O,O-dibutyl hydrogen thiophosphate, compound with 1-octylamine (1:1)

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 21 November 2016 and 29 December 2016

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:

O,O-dibutyl hydrogen thiophosphate, compound with 1-octylamine (1:1)

EC Number:

300-947-2

EC Name:

O,O-dibutyl hydrogen thiophosphate, compound with 1-octylamine (1:1)

Cas Number:

93964-99-9

Molecular formula:

C8H19N.C8H19O3PS

IUPAC Name:

octan-1-amine dibutyl sulfanylphosphonate

impurity 1

Reference substance name:

Octylamine

EC Number:

203-916-0

EC Name:

Octylamine

Cas Number:

111-86-4

Molecular formula:

C8H19N

IUPAC Name:

octan-1-amine

Test material form:

liquid

Specific details on test material used for the study:

Identification: X-19575 Phosphorothioic acid, O,O-dibutyl ester, compd. with 1-octanamine, CASRN 93964-99-9
Nature of Test Item: UVCB
Physical state/Appearance: Amber colored viscous liquid
Batch: X-019575-00-00
Purity: Not applicable (100% as UVCB)
Expiry Date: 01 June 2017
Storage Conditions: Room temperature in darkness

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 hours and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

The Determination of General Physico-Chemical Properties study performed on the test ite (Envigo Study Number BT67DH) indicated that at initial loading rates of 10 and 100 g/L the water solubility was greater than 4 g/L. However preliminary work performed for the aquatic ecotoxicology tests showed that under test conditions it was not possible to obtain a true solution of the test item at a nominal concentration of 100 mg/L. Therefore, due to the limited aqueous solubility under test conditions 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 Tests
The initial range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 10 and 100 mg/L for a period of 72 hours.
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 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 (3.8 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 high growth inhibition, so a second range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 0.010, 0.10 and 1.0 mg/L for a period of 72 hours.
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 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 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 performed from the 1.0 mg/L loading rate to give the 0.10 and 0.010 mg/L loading rates. Due to the low test concentration, it was not practical to prepare the test concentrations as individual WAFs and dilutions were required.
An aliquot (450 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (3.1 mL) to give the required test concentrations of 0.010, 0.10 and 1.0 mg/L loading rate WAF.

Definitive test
Nominal amounts of test item (20, 64 and 200 mg) were each separately added to the surface of 20 liters of culture medium to give the 1.0, 3.2 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 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 1.0, 3.2 and 10 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
Dilutions were made from the 3.2 and 1.0 mg/L loading rate WAFs to give the 0.32 and 0.10 mg/L loading rates, respectively.
An aliquot (1000 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (13.2 mL) to give the required test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L loading rate WAF.

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 ± ºC.
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 ºC 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:

7.6 - 8.0

Nominal and measured concentrations:

Chemical analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from 0.104 to 9.49 mg/L. Chemical analysis of the aged test preparations at 72 hours showed measured test concentrations to range from 0.0976 to 9.72 mg/L.
For chemical analysis, the ion that was monitored by negative mode LC-MS had a m/z of 225. This mass corresponds with the [M-H]- ion of dibutylthiophosphoric acid (C8H19O3PS). 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 dibutylthiophosphoric acid 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 and 1.0 mg/L
Definitive test (nominal): 0.10, 0.32, 1.0, 3.2 and 10 mg/L.

Details on test conditions:

Range-finding test
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 prepared using the WAF methodology and maintained under identical conditions, but not exposed to the test item.
At the start of the range-finding tests, 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
As in the range-finding tests, 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 prepared using the WAF methodology and 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 3.80 x 10^5 cells per mL. Inoculation of 1000 mL of test medium with 13.2 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 initial cell density of 5 x 10^3 cells per mL was at the low end of the acceptable range specified in the OECD Test Guideline. This ensured that the algal cells remained in logarithmic growth throughout the test period.
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.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

3 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.:

1 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.:

1.8 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.:

1 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 significant effect on growth at 0.010 and 0.10 mg/L loading rate WAF. However, growth was observed to be reduced at 1.0, 10 and 100 mg/L loading rate WAF.
Based on this information loading rates of 0.10, 0.32, 1.0, 3.2 and 10 mg/L were selected for the definitive test.

Definitive test

Growth Data
The following results were determined from the data:

Inhibition of growth rate
ErL10 (0 - 72 h) : 1.4 mg/L loading rate WAF
ErL20 (0 - 72 h) : 1.8 mg/L loading rate WAF
ErL50 (0 - 72 h) : 3.0 mg/L loading rate WAF; 95% confidence limits 2.5 – 3.5 mg/L loading rate WAF

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 and all test concentrations using a Williams Multiple Sequential t-test procedure with Levene’s Test on Variance Homogeneity (with residuals). There were no statistically significant differences (p ≥ 0.05), between the control, 0.10, 0.32 and 1.0 mg/L loading rate WAFs, however all other test concentrations were significantly different (p < 0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 1.0 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 3.2 mg/L loading rate WAF.

