Amines, C16-18-(even numbered, saturated and unsaturated) alkyl, O,O-di-Bu phosphorothioates

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:

The study was conducted between 25 November 2016 and 22 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Amines, C16-18-(even numbered, saturated and unsaturated) alkyl, O,O-di-Bu phosphorothioates

Molecular formula:

Not applicable - UVCB

IUPAC Name:

Amines, C16-18-(even numbered, saturated and unsaturated) alkyl, O,O-di-Bu phosphorothioates

Constituent 2

Reference substance name:

C16-18-(even numbered, saturated and unsaturated)-alkylamines

EC Number:

627-034-4

Cas Number:

1213789-63-9

Molecular formula:

R-NH2 R = alkyl mainly C16-18-(even numbered, saturated and unsaturated)-alkyl

IUPAC Name:

C16-18-(even numbered, saturated and unsaturated)-alkylamines

Test material form:

liquid

Specific details on test material used for the study:

Identification: X-19574
Physical state/Appearance: Amber colored viscous liquid
Batch: X-19574-00-00
Purity: 100% UVCB
Expiry Date: 01 June 2017
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Range-finding test:
A sample of each loading rate WAF was taken for chemical analysis at 0 and 72 hours from each range-finding test in order to determine the stability of the test item over the test duration. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test taken from the third range-finding test were analyzed.

Definitive test:
Samples were taken from the control and each loading rate WAF test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. A sample of the 1.0 mg/L loading rate WAF from which the required test concentrations were prepared was also taken for analysis at 0 hours. All 0-Hour test samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

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


Range-finding test

Nominal 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 95 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 (3.4 mL) to give the required test concentrations of 10 and 100 mg/L loading rate WAF.

The results of the initial range-finding test showed significant inhibition of growth occurred at both 10 and 100 mg/L loading rate WAF and so a second range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 0.010, 0.10, 1.0 and 10 mg/L for a period of 72 hours. Due to the need to test at relatively low loading rates, WAFs were prepared at 10 and 1.0 mg/L from which dilutions were made to give further test concentrations of 0.10 and 0.010 mg/L loading rate WAF.
Nominal amounts of test item (20 and 200 mg) were each separately added to the surface of 20 liters of culture medium to give the 1.0 and 10 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 95 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 1.0 and 10 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. A series of dilutions was made from the 1.0 and 10 mg/L loading rate WAFs to give further stock solutions of 0.10 and 0.010 mg/L loading rate WAF. An aliquot (450 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (1.9 mL) to give the required test concentrations of 0.010, 0.10, 1.0 and 10 mg/L loading rate WAF.

The results of the second range-finding test showed significant inhibition of growth occurred at 0.010, 0.10, 1.0 and 10 mg/L loading rate WAF and so a third range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 0.00010, 0.0010, 0.010 and 0.10 mg/L for a period of 72 hours. Due to the need to test at relatively low loading rates, a single WAF was prepared at a nominal loading rate of 1.0 mg/L from which dilutions were made to give the test concentrations of 0.10, 0.010, 0.0010 and 0.00010 mg/L loading rate WAF.
A nominal amount of test item (20 mg) was added to the surface of 20 liters of culture medium to give the 1.0 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 95 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 a 1.0 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. A series of dilutions was made from the 1.0 mg/L loading rate WAF to give further stock solutions of 0.10, 0.010, 0.0010 and 0.00010 mg/L loading rate WAF. An aliquot (450 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (4.0 mL) to give the required test concentrations of 0.00010, 0.0010, 0.010 and 0.10 mg/L loading rate WAF.

Definitive test
Due to the need to test at relatively low loading rates, a single WAF of a nominal loading rate of 1.0 mg/L was prepared from which dilutions were made to give the required test concentrations.
A nominal amount of test item (20 mg) was added to the surface of 20 liters of culture medium to give the 1.0 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 95 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 a 1.0 mg/L loading rate WAF stock solution. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. A series of dilutions was made from the 1.0 mg/L loading rate WAF to give the required test concentrations of 0.10, 0.050, 0.025, 0.0125, 0.00625, 0.00313 and 0.00156 mg/L loading rate WAF. An aliquot (1 litre) of each test concentration was separately inoculated with 5.4 mL of algal suspension.

