A 72-HOUR TOXICITY TEST WITH THE FRESHWATER ALGA

( Pseudokirchneriella subcapitata )

FINAL REPORT- 2 - Project Number 523A-105

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality is made for any information contained in this study on the basis of its

falling within the scope of FIFRA section 10(d) (1)(A), (B), or (C).

Company : _________________________ (Typed Name)

Company Agent : _________________________ (Typed Name)

Title : _________________________

Signature : ________________________________ Date : ____________

### GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

SPONSOR :

# Wildlve International, Ltd. Project Number



### QUALITY ASSURANCE STATEMENT

This study was examined for compliance with Good Laboratory Practice Standards as

published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August

1989; OECD Principles of Good Laboratory Practice (ENVIMCICHEM (98) 17); and Japan MAFF;

1 1 NohSan, Notification No. 6283, Agricultural Production Bureau, 1 October 1999. The dates of all

inspections and audits and the dates that any findings were reported to the Study Director and

Laboratory Management were as follows:

ACTIVITY: DATE REPORTED TO:

DATE CONDUCTED: STUDY DIRECTOR: MANAGEMENT:

Test Substance Preparation August 4,2005 August 4,2005 August 9,2005

Temperature Measurements August 8,2005 August 8,2005 August 9,2005

Protocol August 22,2005 August 22,2005 August 26,2005

Biological Data and Draft Report August 22 & 23, 2005 August 23, 2005 August 26,2005

Final Report August 26,2005 August 26,2005 August 26,2005

All inspections are study-based unless otherwise noted

Date 

# 4 Quality ssurkce Program supervisor

# . Project Number

-5-

REPORT APPROVAL

SPONSOR :- 6 - Project Number- 7 -

TABLE OF CONTENTS

- Continued -

TABLES AND FIGURES

Table 1. Measurements of Total Organic Carbon in Test Solution Samples ......................................18

Table 2. Temperature Measurements ..................................................................................................19

Table 3. pH Measurements.............................................................................................................. ....20

Table 4. Light Intensity Measurements...............................................................................................21

Table 5. Mean Area Under the Growth Curve and Percent Inhibition Over the

72-Hour Exposure Period...................................................................................................... 22

Table 6. Mean Growth Rate and Percent Inhibition Over the 72-Hour Exposure Period...................23

Table 7. EL50 Values Over the 72-Hour Exposure Period.................................................................24

Figure 1. Growth of Pseudokirchneriella subcapitata in the negative control over the 72-hour

exposure period.............................................................................................................. ....... 25

Figure 2. Concentration-response curve for Pseudokirchneriella subcapitata exposed to

the test substance for 72 hours.............................................................................................. 26



### APPENDICES

Appendix 1. Freshwater Algal Medium .......................................................................................... 27

Appendix 2. Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd.

Well Water................................................................................................................... 28

Appendix 3. Cell Density By Replicate Over the 72-Hour Exposure Period................................... 30

Appendix 4. Area Under the Growth Curve By Replicate Over the 72-Hour

Exposure Period........................................................................................................... 31

Appendix 5. Growth Rate By Replicate Over the 72-Hour Exposure Period................................... 32

Appendix 6. Personnel Involved in the Study .................................................................................. 33- 8 -

SUMMARY

SPONSOR :

TITLE : A 72-Hour Toxicity Test with the Freshwater

Alga ( Pseudokirchneriella subcapitata )

:

TEST DATES : Study Initiation : August 3, 2005

Experimental Start (OECD) : August 4, 2005

Experimental Start (EPA) : August 5, 2005

Exposure Termination : August 8, 2005

Experimental Termination : August 11, 2005

LENGTH OF EXPOSURE: 72 Hours Exposure

TEST ORGANISMS : Freshwater Alga ( Pseudokirchneriella subcapitata )

SOURCE OF TEST ORGANISMS : Wildlife International, Ltd. Cultures

Easton, Maryland 21601

TEST CONCENTRATIONS : Nominal WAF Loading Rates

Negative Control

1.8 mg/L

4.1 mg/L

10 mg/L

20 mg/L

45 mg/L

100 mg/L

RESULTS: Based on nominal WAF loading rates:

AREA UNDER THE GROWTH CURVE :

