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

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:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study; restriction due to lack of analytical measurement of test material (nominal concentration used)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

283-392-8

EC Name:

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

Cas Number:

84605-29-8

IUPAC Name:

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

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: liquid at room temperature
- Boiling point: decomposes before boiling
- Vapour pressure: 0.0069 Pa at 25 deg. C
- Henry's law constant (for volatie substances): no data
- Water solubility (under test conditions): 2764 ppm at 22 deg. C
- Solubility in organic solvents: no data
- log Pow: 0.56
- pKa: no data
- Base or acid catalysis of test material: no data
- UV absorption: no data
- Stability of test material at room temperature: stable
- pH dependance on stability: hydrolytically stable at pH 4 and 7 for 5 days at 50 deg. C (OECD 111, Tier 1 preliminary study)

OTHER PROPERTIES (if relevant for this endpoint)
- Toxicity to microorganisms: EC50 > 10,000 mg/L (OECD 209)

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
1) OECD 2000. Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures
2) ECETOC 1996. Aquatic Toxicity Testing of Sparingly Soluble, Volatile and Unstable Substances
- Evidence of undissolved material (e.g. precipitate, surface film, etc): At zero hours all control and test cultures were clear colorless solutions

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: Scenedesmus subspicatus, strain CCAP 276/20
- Source (laboratory, culture collection): Liquid cultures were obtained from the Culture Collection of Algae and Protozoa (CCAP) Institute of Freshwater Ecology, The Ferry House, Far Sawrey, Ambleside, Cumbria.

- Method of cultivation: Cultures were maintained in the laboratory at a temperature of 21 degrees C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

Temperature was maintained at 24C throughout the test

pH:

pH at 0 hours in control and test material solutions ranged from 6.8 to 7.7. pH values at 72 hours in control and test material solutions ranged from 6.9 to 8.4.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks were used. Three flasks each containing 100 mL of test preparations were used for the control and each treatment group. The flasks were plugged with polyurethane foam bungs and incubated at 24 C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at approximately 150 rpm for 72 hours.

- Initial cells density: Preculture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.68 x106 cells per mL. Inoculation of test medium gave an initial cell density of 10^4 cells per mL.

- Control end cells density:
Mean cell density of control at 0 hours was 7.52X10^3 cells per mL
Mean cell density of control at 72 hours was 5.74 x10^5 cells per mL

- No. of vessels per concentration (replicates): triplicate
- No. of vessels per control (replicates): triplicate

GROWTH MEDIUM
The culture medium used for the range finding and definitive tests was the same as that used to maintain the stock culture. Stock solutions of the culture medium was prepared using reverse osmosis purified deionized water. The culture medium contained the following macronutrients: NaNO3, MgCl2.6H2O, CaCl2.2H2O, MgSO4.7H2O, K2HPO4, NaHCO3. The culture medium contained the following micronutrients: H3BO3, MnCl2.4H2O, ZnCl2, FeCl3.6H2O, CoCl2.6H2O, Na2MoO4.2H2O, CuCl2*2H2O; Na2EDTA.2H2O, Na2SeO3.5H2O

- Standard medium used: yes

OTHER TEST CONDITIONS
- Light intensity and quality: 7000 lux continuous illumination

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
Samples were taken at 1, 24, 48, and 72 hours and the cell densities determined using a Coulter Multisizer II Particle Counter.

TEST CONCENTRATIONS

The loading rates to be used in the definitive test were determined by a preliminary range finding test. The range finding test was conducted by exposing Scenedesmus subspicatus cells to a series of nominal loading rates of 10, 100, and 1000 mg/L for a period of 72 hours. After addition of the test material, the culture medium 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. Microscopic observations made on the WAFs indicated that dispersed test material was present in the water column and hence it was considered justifiable to remove the undissolved material by filtering through a glass wool plug (2-4 cm in length). Microscopic observations of the WAFs performed after filtering showed that there were no globules/microdispersions of test material to be present.

The cell densities and percentage inhibition of growth values from the exposure of the alga to the test material during the range finding test showed no effect on growth at 10 mg/L loading rate WAF, however growth was observed to be reduced at 100 and 1000 mg/L loading rate WAFs. Based on this information loading rates of 10, 20, 40, 80, and 160 mg/L were selected for the definitive test.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 20 - 22 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 22 - 26 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes

- Observation of abnormalities (for algal test): 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 10 and 20 mg/L loading rate WAFs; however no intact cells were observed to be present in the test cultures at 40, 80, and 160 mg/L loading rate WAF.

Any other information on results incl. tables

Results based on read across and Justification for read across:

Table. Inhibition of Growth Rate and Biomass

Nominal Loading Rate (mg/L)	Area Under Curve at 72 h	% Inhibition	Growth Rate (0-72 h)	% Inhibition
Control	1.09E7	--	0.060	--
10	1.23E7	[13, increase vs controls]	0.056	7
20	6.84E6	37	0.047	22
40	-3.97E5	104	-0.022	137
80	-4.88E5	104	-0.028	147
160	-5.88E5	105	-0.046	177

Justification for Read Across from EC 283-392 (Source) to EC 270-608-0 (Target)

Common Manufacturing Process: The test substance (EC 270-608-0) and the analogue (EC 283-392-8) 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 a mixture of two alcohols of a similar class - branched alcohols containing C4 and C5 carbons (test substance) and C3 and C6 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 % of the final products. 