Test organisms

Test organisms (species):

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

Details on test organisms:

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 ± 2 °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 10^3 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 10^4 – 10^5 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ± 1 ºC

pH:

7.5 - 8.5

Nominal and measured concentrations:

Analysis of the test preparations at 0 hours showed measured concentrations to range from 0.00097 to 0.11 mg/L. Analysis of the 1.0 mg/L loading rate WAF from which serial dilutions were made to give the required test series gave a measured concentration of 0.97 mg/L. A decline in measured concentrations was observed at 72 hours in the range of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.00071 mg/L, to 0.053 mg/L.
The ion that was monitored by positive mode LC-MS had a m/z of 268. This mass corresponds with the [M+H]+ ion of an unsaturated C18 amine (C18H37N). A reference material was not analyzed to confirm the compound being measured.
The test item concentrations were based on the amount of test item weighed out to prepare the standards. No correction of the test item concentration for the content of the amine component was undertaken. Whilst only the amine component of the test item was measured, the concentration of the standards was in terms of test item (mg/L), as such the calculated values for the test solutions are in the same term (mg/L test item).
As the test item is UVCB in nature, the dissolved portion may have been one or several components of the test item. 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.

Range-finding test 1 (nominal): 10 and 100 mg/L
Range-finding test 2 (nominal): 0.010, 0.10, 1.0 and 10 mg/L
Range-finding test 3 (nominal): 0.00010, 0.0010, 0.010 and 0.10 mg/L

Definitive test (nominal): 0.00156, 0.00313, 0.00625, 0.0125, 0.025, 0.050 and 0.10 mg/L

Details on test conditions:

Range-finding tests
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.
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.

Definitive test
In the definitive test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each 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 9.26 x 10^5 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 10^3 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 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.

Reference substance (positive control):

yes

Remarks:

potassium dichromate. The positive control was conducted between 28 November 2016 and 01 December 2016.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.028 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.013 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.026 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.013 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

Range-finding Tests
The results showed no effect on growth at 0.00010, 0.0010 and 0.010 mg/L loading rate WAF. However, growth was observed to be reduced at 0.10, 1.0, 10 and 100 mg/L loading rate WAF.
Chemical analysis of the 0.010 and 0.10 mg/L loading rate WAF test preparations taken from the third range-finding test showed measured test concentrations of 0.0097 and 0.085 mg/L respectively were obtained at 0 hours. A decline in measured test concentrations was observed at 72 hours to less than the limit of quantification (LOQ) of the analytical method employed, which was determined to be 0.00071 mg/L and 0.020 mg/L for the 0.010 and 0.10 mg/L loading rate WAF test preparations respectively indicating that the test item was unstable over the duration of the test.

Growth Data
From the data it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.
Accordingly the following results were determined from the data:

Inhibition of growth rate
ErL10 (0 - 72 h): 0.025 mg/L loading rate WAF
ErL20 (0 - 72 h): 0.026 mg/L loading rate WAF
ErL50 (0 - 72 h): 0.028 mg/L loading rate WAF*
* It was not possible to calculate 95% confidence limits for the ErL50 value as the data generated did not fit the models available for the calculation of confidence limits.

Where ErLx is the loading rate that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control all loading rates using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences between the control, 0.00156, 0.00313, 0.00625 and 0.0125 mg/L loading rate WAFs (P ≥ 0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 0.0125 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 0.025 mg/L loading rate WAF.

Inhibition of Yield
EyL10 (0 - 72 h): 0.022 mg/L loading rate WAF
EyL20 (0 - 72 h): 0.023 mg/L loading rate WAF
EyL50 (0 - 72 h): 0.026 mg/L loading rate WAF; 95% confidence limits 0.025 – 0.026 mg/L loading rate WAF

Where EyLx is the loading rate that reduced yield by x%.
Statistical analysis of the yield data was carried out as in Section 6.3.3. There were no statistically significant decrease in yield between the control, 0.00156, 0.00313, 0.00625 and 0.0125 mg/L loading rate WAFs (P ≥ 0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 0.0125 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on yield was 0.025 mg/L loading rate WAF.

Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.00156, 0.00313 and 0.00625 mg/L loading rate WAF. Misshapen cells were observed to be present in the 0.0125 mg/L test cultures, clumped cells and some misshapen cells were present in the 0.025 mg/L test cultures, very few misshapen and clumped cells alone with cell debris were observed in the 0.050 mg/L test cultures whilst no intact cells were observed in the 0.10 mg/L test cultures.

Results with reference substance (positive control):

A positive control (Envigo Study Number FP48BQ) conducted between 28 November 2016 and 01 December 2016 used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h) : 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Any other information on results incl. tables

Validation of Mixing Period

Preliminary investigational work indicated that there was a significant increase in the amount of dissolved test item when the preparation period was extended from 24 to 96 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 95 hours followed by a 1-Hour settlement period.

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 194 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.

Nominal cell density of control at 0 hours     :          5.00 x 10³cells per mL

Mean cell density of control at 72 hours       :          9.69 x 10⁵cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 15% 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%.

Water Quality Criteria

The pH values of the control and each test concentration are given in Table 4. Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH value of the control cultures (see Table 4) was observed to increase from pH 7.5 at 0 hours to pH 8.5 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 depths were recorded at the start and end of the mixing period, and were observed to be approximately 1% of the media column height in the control and 1.0 mg/L loading rate.