72-Hour E b C50: 19 mg/L

95% Confidence Limits: 10 and 34 mg/L

72-Hour NOEL: <1.8 mg/L

GROWTH RATE :

72-Hour E

r C50: 72 mg/L

95% Confidence Limits: 66 and 79 mg/L

72-Hour NOEL: 1.8 mg/L- 9 - Project Number

INTRODUCTION

This study was conducted by Wildlife International, Ltd. for

The in-life phase of the definitive

test was conducted from August 5 to 8, 2005. Raw data generated by

I

OBJECTIVE

The objective of this study was to determine the toxicity of the test substance to the

freshwater green alga, Pseudokirchneriella subcapitata , during a 72-hour exposure period.

EXPERIMENTAL DESIGN

The green alga, Pseudokirchneriella subcapitata , was exposed to six water accommodated

fractions (WAF) of the test substance and a negative control (culture medium) under static conditions

for 72 hours. Six replicate test chambers in the control group and three replicate test chambers in

each treatment group were maintained. Nominal loading rates were selected in consultation with the

Sponsor and were based upon the results of an exploratory range finding toxicity test. Nominal WAF

loading rates selected were 1.8, 4.1, 10, 20, 45 and 100 mg per liter (mg/L). Samples of test water

were collected at test initiation and at test termination for analysis of total organic carbon (TOC).

At test initiation an inoculum of the algal cells was added to each test chamber to achieve a

nominal concentration of approximately 10,000 Pseudokirchneriella cells/mL. Samples were

collected from each replicate test chamber at approximately 24-hour intervals during the test to

determine cell densities, which were subsequently used to calculate areas under the growth curve and

growth rates. Areas under the growth curve and growth rates were used to calculate percent inhibition

values relative to the control over the 72-hour exposure period. E b L50 and E

r L50 values were

calculated, when possible, based upon area under the growth curve and growth rate, respectively, for

each 24-hour interval of the exposure period. The 72-hour EL10 and EL20 values were also

calculated for area under the growth curve and growth rate, if applicable. No-observed-effectconcentrations (NOEL) were determined at 72 hours through statistical evaluation of the areas under

the growth curve and growth rates, as well as examination of the concentration-response pattern.- 10 - Project Number

MATERIALS AND METHODS

The study was conducted according to procedures outlined in the protocol, A 72-Hour

Toxicity Test with the Freshwater Alga ( Pseudokirchneriella subcapitata ). The protocol was based

on procedures outlined in Official Journal of the European Communities No. L383, Method C.3.:

Algal Inhibition Test (1); and the working draft of OECD Proposal for Updating Guideline 201:

Freshwater Alga and Cyanobacteria, Growth Inhibition Test (2).

Test Substance

The test substance was received from on April 19, 2005. It was assigned

upon receipt and was stored under ambient

conditions. The test substance, a liquid, was identified as Tall Oil Fatty Acid, Lot number 8488/035.

Test Organism

The freshwater green alga, Pseudokirchneriella subcapitata , was selected as the test species

for this study. The species is representative of an important group of freshwater algae, and was

selected for use in the test based upon a past history of use, and ease of culturing in the laboratory.

Original algal cultures were obtained from the University of Toronto Culture Collection, and had

been maintained in culture medium at Algal cells

used in this test were obtained from cultures that had been actively

growing in culture medium for at least two weeks prior to test initiation. Algal cells for this study

were taken from a culture that had been transferred to fresh media four days prior to test initiation.

The negative control organisms were expected to exhibit exponential growth over the 72-hour

exposure period. Exponential growth phase, defined as the period of growth where the algal cells are

dividing at a constant rate, is indicated by the linear section of the growth curve (Figure 1).

Culture Medium

The algal cells were cultured and tested in freshwater algal medium (1-3). Stock nutrient

solutions were prepared by adding reagent-grade chemicals to purified .

well water. The test medium then was prepared by adding appropriate volumes of the stock nutrient

solutions to purified well water (Appendix 1). The pH was 7.9 and the medium was sterilized by

filtration (0.22 μ m) prior to use. The results of the most recent analyses performed to measure the- 11 - Project Number

concentrations of selected contaminants in the well water used to prepare the algal medium are

presented in Appendix 2.

