 29 29 20/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 38 Rep. 1 29 29 29 21/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 39 Rep. 1 29 29 29 22/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 40 Rep. 1 29 29 29 23/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 41 Rep. 1 29 29 29 24/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 42 Rep. 1 29 29 29 25/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 30 of 112 Table 5a (continued): Lethal and sub-lethal observations - Control Days Eggs/Embryos Larvae Juveniles Alive Number Normal Dead Unfert Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 30 43 Rep. 1 29 29 29 26/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 44 Rep. 1 29 29 29 27/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 45 Rep. 1 29 29 29 28/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 46 Rep. 1 29 29 29 29/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 47 Rep. 1 29 29 29 30/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 48 Rep. 1 29 29 29 01/07/2003 Rep. 2 30 29 1 29 Rep. 3 30 30 30 49 02/07/2003 Rep. 1 Rep. 2 Rep. 3 50 03/07/2003 Rep. 1 Rep. 2 Rep. 3 29 29 29 29 30 30 20 20 20 20 20 20 29 29 30 Alive out of 20 20 20 20 51 04/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 20 18 20 20 2 18 52 05/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 20 20 18 18 18 53 06/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 20 20 18 18 18 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 31 of 112 Table 5a (continued): Lethal and sub-lethal observations - Control Days Eggs/Embryos Larvae Juveniles Alive Number Normal Dead Unfert Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 20 54 07/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 20 20 18 18 18 55 08/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 20 20 18 18 18 56 09/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 20 20 18 18 18 57 10/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 20 20 18 18 18 58 11/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 20 20 18 18 18 59 12/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 19 18 17 20 1 19 1 17 60 13/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 19 18 17 16 20 1 18 1 16 61 14/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 62 15/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 63 16/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 64 17/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 32 of 112 Table 5a (continued): Lethal and sub-lethal observations - Control Days Eggs/Embryos Larvae Juveniles Alive Number Normal Dead Unfert Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 20 65 18/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 66 19/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 67 20/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 68 21/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 69 22/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 70 23/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 71 24/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 72 25/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 73 26/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 74 27/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 75 28/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 33 of 112 Table 5a (continued): Lethal and sub-lethal observations - Control Deform = deformation of the body Mortality and sub-lethal effects in the control Days Eggs/Embryos Larvae Juveniles Alive Number Normal Dead Unfert Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 20 76 29/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 77 30/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 78 31/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 79 01/08/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 80 02/08/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 81 03/08/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 82 04/08/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 83 05/08/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 84 06/08/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 85 07/08/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 Rep. = replicate Unfert= unfertillized RR= Respiration rapide CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 34 of 112 Table 5b: Lethal and sub-lethal observations - 3.13 mg/L nominal Days Eggs/Embryos Number Normal Dead Unfert 0 Rep. 1 60 60 14/05/2003 Rep. 2 60 60 Rep. 3 60 60 1 Rep. 1 60 60 15/05/2003 Rep. 2 60 60 Rep. 3 60 53 7 2 Rep. 1 60 60 16/05/2003 Rep. 2 60 60 Rep. 3 53 53 3 Rep. 1 60 60 17/05/2003 Rep. 2 60 60 Rep. 3 53 53 4 Rep. 1 60 60 18/05/2003 Rep. 2 60 60 Rep. 3 53 53 5 Rep. 1 60 60 19/05/2003 Rep. 2 60 60 Rep. 3 53 53 6 Rep. 1 60 60 20/05/2003 Rep. 2 60 60 Rep. 3 53 53 7 Rep. 1 60 60 21/05/2003 Rep. 2 60 60 Rep. 3 53 49 4 8 Rep. 1 60 59 1 22/05/2003 Rep. 2 60 58 2 Rep. 3 49 48 1 9 Rep. 1 59 57 2 23/05/2003 Rep. 2 58 58 Rep. 3 48 47 1 10 Rep. 1 57 57 24/05/2003 Rep. 2 58 58 Rep. 3 47 47 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Larvae Juveniles Alive Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 60 60 60 60 60 60 53 60 60 53 60 60 53 60 60 53 60 60 53 60 60 53 60 60 49 59 58 48 57 58 47 57 58 47 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 35 of 112 Table 5b (continued): Lethal and sub-lethal observations - 3.13 mg/L nominal Days Eggs/Embryos Number Normal Dead Unfert 11 Rep. 1 57 57 25/05/2003 Rep. 2 58 58 Rep. 3 47 47 12 Rep. 1 57 57 26/05/2003 Rep. 2 58 58 Rep. 3 47 46 1 13 Rep. 1 57 57 27/05/2003 Rep. 2 58 58 Rep. 3 46 46 14 Rep. 1 57 57 28/05/2003 Rep. 2 58 58 Rep. 3 46 46 15 Rep. 1 57 57 29/05/2003 Rep. 2 58 58 Rep. 3 46 46 16 Rep. 1 57 57 30/05/2003 Rep. 2 58 58 Rep. 3 46 46 17 Rep. 1 57 57 31/05/2003 Rep. 2 58 58 Rep. 3 46 46 18 Rep. 1 57 57 01/06/2003 Rep. 2 58 57 1 Rep. 3 46 46 19 Rep. 1 57 57 02/06/2003 Rep. 2 57 57 Rep. 3 46 46 20 Rep. 1 57 57 03/06/2003 Rep. 2 57 57 Rep. 3 46 46 21 Rep. 1 57 57 04/06/2003 Rep. 2 57 56 1 Rep. 3 46 46 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Larvae Juveniles Alive Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 60 57 58 47 57 58 46 57 58 46 57 58 46 57 58 46 57 58 46 57 58 46 57 57 46 57 57 46 57 57 46 57 56 46 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 36 of 112 Table 5b (continued): Lethal and sub-lethal observations - 3.13 mg/L nominal Days 22 05/06/2003 Rep. 1 Rep. 2 Rep. 3 23 06/06/2003 Rep. 1 Rep. 2 Rep. 3 24 07/06/2003 Rep. 1 Rep. 2 Rep. 3 25 08/06/2003 Rep. 1 Rep. 2 Rep. 3 26 09/06/2003 Rep. 1 Rep. 2 Rep. 3 27 10/06/2003 Rep. 1 Rep. 2 Rep. 3 Eggs/Embryos Number Normal Dead Unfert 57 56 1 56 54 2 46 46 56 55 1 54 53 1 46 45 1 55 55 53 51 2 45 44 1 41 41 41 40 1 27 27 21 20 1 10 8 2 12 2 10 6 2 4 27 10/06/2003 Rep. 1 Rep. 2 Rep. 3 28 11/06/2003 Rep. 1 Rep. 2 Rep. 3 29 12/06/2003 Rep. 1 Rep. 2 Rep. 3 30 13/06/2003 Rep. 1 Rep. 2 Rep. 3 31 14/06/2003 Rep. 1 Rep. 2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Larvae Juveniles Alive Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 60 56 54 46 55 53 45 55 51 44 14 14 55 10 10 50 17 17 44 55 55 55 29 29 49 34 34 42 55 55 37 34 36 35 30 30 30 30 30 30 3 1 55 34 36 Alive out of 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 37 of 112 Table 5b (continued): Lethal and sub-lethal observations - 3.13 mg/L nominal Mortality and sub-lethal effects at 3.13 mg/L nominal - 2.18 mg/L measured Days Eggs/Embryos Number Normal Dead Unfert 32 15/06/2003 Rep. 1 Rep.2 Rep. 3 33 16/06/2003 Rep. 1 Rep.2 Rep. 3 34 17/06/2003 Rep. 1 Rep.2 Rep. 3 35 18/06/2003 Rep. 1 Rep.2 Rep. 3 36 19/06/2003 Rep. 1 Rep.2 Rep. 3 37 20/06/2003 Rep. 1 Rep.2 Rep. 3 38 21/06/2003 Rep. 1 Rep.2 Rep. 3 39 22/06/2003 Rep. 1 Rep.2 Rep. 3 40 23/06/2003 Rep. 1 Rep.2 Rep. 3 41 24/06/2003 Rep. 1 Rep.2 Rep. 3 42 25/06/2003 Rep. 1 Rep.2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Larvae Juveniles Alive Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 29 1 29 30 30 30 30 30 30 29 29 29 30 30 30 30 30 30 29 29 29 30 30 30 30 30 30 29 29 29 30 30 30 30 30 30 29 29 29 30 30 30 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 38 of 112 Table 5b (continued): Lethal and sub-lethal observations - 3.13 mg/L nominal Days 43 26/06/2003 Rep. 1 Rep.2 Rep. 3 44 27/06/2003 Rep. 1 Rep.2 Rep. 3 45 28/06/2003 Rep. 1 Rep.2 Rep. 3 46 29/06/2003 Rep. 1 Rep.2 Rep. 3 47 30/06/2003 Rep. 1 Rep.2 Rep. 3 48 01/07/2003 Rep. 1 Rep.2 Rep. 3 49 02/07/2003 Rep. 1 Rep.2 Rep. 3 Eggs/Embryos Number Normal Dead Unfert 50 03/07/2003 Rep. 1 Rep.2 Rep. 3 51 04/07/2003 Rep. 1 Rep.2 Rep. 3 52 05/07/2003 Rep. 1 Rep.2 Rep. 3 53 06/07/2003 Rep. 1 Rep.2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Larvae Juveniles Alive Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 30 30 30 30 29 29 29 30 30 30 30 30 30 29 29 29 30 30 30 30 30 30 29 28 1 28 30 30 30 30 30 30 28 28 28 30 30 30 30 30 30 28 28 28 30 30 30 30 30 30 28 28 28 30 30 30 30 29 28 28 30 30 1 20 20 20 20 20 20 29 28 30 Alive out of 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 19 1 20 20 20 20 20 20 20 20 20 20 20 20 20 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 39 of 112 Table 5b (continued): Lethal and sub-lethal observations - 3.13 mg/L nominal Mortality and