Fatty acids, C16-18 and C18-unsatd., branched and linear, reaction products with diethanolamine

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 21 February 2013 Experimental Completion Date: 20 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N-bis(2-hydroxyethyl)isooctadecan-1-amide

EC Number:

258-193-4

EC Name:

N,N-bis(2-hydroxyethyl)isooctadecan-1-amide

Cas Number:

52794-79-3

Molecular formula:

C22H45NO3

IUPAC Name:

N,N-bis(2-hydroxyethyl)-16-methylheptadecanamide

Constituent 2

Reference substance name:

Isostearamide DEA

IUPAC Name:

Isostearamide DEA

Test material form:

other: amber colored liquid

Details on test material:

TEST ITEM
The integrity of supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor. A Certificate of Analysis provided by the Sponsor is given in the attached Appendix 1.

Identification: Isostearamide DEA
CAS No: 52794-79-3
Description: amber colored liquid
Batch: OE11124 (6/202593/00)
Purity: not applicable – complex mixture
Expiry Date: not provided
Storage Conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Chemical analysis of test loading rates
Samples were taken from the control and the 100 mg/L loading rate WAF test group (replicates R1 - R6 pooled) at 0 and 72 hours for quantitative
analysis. All samples were stored at approximately -20 °C prior to analysis. Duplicate samples were taken at each occasion and stored at
approximately -20 ºC for further analysis if necessary.

The method of analysis, recovery and test preparation analyses are described in the attached Appendix 5.

Test solutions

Vehicle:

no

Details on test solutions:

Validation of mixing period
Pre-study work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF. A WAF of a nominal loading rate of 100 mg/L was prepared, in duplicate, in deionized reverse osmosis water. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and the concentration of the test item in the WAFs was verified by chemical analysis (see the attached Appendix 4).


Range-finding test
Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water
Accommodated Fraction (WAF).

The loading rate to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by
exposing Pseudokirchneriella subcapitata cells to a series of nominal loading rates of 10 and 100 mg/L for a period of 72 hours.
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to
reduce evaporation. Two replicate flasks were prepared for each control and test concentration.

Amounts of test item (20 and 200 mg) were each separately added to the surface of 2 liters of culture medium to give the 10 and 100 mg/L loading
rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore
glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was
removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs. Microscopic inspection of the
WAFs showed no micro-dispersions or undissolved test item to be present.

An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (7.0 mL) to give the required test
concentrations of 10 and 100 mg/L loading rate WAF.

The control group was maintained under identical conditions but not exposed to the test item.

At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter®
Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at
24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

Samples of each loading rate WAF were taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item over the test duration. All samples were stored at approximately -20 °C prior to analysis.


Definitive test
Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/L to confirm that no effect on algal
growth was observed.


Experimental Preparation
An amount of test item (200 mg) was added to the surface of 2 liters of culture medium to give the 100 mg/L loading rate. After the addition of the
test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL
discarded) to give the 100 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be
present.

An aliquot (1 liter) of the WAF was inoculated with algal suspension (5.4 mL) to give the required test concentration of 100 mg/L loading rate WAF.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours (see attached
Appendix 5).

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Test Species
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master
cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1 °C.

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 104 – 105 cells/mL.

A positive control (Harlan Study Number: 41205049) used potassium dichromate as the reference item. Details of the positive control are given in
the attached Appendix 2. The positive control was conducted between 17 September 2012 and 21 September 2012.

Pseudokirchneriella subcapitata is a freshwater unicellular alga, representative of primary producers found in natural waters and can therefore be
considered as an important non-target organism in freshwater ecosystems.

Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

The culture medium is defined in the attached Appendix 3.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

The water hardness (calculated) of the test water will be 0.15 mmol/L (= 15 mg/L as CaCO3 ).

Test temperature:

Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.

pH:

The pH values of the control cultures (see Table 2) were observed to increase from pH 8.0 at 0 hours to pH 7.8 at 72 hours. The pH deviation in the control
cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

The pH of the control and the 100 mg/L loading rate WAF was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.

Dissolved oxygen:

Not recorded.

Salinity:

freshwater used

Nominal and measured concentrations:

The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal loading rates of 10 and 100 mg/L for a period of 72 hours.
Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/L to confirm that no effect on algal growth was
observed.

Details on test conditions:

Exposure conditions
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and 100 mg/L loading rate WAF treatment group.

The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 9.29 x 105 cells per mL. Inoculation of 1 liter of test medium with 5.4 mL of this algal suspension gave an initial nominal cell density of 5 x 103 cells per mL and had no significant dilution effect
on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 °C under continuous
illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.


Physico-chemical measurements
The pH of the control and the 100 mg/L loading rate WAF was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.


Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period.