Test Apparatus

Test chambers were sterile, 250-mL Erlenmeyer flasks plugged with foam stoppers and

contained 100 mL of test or control medium. The test flasks were labeled with the project number,

concentration and replicate, and were indiscriminately positioned daily on a mechanical shaker in an

environmental chamber designed to maintain the desired test temperature throughout the test. The

test flasks were shaken continuously at 100 rpm.

Preparation of Test Concentrations

Test concentrations were WAFs prepared at loading rates of 0.0, 1.8, 4.1, 10, 20, 45 and

100 mg/L. The test substance was added to 4 L of algal growth medium in a 4-L Pyrex aspirator

bottle with tubulation and stirred for approximately 24 hours by means of a Teflon ® coated stir bar on

a magnetic stir plate. Stirring speed was adjusted to produce a vortex depth of approximately 30% of

the test solution height. After mixing, the WAFs were allowed to settle for 1 hour. At mixing

initiation and test initiation, the 4.1, 10, 20, 45 and 100 mg/L test solutions appeared cloudy with

increasing intensity corresponding with increasing concentration. The negative control and the

1.8 mg/L test solutions appeared clear and colorless. At test termination, the 100 mg/L treatment

level had suspended material, remaining test solutions had no visible test material.

Analytical Sampling

Samples of the test solutions were collected at approximately 0 and 72 hours to measure

concentrations total organic carbon (TOC). At test initiation samples were collected from the WAF

preparation vessels for each treatment and control group. At test termination, the biological replicates

from each respective treatment and control group were pooled and then sampled. All samples were

collected in glass vials, acidified with 2N HCl and stored refrigerated until analyzed. TOC was

measured using a Shimadzu Model TOC-5000 or TOC-V CSH total organic carbon analyzer.

Inoculation of Test Chambers

Prior to test initiation, an inoculum of the algal cells was prepared in freshwater algal medium

at a concentration of approximately 1.0 X 10 6 cells/mL. The concentration of algal cells was verified- 12 - Project Number

using a hemacytometer and microscope, and 1.0 mL of the inoculum was added to each test chamber

to achieve a nominal concentration of approximately 10,000 cells/mL at test initiation.

Environmental Conditions

Test chambers were held in an environmental chamber at a temperature of 24 ± 2 ° C. The

temperature of a container of water adjacent to the test chambers in the environmental chamber was

recorded twice daily during the test using a liquid-in-glass thermometer.

The algae were held under continuous cool-white fluorescent lighting throughout the test.

The target light intensity was 6,000 ± 20% lux. Light intensity was measured at five locations

surrounding the test flasks on the shaker table at test initiation. Light intensity was measured using a

SPER Scientific 840006C light meter.

The pH of the medium in each treatment and control group was measured at test initiation

and at test termination using a Thermo Orion Model 525 Aplus pH meter. At test initiation, pH was

measured in the individual batches of test solution prepared for each treatment and the control group.

At test termination, pH was measured in pooled samples of test solution collected from each of the

replicates in each treatment and control group.

Algal Growth Measurements and Observations

Test medium samples were collected from each replicate of the treatment and control group

for the determination of algal cell densities. Samples were collected at approximately 24-hour

intervals during the 72-hour exposure and were held for a maximum of three days under refrigerated

conditions sufficient to inhibit growth until cell counts could be performed. Cell counts were

performed using a hemacytometer and microscope. The samples then were diluted using an electron

solution (Isoton ® ), as needed, to maintain counting accuracy. A small amount of each sample was

loaded onto a hemacytometer and 10 grids were counted. The mean number of cells per grid was

calculated and this value was used to calculate the cell density of the sample. Using this technique,

the minimum quantifiable cell density was 1,000 cells/mL.

Samples of test solution were collected from each of the replicates per treatment and control

group at the end of the test. These samples were pooled within their respective treatments, and

subsamples were removed and examined microscopically for atypical cell morphology (e.g., changes- 13 - Project Number

in cell shape, size or color). Cells in the replicate test chambers also were assessed for aggregations

or flocculations of cells, and adherence of the cells to the test chamber.

Conditions for Validity of the Test

1) The mean cell density in the control flasks increased by a factor greater then 16 within

three days.

2) The pH of the control did not deviate by more than 1.5 units during the test, and

3) The coefficient of variation of average specific growth rate in the control replicates during

the whole test period did not exceed 7%.