sub-lethal effects at 3.13 mg/L nominal - 2.18 mg/L measured Days Eggs/Embryos Number Normal Dead Unfert 54 07/07/2003 Rep. 1 Rep. 2 Rep. 3 55 08/07/2003 Rep. 1 Rep. 2 Rep. 3 56 09/07/2003 Rep. 1 Rep. 2 Rep. 3 57 10/07/2003 Rep. 1 Rep. 2 Rep. 3 58 11/07/2003 Rep. 1 Rep. 2 Rep. 3 59 12/07/2003 Rep. 1 Rep. 2 Rep. 3 60 13/07/2003 Rep. 1 Rep. 2 Rep. 3 61 14/07/2003 Rep. 1 Rep. 2 Rep. 3 62 15/07/2003 Rep. 1 Rep. 2 Rep. 3 63 16/07/2003 Rep. 1 Rep. 2 Rep. 3 64 17/07/2003 Rep. 1 Rep. 2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Larvae Juveniles Alive Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 20 20 20 20 20 20 20 20 20 20 20 20 20 19 20 20 20 1 19 20 20 20 20 19 19 19 20 20 20 20 20 20 19 19 19 20 20 20 20 19 1 20 19 19 19 20 20 20 20 19 19 19 20 20 1 19 19 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 40 of 112 Table 5b (continued): Lethal and sub-lethal observations - 3.13 mg/L nominal Mortality and sub-lethal effects at 3.13 mg/L nominal - 2.18 mg/L measured Days Eggs/Embryos Number Normal Dead Unfert 65 18/07/2003 Rep. 1 Rep. 2 Rep. 3 66 19/07/2003 Rep. 1 Rep. 2 Rep. 3 67 20/07/2003 Rep. 1 Rep. 2 Rep. 3 68 21/07/2003 Rep. 1 Rep. 2 Rep. 3 69 22/07/2003 Rep. 1 Rep. 2 Rep. 3 70 23/07/2003 Rep. 1 Rep. 2 Rep. 3 71 24/07/2003 Rep. 1 Rep. 2 Rep. 3 72 25/07/2003 Rep. 1 Rep. 2 Rep. 3 73 26/07/2003 Rep. 1 Rep. 2 Rep. 3 74 27/07/2003 Rep. 1 Rep. 2 Rep. 3 75 28/07/2003 Rep. 1 Rep. 2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Larvae Juveniles Alive Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 41 of 112 Table 5b (continued): Lethal and sub-lethal observations - 3.13 mg/L nominal Mortality and sub-lethal effects at 3.13 mg/L nominal - 2.18 mg/L measured Days 76 29/07/2003 Rep. 1 Rep. 2 Rep. 3 77 30/07/2003 Rep. 1 Rep. 2 Rep. 3 78 31/07/2003 Rep. 1 Rep. 2 Rep. 3 79 01/08/2003 Rep. 1 Rep. 2 Rep. 3 80 02/08/2003 Rep. 1 Rep. 2 Rep. 3 81 03/08/2003 Rep. 1 Rep. 2 Rep. 3 82 04/08/2003 Rep. 1 Rep. 2 Rep. 3 83 05/08/2003 Rep. 1 Rep. 2 Rep. 3 84 06/08/2003 Rep. 1 Rep. 2 Rep. 3 85 07/08/2003 Rep. 1 Rep. 2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Eggs/Embryos Number Normal Dead Unfert Larvae Juveniles Alive Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 19 19 19 19 19 19 20 20 20 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 42 of 112 Table 5c: Lethal and sub-lethal observations - 6.25 mg/L nominal Days Eggs/Embryos Larvae Juveniles Number Normal Dead Unfert Number Normal Immob Deform Dead Number Normal Immob RR 0 Rep. 1 60 60 14/05/2003 Rep. 2 60 60 Rep. 3 60 60 1 Rep. 1 60 59 1 15/05/2003 Rep. 2 60 60 Rep. 3 60 60 2 Rep. 1 59 59 16/05/2003 Rep. 2 60 60 Rep. 3 60 60 3 Rep. 1 59 59 17/05/2003 Rep. 2 60 60 Rep. 3 60 60 4 Rep. 1 59 59 18/05/2003 Rep. 2 60 60 Rep. 3 60 60 5 Rep. 1 59 59 19/05/2003 Rep. 2 60 60 Rep. 3 60 60 6 Rep. 1 59 59 20/05/2003 Rep. 2 60 60 Rep. 3 60 60 7 Rep. 1 59 58 1 21/05/2003 Rep. 2 60 59 1 Rep. 3 60 59 1 8 Rep. 1 58 58 22/05/2003 Rep. 2 59 57 2 Rep. 3 59 59 9 Rep. 1 58 58 23/05/2003 Rep. 2 57 55 2 Rep. 3 59 58 1 10 Rep. 1 58 57 1 24/05/2003 Rep. 2 55 55 Rep. 3 58 57 1 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Alive Deform Dead out of 60 60 60 60 59 60 60 59 60 60 59 60 60 59 60 60 59 60 60 59 60 60 58 59 59 58 57 59 58 55 58 57 55 57 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 43 of 112 Table 5c (continued): Lethal and sub-lethal observations - 6.25 mg/L nominal Days Eggs/Embryos Number Normal Dead 11 Rep. 1 57 57 25/05/2003 Rep. 2 55 54 1 Rep. 3 57 56 1 12 Rep. 1 57 57 26/05/2003 Rep. 2 54 54 Rep. 3 56 56 13 Rep. 1 57 56 1 27/05/2003 Rep. 2 54 54 Rep. 3 56 56 14 Rep. 1 56 56 28/05/2003 Rep. 2 54 54 Rep. 3 56 56 15 Rep. 1 56 55 1 29/05/2003 Rep. 2 54 54 Rep. 3 56 56 16 Rep. 1 55 55 30/05/2003 Rep. 2 54 54 Rep. 3 56 56 17 Rep. 1 55 55 31/05/2003 Rep. 2 54 54 Rep. 3 56 56 18 Rep. 1 55 55 01/06/2003 Rep. 2 54 54 Rep. 3 56 56 19 Rep. 1 55 55 02/06/2003 Rep. 2 54 54 Rep. 3 56 56 20 Rep. 1 55 54 1 03/06/2003 Rep. 2 54 53 1 Rep. 3 56 56 21 Rep. 1 54 54 04/06/2003 Rep. 2 53 53 Rep. 3 56 56 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Unfert Larvae Number Normal Immob Deform Dead Juveniles Number Normal Immob RR Alive Deform Dead out of 60 57 54 56 57 54 56 56 54 56 56 54 56 55 54 56 55 54 56 55 54 56 55 54 56 55 54 56 54 53 56 54 53 56 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 44 of 112 Table 5c (continued): Lethal and sub-lethal observations - 6.25 mg/L nominal Days 22 05/06/2003 23 06/06/2003 24 07/06/2003 25 08/06/2003 26 09/06/2003 27 10/06/2003 Eggs/Embryos Larvae Juveniles Number Normal Dead Unfert Number Normal Immob Deform Dead Number Normal Immob RR Rep. 1 54 53 1 Rep. 2 53 52 1 Rep. 3 56 55 1 Rep. 1 53 53 Rep. 2 52 52 Rep. 3 55 55 Rep. 1 53 53 Rep. 2 52 51 1 Rep. 3 55 55 Rep. 1 53 51 2 Rep. 2 51 48 3 Rep. 3 55 55 Rep. 1 29 29 Rep. 2 37 37 Rep. 3 31 29 2 22 22 11 11 24 24 Rep. 1 3 3 48 47 1 Rep. 2 5 2 3 43 43 Rep. 3 7 2 5 46 46 27 10/06/2003 Rep. 1 Rep. 2 Rep. 3 28 11/06/2003 Rep. 1 Rep. 2 Rep. 3 29 12/06/2003 Rep. 1 Rep. 2 Rep. 3 30 13/06/2003 Rep. 1 Rep. 2 Rep. 3 31 14/06/2003 Rep. 1 Rep. 2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 Alive Deform Dead out of 60 53 52 55 53 52 55 53 51 55 51 48 55 51 48 53 48 43 46 Alive out of 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 45 of 112 Table 5c (continued): Lethal and sub-lethal observations - 6.25 mg/L nominal Mortality and sub-lethal effects at 6.25 mg/L nominal - 4.48 mg/L measured Days Eggs/Embryos Larvae Juveniles Number Normal Dead Unfert Number Normal Immob Deform Dead Number Normal Immob RR 32 15/06/2003 Rep. 1 Rep. 2 Rep. 3 30 30 30 30 30 30 33 16/06/2003 Rep. 1 Rep. 2 Rep. 3 30 30 30 30 30 30 34 17/06/2003 Rep. 1 Rep. 2 Rep. 3 30 30 30 30 30 30 35 18/06/2003 Rep. 1 Rep. 2 Rep. 3 30 30 30 30 30 30 36 19/06/2003 Rep. 1 Rep. 2 Rep. 3 30 30 30 30 30 30 37 20/06/2003 Rep. 1 Rep. 2 Rep. 3 30 30 30 30 30 30 38 21/06/2003 Rep. 1 Rep. 2 Rep. 3 30 30 30 30 30 30 39 22/06/2003 Rep. 1 Rep. 2 Rep. 3 30 29 1 30 30 30 30 40 23/06/2003 Rep. 1 Rep. 2 Rep. 3 29 29 30 30 30 30 41 24/06/2003 Rep. 1 Rep. 2 Rep. 3 29 29 30 30 30 30 42 25/06/2003 Rep. 1 Rep. 2 Rep. 3 29 29 30 30 30 30 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Alive Deform Dead out of 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 29 30 30 29 30 30 29 30 30 29 30 30 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 46 of 112 Table 5c (continued): Lethal and sub-lethal observations - 6.25 mg/L nominal Days 43 26/06/2003 44 27/06/2003 45 28/06/2003 46 29/06/2003 47 30/06/2003 48 01/07/2003 49 02/07/2003 Eggs/Embryos Larvae Juveniles Number Normal Dead Unfert Number Normal Immob Deform Dead Number Normal Immob RR Rep. 1 Rep. 2 Rep. 3 29 29 30 30 30 30 Rep. 1 Rep. 2 Rep. 3 29 29 30 29 1 30 29 1 Rep. 1 Rep. 2 Rep. 3 29 29 29 29 29 29 Rep. 1 Rep. 2 Rep. 3 29 29 29 29 29 29 Rep. 1 Rep. 2 Rep. 3 29 29 29 29 29 29 Rep. 1 Rep. 2 Rep. 3 29 29 29 29 29 29 Rep. 1 Rep. 2 Rep. 3 29 29 29 29 29 28 1 50 03/07/2003 Rep. 1 Rep. 2 Rep. 3 51 04/07/2003 Rep. 1 Rep. 2 Rep. 3 52 05/07/2003 Rep. 1 Rep. 2 Rep. 3 53 06/07/2003 Rep. 1 Rep. 2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 19 20 20 20 20 19 19 Alive Deform Dead out of 30 29 30 30 29 29 29 29 29 29 29 29 29 29 29 29 29 29 29 29 29 28 Alive out of 20 20 20 20 20 20 20 20 20 1 19 20 20 19 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 47 of 112 Table 5c (continued): Lethal and sub-lethal observations - 6.25 mg/L nominal Days Eggs/Embryos Larvae Juveniles Number Normal Dead Unfert Number Normal Immob Deform Dead Number Normal Immob RR 54 07/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 19 19 55 08/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 19 19 56 09/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 19 19 57 10/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 19 19 58 11/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 19 19 59 12/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 19 19 60 13/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 19 19 61 14/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 19 19 62 15/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 19 19 63 16/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 19 19 64 17/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 19 19 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Alive Deform Dead out of 20 20 20 19 20 20 19 20 20 19 20 20 19 20 20 19 20 20 19 20 20 19 20 20 19 20 20 19 20 20 19 20 20 19 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 48 of 112 Table 5c (continued): Lethal and sub-lethal observations - 6.25 mg/L nominal Days Eggs/Embryos Larvae Juveniles Number Normal Dead Unfert Number Normal Immob Deform Dead Number Normal Immob RR 65 18/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 19 19 66 19/07/2003 Rep. 1 Rep. 2 Rep. 3 20 19 20 20 19 19 67 20/07/2003 Rep. 1 Rep. 2 Rep. 3 19 19 20 20 19 19 68 21/07/2003 Rep. 1 Rep. 2 Rep. 3 19 19 20 20 19 19 69 22/07/2003 Rep. 1 Rep. 2 Rep. 3 19 19 20 20 19 19 70 23/07/2003 Rep. 1 Rep. 2 Rep. 3 19 19 20 19 19 19 71 24/07/2003 Rep. 1 Rep. 2 Rep. 3 19 19 19 19 19 19 72 25/07/2003 Rep. 1 Rep. 2 Rep. 3 19 19 19 19 19 19 73 26/07/2003 Rep. 1 Rep. 2 Rep. 3 19 19 19 19 19 19 74 27/07/2003 Rep. 1 Rep. 2 Rep. 3 19 19 19 19 19 19 75 28/07/2003 Rep. 1 Rep. 2 Rep. 