 

Impurities: The level of impurities in the submission substance and the analogue is minimal (0.09 % wt and < 0.15% wt, respectively). Impurities have been identified as residual, unreacted alcohols from the production of the phosphorodithioic acid ester intermediates (isobutyl alcohol and pentyl alcohol isomers in the submission substance and isopropyl and 1,3-dimethylbutyl alcohols in the analogue).

 

Same Chemical Category: The submission substance (EC 270-608-0) and the analogue (EC 283-392-8), 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 test substance (EC 270-608-0) and the analogue (EC 283-392-8) 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 test substance and the analogue are related to the alkyl (R) groups of the alkyldithiophosphate ligands.

 

Both substances contain a distribution of several different zinc dialkyldithiophosphates (ZDDPs). 

The type and distribution of the zinc dialkyldithiophosphates is determined by the alcohol mixture and charge ratios of the alcohols used in the manufacturing process.

 

The test substance (EC 270-608-0) is a mixture of ZDDPs containing all isobutyldithiophosphate ligands, all pentyldithiophosphate ligands, and components containing both isobutyldithiophosphate and isomeric pentyldithiophosphate ligands resulting in a multicomponent substance with a molecular weight range of 548 – 604 (for monomers).            

 

The analogue (EC 283-392-8) is a multicomponent mixture of ZDDPs containing all isobutyl dithiophosphate ligands, all pentyl dithiophosphates ligands and ZDDPs containing both isobutyl and isomeric pentyl dithiophosphates with a molecular weight range of 492 – 660 (for 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.

Similarity of Physicochemical Properties: In addition to the structural similarities, similar physicochemical properties further support the justification for read across from the analogue. Both the test substance and analogue have similar physical states, densities and the same order of magnitude of vapour pressures and partition coefficients (logPow). Both were shown to be hydrolytically stable at pH 4, 7 and 9 in an OECD 111 preliminary hydrolysis study.

 

In evaluating the evidence for read across, significant consideration was given to water solubility. Water solubility studies conducted on the test substance and the analogue show the lower molecular weight ZDDP monomers are preferrentially dissolved in water with solubility decreasing with increasing alkyl chain lengths (molecular weights) of the alkyldithiophosphate ligands.  In this respect, the water soluble composition of the test substance (EC 270-608-0) and the analogue (EC 283-392-8) are considered to be sufficiently close to reasonably expect similar effects.  The slightly lower molecular weight of the water solubles in the analogue would represent the “worst case” with respect to bioavailability and effects on algal growth.

 

                                Data Matrix for Read Across from Analogue                                       

Property	Submission Substance (Target)	Analogue  (Data Source)
EC	270-608-0	283-392-8
CAS	68457-79-4	84605-29-8
Chemical Name	Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts	Phosphorodithioic acid, mixed O,O-bis(1,3-dimethylbutyl and iso-Pr) esters, zinc salts
Physical	Viscous liquid	Viscous liquid
Boiling Point	Decomposes before boiling	Decomposes before boiling
Density @ 15.6 deg. C  (ASTM D4052)	1.17 mg/L	1.2 mg/L
Vapour Pressure @ 25 C   (EU method A.4)	0.0025 Pa	0.0069 Pa
Water Solubility @ 22 deg. C (OECD 105)	1658 mg/L	2764 mg/L
Identity of water soluble components (% = GC area %)	ZDDPs containing 76% isobutyl (C4) dithiophosphate ligands + 24% containing mixed isobutyl (C4) and pentyl (C5) dithiophosphate ligands	ZDDP containing 95% isopropyl (C3) dithiophosphate ligands + 5% containing mixed isopropyl and 1,3 -dimethylbutyl dithiophosphate ligands
Average molecular weight of water solubles (weighted average based on GC peak area %)	562	500.4
Partition Coefficient, logPow (OECD 107)	0.69	0.56
Hydrolysis as a function of pH (OECD 111, Tier 1 preliminary study at pH 4,7 and 9)	Hydrolytically stable at pH 4, 7 and 9	Hydrolytically stable at pH 4, 7 and 9

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Scenedesmus subspicatus to the test material gave an EbL50 (72h) value of 21 mg/L loading rate WAF and an ErL50 (0-72hr) value of 24 mg/L loading rate WAF. The No Observed Effect Loading rate was 10 mg/L loading rate WAF.

Executive summary:

Introduction. A study was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No. 201, “Alga Growth Inhibition Test”. 

 

Methods:

Following a preliminary range finding test, the alga were exposed to Water Accommodated Fractions of test material over a range of nominal loading rates of 10, 20, 40, 80, and 160 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 degrees 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:

Exposure ofScenedesmus subspicatusto the test material gave an EbL50 (72h) value of 21 mg/L loading rate WAF and an ErL50 (0-72hr) value of 24 mg/L loading rate WAF. The No Observed Effect Loading rate was 10 mg/L loading rate WAF.