Statistical Analyses

The calculation of areas under the growth curve, growth rates and percent inhibition values,

as well as all statistical analyses, were conducted using “The SAS System for Windows”, Version

8.02 (4) or TOXSTAT version 3.5 (5). Area under the growth curve was calculated for each replicate

of the control and treatment groups using the following formula:

A = ((N 1 -N

o )/2)(t 1 )+((N 1 +N 2 -2N

o )/2)(t 2 -t 1 ) +...+((N n-1 +N

n -2N

o )/2)(t

n -t n-1 )

where: A = Area under the growth curve

N

o = Mean number of cells/mL at t

o N 1 = Mean measured number of cells/mL at t 1

N 2 = Mean measured number of cells/mL at t 2

N

n = Mean measured number of cells/mL at t

n

t 1 = Time of first measurement after beginning of test (hours)

t 2 = Time of second measurement after beginning of test (hours)

t

n = Time of n th measurement after beginning of test (hours)

Growth rate was calculated for each replicate of the control and treatment groups using the

following formula:

Growth Rate =

1 2 1 2

t

- t N ln

- N ln

where: N 1 = Mean measured number of cells/mL at t 1

N 2 = Mean measured number of cells/mL at t 2 t = Time of measurement (hours)- 14 - Project Number

Inhibition values were calculated for each treatment group as the percent reduction in area

under the growth curve and growth rate relative to the negative control replicates using the following

formula:

Percent Inhibition = Mean Control Response - Mean Treatment Response X 100

Mean Control Response

Areas under the growth curve and growth rates were analyzed statistically to estimate EL50

values, (i.e., the theoretical loading rate that would produce a 50% reduction in area under the growth

curve (E b L50) and growth rate (E

r L50) in comparison to the negative control) and the corresponding

95% confidence limits for each 24-hour exposure interval. In addition the 72-hour EL10 and EL20

values were calculated for the area under the growth curve and growth rate. The EL10, EL20 and

EL50 values were calculated by non-linear regression or linear interpolation (6, 7). The area under

the growth curve and growth rate data were evaluated for normality and homogeneity of variance

( p =0.05) using the Shapiro-Wilk’s and Levene’s tests, respectively. If the data were normal with

homogeneous variances, the treatment groups were compared to the negative control using ANOVA

and Dunnett’s test ( p =0.05). If the assumptions of normality and homogeneity of variances were not

met an attempt was made to correct the condition by log transformation of the data. If the data failed

the assumptions of normality and/or homogeneity of variances and transformations would not correct

the problem Dunnett’s test was still used to make the comparisons. The results of the statistical

analyses, as well as an evaluation of the concentration-response pattern, were used to determine the

NOEL relative to each parameter at 72 hours.

RESULTS AND DISCUSSION

Measurement of TOC in Test Solutions

Results of analyses for TOC in the test solutions are presented in Table 1. Measurements of

TOC at test initiation ranged from 1.6 to 48 mgC/L. By test termination (Day 3), measurements of

TOC ranged from 2.7 to 36.0 mgC/L. TOC content typically increased with increasing test substance

concentration, and was generally consistent over the test period.

Observations and Measurements

Measurements of temperature, pH and light intensity are presented in Tables 2, 3 and 4,

respectively. Temperatures ranged from 23.1 to 23.3°C and were within the 24 ± 2°C range- 15 - Project Number

established for the test. Measurements of pH ranged from 7.7 to 7.9 in the test solutions at the

beginning of the test period and ranged from 7.6 to 8.1 at test termination. The pH tended to increase

relative to increases in algal densities, which is typical for tests conducted with Pseudokirchneriella

subcapitata . The light intensity ranged from 5030 to 7120 lux, which was within the desired range of

6,000 ± 20% lux.

The toxicity of the test substance to Pseudokirchneriella subcapitata was determined by

evaluating changes in cell density over a 72-hour exposure period. Cell densities were used to

calculate areas under the growth curve and growth rates for each 24-hour interval of exposure. Mean

areas under the growth curve and growth rates with the corresponding percent inhibition values are

presented in Tables 5 and 6, respectively. E b L50 and E

r L50 values and the corresponding 95%

confidence limits for area under the growth curve and growth rates, respectively, are presented in

Table 7. Individual replicate data for each growth parameter are presented in Appendices 3, 4 and 5,

while mean cell densities are illustrated graphically in Figure 1 for the negative control and for the

negative control and each test concentration in Figure 2. Changes in mean cell density in the negative

control replicates over the 72-hour exposure period indicated that exponential growth of cells

occurred in those replicates (Figure 1).