3 19 19 19 19 19 19 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Alive Deform Dead out of 20 20 20 19 1 19 20 19 19 20 19 19 20 19 19 20 19 19 1 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 49 of 112 Table 5c (continued): Lethal and sub-lethal observations - 6.25 mg/L nominal Days Eggs/Embryos Larvae Juveniles Number Normal Dead Unfert Number Normal Immob Deform Dead Number Normal Immob RR 76 29/07/2003 Rep. 1 Rep.2 Rep. 3 19 19 19 19 19 19 77 30/07/2003 Rep.1 Rep.2 Rep. 3 19 19 19 19 19 19 78 31/07/2003 Rep. 1 Rep.2 Rep. 3 19 19 19 19 19 19 79 01/08/2003 Rep. 1 Rep.2 Rep. 3 19 19 19 19 19 19 80 02/08/2003 Rep. 1 Rep.2 Rep. 3 19 19 19 19 19 19 81 03/08/2003 Rep. 1 Rep.2 Rep. 3 19 19 19 19 19 19 82 04/08/2003 Rep. 1 Rep.2 Rep. 3 19 19 19 19 19 19 83 05/08/2003 Rep. 1 Rep.2 Rep. 3 19 19 19 19 19 19 84 06/08/2003 Rep. 1 Rep.2 Rep. 3 19 19 19 19 19 19 85 07/08/2003 Rep. 1 Rep.2 Rep. 3 19 19 19 19 19 19 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Alive Deform Dead out of 20 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 50 of 112 Table 5d: Lethal and sub-lethal observations - 12.5 mg/L nominal Days Eggs/Embryos Larvae Number Normal Dead Unfert Number Normal Immob Deform Dead 0 Rep. 1 60 60 14/05/2003 Rep. 2 60 60 Rep. 3 60 60 1 Rep. 1 60 60 15/05/2003 Rep. 2 60 60 Rep. 3 60 57 3 2 Rep. 1 60 60 16/05/2003 Rep. 2 60 60 Rep. 3 57 57 3 Rep. 1 60 60 17/05/2003 Rep. 2 60 60 Rep. 3 57 57 4 Rep. 1 60 60 18/05/2003 Rep. 2 60 60 Rep. 3 57 56 1 5 Rep. 1 60 60 19/05/2003 Rep. 2 60 60 Rep. 3 56 56 6 Rep. 1 60 60 20/05/2003 Rep. 2 60 60 Rep. 3 56 56 7 Rep. 1 60 59 1 21/05/2003 Rep. 2 60 60 Rep. 3 56 55 1 8 Rep. 1 59 59 22/05/2003 Rep. 2 60 58 2 Rep. 3 55 55 9 Rep. 1 59 56 3 23/05/2003 Rep. 2 58 57 1 Rep. 3 55 55 10 Rep. 1 56 56 24/05/2003 Rep. 2 57 57 Rep. 3 55 53 2 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Juveniles Alive Number Normal Immob RR Deform Dead out of 60 60 60 60 60 60 57 60 60 57 60 60 57 60 60 56 60 60 56 60 60 56 59 60 55 59 58 55 56 57 55 56 57 53 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 51 of 112 Table 5d (continued): Lethal and sub-lethal observations - 12.5 mg/L nominal Days Eggs/Embryos Larvae Number Normal Dead Unfert Number Normal Immob Deform Dead 11 Rep. 1 56 56 25/05/2003 Rep. 2 57 57 Rep. 3 53 53 12 Rep. 1 56 55 1 26/05/2003 Rep. 2 57 57 Rep. 3 53 52 1 13 Rep. 1 55 55 27/05/2003 Rep. 2 57 57 Rep. 3 52 51 1 14 Rep. 1 55 55 28/05/2003 Rep. 2 57 57 Rep. 3 51 51 15 Rep. 1 55 55 29/05/2003 Rep. 2 57 56 1 Rep. 3 51 51 16 Rep. 1 55 55 30/05/2003 Rep. 2 56 56 Rep. 3 51 51 17 Rep. 1 55 55 31/05/2003 Rep. 2 56 56 Rep. 3 51 51 18 Rep. 1 55 55 01/06/2003 Rep. 2 56 56 Rep. 3 51 51 19 Rep. 1 55 54 1 02/06/2003 Rep. 2 56 56 Rep. 3 51 51 20 Rep. 1 54 54 03/06/2003 Rep. 2 56 55 1 Rep. 3 51 50 1 21 Rep. 1 54 54 04/06/2003 Rep. 2 55 54 1 Rep. 3 50 50 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Juveniles Alive Number Normal Immob RR Deform Dead out of 60 56 57 53 55 57 52 55 57 51 55 57 51 55 56 51 55 56 51 55 56 51 55 56 51 54 56 51 54 55 50 54 54 50 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 52 of 112 Table 5d (continued): Lethal and sub-lethal observations - 12.5 mg/L nominal Days 22 05/06/2003 23 06/06/2003 24 07/06/2003 25 08/06/2003 26 09/06/2003 27 10/06/2003 Eggs/Embryos Number Normal Dead Unfert Rep. 1 54 54 Rep. 2 54 54 Rep. 3 50 49 1 Rep. 1 54 53 1 Rep. 2 54 54 Rep. 3 49 49 Rep. 1 53 53 Rep. 2 54 52 2 Rep. 3 49 48 1 Rep. 1 53 53 Rep. 2 52 52 Rep. 3 47 45 2 Rep. 1 30 30 Rep. 2 39 39 Rep. 3 25 25 Rep. 1 9 Rep. 2 9 Rep. 3 4 2 7 4 5 2 2 27 10/06/2003 Rep. 1 Rep. 2 Rep. 3 28 11/06/2003 Rep. 1 Rep. 2 Rep. 3 29 12/06/2003 Rep. 1 Rep. 2 Rep. 3 30 13/06/2003 Rep. 1 Rep. 2 Rep. 3 31 14/06/2003 Rep. 1 Rep. 2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Larvae Number Normal Immob Deform Dead 1 1 23 23 13 13 21 21 44 44 43 43 42 40 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 1 1 Juveniles Alive Number Normal Immob RR Deform Dead out of 60 54 54 49 53 54 49 53 52 48 53 52 46 53 52 46 44 43 41 Alive out of 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 53 of 112 Table 5d (continued): Lethal and sub-lethal observations - 12.5 mg/L nominal Mortality and sub-lethal effects at 12.5 mg/L nominal - 10.7 mg/L measured Days Eggs/Embryos Number Normal Dead Unfert 32 15/06/2003 Rep. 1 Rep. 2 Rep. 3 33 16/06/2003 Rep. 1 Rep. 2 Rep. 3 34 17/06/2003 Rep. 1 Rep. 2 Rep. 3 35 18/06/2003 Rep. 1 Rep. 2 Rep. 3 36 19/06/2003 Rep. 1 Rep. 2 Rep. 3 37 20/06/2003 Rep. 1 Rep. 2 Rep. 3 38 21/06/2003 Rep. 1 Rep. 2 Rep. 3 39 22/06/2003 Rep. 1 Rep. 2 Rep. 3 40 23/06/2003 Rep. 1 Rep. 2 Rep. 3 41 24/06/2003 Rep. 1 Rep. 2 Rep. 3 42 25/06/2003 Rep. 1 Rep. 2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Larvae Number Normal Immob Deform Dead 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 29 1 30 30 30 30 29 29 30 30 30 29 1 29 29 30 30 29 29 29 29 Juveniles Alive Number Normal Immob RR Deform Dead out of 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 29 30 30 29 30 29 29 30 29 29 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 54 of 112 Table 5d (continued): Lethal and sub-lethal observations - 12.5 mg/L nominal Days 43 26/06/2003 44 27/06/2003 45 28/06/2003 46 29/06/2003 47 30/06/2003 48 01/07/2003 49 02/07/2003 Eggs/Embryos Number Normal Dead Unfert Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 50 03/07/2003 Rep. 1 Rep. 2 Rep. 3 51 04/07/2003 Rep. 1 Rep. 2 Rep. 3 52 05/07/2003 Rep. 1 Rep. 2 Rep. 3 53 06/07/2003 Rep. 1 Rep. 2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Larvae Number Normal Immob Deform Dead 30 30 29 29 29 29 30 30 29 28 1 29 29 30 30 28 28 29 29 30 30 28 28 29 29 30 30 28 28 29 29 30 30 28 28 29 29 30 30 28 28 29 28 1 Juveniles Alive Number Normal Immob RR Deform Dead out of 30 30 29 29 30 28 29 30 28 29 30 28 29 30 28 29 30 28 29 20 20 20 20 20 20 30 28 28 Alive out of 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 18 20 20 20 2 18 20 20 20 20 18 17 1 18 20 20 20 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 55 of 112 Table 5d (continued): Lethal and sub-lethal observations - 12.5 mg/L nominal Days Eggs/Embryos Number Normal Dead Unfert 54 07/07/2003 Rep. 1 Rep. 2 Rep. 3 55 08/07/2003 Rep. 1 Rep. 2 Rep. 3 56 09/07/2003 Rep. 1 Rep. 2 Rep. 3 57 10/07/2003 Rep. 1 Rep. 2 Rep. 3 58 11/07/2003 Rep. 1 Rep. 2 Rep. 3 59 12/07/2003 Rep. 1 Rep. 2 Rep. 3 60 13/07/2003 Rep. 1 Rep. 2 Rep. 3 61 14/07/2003 Rep. 1 Rep. 2 Rep. 3 62 15/07/2003 Rep. 1 Rep. 2 Rep. 3 63 16/07/2003 Rep. 1 Rep. 2 Rep. 3 64 17/07/2003 Rep. 1 Rep. 2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Larvae Number Normal Immob Deform Dead Juveniles Alive Number Normal Immob RR Deform Dead out of 20 20 20 18 17 1 20 19 20 18 1 19 20 20 20 18 17 1 18 19 19 19 20 20 18 16 2 19 18 20 18 1 18 20 20 20 18 16 2 18 18 18 18 20 20 20 18 15 2 1 18 18 18 18 20 20 18 17 18 18 20 1 17 18 20 20 17 15 18 18 20 2 15 18 20 20 20 15 15 15 18 18 18 20 20 15 14 18 18 20 1 14 18 20 20 20 14 14 14 18 17 1 18 20 20 20 14 14 14 18 17 1 18 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 56 of 112 Table 5d (continued): Lethal and sub-lethal observations - 12.5 mg/L nominal Mortality and sub-lethal effects at 12.5 mg/L nominal - 10.7 mg/L measured Days Eggs/Embryos Number Normal Dead Unfert 65 18/07/2003 Rep. 1 Rep. 2 Rep. 3 66 19/07/2003 Rep. 1 Rep. 2 Rep. 3 67 20/07/2003 Rep. 1 Rep. 2 Rep. 3 68 21/07/2003 Rep. 1 Rep. 2 Rep. 3 69 22/07/2003 Rep. 1 Rep. 2 Rep. 3 70 23/07/2003 Rep. 1 Rep. 2 Rep. 3 71 24/07/2003 Rep. 1 Rep. 2 Rep. 3 72 25/07/2003 Rep. 1 Rep. 2 Rep. 3 73 26/07/2003 Rep. 1 Rep. 2 Rep. 3 74 27/07/2003 Rep. 1 Rep. 2 Rep. 3 75 28/07/2003 Rep. 1 Rep. 2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Larvae Number Normal Immob Deform Dead Juveniles Alive Number Normal Immob RR Deform Dead out of 20 20 20 14 14 18 17 20 14 1 17 20 20 20 14 14 14 17 17 17 20 20 20 14 14 14 17 17 17 20 20 20 14 14 14 17 17 17 20 20 20 14 14 14 17 17 17 20 20 20 14 14 14 17 17 17 20 20 20 14 14 14 17 17 17 20 20 20 14 14 14 17 17 17 20 20 20 14 14 14 17 17 17 20 20 20 14 14 14 17 17 17 20 19 14 13 17 17 1 19 1 13 17 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 57 of 112 Table 5d (continued): Lethal and sub-lethal observations - 12.5 mg/L nominal Mortality and sub-lethal effects at 12.5 mg/L nominal - 10.7 mg/L measured Days Eggs/Embryos Number Normal Dead Unfert 76 29/07/2003 Rep. 1 Rep. 2 Rep. 3 77 30/07/2003 Rep. 1 Rep. 2 Rep. 3 78 31/07/2003 Rep. 1 Rep. 2 Rep. 3 79 01/08/2003 Rep. 1 Rep. 2 Rep. 3 80 02/08/2003 Rep. 1 Rep. 2 Rep. 3 81 03/08/2003 Rep. 1 Rep. 2 Rep. 3 82 04/08/2003 Rep. 1 Rep. 2 Rep. 3 83 05/08/2003 Rep. 1 Rep. 2 Rep. 3 84 06/08/2003 Rep. 1 Rep. 2 Rep. 3 85 07/08/2003 Rep. 1 Rep. 2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Larvae Number Normal Immob Deform Dead Juveniles Alive Number Normal Immob RR Deform Dead out of 20 19 19 19 13 13 13 17 17 17 19 19 19 13 13 13 17 17 17 19 18 13 13 17 17 1 18 13 17 18 18 18 13 13 13 17 17 17 18 18 18 13 13 13 17 17 17 18 18 18 13 13 13 17 17 17 18 18 18 13 13 13 17 17 17 18 18 18 13 13 13 17 17 17 18 18 18 13 13 13 17 17 17 18 18 18 13 13 13 17 17 17 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 58 of 112 Table 5e: Lethal and sub-lethal observations - 25.0 mg/L nominal Days Eggs/Embryos Number