Chemical analysis of test loading rates
Samples were taken from the control and the 100 mg/L loading rate WAF test group (replicates R1 - R6 pooled) at 0 and 72 hours for quantitative
analysis. All samples were stored at approximately -20 °C prior to analysis. Duplicate samples were taken at each occasion and stored at
approximately -20 ºC for further analysis if necessary.

The method of analysis, recovery and test preparation analyses are described in the attached Appendix 5.

The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

The culture medium is defined in the attached Appendix 3.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on WAF loading

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on WAF loading

Effect concentrations 3

Duration:

72 h

Dose descriptor:

other: EyL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of yield

Remarks on result:

other: Based on WAF loading

Effect concentrations 4

Duration:

72 h

Dose descriptor:

other: NOELy

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of yield

Remarks on result:

other: Based on WAF loading

Details on results:

RESULTS
Validation of Mixing Period
Pre-study work (see attached Appendix 4) indicated that there was no significant increase in the measured test concentration present in the WAF by extending the preparation period for longer than 24 hours.


Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the
range-finding test are given in Table 1.

The results showed no effect on growth at 10 and 100 mg/L loading rate WAF.

Based on this information a single loading rate of six replicates of 100 mg/L, using a stirring period of 23 hours followed by a 1-Hour standing
period, was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.


Definitive Test
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.

The mean cell densities versus time for the definitive test are presented in the attached Figure 1.


Validation criteria
The following data show that the cell concentration of the control cultures increased by a factor of 132 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours : 6.30 x 103 cells per mL
Mean cell density of control at 72 hours : 8.28 x 105 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 7% and hence satisfied the validation
criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 1% and hence satisfied the
validation criterion given in the OECD Guideline which states that this must not exceed 7%.


Growth data
From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not
affected by the presence of the test item over the 72-Hour exposure period.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Accordingly the following results were determined from the data:


Inhibition of growth rate
ErL*10 (0 - 72 h) : >100 mg/L loading rate WAF
ErL*20 (0 - 72 h) : >100 mg/L loading rate WAF
ErL*50 (0 - 72 h) : >100 mg/L loading rate WAF

where ErL*x is the loading rate that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and 100 mg/L loading rate WAF test group using a Student’s t-test
incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences (P≥0.05), between the control and 100 mg/L loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/L loading rate WAF.


4.3.2.2 Inhibition of yield
EyL*10 (0 - 72 h) : >100 mg/L loading rate WAF
EyL*20 (0 - 72 h) : >100 mg/L loading rate WAF
EyL*50 (0 - 72 h) : >100 mg/L loading rate WAF

where EyL*x is the loading rate that reduced yield by x%.

Statistical analysis of the yield data was carried out as in Section 4.3.2.1. There were no statistically significant differences (P≥0.05), between the
control and 100 mg/L loading rate WAF and therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/L loading rate WAF.


Observations on cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.


Observations on test item solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.

At the start of stirring the 100 mg/L loading rate WAF was observed to have formed a clear colorless media column with an oily droplet of the test
item at the surface. After stirring, and following a 1-Hour standing period, the WAF was observed to have formed a clear colorless media column
with a single lump of test item at the surface. Microscopic examination of the WAF showed there to be no micro-dispersions of test item present.

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and test
cultures were observed to be green dispersions.


Physico-chemical measurements
The pH values of the control and the 100 mg/L loading rate WAF are given in Table 2. Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH value of the control cultures (see Table 2) was observed to range from pH 8.0 at 0 hours to pH 7.8 at 72 hours. The pH deviation in the
control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.


Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface
(see Table 5).


Chemical analysis of test loading rates
Analysis of the 100 mg/L loading rate WAF at 0 and 72 hours (see Appendix 5) showed measured test concentrations of less than the limit of
quantitation (LOQ) of the analytical method employed, which was determined to be 0.070 mg/L were obtained.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Results with reference substance (positive control):

The results from the positive control with potassium dichromate were within the normal ranges for this reference item (see Appendix 2).

Duration Endpoint Effect conc. Nominal/Measured Conc. based on Basis for effect Remarks (e.g. 95% CL)
72 h other: ErC50 1.1 mg/L nominal other: Positive control other: 95% confidence limits 1.0 – 1.3 mg/L
72 h other: EyC50 0.7 mg/L nominal other: Positive control other:
72 h NOEC 0.5 mg/L nominal other: Positive control growth rate
72 h NOEC 0.25 mg/L nominal other: Positive control other: yield
72 h LOEC 1 mg/L nominal other: Positive control growth rate
72 h LOEC 0.5 mg/L nominal other: Positive control other: Yield

Reported statistics and error estimates:

A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data
after 72 hours for the control and the 100 mg/L loading rate to determine any statistically significant differences between the test and control
groups.
All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Any other information on results incl. tables

Tables

Table1     Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Loading Rate (mg/L)		Cell Densities* (cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	7.49E+03	1.19E+06	-	-
	R2	6.27E+03	1.17E+06
	Mean	6.88E+03	1.18E+06
10	R1	5.60E+03	8.73E+05	1	14
	R2	7.34E+03	1.17E+06
	Mean	6.47E+03	1.02E+06
100	R1	7.02E+03	1.51E+06	[4]	[5]
	R2	5.12E+03	9.58E+05
	Mean	6.07E+03	1.23E+06

 

*Cell densities represent the mean number of cells per mL calculated from thean of the cell counts from 3 counts for each of the replicate flasks.