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 the mixing period the 1.0 mg/l loading rate WAF was observed to have formed a clear colorless media column with an oily droplet of test item at the media surface. After stirring, and following a 1-Hour standing period, the 1.0 mg/L loading rate WAF was observed to have formed a clear colorless media column with an oily film of test item floating at the media 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, 0.00156, 0.00313, 0.00625 and 0.0125 mg/L loading rate WAF test cultures were observed to be green dispersions. The 0.025 mg/L loading rate WAF test cultures were pale green dispersions whilst the 0.050 and 0.10 mg/L loading rate WAF test cultures were clear colorless solutions.

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	0.073		9.48E+05
	R2	0.071		8.49E+05
	R3	0.075		1.11E+06
	R4	0.073	-	9.63E+05	-
	R5	0.073		9.61E+05
	R6	0.073		9.51E+05
	Mean	0.073		9.64E+05
	SD	0.001		8.47E+04
0.00156	R1	0.078	[7]	1.36E+06
	R2	0.075	[3]	1.11E+06
	R3	0.076	[4]	1.18E+06
	Mean	0.076	[5]	1.22E+06	[27]
	SD	0.002		1.29E+05
0.00313	R1	0.076	[4]	1.18E+06
	R2	0.073	0	9.78E+05
	R3	0.075	[3]	1.09E+06
	Mean	0.075	[2]	1.08E+06	[12]
	SD	0.002		1.02E+05
0.00625	R1	0.075	[3]	1.13E+06
	R2	0.076	[4]	1.15E+06
	R3	0.074	[1]	1.05E+06
	Mean	0.075	[3]	1.11E+06	[15]
	SD	0.001		4.98E+04
0.0125	R1	0.074	[1]	1.01E+06
	R2	0.074	[1]	1.01E+06
	R3	0.073	0	9.39E+05
	Mean	0.074	[1]	9.86E+05	[2]
	SD	0.001		4.11E+04
0.025	R1	0.070	4	7.47E+05
	R2	0.062	15	4.14E+05
	R3	0.064	12	4.84E+05
	Mean	0.065	10	5.48E+05	43
	SD	0.004		1.76E+05
0.050	R1	-0.014	119	-3.16E+03
	R2	-0.012	116	-2.89E+03
	R3	-0.015	121	-3.34E+03
	Mean	-0.014	119	-3.13E+03	100
	SD	0.002		2.26E+02
0.10	R1	-0.015	121	-3.24E+03
	R2	-0.019	126	-3.72E+03
	R3	-0.022	130	-3.99E+03
	Mean	-0.019	126	-3.65E+03	100
	SD	0.004		3.81E+02

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

R₁-R₆= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to the controls]

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results:

Inhibition of growth rate
ErL10 (0 - 72 h): 0.025 mg/L loading rate WAF
ErL20 (0 - 72 h): 0.026 mg/L loading rate WAF
ErL50 (0 - 72 h): 0.028 mg/L loading rate WAF*
* It was not possible to calculate 95% confidence limits for the ErL50 value as the data generated did not fit the models available for the calculation of confidence limits.
NOEL: 0.0125 mg/L
LOEL: 0.025 mg/L

Inhibition of Yield
EyL10 (0 - 72 h): 0.022 mg/L loading rate WAF
EyL20 (0 - 72 h): 0.023 mg/L loading rate WAF
EyL50 (0 - 72 h): 0.026 mg/L loading rate WAF; 95% confidence limits 0.025 – 0.026 mg/L loading rate WAF
NOEL: 0.0125 mg/L
LOEL: 0.025 mg/L

Executive 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 preliminary range-finding tests and initial experiments,Pseudokirchneriella subcapitatawas exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 0.00156, 0.00313, 0.00625, 0.0125, 0.025, 0.050 and 0.10 mg/L (three replicate flasks per concentration) 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

     

Chemical analysis was based on measurement of an unsaturated C18 amine component of the test item. No reference item was analyzed to confirm the compound measured.

Analysis of the test preparations at 0 hours showed measured concentrations to range from 0.00097 to 0.11 mg/L. Analysis of the 1.0 mg/L loading rate WAF from which serial dilutions were made to give the required test series gave a measured concentration of 0.97 mg/L.  A decline in measured concentrations was observed at 72 hours in the range of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.00071 mg/L, to 0.053 mg/L. 

As the test item is UVCB in nature, the dissolved portion may have been one or several components of the test item. 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.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Response Variable	EL10 (mg/L)	EL20 (mg/L)	EL50 (mg/L)	95% Confidence Limits (mg/L Loading Rate WAF)			No Observed Effect Loading Rate (NOEL) (mg/L)	Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Growth Rate	0.025	0.026	0.028	Not determined			0.0125	0.025
Yield	0.022	0.023	0.026	0.025	-	0.026	0.0125	0.025