Normal Dead Unfert Deform 0 Rep. 1 60 60 14/05/2003 Rep. 2 60 60 Rep. 3 60 60 1 Rep. 1 60 60 15/05/2003 Rep. 2 60 60 Rep. 3 60 59 1 2 Rep. 1 60 60 16/05/2003 Rep. 2 60 60 Rep. 3 59 59 3 Rep. 1 60 60 17/05/2003 Rep. 2 60 60 Rep. 3 59 59 4 Rep. 1 60 60 18/05/2003 Rep. 2 60 60 Rep. 3 59 59 5 Rep. 1 60 60 19/05/2003 Rep. 2 60 60 Rep. 3 59 59 6 Rep. 1 60 60 20/05/2003 Rep. 2 60 60 Rep. 3 59 58 1 7 Rep. 1 60 60 21/05/2003 Rep. 2 60 60 Rep. 3 58 58 8 Rep. 1 60 60 22/05/2003 Rep. 2 60 60 Rep. 3 58 58 9 Rep. 1 60 59 1 23/05/2003 Rep. 2 60 59 1 Rep. 3 58 57 1 10 Rep. 1 59 59 24/05/2003 Rep. 2 59 59 Rep. 3 57 57 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Larvae Juveniles Alive Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 60 60 60 60 60 60 59 60 60 59 60 60 59 60 60 59 60 60 59 60 60 58 60 60 58 60 60 58 59 59 57 59 59 57 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 59 of 112 Table 5e (continued): Lethal and sub-lethal observations - 25.0 mg/L nominal Days Eggs/Embryos Larvae Juveniles Alive Number Normal Dead Unfert Deform Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 60 11 Rep. 1 59 59 59 25/05/2003 Rep. 2 59 59 59 Rep. 3 57 57 57 12 Rep. 1 59 59 59 26/05/2003 Rep. 2 59 59 59 Rep. 3 57 57 57 13 Rep. 1 59 59 59 27/05/2003 Rep. 2 59 59 59 Rep. 3 57 57 57 14 Rep. 1 59 59 59 28/05/2003 Rep. 2 59 59 59 Rep. 3 57 57 57 15 Rep. 1 59 59 59 29/05/2003 Rep. 2 59 58 1 58 Rep. 3 57 57 57 16 Rep. 1 59 59 59 30/05/2003 Rep. 2 58 58 58 Rep. 3 57 57 57 17 Rep. 1 59 59 59 31/05/2003 Rep. 2 58 57 1 57 Rep. 3 57 57 57 18 Rep. 1 59 59 59 01/06/2003 Rep. 2 57 57 57 Rep. 3 57 57 57 19 Rep. 1 59 59 59 02/06/2003 Rep. 2 57 57 57 Rep. 3 57 56 1 56 20 Rep. 1 59 59 59 03/06/2003 Rep. 2 57 56 1 56 Rep. 3 56 56 56 21 Rep. 1 59 59 59 04/06/2003 Rep. 2 56 55 1 55 Rep. 3 56 56 56 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 60 of 112 Table 5e (continued): Lethal and sub-lethal observations - 25.0 mg/L nominal Days Eggs/Embryos Larvae Juveniles Alive Number Normal Dead Unfert Deform Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 60 22 Rep. 1 59 59 59 05/06/2003 Rep. 2 55 55 55 Rep. 3 56 56 56 23 Rep. 1 59 59 59 06/06/2003 Rep. 2 55 54 1 54 Rep. 3 56 56 56 24 Rep. 1 59 58 1 58 07/06/2003 Rep. 2 54 53 1 53 Rep. 3 56 56 56 25 Rep. 1 56 56 2 2 58 08/06/2003 Rep. 2 52 52 1 1 53 Rep. 3 56 55 1 55 26 Rep. 1 37 35 2 21 21 56 09/06/2003 Rep. 2 35 35 18 18 53 Rep. 3 34 33 1 21 21 54 27 Rep. 1 6 10/06/2003 Rep. 2 11 Rep. 3 9 27 10/06/2003 Rep. 1 Rep. 2 Rep. 3 1 5 7 4 5 4 50 50 42 41 1 45 44 1 30 30 30 30 30 30 50 42 45 Alive out of 30 30 30 30 28 Rep. 1 30 30 30 11/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 29 Rep. 1 30 30 30 12/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 30 Rep. 1 30 30 30 13/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 31 Rep. 1 30 30 30 14/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 61 of 112 Table 5e (continued): Lethal and sub-lethal observations - 25.0 mg/L nominal Mortality and sub-lethal effects at 25.0 mg/L nominal - 20.9 mg/L measured Days Eggs/Embryos Larvae Juveniles Alive Number Normal Dead Unfert Deform Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 30 32 Rep. 1 30 30 30 15/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 33 Rep. 1 30 30 30 16/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 34 Rep. 1 30 30 30 17/06/2003 Rep. 2 30 30 30 Rep. 3 30 30 30 35 Rep. 1 30 30 30 18/06/2003 Rep. 2 30 29 1 29 Rep. 3 30 30 30 36 Rep. 1 30 30 30 19/06/2003 Rep. 2 29 29 29 Rep. 3 30 30 30 37 Rep. 1 30 30 30 20/06/2003 Rep. 2 29 29 29 Rep. 3 30 30 30 38 Rep. 1 30 30 30 21/06/2003 Rep. 2 29 29 29 Rep. 3 30 29 1 29 39 Rep. 1 30 30 30 22/06/2003 Rep. 2 29 29 29 Rep. 3 29 28 1 28 40 Rep. 1 30 30 30 23/06/2003 Rep. 2 29 29 29 Rep. 3 28 28 28 41 Rep. 1 30 30 30 24/06/2003 Rep. 2 29 29 29 Rep. 3 28 28 28 42 Rep. 1 30 30 30 25/06/2003 Rep. 2 29 29 29 Rep. 3 28 28 28 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 62 of 112 Table 5e (continued): Lethal and sub-lethal observations - 25.0 mg/L nominal Days Eggs/Embryos Larvae Juveniles Alive Number Normal Dead Unfert Deform Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 30 43 Rep. 1 30 30 30 26/06/2003 Rep. 2 29 29 29 Rep. 3 28 28 28 44 Rep. 1 30 30 30 27/06/2003 Rep. 2 29 29 29 Rep. 3 28 28 28 45 Rep. 1 30 30 30 28/06/2003 Rep. 2 29 29 29 Rep. 3 28 28 28 46 Rep. 1 30 30 30 29/06/2003 Rep. 2 29 29 29 Rep. 3 28 28 28 47 Rep. 1 30 30 30 30/06/2003 Rep. 2 29 29 29 Rep. 3 28 28 28 48 Rep. 1 30 30 30 01/07/2003 Rep. 2 29 29 29 Rep. 3 28 28 28 49 02/07/2003 Rep. 1 Rep. 2 Rep. 3 50 03/07/2003 Rep. 1 Rep. 2 Rep. 3 30 30 29 28 28 28 1 20 20 20 20 20 20 30 28 28 Alive out of 20 20 20 20 51 04/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 20 18 20 20 2 18 52 05/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 19 1 20 18 18 18 53 06/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 20 19 1 20 18 17 1 18 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 63 of 112 Table 5e (continued): Lethal and sub-lethal observations - 25.0 mg/L nominal Deform = deformation of the body Mortality and sub-lethal effects at 25.0 mg/L nominal - 20.9 mg/L measured Days Eggs/Embryos Larvae Juveniles Alive Number Normal Dead Unfert Deform Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 20 54 07/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 19 18 16 20 1 19 2 16 55 08/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 19 19 19 16 16 16 56 09/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 19 19 19 16 16 16 57 10/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 19 19 19 16 16 16 58 11/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 19 19 19 16 16 16 59 12/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 19 19 19 16 16 16 60 13/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 19 19 19 16 16 16 61 14/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 19 19 19 16 16 16 62 15/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 19 18 16 16 20 1 18 16 63 16/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 64 17/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 Rep. = replicate Unfert= unfertillized RR= Respiration rapide CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 64 of 112 Table 5e (continued): Lethal and sub-lethal observations - 25.0 mg/L nominal Deform = deformation of the body Mortality and sub-lethal effects at 25.0 mg/L nominal - 20.9 mg/L measured Days Eggs/Embryos Larvae Juveniles Alive Number Normal Dead Unfert Deform Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 20 65 18/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 66 19/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 67 20/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 68 21/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 69 22/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 16 16 16 70 23/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 18 18 16 15 20 18 1 15 71 24/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 15 15 15 72 25/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 15 15 15 73 26/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 15 15 15 74 27/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 15 15 15 75 28/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 15 15 15 Rep. = replicate Unfert= unfertillized RR= Respiration rapide CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 65 of 112 Table 5e (continued): Lethal and sub-lethal observations - 25.0 mg/L nominal Deform = deformation of the body Mortality and sub-lethal effects at 25.0 mg/L nominal - 20.9 mg/L measured Days Eggs/Embryos Larvae Juveniles Alive Number Normal Dead Unfert Deform Number Normal Immob Deform Dead Number Normal Immob RR Deform Dead out of 20 76 29/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 15 15 15 77 30/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 15 15 15 78 31/07/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 15 15 15 79 01/08/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 15 15 15 80 02/08/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 15 15 15 81 03/08/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 15 15 15 82 04/08/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 15 15 15 83 05/08/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 15 15 15 84 06/08/2003 Rep. 1 Rep. 2 Rep. 3 20 20 20 18 18 18 15 15 15 85 07/08/2003 Rep. 1 Rep. 2 Rep. 3 20 19 1 20 18 18 18 15 15 15 Rep. = replicate Unfert= unfertillized RR= Respiration rapide CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 66 of 112 Table 5f: Lethal and sub-lethal observations - 50.0 mg/L nominal Days Eggs/Embryos Number Normal Dead 0 Rep. 1 60 60 14/05/2003 Rep. 2 60 60 Rep. 3 60 60 1 Rep. 1 60 60 15/05/2003 Rep. 2 60 60 Rep. 3 60 56 4 2 Rep. 1 60 60 16/05/2003 Rep. 2 60 60 Rep. 3 56 54 2 3 Rep. 1 60 60 17/05/2003 Rep. 2 60 60 Rep. 3 54 54 4 Rep. 1 60 60 18/05/2003 Rep. 2 60 60 Rep. 3 54 54 5 Rep. 1 60 60 19/05/2003 Rep. 2 60 60 Rep. 3 54 54 6 Rep. 1 60 60 20/05/2003 Rep. 2 60 60 Rep. 3 54 53 1 7 Rep. 1 60 59 1 21/05/2003 Rep. 2 60 60 Rep. 3 53 52 1 8 Rep. 1 59 57 2 22/05/2003 Rep. 2 60 60 Rep. 3 52 52 9 Rep. 1 57 57 23/05/2003 Rep. 2 60 60 Rep. 3 52 51 1 10 Rep. 1 57 56 1 24/05/2003 Rep. 2 60 60 Rep. 3 51 50 1 