R₁and R₂ = Replicates 1 and 2

[Increase in growth compared to controls]

Table 2     Cell Densities and pH Values in the Definitive Test 

 

Nominal Loading Rate (mg/L)		pH	Cell Densities* (cells per mL)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	8.0	7.54E+03	2.55E+04	1.18E+05	7.70E+05	7.8
	R2		5.70E+03	2.99E+04	1.60E+05	9.11E+05
	R3		4.88E+03	2.73E+04	1.50E+05	8.82E+05
	R4		6.10E+03	2.67E+04	1.52E+05	7.55E+05
	R5		6.85E+03	2.74E+04	1.11E+05	7.68E+05
	R6		6.72E+03	2.64E+04	1.53E+05	8.83E+05
	Mean		6.30E+03	2.72E+04	1.41E+05	8.28E+05
100	R1	7.7	6.26E+03	2.43E+04	1.02E+05	6.66E+05	7.8
	R2		5.52E+03	2.66E+04	1.32E+05	8.04E+05
	R3		5.58E+03	2.41E+04	1.01E+05	8.09E+05
	R4		6.10E+03	2.58E+04	1.32E+05	8.42E+05
	R5		5.39E+03	2.76E+04	1.40E+05	8.50E+05
	R6		7.28E+03	2.64E+04	1.58E+05	9.03E+05
	Mean		6.02E+03	2.58E+04	1.28E+05	8.12E+05

*Cell densities represent thean number of cells per mL calculated from thean of the cell counts from 3 counts for each of the replicate flasks.

R₁ - R₆ = Replicates 1 to 6

Table 3     Daily Specific Growth Rates for the Control Cultures in the Definitive Test

                                                                                                                                                

		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.068	0.064	0.078
	R2	0.075	0.070	0.072
	R3	0.071	0.071	0.074
	R4	0.070	0.072	0.067
	R5	0.071	0.058	0.081
	R6	0.069	0.073	0.073
	Mean	0.071	0.068	0.074

 

R₁ - R₆ = Replicates 1 to 6

Table 4     Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.070		7.62E+05
	R2	0.072		9.05E+05
	R3	0.072		8.77E+05
	R4	0.070	-	7.49E+05	-
	R5	0.070		7.62E+05
	R6	0.072		8.76E+05
	Mean	0.071		8.22E+05
	SD	0.001		7.15E+04
100	R1	0.068	4	6.60E+05
	R2	0.071	0	7.98E+05
	R3	0.071	0	8.03E+05
	R4	0.071	0	8.36E+05
	R5	0.071	0	8.44E+05
	R6	0.072	[1]	8.96E+05
	Mean	0.071	1	8.06E+05	2
	SD	0.001		7.97E+04

 

*In accordance with the OECD test guideline only the mean value for yield is calculated

R₁ – R₆ = Replicates 1 to 6

SD = Standard Deviation

[Increase in growth as compared to controls]

Table 5     Vortex Depth Measurements at the Start and End of the Mixing Period

	Nominal Loading Rate (mg/L)
	Control		100
	*	+	*	+
Height of Media Column (cm)	9.5	9.5	11.5	11.5
Depth of Vortex (cm)	~ 0.2	~ 0.2	~ 0.2	~ 0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present

* = Start of mixing period                                                                                                                                                                    

+ = End of mixing period

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

CONCLUSION
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and results show 72-hour EL50 values (based on inhibition of both growth and yield) of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF in both cases.

Executive summary:

SUMMARY

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

 

 

Methods

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

 

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of the test item, as a limit test at a single nominal loading rate of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

 

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

 

 

Results

Exposure of Pseudokirchneriella subcapitata to the test item gave EL*₅₀ values (based on inhibition of both growth and yield) of greater than 100 mg/L loading rate WAF.  The No Observed Effect Loading Rate was 100 mg/L loading rate WAF in both cases.

 

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

 

Analysis of the 100 mg/L loading rate WAF at 0 and 72 hours showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed, which was determined to be 0.070 mg/L were obtained.

 

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

 

 

EL = Effective Loading Rate