Inhibition of Yield
EyL10 (0 - 72 h) : 1.1 mg/L loading rate WAF
EyL20 (0 - 72 h) : 1.3 mg/L loading rate WAF
EyL50 (0 - 72 h) : 1.8 mg/L loading rate WAF ; 95% confidence limits 1.6 – 1.9 mg/L loading rate WAF

Where: EyLx is the loading rate that reduced yield by x%.
There were no statistically significant differences (p ≥ 0.05), between the control, 0.10, 0.32 and 1.0 mg/L loading rate WAFs, however all other test concentrations were significantly different (p < 0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 1.0 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 3.2 mg/L loading rate WAF.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 112 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 : 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 5.58 x 10^5 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 5% 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 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

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 0.10, 0.32, 1.0 and 3.2 mg/L loading rate WAFs, however no intact cells were observed to be present in the test cultures at 10 mg/L loading rate WAF.

Results with reference substance (positive control):

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

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 no significant increase in the amount of dissolved test item when the preparation period was extended for longer than 24 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.

Water Quality Criteria

Temperature was maintained at 24 ± 1 ºC throughout the test.

Vortex Depth Measurements

The vortex depths were recorded at the start and end of the mixing period, and were observed to be approximately 1 to 2% of the water column height.

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 be a clear colorless media column with a slight oily sheen on the surface. The 3.2 mg/L loading rate WAF was observed to be a clear colorless media column with an oily sheen on the surface. The 10 mg/L loading rate WAF was observed to be a clear colorless media column with an oily sheen and some small globules of test item on the surface. These observations confirmed the limited solubility of the test item under test conditions.

After 23 hours stirring and a 1-Hour standing period all WAF loading rates were observed to remain the same. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.

At the start of the test, the control and all loading rate WAF test cultures were observed to be clear colorless solutions.

At 72 hours, the control, 0.10, 0.32 and 1.0 mg/L loading rate WAF test cultures were observed to be pale green dispersions. The 3.2 mg/L loading rate WAF test cultures were observed to be extremely pale green dispersions. The 10 mg/L loading rate WAF test cultures were observed to be 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	0.066		5.69E+05
	R2	0.066		5.72E+05
	R3	0.066		5.68E+05
	R4	0.063	-	4.48E+05	-
	R5	0.065		5.29E+05
	R6	0.067		6.29E+05
	Mean	0.066		5.53E+05
	SD	0.001		6.06E+04
0.10	R1	0.068	[3]	6.44E+05
	R2	0.065	2	5.53E+05
	R3	0.066	0	5.75E+05
	Mean	0.066	[0]	5.91E+05	[7]
	SD	0.002		4.76E+04
0.32	R1	0.066	0	5.81E+05
	R2	0.068	[3]	6.49E+05
	R3	0.063	5	4.55E+05
	Mean	0.066	1	5.62E+05	[2]
	SD	0.003		9.86E+04
1.0	R1	0.064	3	4.80E+05
	R2	0.065	2	5.46E+05
	R3	0.065	2	5.18E+05
	Mean	0.065	2	5.15E+05	7
	SD	0.001		3.30E+04
3.2	R1	0.031	53	4.03E+04
	R2	0.028	58	3.30E+04
	R3	0.028	58	3.36E+04
	Mean	0.029	56	3.56E+04	94
	SD	0.002		4.07E+03
10	R1	0.009	86	4.54E+03
	R2	0.000	100	5.60E+01
	R3	0.003	95	1.10E+03
	Mean	0.004	94	1.90E+03	100
	SD	0.005		2.34E+03

*       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 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): 1.4 mg/L loading rate WAF
ErL20 (0 - 72 h): 1.8 mg/L loading rate WAF
ErL50 (0 - 72 h): 3.0 mg/L loading rate WAF; 95% confidence limits 2.5 – 3.5 mg/L loading rate WAF
NOEL: 1.0 mg/L
LOEL: 3.2 mg/L

Inhibition of Yield
EyL10 (0 - 72 h): 1.1 mg/L loading rate WAF
EyL20 (0 - 72 h): 1.3 mg/L loading rate WAF
EyL50 (0 - 72 h): 1.8 mg/L loading rate WAF; 95% confidence limits 1.6 – 1.9 mg/L loading rate WAF
NOEL: 1.0 mg/L
LOEL: 3.2 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…

Preliminary solubility work performed indicated that under test conditions the test item was not readily soluble. Therefore, due to the limited 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,Pseudokirchneriella subcapitatawas exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 0.10, 0.32, 1.0, 3.2 and 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 the dibutylthiophosphoric acid component of the test item. No reference material was analyzed to confirm the compound measured.

Chemical analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from 0.104 to 9.49 mg/L. Chemical analysis of the aged test preparations at 72 hours showed measured test concentrations to range from 0.0976 to 9.72 mg/L.

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 dibutylthiophosphoric acid 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.

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)			No Observed Effect Loading Rate (NOEL) (mg/L)	Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Growth Rate	1.4	1.8	3.0	2.5	-	3.5	1.0	3.2
Yield	1.1	1.3	1.8	1.6	-	1.9	1.0	3.2