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Unfert Larvae Number Normal Immob Deform Dead Juveniles Number Normal Immob RR Alive Deform Dead out of 60 60 60 60 60 60 56 60 60 54 60 60 54 60 60 54 60 60 54 60 60 53 59 60 52 57 60 52 57 60 51 56 60 50 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 67 of 112 Table 5f (continued): Lethal and sub-lethal observations - 50.0 mg/L nominal Days Eggs/Embryos Number Normal Dead 11 Rep. 1 56 56 25/05/2003 Rep. 2 60 60 Rep. 3 50 50 12 Rep. 1 56 56 26/05/2003 Rep. 2 60 60 Rep. 3 50 49 1 13 Rep. 1 56 56 27/05/2003 Rep. 2 60 60 Rep. 3 49 49 14 Rep. 1 56 56 28/05/2003 Rep. 2 60 60 Rep. 3 49 49 15 Rep. 1 56 56 29/05/2003 Rep. 2 60 60 Rep. 3 49 49 16 Rep. 1 56 56 30/05/2003 Rep. 2 60 59 1 Rep. 3 49 49 17 Rep. 1 56 56 31/05/2003 Rep. 2 59 59 Rep. 3 49 48 1 18 Rep. 1 56 56 01/06/2003 Rep. 2 59 59 Rep. 3 48 48 19 Rep. 1 56 56 02/06/2003 Rep. 2 59 59 Rep. 3 48 46 2 20 Rep. 1 56 56 03/06/2003 Rep. 2 59 59 Rep. 3 46 46 21 Rep. 1 56 56 04/06/2003 Rep. 2 59 58 1 Rep. 3 46 45 1 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Unfert Larvae Number Normal Immob Deform Dead Juveniles Number Normal Immob RR Alive Deform Dead out of 60 56 60 50 56 60 49 56 60 49 56 60 49 56 60 49 56 59 49 56 59 48 56 59 48 56 59 46 56 59 46 56 58 45 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 68 of 112 Table 5f (continued): Lethal and sub-lethal observations - 50.0 mg/L nominal Days 22 05/06/2003 23 06/06/2003 24 07/06/2003 25 08/06/2003 26 09/06/2003 27 10/06/2003 Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Eggs/Embryos Number Normal Dead Unfert 56 56 58 58 45 45 56 56 58 58 45 44 1 56 56 58 58 44 41 3 56 54 2 58 58 40 40 24 24 37 37 19 18 1 1 1 9 3 6 3 2 1 27 10/06/2003 Rep. 1 Rep. 2 Rep. 3 28 11/06/2003 Rep. 1 Rep. 2 Rep. 3 29 12/06/2003 Rep. 1 Rep. 2 Rep. 3 30 13/06/2003 Rep. 1 Rep. 2 Rep. 3 31 14/06/2003 Rep. 1 Rep. 2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Larvae Juveniles Number Normal Immob Deform Dead Number Normal Immob RR 1 1 30 30 21 21 22 22 53 53 49 49 37 37 30 30 30 30 30 30 30 30 30 30 30 29 1 30 30 30 30 29 29 30 30 30 30 29 29 30 30 30 30 29 29 Alive Deform Dead out of 60 56 58 45 56 58 44 56 58 41 54 58 41 54 58 40 53 49 37 Alive out of 30 30 30 30 30 30 29 30 30 29 30 30 29 30 30 29 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 69 of 112 Table 5f (continued): Lethal and sub-lethal observations - 50.0 mg/L nominal Mortality and sub-lethal effects at 50.0 mg/L nominal - 40.0 mg/L measured Days Eggs/Embryos Number Normal Dead Unfert 32 15/06/2003 Rep. 1 Rep. 2 Rep. 3 33 16/06/2003 Rep. 1 Rep. 2 Rep. 3 34 17/06/2003 Rep. 1 Rep. 2 Rep. 3 35 18/06/2003 Rep. 1 Rep. 2 Rep. 3 36 19/06/2003 Rep. 1 Rep. 2 Rep. 3 37 20/06/2003 Rep. 1 Rep. 2 Rep. 3 38 21/06/2003 Rep. 1 Rep. 2 Rep. 3 39 22/06/2003 Rep. 1 Rep. 2 Rep. 3 40 23/06/2003 Rep. 1 Rep. 2 Rep. 3 41 24/06/2003 Rep. 1 Rep. 2 Rep. 3 42 25/06/2003 Rep. 1 Rep. 2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Larvae Juveniles Number Normal Immob Deform Dead Number Normal Immob RR 30 30 30 30 29 29 30 30 30 30 29 29 30 30 30 30 29 29 30 30 30 30 29 29 30 30 30 30 29 29 30 30 30 30 29 29 30 29 1 30 30 29 29 29 28 1 30 30 29 29 28 28 30 30 29 28 1 28 28 30 30 28 28 28 27 30 30 28 27 1 1 Alive Deform Dead out of 30 30 30 29 30 30 29 30 30 29 30 30 29 30 30 29 30 30 29 29 30 29 28 30 29 28 30 28 28 30 28 27 30 28 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 70 of 112 Table 5f (continued): Lethal and sub-lethal observations - 50.0 mg/L nominal Days 43 26/06/2003 44 27/06/2003 45 28/06/2003 46 29/06/2003 47 30/06/2003 48 01/07/2003 49 02/07/2003 Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Eggs/Embryos Number Normal Dead Unfert 50 03/07/2003 Rep. 1 Rep. 2 Rep. 3 51 04/07/2003 Rep. 1 Rep. 2 Rep. 3 52 05/07/2003 Rep. 1 Rep. 2 Rep. 3 53 06/07/2003 Rep. 1 Rep. 2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Immob = immobile animal Deform = deformation of the body Larvae Juveniles Number Normal Immob Deform Dead Number Normal Immob RR 27 27 30 30 28 27 1 27 27 30 29 28 27 1 1 27 27 29 29 28 27 1 27 27 29 29 28 27 1 27 27 29 29 28 27 1 27 27 29 29 28 27 1 27 27 29 29 28 27 1 20 20 20 20 20 19 20 19 20 20 20 19 19 19 20 20 20 19 19 17 2 20 19 1 20 18 1 Alive Deform Dead out of 30 27 30 28 27 29 28 27 29 28 27 29 28 27 29 28 27 29 28 27 29 28 Alive out of 20 20 20 1 20 1 19 20 1 20 19 20 1 20 19 20 1 20 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 71 of 112 Table 5f (continued): Lethal and sub-lethal observations - 50.0 mg/L nominal Deform = deformation of the body Mortality and sub-lethal effects at 50.0 mg/L nominal - 40.0 mg/L measured Days Eggs/Embryos Number Normal Dead Unfert 54 07/07/2003 Rep. 1 Rep. 2 Rep. 3 55 08/07/2003 Rep. 1 Rep. 2 Rep. 3 56 09/07/2003 Rep. 1 Rep. 2 Rep. 3 57 10/07/2003 Rep. 1 Rep. 2 Rep. 3 58 11/07/2003 Rep. 1 Rep. 2 Rep. 3 59 12/07/2003 Rep. 1 Rep. 2 Rep. 3 60 13/07/2003 Rep. 1 Rep. 2 Rep. 3 61 14/07/2003 Rep. 1 Rep. 2 Rep. 3 62 15/07/2003 Rep. 1 Rep. 2 Rep. 3 63 16/07/2003 Rep. 1 Rep. 2 Rep. 3 64 17/07/2003 Rep. 1 Rep. 2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Larvae Juveniles Number Normal Immob Deform Dead Number Normal Immob RR 19 18 20 20 20 18 18 18 20 20 19 17 1 18 18 20 20 19 18 18 18 20 20 19 18 18 18 20 20 19 18 18 18 20 20 19 17 18 18 20 20 18 17 18 18 20 19 1 18 16 18 18 20 20 16 16 18 18 20 20 16 16 18 18 20 20 16 16 Alive Deform Dead out of 20 1 18 20 1 1 19 18 20 1 19 18 20 1 19 18 20 1 19 18 20 1 19 18 20 1 1 18 18 20 1 18 18 20 2 16 18 20 16 18 20 16 18 20 16 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 72 of 112 Table 5f (continued): Lethal and sub-lethal observations - 50.0 mg/L nominal Deform = deformation of the body Mortality and sub-lethal effects at 50.0 mg/L nominal - 40.0 mg/L measured Days Eggs/Embryos Number Normal Dead Unfert 65 18/07/2003 Rep. 1 Rep. 2 Rep. 3 66 19/07/2003 Rep. 1 Rep. 2 Rep. 3 67 20/07/2003 Rep. 1 Rep. 2 Rep. 3 68 21/07/2003 Rep. 1 Rep. 2 Rep. 3 69 22/07/2003 Rep. 1 Rep. 2 Rep. 3 70 23/07/2003 Rep. 1 Rep. 2 Rep. 3 71 24/07/2003 Rep. 1 Rep. 2 Rep. 3 72 25/07/2003 Rep. 1 Rep. 2 Rep. 3 73 26/07/2003 Rep. 1 Rep. 2 Rep. 3 74 27/07/2003 Rep. 1 Rep. 2 Rep. 3 75 28/07/2003 Rep. 1 Rep. 2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Larvae Juveniles Number Normal Immob Deform Dead Number Normal Immob RR 18 18 20 20 16 16 18 18 20 20 16 16 18 18 20 20 16 16 18 18 20 19 1 16 16 18 17 20 19 16 16 17 17 19 19 16 16 17 17 19 19 16 16 17 17 19 19 16 16 17 17 19 19 16 16 17 17 19 19 16 16 17 17 19 19 16 16 Alive Deform Dead out of 20 18 20 16 18 20 16 18 20 16 18 20 16 1 17 1 19 16 17 19 16 17 19 16 17 19 16 17 19 16 17 19 16 17 19 16 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 73 of 112 Table 5f (continued): Lethal and sub-lethal observations - 50.0 mg/L nominal Deform = deformation of the body Mortality and sub-lethal effects at 50.0 mg/L nominal - 40.0 mg/L measured Days Eggs/Embryos Number Normal Dead Unfert 76 29/07/2003 Rep. 1 Rep. 2 Rep. 3 77 30/07/2003 Rep. 1 Rep. 2 Rep. 3 78 31/07/2003 Rep. 1 Rep. 2 Rep. 3 79 01/08/2003 Rep. 1 Rep. 2 Rep. 3 80 02/08/2003 Rep. 1 Rep. 2 Rep. 3 81 03/08/2003 Rep. 1 Rep. 2 Rep. 3 82 04/08/2003 Rep. 1 Rep. 2 Rep. 3 83 05/08/2003 Rep. 1 Rep. 2 Rep. 3 84 06/08/2003 Rep. 1 Rep. 2 Rep. 3 85 07/08/2003 Rep. 1 Rep. 2 Rep. 3 Rep. = replicate Unfert= unfertillized RR= Respiration rapide Larvae Juveniles Number Normal Immob Deform Dead Number Normal Immob RR 17 17 19 19 16 16 17 17 19 19 16 16 17 17 19 19 16 16 17 17 19 19 16 16 17 17 19 19 16 16 17 17 19 19 16 16 17 17 19 19 16 16 17 17 19 19 16 16 17 17 19 19 16 16 17 17 19 19 16 16 Alive Deform Dead out of 20 17 19 16 17 19 16 17 19 16 17 19 16 17 19 16 17 19 16 17 19 16 17 19 16 17 19 16 17 19 16 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 74 of 112 Table 6: Cumulative mortality at embryo, larval and juvenile stages 0 Replicate (rep.) Fertilization Unfertilized eggs out of 60 % unfertilized eggs 0 123 003 005 3.13 2.18 123 0 10 4 0 17 7 Mortality Nominal concentration (mg/L) 6.25 12.5 Measured concentration (mg/L) 4.48 10.7 123 123 335 558 752 12 8 3 25.0 20.9 123 544 877 50.0 40.0 123 061 0 10 2 Eggs/embryos Eggs/embryos out of 60 % of fertilized eggs % of fertilized eggs (rep. mean) 10 10 13 17 17 23 19 5 13 20 8 26 36 23 9 14 9 16 25 16 19 9 12 16 17 22 28 22 5 14 11 9 25 20 18 7 5 21 12 9 36 19 Larvae Larvae out of 30 1 1 0 Larvae % 330 Larvae % 2 (replicate mean) 120 370 3 112 337 4 022 077 5 022 077 5 312 10 3 7 7 Juveniles Juveniles out of 20 Juveniles % Juveniles % (replicate mean) Hatching success % Mean hatching success % Post-hatch success % Mean post-hatch success % 024 0 10 20 10 83 83 77 81 97 87 80 88 110 550 3 111 555 5 92 74 64 84 75 84 92 88 100 92 92 88 273 10 35 15 20 025 0 10 25 12 83 78 72 91 75 80 90 58 78 100 83 68 314 15 5 20 13 88 91 64 75 92 73 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 75 of 112 Table 7: Time to hatch and number hatched larvae in each replicate Replicate 0 0 123 3.13 2.18 123 Nominal concentration (mg/L) 6.25 12.5 Measured concentration (mg/L) 4.48 10.7 123 123 25.0 20.9 123 50.0 40.0 123 First day of hatching Last day of hatching Time to hatch 24 24 24 26 26 26 222 25 25 25 26 27 27 122 26 26 26 27 27 27 111 26 26 25 27 27 27 112 25 25 26 27 27 27 221 26 26 25 27 