Phosphorodithioic acid, O,O-bis(2-methylpropyl) ester, compd. with N,N-dimethylmethanamine (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:

27 September - 8 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also in accordance with GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

O,O-bis(2-methylpropyl) sulfanidylphosphonothioate; trimethylazanium

EC Number:

700-910-4

Cas Number:

1354201-99-2

Molecular formula:

C11H28NO2PS2

IUPAC Name:

O,O-bis(2-methylpropyl) sulfanidylphosphonothioate; trimethylazanium

Test material form:

solid

Remarks:

pale yellow glassy solid

Specific details on test material used for the study:

- Name of test material (as cited in study report): S-10713
- Substance type: Straw color, lump solid
- Physical state: solid
- Lot/batch No.: AQ11216TZ
- Expiration date of the lot/batch: July 2013
- Stability: Stable under normal conditions
- Storage condition of test material: Room temperature; in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Chemical confirmation of the range-finding and definitive exposure concentrations was conducted by Investigative Science Incorporated (ISI - 1050 Cooke Boulevard, Unit 2 Burlington, Ontario L7T 4A8 Canada). Subsamples of the test and control solutions, collected at 0- and 72-hours (pooled replicates), were saved for analytical verification of the nominal concentrations. At both time periods, subsamples of the test concentrations and control solutions were collected in ~ 20 mL glass vials. The samples for chemical confirmation were refrigerated at AquaTox and transported in a cooler to ISI where they were refrigerated pending analysis.

Test solutions

Vehicle:

no

Details on test solutions:

- For the range-finding test a 500 ml solution of the highest concentration (i.e., 1000 mg/L) was prepared by adding 0.50009 g of S-10713 into 500 mL of filter-sterilized algal nutrient medium in an appropriate container and stirring for 1 hour at room temperature. Preparation of the remaining test solutions was completed at this time. All lower concentrations of the test item (including 100, 10, 1, 0.1 and 0.01 mg/L) were prepared using a ten-fold serial dilution.

- The definitive test consisted of a Multiple Concentration test. Six exposure concentrations were prepared by adding 2.0002 g of S-10713 into 1000 mL of filter-sterilized algal nutrient medium in an appropriate container and stirring for 1 hour at room temperature. This 2000 mg/L solution was then used to make half by half dilutions for the concentration series (2000, 1000, 500, 250, 125 and 62.5 mg/L of S-10713). One algal nutrient medium control was included (0.0 mg/L S-10713).

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Source (laboratory, culture collection): :
Pseudokirchneriella subcapitata has been cultured at AquaTox’s Ecotoxicity Laboratory (Guelph, ON) since August 1999. The P. subcapitata culture was obtained from the University of Texas (UTEX 1648).
- Age of inoculum (at test initiation): 3 – 7 days old and in exponential growth
- Method of cultivation:
Cultures are aseptically transferred twice weekly (typically from 3 – 7 day old donors) and maintained in temperature and light controlled environments isolated from all testing. The axenic nature of the stock culture is verified by plating on Trypticase Soy Agar (TSA) and Plate Count Agar (PCA). Algal growth curves are conducted semi-annually to ensure that algae are in an exponential growth phase and are suitable for testing

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test.
- Any deformed or abnormal cells observed: Not reported

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

TOC < 2mg/L

Test temperature:

Both tests: 23 ± 1 °C

pH:

Range finding test:
- Initial: 6.53-7.36
- Final: 7.51-7.93

Definitive test:
- Initial: 4.83-7.36
- Final: 4.90-7.47

Nominal and measured concentrations:

- Results were calculated using nominal concentrations since the difference between nominal and measured values was <20%.
Please see data provided in point: "Any other information on results incl. tables".

Details on test conditions:

TEST SYSTEM (definitive test)
- Test vessel: Clear glass 250-mL Erlenmeyer flasks covered with Jaece® non-toxic foam plugs
- Material, size, headspace, fill volume: Glass, with a capacity of 250 mL and filled with 50 ml test solution
- Initial cells density: 7600 cells/mL (5000-10000 cells/ml)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

