Phosphorodithioic acid, O,O-bis(2-methylpropyl) ester, compd. with N,N-dimethylmethanamine (1:1)

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 September - 6 October 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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 202 (Daphnia sp. Acute Immobilisation 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:

- Concentrations (nominal):
Range finding test - Control, 1, 10, 100 and 1000 mg/L
Definitive tests:
Nr. 1: 0, 10, 20, 40, 80, 160 and 320 mg/L
Nr. 2: 0, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 mg/L

- Sampling method:
For the range-finding and definitive tests, samples were collected from pooled control and exposure solutions at the beginning and end of the test in 20 mL vials (pre-cleaned glass with Teflon lined caps) provided by Investigative Science Incorporated laboratory (ISI - 1050 Cooke Boulevard, Unit 2 Burlington, Ontario L7T 4A8 Canada) and pre-charged with a preservative (nitric acid). The vials were filled, leaving some headspace. A single set of samples were collected from the range-finding test and duplicate samples were collected from the definitive tests. One set of the duplicate samples was retained by AquaTox for use as backup in the event that a sample was lost, damaged, or if re-analysis was required.

- Sample storage conditions before analysis:
The 0-hour and 48-hour samples were stored in the dark at 4 ± 2°C prior to being picked up for analysis. The samples were kept cool (packed in a cooler with an ice pack) while being delivered to the analytical laboratories for analysis.

Test solutions

Vehicle:

no

Details on test solutions:

- All S-10713 test solutions for the range-finding and definitive test were prepared on the basis of the whole product concentration (i.e., without correction for purity) and without the use of any solubilizing agents.

- Test concentrations for the range-finding test were based on a logarithmic series and included nominal S-10713 concentrations of 1, 10, 100 and 1000 mg/L plus a dilution water control (100% laboratory water). The solution containing the highest nominal S-10713 concentration of 1000 mg/L was prepared by weighing out 0.5003 g of the test item into a 500 mL volumetric flask and making this up to a 500 mL volume using dilution water. Gentle stirring was applied for approximately 1. 5 hours. All lower concentrations were prepared using a ten-fold serial dilution beginning with the 1000 mg/L test solution.

- Test concentrations for the definitive tests were based on a geometric series. The initial test included nominal S-10713 concentrations of 10, 20, 40, 80, 160 and 320 mg/L plus a dilution water control (i.e., 100% laboratory water). The solution containing the highest nominal S-10713 concentration of 320 mg/L was prepared by weighing out 0.3201 g of the test item into a 1 L volumetric flask and making this up to a 1000 mL volume using dilution water. Gentle stirring was applied for approximately 1. 5 hours. All lower concentrations were prepared by serial dilution, using a 0.5 times dilution factor, beginning with the 320 mg/L test solution.

- The second definitive test was conducted using nominal S-10713 concentrations of 15.6, 31.3, 62.5, 125, 250, 500, 1000, and 2000 mg/L. The 1000 and 2000 mg/L solutions were prepared individually, followed by gentle stirring for approximately 1. 5 hours. The 2000 mg/L solution was prepared by weighing 1.0005 g of S-10713 into a 500 mL volumetric flash and making this up to a 500 mL volume using laboratory dilution water. The 1000 mg/L solution was prepared by weighing 1.0009 g of S-10713 into a 1 L volumetric flash and making this up to a 1000 mL volume using laboratory dilution water. All lower concentrations were prepared by serial dilution, using a 0.5 times dilution factor, beginning with the 1000 mg/L solution.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Source: The organisms were originally obtained from the Ministry of the Environment (Toronto, Ontario, Canada). The population has been cultured continuously in AquaTox’s laboratory since 1988.
- Age at study initiation: First instar, neonate (<24 h old)
- Feeding during test: None

ACCLIMATION
- Acclimation conditions (same as test or not): yes

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

~ 200 mg/L as CaCO3

Test temperature:

20 ± 2 °C

pH:

Definitive test Nr. 2 :
- Initial: 7.5-8.4
- Final: 8.0-8.5

Dissolved oxygen:

Definitive test Nr. 2 (mg/L) :
- Initial: 8.6-8.5
- Final: 8.2-8.8

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 Nr. 2)
- Test vessel: Glass beaker
- Material, size, headspace, fill volume: Glass, with a capacity of 250 ml and filled with 100 ml of test solution
- Aeration: None
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Loading rate: Approximately 20 mL/daphnid