27 27 112 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 76 of 112 Table 8: Individual weight of surviving fish at the end of the test (mg) Replicate 1 Animals number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Mean Replicate 2 Animals number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Mean 0 0 834 527 730 1040 590 489 479 501 620 668 801 446 872 491 575 574 470 472 615 597 619.6 Test item nominal concentrations (mg/L) 3.13 6.25 12.5 25.0 Test item measured concentrations (mg/L) 2.18 4.48 10.7 20.9 601 707 476 596 592 630 543 619 869 721 734 847 544 655 822 568 640 755 655 573 668 774 660 676 836 607 634 657 1130 587 457 535 686 661 729 445 389 526 551 860 493 640 698 969 457 693 850 724 617 662 675 891 727 850 649 398 442 521 833 714 485 645 512 669 580 526 564 631 594 529 903 221 940 557 739 652 646.8 644.5 663.6 649.2 675 746 647 550 933 718 832 638 713 419 803 808 548 1187 817 837 631 657 731.1 933 591 684 642 764 737 656 698 518 810 758 857 983 653 681 622 839 633 702 724.3 480 547 556 620 650 708 666 653 601 520 693 626 627 697 573 565 596 587 762 617.2 866 826 914 913 569 388 629 752 512 899 658 506 743 705.8 544 847 703 628 890 527 771 586 672 583 604 436 808 834 623 236 507 721 640.0 50.0 40.0 728 626 746 961 937 733 651 929 786 604 638 671 741 475 561 598 645 707.6 559 605 805 1003 635 451 782 516 685 554 649 851 1043 746 495 605 706 638 785 690.2 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 77 of 112 Table 8 (continued): Individual weight of surviving fish at the end of the test (mg) 0 Replicate 3 0 Animals number 1 1039 2 937 3 850 4 827 5 729 6 719 7 697 8 855 9 928 10 1250 11 909 12 797 13 672 14 927 15 897 16 787 17 18 19 20 Mean 863.8 Mean of replicate means 738.1 Mean of individual weights 729.1 Test item nominal concentrations (mg/L) 3.13 6.25 12.5 25.0 Test item measured concentrations (mg/L) 2.18 4.48 10.7 20.9 565 441 504 624 530 701 728 513 616 555 708 1059 493 817 664 463 590 541 815 658 629.3 666.8 666.1 550 588 376 977 474 799 901 485 611 720 515 766 593 695 635 605 748 454 562 634.4 632.1 632.1 972 609 578 722 708 674 744 963 658 605 861 717 767 805 647 526 673 719.4 696.2 694.8 852 681 639 722 679 771 884 743 709 1170 708 617 779 869 722 769.7 686.3 680.2 50.0 40.0 630 719 461 686 794 767 1159 842 838 694 646 903 885 392 648 560 726.5 708.1 707.1 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 78 of 112 Table 9: Individual length of surviving fish at the end of the test (mm) Replicate 1 Animals number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Mean Replicate 2 Animals number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Mean Test item nominal concentrations (mg/L) 0 3.13 6.25 12.5 25.0 50.0 Test item measured concentrations (mg/L) 0 2.18 4.48 10.7 20.9 40.0 42 35 34 31 34 39 37 36 35 34 36 36 44 37 37 37 38 37 49 34 35 38 36 39 42 34 38 34 36 37 36 33 36 35 35 37 32 37 36 34 36 34 33 42 33 30 34 40 35 32 35 36 29 37 37 29 32 34 42 34 39 34 35 40 39 35 33 29 36 40 35 37 40 34 40 35 40 34 33 38 37 37 29 29 34 31 35 37 36 32 35 32 36 34 37 34 33 35 32 35 33 34 30 34 32 40 23 37 40 33 34 35 37 36.8 34.5 35.1 35.6 35.0 35.6 37 40 32 38 34 32 37 35 31 39 39 36 34 35 34 37 35 38 33 35 35 40 38 41 39 37 36 32 40 35 36 36 36 30 34 30 38 36 34 34 39 39 37 36 35 36 34 32 39 32 34 34 36 37 29 39 30 39 32 36 40 35 35 35 36 38 37 41 34 30 32 39 33 39 36 38 38 44 44 33 37 36 36 39 37 35 36 28 39 32 37 26 34 36 38 36 34 38 37 36 34 37 35 37 39 39 36.9 36.3 34.7 35.5 35.3 36.2 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 79 of 112 Table 9 (continued): Individual length of surviving fish at the end of the test (mm) Replicate 3 Animals number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Test item nominal concentrations (mg/L) 0 3.13 6.25 12.5 25.0 50.0 Test item measured concentrations (mg/L) 0 2.18 4.48 10.7 20.9 40.0 41 34 33 40 38 34 39 32 35 36 34 38 36 33 30 34 32 31 38 36 39 35 35 36 38 33 32 36 34 35 37 34 37 34 36 35 35 38 37 39 37 43 37 34 32 40 36 39 37 35 35 35 37 37 43 34 38 36 40 34 39 35 33 37 38 32 34 41 39 35 33 41 34 31 36 37 32 38 40 39 37 37 39 26 40 35 37 36 36 34 40 33 34 34 30 36 39 36 34 32 37 34 37 Mean 38.0 35.1 35.2 36.3 35.8 35.2 Mean of replicate means 37.2 35.3 35.0 35.8 35.4 35.6 Mean of individual lengths 37.2 35.3 35.0 35.8 35.3 35.7 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 80 of 112 Table 10: Mean weight and length of surviving fish at the end of the test Length and weight of fish Nominal concentration (mg/L) 0 3.13 6.25 12.5 25.0 50.0 Replicate 0 123 2.18 123 Measured concentration (mg/L) 4.48 10.7 123 123 20.9 123 40.0 123 Number of fish 20 18 16 19 19 20 19 19 19 18 13 17 20 18 15 17 19 16 Mean weight (mg) Mean length (mm) 619.6 731.1 863.8 646.8 724.3 629.3 644.5 617.2 634.4 663.6 705.8 719.3 649.2 640.0 769.7 707.6 690.2 726.5 36.8 36.9 38.0 34.5 36.3 35.1 35.1 34.7 35.2 35.6 35.5 36.3 35.0 35.3 35.8 35.6 36.2 35.2 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 81 of 112 APPENDICES CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 82 of 112 1. Analytical certificate and composition of the test item CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 83 of 112 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 84 of 112 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 85 of 112 2. Typical water quality characteristics CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 86 of 112 CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 87 of 112 3. Chemical analysis of test solutions CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 88 of 112 CHEMICAL ANALYSIS Principle An aliquot of each sample was diluted and analyzed by Ion Chromatography with Electrochemical Detection (Conductimetry). The concentrations of AMMONIUM PERFLUOROOCTANOATE (APFO) were determined from a calibration curve of peak area against concentration of AMMONIUM PERFLUOROOCTANOATE (APFO) in standard solutions. Sample preparation All samples were mixed. If necessary, samples were then diluted with Milli-Q to achieve concentrations in the range 1-100 mg/L of AMMONIUM PERFLUOROOCTANOATE (APFO). Chromatographic conditions Pump : GP 50 Gradient Pump (Dionex) Mobile phase : phase A: aqueous sodium hydroxide solution 50 mM* phase B: Milli-Q water phase C: acetonitrile * Aqueous sodium hydroxide solution 50 mM: 2.6 mL of sodium hydroxide solution at 46/48% (Fisher Chemicals, Ref.: S/4930/05) was added to 1 L of Milli-Q Water. The obtained solution was degassed with helium (10 min). Time (min) 0 5 12 13 25 phase A (%) 10 40 40 10 10 phase B (%) 85 0 0 85 85 phase C (%) 5 60 60 5 5 curve 6 6 - Flow rate Precolumn Column Temperature Detector : 1 mL/min : Ionpac ATC-3, 4 mm (Dionex) : Ionpac AS16 (Dionex) length = 250 mm, inner diameter = 4 mm : 30C : ED 50 Electrochemical Detector (Dionex) Conductimetry after eluent neutralization CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 89 of 112 Neutralization : ASRS Ultra (Dionex), electric current 70 mA or AMMS (Dionex) Injector Injected volume : AS50 (Dionex), at 10C : 25 L Data acquisition software : Multichrom 2 (Fisons Instruments) Retention time : APFO, approx. 9 min Analysis time : 30 min Calibration curve Peak areas were determined for standard solutions ranging from 1 to 100 mg/L of AMMONIUM PERFLUOROOCTANOATE (APFO) (six levels). A calibration curve was obtained by plotting test item peak areas against concentrations. The regression analysis of the calibration data gave an equation of the following form: where Y = aX + b Y = test item peak area (Vs) X = concentration of test item (mg/L) a = slope value b = intercept Assay Samples of AMMONIUM PERFLUOROOCTANOATE (APFO) were analyzed by Ion Chromatography with Electrochemical Detection (Conductimetry). One dilution was prepared for each sample and one injection (of 25-L aliquots) was performed for each final dilution. The peak area was determined for each sample. The concentration of AMMONIUM PERFLUOROOCTANOATE (APFO) in each sample was calculated using the equation obtained from the calibration data. All the results are expressed as mg/L of APFO. CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 90 of 112 Table A: Concentration of AMMONIUM PERFLUOROOCTANOATE (APFO) in the test solutions during the study (mg/L) Measured (mg/L) (1) Nominal (mg/L) Day 0 Day 2 Day 5 Day 7 Day 12 Day 20 Day 22 Test solutions 3.13 2.95 (94) 2.90 (93) 2.76 (88) 3.41 (109) 3.33 (106) 1.51 (48) 3.48 (111) 6.25 6.25 (100) 5.51 (88) 5.23 (84) 7.34 (117) 7.44 (119) 4.98 (80) 4.88 (78) 12.5 12.0 (96) 10.9 (87) 10.6 (85) 13.1 (105) 12.3 (98) 10.2 (82) 13.3 (106) 25.0 29.7 (119) 23.6 (94) 24.6 (98) 23.0 (92) 24.3 (97) 16.6 (66) 27.1 (108) 50.0 50.7 (101) 47.8 (96) 46.3 (93) 43.6 (87) 41.7 (83) 43.0 (86) 53.2 (106) Stock solutions 187.5 937.5 160 (85) 154 (82) 162 (86) 160 (85) 877 (94) 864 (92) 860 (92) 898 (96) Nominal (mg/L) Day 28 Day 37 Day 44 Day 51 Day 54 Day 58 Day 63 Test solutions 3.13 2.97 (95) 1.74 (56) 2.54 (81) 1.59 (51) 1.46 (47) 2.11 (67) 1.61 (51) 6.25 7.86 (126) 5.23 (84) 3.91 (63) 4.42 (71) 3.70 (59) 4.71 (75) vd nc 12.5 17.6 (141) 11.6 (93) 21.9 (2) (175) 8.47 (68) 9.00 (72) 9.79 (78) 11.4 (91) 25.0 16.6 (66) 11.7 (47) 19.5 (78) 19.5 (78) 21.5 (86) 22.5 (90) 21.7 (87) 50.0 66.9 (134) 25.9 (52) vd nc 44.0 (88) 34.5 (69) 23.1 (46) 36.7 (73) Stock solutions 750 3750 748 (100) 767 (102) 686 (91) 3587 (96) 3688 (98) 3463 (92) CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 91 of 112 Table A (continued): Concentration of AMMONIUM PERFLUOROOCTANOATE (APFO) in the test solutions during the study (mg/L) Measured (mg/L) (1) Nominal (mg/L) Day 68 Day 70 Day 72 Day 75 Day 79 Day 82 Day 84 Test solutions 3.13 1.93 (62) 1.59 (51) 2.30 (73) 2.37 (76) 1.89 (60) 2.90 (93) 2.30 (73) 6.25 3.21 (51) 3.67 (59) vd nc 2.86 (46) 3.02 (48) 4.61 (74) 3.69 (59) 12.5 9.34 (75) 10.7 (86) 8.54 (68) 7.21 (58) 10.1 (81) 9.21 (74) vd nc 25.0 20.1 (80) 20.9 (84) 20.5 (82) 20.6 (82) 23.8 (95) 24.4 (98) 23.1 (92) 50.0 43.3 (87) 41.2 (82) 31.7 (63) 35.1 (70) 45.3 (91) 49.4 (99) 42.0 (84) Stock solutions 750 3750 714 (95) 706 (94) 3630 (97) 3517 (94) 734 (98) 827 (110) 740 (99) 743 (99) 609 (81) 3449 (92) 3850 (103) 3489 (93) 3745 (100) 3158 (84) Nominal (mg/L) Test solutions 3.13 6.25 12.5 25.0 50.0 Harmonic mean 2.18 4.48 10.7 20.9 40.0 CV% accuracy 31 70 33 72 31 85 18 84 24 80 Stock solutions 187.5 937.5 750 3750 159 875 723 3548 2 85 2 93 8 96 5 95 (1): numbers in brackets represent percentages of the nominal concentration (2): the important variation of this concentration compared to the nominal value was explained by a malfunctionning of the peristaltic pump used for the stock solution. According to calibration data, this pump delivered on day 49 a flow rate approximately twice higher than the target flow rate (adjusted on day 43). vd: value discarded nc: not calculated CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 92 of 112 VALIDATION OF THE ANALYTICAL METHOD The validation of the analytical method was performed according to CIT Standard Operating Procedures. The specificity, limit of quantification, linearity, repeatability of injections, accuracy and precision (Coefficient of Variation: CV %) of the analytical method were determined. Specificity The specificity of the analytical method was demonstrated as follows: analysis of a standard solution of AMMONIUM PERFLUOROOCTANOATE (APFO) in Milli-Q water, analysis of Milli-Q water or test water (LC, M4 or dechlorinated:desionized water) without dilution. No relevant interference between the test item peak and Milli-Q water or test water was observed on chromatograms. Limit of quantification The limit of quantification of the analytical method was established as 1 mg/L for a standard solution of APFO. This limit corresponds to a limit of quantification of 1 mg/L for the test item in aqueous phase. Linearity Linearity was checked by analysis of three different sets of six standard solutions containing 1, 5, 10, 20, 50 and 100 mg/L of AMMONIUM PERFLUOROOCTANOATE (APFO) in Milli-Q water. Satisfactory linearity was demonstrated in the range 1 to 100 mg/L since the coefficients of determination obtained were higher than 0.999. Repeatability of injections Replicate analysis (n = 10) of a solution containing 50 mg/L of the test item gave satisfactory results since the coefficients of variation values obtained were as follows: 1% based on peak height, 1% based on peak area. Based on these results, the repeatability of injections of the analytical method was validated taking into account peak area. CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 93 of 112 Accuracy and precision Six analyses of solutions containing 2.03 and 50.7 mg/L of the test item in Milli-Q water were carried out. The accuracy and the precision (CV%) obtained were as follows: Concentration (mg/L) Nominal Mean measured* 2.03 50.7 2.02 53.7 *: mean values of six determinations Dilution CV factor (%) 1 6 1 2 Accuracy (%) 99 106 Conclusion The analytical method was validated and considered to be suitable for the analysis of the samples of the study. CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 94 of 112 4. NOEC determination based on mortality of eggs/embryons CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 95 of 112 NOEC determination based on cumulative mortality of eggs/embryos per replicate CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 96 of 112 NOEC determination based on cumulative mortality of eggs/embryos per replicate (continued) CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 97 of 112 5. NOEC determination based on mortality of larvae CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 98 of 112 NOEC determination based on cumulative mortality of larvae per replicate CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 99 of 112 NOEC determination based on cumulative mortality of larvae per replicate (continued) CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 100 of 112 6. NOEC determination based on mortality of juvenile fish CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 101 of 112 NOEC determination based on cumulative mortality of juvenile fish per replicate CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 102 of 112 NOEC determination based on cumulative mortality of juvenile fish per replicate (continued) CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 103 of 112 NOEC determination based on cumulative mortality of juvenile fish per replicate (continued) CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 104 of 112 7. NOEC determination based on animal weight and length CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 105 of 112 December 2, 2003 STATISTICAL ANALYSIS OF FISH LENGTH AND WEIGHT DATA CIT Study Number 22659 ECP (Carried out on behalf of the sponsor by John W. Green, Ph.D., Ph.D., Principal Research Biostatistician, E I Dupont de Nemours and Co., Newark, DE, USA) INTRODUCTION The purpose of this study was to determine the toxicity of the test compound to rainbow trout. The experimental protocol in the study was designed to satisfy OECD Guideline 210. The data were analyzed to determine the toxicity of the test substance. The NOEC, LOEC and MATC were calculated with respect to body length and weight of surviving adults at the end of the experiment. Endpoints were observed at each of five nominal concentrations (3.13, 6.25, 12.5, 25, and 50 ppm), and a water control. Three replicate vessels with twenty trouts each were tested at each test concentration and each control. STATISTICAL METHODOLOGY The NOEC for each response was determined through the use of trend tests (Jonckheere trend test) wherever appropriate. These trend tests assume monotonicity in the concentration-response. Parametric, or if appropriate, non-parametric tests, were used to determine if there was significant departure from monotonicity. Other multiple comparison methods were used for non-monotone data. Continuous or reproduction data (length, weight) were evaluated for normality using the Shapiro-Wilk test (3) while homogeneity of variance was assessed by Levene's test.(4) Data found (2 ) to be both normal and homogenous were evaluated in the context of ANOVA. Monotonic concentration-response data were evaluated using the Jonckheere -Terpstra trend test.(5) Non-monotonic concentration-response data were assessed using the Dunnett method (2) if the data were normally distributed and homogeneous, and using the Tamhane-Dunnett (or T3) method (2) if the data were normally distributed but heterogeneous. If factors in addition to concentration were considered, such as the number of specimens in the test chamber at test start, then the Jonckheere test was not appropriate and mult(4i-)factor ANOVA was used for analysis. Non-normal data were analyzed by alternative methods. If possible, a transformation was found to obtain normality. Otherwise, a Kruskal-Wallis test (5) replaced the ANOVA, Dunn's test (2) replaced Dunnett's, and the Jonckheere-Terpstra test was still applicable, except as noted. Outliers were determined by the Tukey outlier rule and, if present, their effect on the conclusions was determined. If massive ties were present in a continuous response, exact (6) permutation data analysis methods were used for analysis. An exact version of the Kruskal-Wallis test was used for analysis, followed by exact versions of Jonckheere-Terpstra or separate Mann-Whitney (5) (or, equivalently, Wilcoxon) comparisons of each treatment group to the control, using a Bonferonni correction. CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 106 of 112 The No Observable Effect Concentration (NOEC) was defined as the highest measured concentration at or below which no statistically significant effect was observed. The Lowest Observed Effect Concentration (LOEC) was the lowest measured concentration at which a statistically significant effect occurred. The maximum acceptable toxicant concentration (MATC) was defined as the geometric mean of the NOEC and LOEC. DISCUSSION OF STATISTICAL TESTS Length was not normally distributed and no normalizing transformation was found. Consequently, a non-parametric analysis was done. A test for dose-response monotonicity for length indicated significant departure from the expected monotone dose-response. Consequently, no trend analysis is reported. Based on the pairwise Dunn's test, the 3.13 and 6.25 ppm median lengths were significantly smaller than the control median, but the 12.5, 25, and 50 ppm median lengths were not significantly smaller than the control. Given that the mean or median length was not significantly smaller than the control at 50, 25 or 12.5 ppm and there is no trend evident formally or visually in the dose-response, the statistically significant findings at the two lowest concentrations are considered chance occurrences. The NOEC was, accordingly, above 50 ppm. Weight was not normally distributed when the fish was treated as the unit of analysis, so a non-parametric analysis was done. A test for dose-response monotonicity for weight indicated significant departure from the expected monotone dose-response. Consequently, no trend analysis is reported. From Dunn's test, no treatment mean was significantly different from the control. The NOEC for weight, was, accordingly, above 50 ppm. ENDPOINT Length Weight SUMMARY TABLE OF STATISTICAL ENDPOINTS NOEC MATC LOEC STATISTICAL TEST (ppm) (ppm) (ppm) > 50 * * Dunn's > 50 * * Dunn's * not calculated/could not be adequately determined CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 107 of 112 REFERENCES 1. Agresti, A. 1990. Categorical Data Analysis, Wiley, New York. 2. Hochberg, Y. and A.C. Tamhane. 