Phosphorodithioic acid, mixed O,O-bis(sec-Bu and 1,3-dimethylbutyl) esters, zinc salts

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:

17 May 2005 - 14 July 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, without deviations from standard test guidelines. Test material was not measured analytically

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

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:

Phosphorodithioic acid, mixed O,O-bis(sec-Bu and 1,3-dimethylbutyl) esters, zinc salts

EC Number:

272-238-5

EC Name:

Phosphorodithioic acid, mixed O,O-bis(sec-Bu and 1,3-dimethylbutyl) esters, zinc salts

Cas Number:

68784-31-6

Molecular formula:

C(16-24) H(36-42) O4 P2 S4 Zn

IUPAC Name:

Phosphorodithioic acid, mixed O,O-bis(sec-Bu and 1,3-dimethylbutyl) esters, zinc salts

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

10, 18, 32, 56, and 100 mg/L loading rate WAF

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low aqueous solubility and complex nature of the test material for the purpose of the definitive test the test material was prepared as a Water Accommodated Fraction. Amounts of test material were each separately to the surface of 2.5 liters of reconstituted water to give the 10, 18, 32, 56 and 100 mg/L loading rates. After the addition of the test material, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. These were stirred for 24 hours. The stirring was stopped and the mixtures allowed to stand for 1 hour. The aqueous phase or WAF was removed by mid depth siphoning. Microscopic inspection of the WAFs showed no microdispersions or undissolved test material to be present.

- Differential loading: Preparation of test solutions with individually prepared loading rates
- Controls: Untreated negative control; and reference/positive control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): water
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): After siphoning and for the duration of the test the 10, 18, 32, 56, and 100 mg/L loading rates were observed to be clear colorless solutions. Microscopic inspection of the WAFs showed no microdispersions or undissolved test material to be present.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water Flea

- Source: In house laboratory cultures with a known history
- Age at study initiation (mean and range, SD): less than 24 h old
- Weight at study initiation (mean and range, SD): no data available.
- Length at study initiation (length definition, mean, range and SD): no data available.
- Valve height at study initiation, for shell deposition study (mean and range, SD): no data available.
- Peripheral shell growth removed prior to test initiation: no data available.
- Method of breeding: parthenogenises
- Feeding during test : The daphnids received no food during exposure
- Food type: Not applicable.
- Amount: Not applicable.
- Frequency: Not applicable.

ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not): Not reported
- Type and amount of food: Each culture was fed daily with a suspension of freshwater algae
- Feeding frequency: daily
- Health during acclimation (any mortality observed): Culture conditions ensured that reproduction was by parthenogenesis. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

QUARANTINE (wild caught)
- Duration: Not applicable.
- Health/mortality: Not applicable.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim for 15 seconds after gentle agitation.

Test conditions

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3

Test temperature:

19.1ºC to 20.8ºC

pH:

pH values ranged from 7.6 to 8.0 in controls and all test concentrations at the beginning of the test and ranged from 7.8 to 8.0 at the end of the 48 duration.

Dissolved oxygen:

There were no treatment related differences in oxygen concentration (range from 8.5 to 8.6 at start of test and 8.8 to 8.9 at end of test)

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal loading rates of 10, 18, 32, 56, and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mLglass jars containing approximately 200 mL of test solution
- Type (delete if not applicable): glass jars covered to reduce evaporation
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): static.

- No. of organisms per vessel: ten first instar daphnids per vessel
- No. of vessels per concentration (replicates): 2 vessels
- No. of vessels per control (replicates): 2 vessels
- No. of vessels per vehicle control (replicates): Not applicable.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water used for both the range finding and definitive tests was the same as that used to maintain the stock animals. All chemicals were of analytical grade (CaCl2.2H2O; MgSO4.7H2O; NaHCO3; KCl) and deionised water was used to prepare the reconstituted water.
- Total organic carbon: NDA
- Particulate matter: NDA
- Metals: NDA
- Pesticides: NDA
- Chlorine: NDA
- Alkalinity: NDA
- Ca/mg ratio: NDA
- Conductivity: NDA
- Culture medium different from test medium: No difference
- Intervals of water quality measurement: N/A

OTHER TEST CONDITIONS
- Adjustment of pH: NA
- Photoperiod: 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods
- Light intensity: NDA

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
At 24 hours and 48 hours, the criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim within approximately 15 seconds after gentle agitation of the test vessel.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: NA
- Range finding study. The loading rates to be used in the definitive test were determined by a preliminary range finding test. In the range finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10, and 100 mg/L. The test material was prepared as a WAF. Ten daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 21 degrees C.
- Test concentrations:
- Results used to determine the conditions for the definitive study: No immobilization was observed at the 1 and 10 mg/L loading rate WAFs. However immobilization was observed at the 100 mg/L loading rate WAF. Based on this information loading rates of 10, 18, 32, 56 and 100 mg/L using a stirring period of 24 hours followed by a one hour standing period were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 65 - 87 mg/L

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: NDA
- Observations on body length and weight: NDA
- Other biological observations: NDA
- Mortality of control: 0
- Other adverse effects control: None noted
- Abnormal responses: NDA
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
- Effect concentrations exceeding solubility of substance in test medium: N/A

Results with reference substance (positive control):

EC50/LC50: A positive control using potassium dichromate as the reference material was conducted using test concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. Exposure conditions for the positive control were the same as to those used in the definitive tests. Inspection of immobilisation results gave a 48 hour EC50 of 1.2 mg/L with 95% confidence limits from 1.0 to 1.3 mg/L. The No Observed Effect Concentration after 24 and 48 hours were 1.0 mg/L and 0.56 mg/L respectively. The results from the positive control with potassium dichromate were within the normal range for this reference material.

Reported statistics and error estimates:

An estimate of the EL50 value at 24 hours was given by inspection of the immobilization data. The EL50 value and associated confidence limits at 48 hours were calculated by the maximum likelihood probit method using the ToxCalc computer software package.

Any other information on results incl. tables

Table. Cumulative Immobilisation Data in the Definitive Test

Nominal Loading Rate (mg/L)	Cumulative Immobilised Daphnia (10 per replicate)
	24 hours				48 hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
10	0	0	0	0	0	0	0	0
18	0	0	0	0	0	0	0	0
32	0	0	0	0	0	0	0	0
56	0	0	0	0	2	1	3	15
100	0	2	2	10	8	9	17	85

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL*50 value of 75 mg/L nominal loading rate with 95% confidence limits of 65 to 87 mg/L. The No Observed Effect Loading rate was 32 mg/l nominal loading rate.

Executive summary:

Introduction.A study was performed to assess the acute toxicity of the test material toDaphnia magna. The method followed that described in the OECD 202 Guidelines, “Daphnia sp, Acute Immobilisation Test and Reproduction Test”.

Methods.  Following a preliminary range finding test, twenty daphnids (2 replicates of 10 animals) were exposed to water accommodated fractions of the test material over a range of nominal loading rates of 10, 18, 32, 56, and 100 mg/L for 48 hours at a temperature of 21 degrees C under static test conditions. The number of immobilized Daphnia were recorded after 24 and 48 hours.

 

Results.The 48-Hour EL50 for the test material to Daphnia magna based on nominal loading rates was 75 mg/L with 95% confidence limits of 65 to 87 mg/L. The No Observed Effect Loading rate was 32 mg/l nominal loading rate WAF.

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