After 72 hours of exposure, inhibition of growth rate in the 1.8, 4.1, 10, 20, 45 and 100 mg/L

treatment groups was 3.8, 6.9, 11, 16, 13 and 85%, respectively, relative to the negative control.

Treatment related effects for growth rate were apparent in the five highest test concentrations.

Dunnett’s test indicated that growth rate was significantly reduced ( p <0.05) in the 4.1, 10, 20, 45 and

100 mg/L treatment levels. Consequently, the 72-hour NOEL for growth rate was 1.8 mg/L.

After 72 hours of exposure, inhibition of area under the growth curve (biomass) in the 1.8,

4.1, 10, 20, 45 and 100 mg/L treatment groups was 19, 31, 43, 55, 53 and 98% respectively, relative

to the negative control. Treatment related effects for biomass were apparent in all test concentrations.

Dunnett’s test indicated that biomass was significantly reduced ( p <0.05) in the 1.8, 4.1, 10, 20, 45

and 100 mg/L treatment levels. Consequently, the 72-hour NOEL for biomass was <1.8 mg/L.

After 72 hours of exposure, there were no signs of adherence of cells to the test chambers or

aggregation/flocculation of algae in the controls or in any treatment group. There were no noticeable

changes in cell morphology in any of the tested concentrations when compared to the control.- 16 - Project Number

CONCLUSIONS

Pseudokirchneriella subcapitata were exposed to six nominal water accommodated fractions

(WAF) of dibutylamine/TOFA and evaluated for effects on area under the growth curve and growth

rate. Area under the growth curve was the most sensitive endpoint, as defined by the lowest EL50

value. The 72-hour E b L50, based on area under the growth curve, was 19 mg/L, with a 95%

confidence interval of 10 and 34 mg/L. The 72-hour E

r L50 value based on growth rate were 72 mg/L

with a 95% confidence intervals of 66 and 79 mg/L. The 72-hour NOEL, based on biomass and

growth rate was <1.8 mg/L and 1.8 mg/L, respectively.- 17 - Project Number

REFERENCES

1 Official Journal of the European Communities. 1992. No. L383. Method C.3. Algal

Inhibition Test.

2 OECD . 2004. Working draft of OECD Proposal for Updating Guideline 201: Freshwater Alga

and Cyanobacteria, Growth Inhibition Test . Circulated 9 April 2004.

3 ASTM Standard Guide 1218-90E . 1990. S tandard Guide for Conducting Static 96-Hour

Toxicity Tests with Microalgae . American Society for Testing and Materials. Philadelphia,

Pennsylvania.

4 The SAS System for Windows . 1999. Version 8.02, TS Level 00M0. SAS Institute Inc., Cary,

North Carolina.

5 West, Inc. and D.D. Gulley. TOXSTAT Version 3.5. Copyright 1996. Western EcoSystems

Technology, Inc., Cheyenne, Wyoming.

6 Bruce, Robert D. and Donald J. Versteeg. 1992. A Statistical Procedure for Modeling

Continuous Toxicity Data. Environmental Toxicology and Chemistry. 11: 1485-1494.

7 Norberg-King, T.J. . 1993. A Linear Interpolation Method for Sublethal Toxicity: The

Inhibition Concentration (ICp) Approach. Version 2.0 U.S. Environmental Protection Agency.