1987. Multiple Comparison Procedures, Wiley, New York. 3. Shapiro, S.S. and M.B Wilk. 1965. An analysis of variance test for normality (complete samples). Biometrika 52: 591-611. 4. Box, G.E.P. 1953. Non-normality and tests on variances. Biometrika 40: 318-335. 5. Lehmann, E. I. 1975. Nonparametrics: Statistical Methods Based on Ranks. Holden-Day, San Francisco. 6. Mehta, C. and N. Patel. Cambridge, MA. 1992. StatXact-Turbo, Cytel Software Corporation, 7. Draper, N.R. and H. Smith. 1981. Applied Regression Analysis, 2nd edition, Wiley, New York. STATISTICAL ANALYSIS: Length CONC 0.00 3.13 6.25 12.50 25.00 50.00 LENGTH 37.1852 35.2759 35.0175 35.8333 35.3208 35.6731 PERCENT CHANGE FROM CONTROL PCTCHNGL WEIGHT PCTCHNGW 0.0 5.1 ** 5.8 ** 3.6 5.0 4.1 729.074 666.138 632.053 694.771 680.170 707.058 0.0 8.6 13.3 4.7 6.7 3.0 DOSE DOSEVAL 1 0 2 3.13 3 6.25 4 12.5 5 25 6 50 STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 15:13 MONDAY 27OCT03 ECOSTATS MEASUREMENTS FROM DATASET ELS GROUP STATISTICS FOR LENGTH BY DOSE 11:35 Monday, October 27, 2003 COUNT MEAN MEDIAN STD_DEV STD_ERR 54 37.1852 37 3.65531 0.49742 58 35.2759 35 2.82714 0.37122 57 35.0175 35 2.31834 0.30707 48 35.8333 36 2.67640 0.38631 53 35.3208 36 3.42375 0.47029 52 35.6731 36 3.62303 0.50242 STATISTICAL ANALYSIS LIST FILE CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 108 of 112 REPORT CREATED ON 15:13 MONDAY 27OCT03 SHAPIRO-WILK TEST OF NORMALITY OF LENGTH FISH: FULL DATA OBS STD SKEW KURT SW_STAT P_VALUE SIGNIF 322 3.09777 -0.21029 1.27715 0.98721 .006038444 ** --------------------------------------------------------------------- Outliers & Influential Observations LENGTH FROM FULL DATA 11:35 Monday, October 27, 2003 Obs FISH DOSE DOSEVAL GROUP OBSER Pred SE_PRED 1 4 2 18 3 10 4 18 5 16 6 13 7 15 8 7 9 14 1 0 1 0 1 0 5 25 5 25 6 50 6 50 6 50 6 50 I 49 I 30 I 29 V 23 V 26 VI 44 VI 28 VI 43 VI 26 37.1852 37.1852 37.1852 35.3208 35.3208 35.6731 35.6731 35.6731 35.6731 0.42488 0.42488 0.42488 0.42886 0.42886 0.43297 0.43297 0.43297 0.43297 Obs L95M U95M Resid LB UB 1 36.3492 38.0211 11.8148 -6.90491 7.04665 2 36.3492 38.0211 -7.1852 -6.90491 7.04665 3 36.3492 38.0211 -8.1852 -6.90491 7.04665 4 34.4770 36.1645 -12.3208 -6.90491 7.04665 5 34.4770 36.1645 -9.3208 -6.90491 7.04665 6 34.8212 36.5249 8.3269 -6.90491 7.04665 7 34.8212 36.5249 -7.6731 -6.90491 7.04665 8 34.8212 36.5249 7.3269 -6.90491 7.04665 9 34.8212 36.5249 -9.6731 -6.90491 7.04665 NOTE The data was NOT normally distributed. A nonparametric analysis will be performed. CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 109 of 112 STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 15:13 MONDAY 27OCT03 Kruskal-Wallis Test on LENGTH ECOSTATS MEASUREMENTS FOR FULL DATA 11:35 Monday, October 27, 2003 The NPAR1WAY Procedure Wilcoxon Scores (Rank Sums) for Variable RESPONSE Classified by Variable DOSE Sum of Expected Std Dev Mean DOSE N Scores Under H0 Under H0 Score ---------------------------------------------------------------- 1 54 10766.50 8721.00 620.225295 199.379630 2 58 8437.00 9367.00 637.971323 145.465517 3 57 7894.00 9205.50 633.644344 138.491228 4 48 8031.00 7752.00 591.263538 167.312500 5 53 8345.00 8559.50 615.600937 157.452830 6 52 8529.50 8398.00 610.898068 164.028846 Average scores were used for ties. Kruskal-Wallis Test Chi-Square 14.6503 DF 5 Pr > Chi-Square 0.0120 ------------------------------------------------------------------- STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 15:13 MONDAY 27OCT03 Modified Dunn's Multiple Comparisons (Two-sided) on LENGTH ECOSTATS MEASUREMENTS FOR FULL DATA USING ALPHA=.05 11:35 Monday, October 27, 2003 DOSE DOSEVAL COUNT N0 MRANK ABS_DIFF CRIT_05 CRIT_01 SIGNIF P_VAL 1 0 54 2 3.13 58 3 6.25 57 4 12.5 48 5 25 53 6 50 52 54 199.380 54 145.466 54 138.491 54 167.313 54 157.453 54 164.029 0.0000 45.8615 55.0202 53.9141 45.0639 54.0633 * 60.8884 45.2540 54.2915 ** 32.0671 47.2730 56.7136 41.9268 46.0773 55.2791 35.3508 46.3004 55.5467 . 0.005 0.001 0.201 0.047 0.123 ------------------------------------------------------------------- CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 110 of 112 STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 15:13 MONDAY 27OCT03 MONOTONICITY CHECK OF LENGTH - FULL DATA DOSES 0, 3.13, 6.25, 12.5, 25, 50 ppm PARM P_T SIGNIF DOSE TREND 0.11636 DOSE QUAD 0.01005 * NOTE ***** WARNING ***** A nonlinear trend was detected. Jonckheere trend test assumptions are violated. Jonckheere test will not be done. STATISICAL ANALYSIS: Weight STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 15:14 MONDAY 27OCT03 ECOSTATS MEASUREMENTS FROM DATASET ELS GROUP STATISTICS FOR WEIGHT BY DOSE 11:35 Monday, October 27, 2003 DOSE DOSEVAL COUNT MEAN MEDIAN STD_DEV STD_ERR 1 0 2 3.13 3 6.25 4 12.5 5 25 6 50 54 729.074 718.5 185.275 25.2127 58 666.138 647.5 156.658 20.5702 57 632.053 626.0 110.684 14.6605 48 694.771 674.5 141.942 20.4876 53 680.170 679.0 167.618 23.0241 52 707.058 685.5 158.493 21.9790 11:35 Monday, October 27, 2003 STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 15:14 MONDAY 27OCT03 SHAPIRO-WILK TEST OF NORMALITY OF WEIGHT FISH: FULL DATA STD SKEW KURT SW_STAT P_VALUE SIGNIF 153.802 0.39272 0.71751 0.98666 .004545086 ** -------------------------------------------------------------------- CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 111 of 112 Outliers & Influential Observations WEIGHT FROM FULL DATA 11:35 Monday, October 27, 2003 Obs FISH DOSE DOSEVAL GROUP OBSER Pred SE_PRED 1 14 2 10 3 8 4 12 5 18 6 16 7 10 8 7 1 0 I 1 0 I 2 3.13 II 2 3.13 II 5 25 V 5 25 V 5 25 V 6 50 VI 1187 1250 1130 1059 221 236 1170 1159 729.074 729.074 666.138 666.138 680.170 680.170 680.170 707.058 21.0948 21.0948 20.3544 20.3544 21.2928 21.2928 21.2928 21.4966 Obs L95M U95M Resid LB UB 1 687.570 770.578 457.926 -386.266 374.075 2 687.570 770.578 520.926 -386.266 374.075 3 626.091 706.185 463.862 -386.266 374.075 4 626.091 706.185 392.862 -386.266 374.075 5 638.276 722.063 -459.170 -386.266 374.075 6 638.276 722.063 -444.170 -386.266 374.075 7 638.276 722.063 489.830 -386.266 374.075 8 664.763 749.352 451.942 -386.266 374.075 NOTE The data was NOT normally distributed. A nonparametric analysis will be performed. STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 15:14 MONDAY 27OCT03 Kruskal-Wallis Test on WEIGHT ECOSTATS MEASUREMENTS FOR FULL DATA 11:35 Monday, October 27, 2003 The NPAR1WAY Procedure Wilcoxon Scores (Rank Sums) for Variable RESPONSE Classified by Variable DOSE Sum of Expected Std Dev Mean DOSE N Scores Under H0 Under H0 Score -------------------------------------------------------------------- 1 54 9897.00 8721.00 624.124466 183.277778 2 58 8589.00 9367.00 641.982058 148.086207 3 57 7340.00 9205.50 637.627876 128.771930 4 48 8259.50 7752.00 594.980635 172.072917 5 53 8816.00 8559.50 619.471036 166.339623 6 52 9101.50 8398.00 614.738601 175.028846 Average scores were used for ties. Kruskal-Wallis Test CIT/Study No. 22659 ECP/AMMONIUM PERFLUOROOCTANOATE (APFO)/APME 112 of 112 Chi-Square DF Pr > Chi-Square 13.0639 5 0.0228 ---------------------------------------------------------------- STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 15:14 MONDAY 27OCT03 Modified Dunn's Multiple Comparisons (Two-sided) on WEIGHT ECOSTATS MEASUREMENTS FOR FULL DATA USING ALPHA=.05 11:35 Monday, October 27, 2003 DOSE DOSEVAL COUNT N0 MRANK ABS_DIFF CRIT_05 CRIT_01 SIGNIF P_VAL 1 0 54 54 183.278 0.0000 46.1498 55.3661 . 2 3.13 58 54 148.086 35.1916 45.3472 54.4032 0.11403 3 6.25 57 54 128.772 54.5058 45.5385 54.6328 * 0.00512 4 12.5 48 54 172.073 11.2049 47.5702 57.0701 1.00000 5 25 53 54 166.340 16.9382 46.3670 55.6267 0.86681 6 50 52 54 175.029 8.2489 46.5915 55.8960 1.00000 ---------------------------------------------------------------------- STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 15:14 MONDAY 27OCT03 MONOTONICITY CHECK OF WEIGHT - FULL DATA DOSES 0, 3.13, 6.25, 12.5, 25, 50 ppm PARM P_T SIGNIF DOSE TREND 0.81925 DOSE QUAD 0.01618 * NOTE ***** WARNING ***** A nonlinear trend was detected. Jonckheere trend test assumptions are violated. Jonckheere test will not be done.