GROWTH MEDIUM
- Standard medium used: yes
Water used for preparation of the nutrient medium for culture and testing of P. subcapitata was Nanopure II water (TOC < 2.0 mg/L) that was free of particles, ions, organic molecules and microorganisms greater than 0.45 μm in diameter.
Preparation of the medium was conducted according to Environment Canada (2007). The Environment Canada (2007) medium meets the nutrient requirements outlined in OECD (2011). In our laboratory, the Environment Canada growth medium has been used successfully for culturing and testing with P. subcapitata since August 1999.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water used for preparation of the nutrient medium for culture and testing of P. subcapitata was Nanopure II water (TOC < 2.0 mg/L) that was free of particles, ions, organic molecules and microorganisms greater than 0.45 μm in diameter.
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions:
The nutrient medium was filter-sterilized prior to use in cultures and in testing.
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality:
Cool-white fluorescent
4440 – 8880 Lux (Measured at the surface of the liquid in the flasks) for testing (OECD, 2011)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Conducted using a haemocytometer and a phase-contrast microscope (at 100 – 200 times magnification). At 0, 24, 48 and 72 h.
- Other:
Changes in cell development or appearance, such as cell clumping, cell morphology, cell color, cell shape, cell size, etc. (or lack thereof)
Additional observations, such as sedimentation of the test solution, precipitation of cells, solution appearance / coloration, or other abnormalities

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
Range finding test - 10
Definitive test - 2

- Range finding study
- Test concentrations:
Control, 0.01, 0.1, 1, 10, 100 and 1000 mg/L

- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

Sodium chloride

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

125 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1 491 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 1149- 1791

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1 048 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 921-1135

Results with reference substance (positive control):

The 96 h EC25 (cell count) for sodium chloride was 990 mg/L (95% CL: 872-1105 mg/L).

Reported statistics and error estimates:

For all concentrations and the controls, cell yield (72-hour) and average specific growth rate (0 to 3 days) were calculated for each replicate and then averaged. Percent inhibition and % C.V. were also calculated for cell yield and average specific growth rate. For the control flasks, the section-by-section specific growth rates (e.g., 0-1, 1-2, and 2-3 days) and mean % C.V. were determined. Concentration-response curves were plotted for cell yield and average specific growth rate. Test results were reported in terms of the Effective Concentration (i.e. EC50), referring to the concentration that causes an adverse effect (in this case, immobility) in 50% of the exposed population) within a specified period (i.e., 24, 48, 72 hours).

Statistical analyses for the Cell Yield No Observable Effect Concentration (NOEC) and Lowest Observable Effect Concentration (LOEC) endpoints were conducted using Williams Multiple Comparison Test. Statistical analyses for the Average Specific Growth Rate NOEC/ LOEC endpoints were conducted using Jonckheere- Terpstra Step-Down Test. Calculations of Cell Yield ECX values were conducted using were conducted using Linear Interpolation. Average Specific Growth Rate ECx values were calculated using Non-Linear Regression.

Any other information on results incl. tables

- Measured Exposure Concentrations of S-10713:

Table 1. Summary of Definitive Analytical Test Results Including Measured Exposure Concentrations of the Test Item S-10713 (0 and 72 hours)
Nominal Concentration (mg/L)	0 hour	72 hour
2000	1938.7	2022.1
1000	995.3	1017
500	490.9	496.2
250	250.5	252.6
125	123.3a)	125.8a)
62.5	62.2	61.9
0	<MDLb)	<MDLb)

a) This is the average of duplicate analysis

b) Method Detection Limit for S-10713 = 0.7 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In the definitive test the 72-hour EC50s for S-10713 based on cell yield and average specific growth rate were 1119 and 1491 mg/L, respectively. The NOEC and LOEC for cell yield were 250 and 500 mg/L, respectively. The NOEC and LOEC for average specific growth rate were 125 and 250 mg/L S-10713, respectively.

Executive summary:

The toxicity of S-10713 to aquatic algae was assessed according to OECD guideline No. 201 and GLP principles. Nominal exposure concentrations were the following:

Range finding test: control, 0.01, 0.1, 1.0, 10, 100 and 1000 mg/L;

Definitive test: control, 62.5, 125, 250, 500, 1000, 2000 mg/L.

Measured concentrations relative to nominal concentrations varied less than 20% for both tests. Therefore, results are presented in nominal concentrations.

In the definitive test S-10713 significantly reduced growth rate and inhibited the yield of the freshwater algae species Pseudokirchneriella subcapitata at a concentration of 250 and 500 mg/L, respectively.

The EC50 for the average specific growth rate (ErC50: 0-72h) was 1491 mg/L with a NOEC of 125 mg/L and an EC10 of 1048 mg/L. For the cell yield, the EC50 (EyC50: 0-72h) was 1119 mg/L with a NOEC 250 mg/L.

All criteria for acceptability of the test were met and the present toxicity study is classified as reliable without restrictions according to OECD guideline No. 201. The pH in the definitive test decreased significantly with increasing test concentrations. At the highest test concentration (2000 mg/L) the measured pH was 4.83 (start) and 4.9 (end). The low pH might cause, at least partially, the strong effects seen at the highest test concentration.