National Effluent Toxicity Assessment Center. Duluth, Minnesota. Technical Report 03-93.- 18 - Project Number

Table 1

Measurements of Total Organic Carbon in Test Solution Samples

TOC (mg C/L) 1 Nominal WAF

Loading Rate

(mg/L) 0 Hour

New Solution 72 Hours

Old Solution

Negative

Control 1.6 3.7

1.8 2.7 3.3

4.1 2.5 2.7

10 4.1 5.2

20 9.0 11.7

45 14 15.9

100 48 36.0

1 The limit of quantitation was 1.0 mg C/L.- 19 - Project Number

Table 2

Temperature Measurements

Temperature ( ° C) 1

Time

(Days) Measurement 1 Measurement 2

0 23.1 23.3

1 23.3 23.2

2 23.2 23.3

3 23.2 23.2

1 Temperature measured twice daily in a container of water located adjacent to the test chambers

using a calibrated hand-held liquid-in-glass thermometer. Measurements typically are taken no

less than 4 hours apart, with the exception of test termination.- 20 - Project Number

Table 3

pH Measurements

pH Measurements Nominal WAF

Loading Rate

(mg/L) Day 0 1 Day 3 2

Negative Control 7.8 8.1

1.8 7.7 8.1

4.1 7.7 7.9

10 7.7 7.9

20 7.8 7.8

45 7.8 7.9

100 7.9 7.6

1 Day 0 samples were collected from the individual batches of test solution prepared for

each treatment and control group at test initiation.

2 Day 3 samples were composites of test solution collected from each of the replicates per

treatment and control group at test termination.- 21 - Project Number

Table 4

Light Intensity Measurements

Time Light Intensity (lux) 1

Day 0 5030 5150 7120 5890 5130

1 Measurements were taken at five locations surrounding the shaker table.- 22 -Project NumberTable 5Mean Area Under the Growth Curve and Percent Inhibition Over the 72-Hour Exposure Period24 Hours 48 Hours 72 HoursNominal WAFLoading Rate(mg/L)MeanArea1PercentInhibition1,2MeanArea1PercentInhibition1,2MeanArea1PercentInhibition1,2Negative Control 344,000 -- 3,902,000 -- 21,836,000 --1.8 172,000 50 2,996,000 23 17,788,000* 194.1 232,000 33 2,532,000 35 15,120,000* 3110 296,000 14 2,160,000 45 12,408,000* 4320 140,000 59 1,576,000 60 9,732,000* 5545 160,000 53 1,468,000 62 10,276,000* 53100 44,000 87 156,000 96 364,000* 981Calculations were performed using SAS Version 8.02. Manual calculations may differ slightly.2Percent inhibition was calculated relative to the negative control replicates.* Statistically significant difference (p<0.05) at 72 hours from the negative control replicates using Dunnett’s test.- 23 -Project NumberTable 6Mean Growth Rate and Percent Inhibition Over the 72-Hour Exposure Period24 Hours 48 Hours 72 HoursNominal WAFLoading Rate(mg/L)MeanGrowth Rate1PercentInhibition1,2MeanGrowth Rate1PercentInhibition1,2MeanGrowth Rate1PercentInhibition1,2Negative Control 0.0546 -- 0.0691 -- 0.0668 --1.8 0.0370 32 0.0651 5.8 0.0642 3.84.1 0.0448 18 0.0604 13 0.0622* 6.910 0.0511 6.4 0.0551 20 0.0596* 1120 0.0313 43 0.0513 26 0.0564* 1645 0.0352 35 0.0490 29 0.0578* 13100 0.0110 80 0.0097 86 0.0100* 851Calculations were performed using SAS Version 8.02. Manual calculations may differ slightly.2Percent inhibition was calculated relative to the negative control replicates.* Statistically significant difference (p<0.05) at 72 hours from the negative control replicates using Dunnett’s test.- 24 -Project NumberTable 7EL Values Over the 72-Hour Exposure PeriodArea Under the Growth Curve Growth RateTimeEbL10(95% ConfidenceInterval)EbL20(95% ConfidenceInterval)EbL50(95% ConfidenceInterval)ErL10(95% ConfidenceInterval)ErL20(95% ConfidenceInterval)ErL50(95% ConfidenceInterval)24 Hours NA NA17 mg/L(9.4 – 78 mg/L)NA NA57 mg/L(35 – 94 mg/L)48 Hours NA NA13 mg/L(7.7 – 23 mg/L)NA NA64 mg/L(55 – 73 mg/L)72 Hours2.7 mg/L(0.73 – 9.7 mg/L)5.2 mg/L(1.9 – 15 mg/L)19 mg/L(10 – 34 mg/L)47 mg/L(39 – 57 mg/L)55 mg/L(46 – 64 mg/L)72 mg/L(66 – 79 mg/L)195% confidence limits could not be calculated with the data obtained.- 29 - Project Number