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The source of water used for D. magna culturing and testing was moderately hard groundwater (~300 mg/L as CaCO3) obtained from an aquifer in Aberfoyle, Ontario, Canada. For culture and testing purposes with D. magna, the groundwater was filtered through a 20 micron cellulose-acetate filter and diluted with Reverse Osmosis water to achieve a hardness of ~200 mg/L (as CaCO3).
A continuous supply of oil-free compressed air was provided to bring the pH and concentrations of dissolved oxygen and other gases into equilibrium with air. The concentration of dissolved oxygen in the water was maintained at >80% of the air saturation value.
- Culture medium different from test medium: No
- Intervals of water quality measurement:
Dissolved oxygen, pH, conductivity at start and end of test, temperature and visual observations at 0, 24 and 48 hours, dilution water hardness at 0 hours.

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light, 8-hour dark (with 30 minute transition period)
- Light intensity: 400 to 800 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mobility

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
Range finding test: 10
Definitive test:
Nr. 1: 2
Nr. 2: 2

- Range finding study
- Test concentrations: control, 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

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

121 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 85-171

Details on results:

- Complete immobility of D. magna (i.e., 100%) was observed within the first 24 hours at nominal S-10713 concentrations of 1000 and 2000 mg/L, with partial immobility at the 500 and 250 mg/L concentrations of 55% and 5% respectively. No immobility of daphnids was observed at lower concentrations within the first 24 hours. At test termination, partial immobility of daphnids (20% to 80%) was observed at intermediate exposure concentrations from 5.6 mg/L to 500 mg/L, with complete immobility at higher concentrations (≥ 1000 mg/L) and no immobility at lower concentrations (≤ 7.8 mg/L).

Results with reference substance (positive control):

The 48-hour LC50 for the Daphnia magna reference toxicant (sodium chloride) test was 6.0 mg/L (95% CL: 5.4 - 6.4 mg/L)

Reported statistics and error estimates:

- The CETIS© (2007) program was used to calculate the 24- and 48-hour EC50s (and 95% Confidence Limits) according to the methods of Linear Regression and Spearman-Karber, respectively.

Any other information on results incl. tables

Table 1. Test substance (S-10713) water concentrations in the range-finding test.

Nominal Concentration (mg/L)	Measured Concentration (mg/L)		Measured Concentration as a Percentage of Nominal (%)
	0 hour	48 hour	0 hour	48 hour
1000	1026	1089	102.6	108.9
100	101.9^a	108.2	101.9	108.2
10	8.9	9.5	89	95
1	0.9	1.0	90	100
Control	0	<MDL^b	N/A^c	N/A

^a Average of duplicate analysis. RPD = 1.2%

^b Method Detection Limit = 0.7 mg/L
^c Not Applicable

Table 2. Summary of the Definitive Test Results of test # 2. Including Measured Exposure Concentrations of S-10713 (after 0 and 48 hours) and Immobility of D. magna (after 24 and 48 hours).
Nominal Concentration (mg/L)	Measured Concentration (mg/L)		Measured Concentration as a Percentage of Nominal (%)		Immobility
					(%)
	T= 0 h	T= 48 h	T= 0 h	T= 48 h	T= 24 h	T= 48 h
2000	2029.6	2074.5	101.5	103.7	100	100
1000	1001.2	1027.8	100.1	102.8	100	100
500	502	515.8	100.4	103.2	55	80
250	251.2	257.5	100.5	103	5	20
125	124.0^a	129.1^b	99.2	103.3	0	45
62.5	61.8	63.7	98.9	101.9	0	50
31.3	30	32	96	102.4	0	40
15.6	15.3	15.8	97.9	101.1	0	20
7.8	8.4	7	107.5	89.6	0	0
0	< MDL^c	< MDL	N/A^d	N/A	0	0

^a Average of duplicate analysis. RPD = 1.2%
^b Average of duplicate analysis. RPD = 1.4%
^c Method Detection Limit = 0.7 mg/L
^d Not Applicable

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In the definitive test Nr. 2 the 24-hour and 48-hour EC50s for S-10713 were estimated to be 463 mg/L and 121 mg/L, respectively.

Executive summary:

The toxicity of S-10713 to aquatic invertebrates was assessed according to OECD guideline No. 202 and GLP principles. Nominal exposure concentrations were the following: 

Range finding test - Control, 1, 10, 100 and 1000 mg/L 

Definitive tests: 

Nr. 1: 0, 10, 20, 40, 80, 160 and 320 mg/L; 

Nr. 2: 0, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 mg/L. 

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

In the definitive test Nr. 2 the 48 h EC50 (immobility) for S-10713 was 121 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. 202.