Phosphorodithioic acid, mixed O,O-bis(2-ethylhexyl and iso-Bu and pentyl) esters, zinc salts

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study; restriction due to lack of analytical monitoring of test material and read-across.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

not specified

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts

EC Number:

270-608-0

EC Name:

Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts

Cas Number:

68457-79-4

IUPAC Name:

zinc bis(O,O-diisobutyl dithiophosphate)

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Concentrations: 0, 26, 64, 400 and 1000 mg/L Water Soluble Fractions

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test material solutions were prepared as Water Soluble Fractions (WSF). For each test concentration an appropriate aliquot by weight of the test material was added to dilution water in a glass aquarium. Each aquarium was fully covered and the solution was gently stirred overnight (24 hours) using a magnetic stirrer with a Teflon coated stir bar. Following an additional 24 hours of settling, the WSF of each concentration was separated from floating or settled material. Greater than 90% of the aged (approximately 24 hours old) test solution was replaced daily in each aquarium with freshly prepared test solution following the method described above.


- Controls: untreated negative control; reference toxicant

- Evidence of undissolved material (e.g. precipitate, surface film, etc): WSF were separated from floating or settled material and the solutions were observed to be colorless.

Test organisms

Test organisms (species):

Cyprinodon variegatus

Details on test organisms:

TEST ORGANISM
- Common name: Sheepshead minnow
- Strain: Cyprinodon variegatus
- Source: A commercial supplier in Massachusetts
- Age at study initiation (mean and range, SD): Test fish were 31 days old at the time of test initiation
- Length at study initiation (length definition, mean, range and SD): mean =14.2 mm, range 11-18 mm, N=30
- Weight at study initiation (mean and range, SD): mean wet weight of 0.052 g; range 0.02 to 0.09 g; N=30

- Feeding during test: not fed during exposure

ACCLIMATION
- Acclimation period: Minimum of 7 days
- Acclimation conditions (same as test or not): Fish were held in glass aquaria or fiberglass holding tanks under a photoperiod of 16 hours light and 8 hours darkness. A closed loop recirculatng filtration system provided natural filtered (5 um) seawater with a salinity range of 32 to 36 ‰, a pH range of 7.5, and a dissolved oxygen concentration range of 96% of saturation. The temperature range in the holding trank was 23 to 24 degrees C.
- Type and amount of food: Fish were fed less than 48 hours old Artemia nauplii, ad libitum, daily until the day prior to testing
- Feeding frequency: ad libitum
- Health during acclimation (any mortality observed): There was no mortality during the seven days prior to testing

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

Study design

Test type:

semi-static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Test temperature:

22 degrees C

pH:

No treatment related differences in pH. Mean pH values ranged from 7.6 to 7.8 in control and test solutions.

Dissolved oxygen:

No treatment related differences in DO. All solutions measured were greater than 60% of saturation and the range at the start of the test was from 6.9 to 7.8 mg/L

Salinity:

32%

Nominal and measured concentrations:

Nominal concentrations of 0, 26, 64, 160, 400, and 1000 mg/L WSF

Details on test conditions:

TEST SYSTEM
- Test vessel: 18.9 L glass covered glass aquaria that contained 5 L of test solution
- Type (delete if not applicable): covered
- Material, size, headspace, fill volume: 5 L of test solution with a test solution depth of 6.0 cm with a surface area of 985 cm2.
- Aeration: no auxiliary aeration
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates):
- Biomass loading rate: Ten sheepshead minnows selected impartially from the holding tank were placed in each test aquaria within 10 minutes after the test solutions had been prepared. The resulting test organism loading concentration was 0.104 grams of biomass per liter of test solution.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution was used was natural seawater collected from Cape Cod Canal, Bourne, Massachusetts. The seawater was filtered through a 5 um porosity polypropylene core filter and an activated carbon canister and stored for a period of 1 to 4 days prior to use.

- Culture medium different from test medium: No difference, the water in the culture system was the same type of water used in the bioassay. Fish were acclimated to test conditions prior to the study
- Intervals of water quality measurement: Daily for temperature, pH, DO, salinity

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 hours light and 8 hours dark
- Light intensity: 25 foot candles with a color rendering index greater than 91 by a combination of Sylvania Growlux and Cool White fluorescent bulbs.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : All aquaria were examined after 0, 24, 48, 72, and 96 hours of exposure as follows: mortalities were recorded, dead fish were removed, and observations of the fish and physical characteristics of the test solutions were recorded.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: NA
- Range finding study
A preliminary study was conducted using 100% WSF at 10,000 mg/L nominal concentrations for a minimum of 48 hours and a maximum of 96 hours with daily renewal of the test and control solutions. This study was conducted with duplicate exposure of ten fish each and included a control exposure.
- Test concentrations: 100% WSF at 10,000 mg/L nominal concentrations
- Results used to determine the conditions for the definitive study: The 96 hour mortality of the fish tested at 100% Water Soluble Fraction was 100%. Based on this result, the definitive test was deemed necessary.

Reference substance (positive control):

yes

Remarks:

sodium lauryl sulfate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

46 mg/L

Nominal / measured:

nominal

Basis for effect:

mortality (fish)

Remarks on result:

other: 33 - 61 mg/L

Effect concentrations 2

Duration:

96 h

Dose descriptor:

other: NOEL

Effect conc.:

< 26 mg/L

Nominal / measured:

nominal

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: The NOEL was reported to be less than 26 mg/L, the lowest concentration tested, and this endpoint is based on observations of physical and behavioral abnormalities including lethargy, loss of equilibrium, and darkened pigmentation.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no insoluble materials were reported in the test solutions

Results with reference substance (positive control):

- Results with reference substance valid? Yes. A reference test with sodium lauryl sulfate was conducted with the test fish and the resulting 96 hour LC50 was 1.3 mg/L with a 95% confidence interval of 0.72 to 2.0 mg/L.

Reported statistics and error estimates:

The concentrations tested and the corresponding mortality data derived from the definitive toxicity test were used to estimate the medial LC50 values and 95% confidence intervals using standard statistical computations and software. The statistical methods available were probit analysis, nonlinear interpolation, and moving average analysis.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table 1. Cumulative Mortality Data

WSF Test Concentration mg/L	Replicate	Cumulative Mortality (%) at Hour   24          48      72      96
1000	R1 R2	100 100	100 100	100 100	100 100
400	R1 R2	100 100	100 100	100 100	100 100
160	R1 R2	50 50	80 90	100 90	100 90
64	R1 R2	30 60	50 70	50 80	60 80
26	R1 R2	0 0	0 40	0 40	0 40
0, Control	R1 R2	0 0	0 0	0 0	0 0

 

 

Table 2. Statistical Analysis of Mortality Data

Exposure Period (hours)	LC50 (mg/L)	95% Confidence Limits (mg/L)
24	100	76 to 140
48	54	37 to 74
72	48	34 to 64
96	46	33 to 61

Justification for Read Across from Analogue EC 270-608-0

Common Manufacturing Process:The submission substance (EC 273-527-9) and the analogue (EC 270-608-0) are produced under a common manufacturing process in which a phosphorodithioic acid ester intermediate, (RO)₂PS₂H, is produced by the reaction of phosphorus pentasulfide with an alcohol or a mixture of two alcohols of a similar class - branched alcohol containing C8, C5 and C4 carbons (submission substance) and C4 and C5 carbons (analogue). The intermediate is neutralized with zinc oxide to produce the final multicomponent substance. The reaction is performed in the presence of a highly refined base oil which accounts for 8 – 10.3 % of the final products.

Impurities:The level of impurities in the submission substance and the analogue (data source) is minimal. Impurities have been identified as residual, unreacted alcohols from the production of the phosphorodithioic acid ester intermediates (isobutanol, pentanol and 2-ethylhexanol in the submission substance and isobutanol and pentanol in the analogue).

Same Chemical Category:The submission substance (EC 273-527-9) and the analogue (EC 270-608-0), generically referred to as ZDDPs, have been shown to have sufficient structural similarities to be included in the Zinc Dialkydithiophosphate Category (ZDDPs) in the United States Environmental Protection Agency High Production Volume (HPV) Chemical Challenge Program.

Structural Similarity:The primary feature accounting for the similarity of the submission substance (EC 273-527-9) and the analogue (EC 270-608-0) is the common organometallic core structure consisting of a central zinc metal bonded to four alkyldithiophosphate esters (ligands) by coordinate covalent bonds -Zn[(S₂P(OR)₂]_2.Structural variations between the submission substance and the analogue are related to the alkyl (R) groups of the alkyldithiophosphate ligands.

The analogue/data source (EC 270-608-0) is a multicomponent mixture of ZDDP monomers and dimers containing isobutyl dithiophosphate ligands, pentyl dithiophosphate ligands, and mixtures of isobutyl and pentyl dithiophosphate ligands with a molecular weight range of 548 – 604 (monomer).

The submission substance (EC 273-527-9) is a multicomponent mixture of ZDDP monomers and dimers containing isobutyl dithiophosphate ligands, pentyl dithiophosphate ligands, 2-ethylhexyl dithiophosphates ligands and mixtures of isobutyl, pentyl and 2-ethylhexyl dithiophosphate ligands with a molecular weight range of 548 – 772 (monomer).

Tanimoto Fingerprint (ToxMatch Version 1.06 software) gives a similarity index greater than 0.8 (values range from 0, no similarity to 1, identical). Peer reviewed literature indicates that values greater than 0.6 are significantly similar.DSSTox similarity was 80% between the submission substance and the analogue.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the saltwater fish, Cyprinodon variegatus, was investigated and resulted in a 96 hour LC50 of 46 mg/L and a NOEL of less than 26 mg/L.

Executive summary:

Introduction.  A study was performed to assess the acute toxicity of the test material to sheepshead minnow under static renewal test conditions using OECD 203 Guidelines.

 

Methods. Following preliminary range finding studies fish were exposed in groups of ten to water soluble fractions of the test material at nominal test concentrations of 0, 26, 64, 160, 400, and 1000 mg/L. The test was conducted under static renewal conditions for 96 hours at a temperature of 22 degrees C. The number of fish mortalities and any sublethal effects were recorded after 24, 48, 72, and 96 hours. 

 

Results. The acute toxicity of the test material to the saltwater fish,Cyprinodon variegatuswas investigated and resulted in a 96 hour LC50 of 46 mg/L. A reference toxicity test was conducted with sodium lauryl sulfate resulting in a 96 hour LC50 of 1.3 mg/L.