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Diss Factsheets

Administrative data

activated sludge respiration inhibition testing
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
11 November 1993
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study. Restriction due to lack of chemical analysis of test material

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
Phosphorodithioic acid, mixed O,O-bis(1,3-dimethylbutyl and iso-Pr) esters, zinc salts
EC Number:
EC Name:
Phosphorodithioic acid, mixed O,O-bis(1,3-dimethylbutyl and iso-Pr) esters, zinc salts
Cas Number:
Molecular formula:
Not applicable
Phosphorodithioic acid, mixed O,O-bis(1,3-dimethylbutyl and iso-Pr) esters, zinc salts

Sampling and analysis

Analytical monitoring:
Details on sampling:
Concentrations: Nominal test concentrations were prepared at 1, 10, 100, 1,000 and 10,000 mg/L
- Sampling method: The test material was not monitored in the test solutions

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Test Material preparation
The testing system consisted of two units: 1) a system used to expose the sludge inoculum to the test material; and 2) a system to measure the rate of oxygen consumption of the sludge in a closed bottle. The sludge / microbial inoculum were exposed to the test material in flint glass bottles. Low pressure oil free air was delivered to each test vessel using a glass pipet. All of the test vessels were held in a water jacketed incubator in the dark during the three hour exposure period. The incubator maintained a constant temperature of 20 degrees C. To measure the rate of oxygen consumption for the activated sludge after exposure to the test materials a dissolved oxygen meter was used. The oxygen meter was fitted with a BOD probe with a standard sensitivity membrane and an external output to a strip chart recorder. To measure the consumption of oxygen, a sub sample of the exposed sludge was placed in a clean BOD bottle, the change in oxygen concentration over time was measured with a meter and probe and the results were recorded on a strip chart recorder.

Preparation of Inoculum
Activated sludge with associated aerobic organisms were obtained from a domestic wastewater treatment plant that serves only a residential subdivision. The sludge was aerated with low pressure oil free air and was fed a synthetic sewage feed at a rate of 50 mg per liter. The synthetic sewage medium contained peptone, beef extract, urea, NaCl, CaCl2, MgSO4, and K2HPO4. Triplicate samples of the mixed sludge were dried at 100 degrees C in a drying oven until a constant weight was achieved. Based on these results the sludge was diluted to produce a dry weight per unit volume concentration of 4 g/L.

Reference Material preparation
For the purpose of the test 3,5-dichlorophenol was used as a reference material, and was prepared at a nominal concentration of 500 ppm.

Test organisms

Test organisms (species):
other: activated sludge of a domestic residential sewage
Details on inoculum:
Activated sludge was obtained from the Cottonwood Subdivision Wastewater Treatment Plant located in Franklin, Tennessee. This wastewater treatment plant serves only the residential subdivision and its community pool. One gallon of activated sludge was collected by the plant operator, picked up on the day of collection and transported to the testing laboratory.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
3 h

Test conditions

no data
Test temperature:
20 degrees C
no data
Dissolved oxygen:
Change in O2 and O2 consumption measured
not applicable/freshwater
Nominal and measured concentrations:
Definitive Test: 0, 1, 10, 100, 1,000, 10,000 mg/L nominal
Details on test conditions:
To initiate the test, the first test vessel was used as the first control in the testing series. The synthetic sewage feed, dechlorinated tap water, and sludge inoculum were added to the vessel. The vessel was immediately placed in the temperature controlled incubator and aerated. Fifteen minutes later the reference toxicant series was begun. In a separate test vessel, the sewage feed was added followed by dechlorinated water, reference toxicant stock solution, and sludge inoculum. As with the first control, the vessel was immediately placed in the temperature controlled incubator and aerated. The nominal reference toxicant concentrations were 10, 20, and 40 ppm respectively.
Once the series of three reference toxicant test vessels was started, test vessels containing the test material was prepared. As the test material was considered insoluble, no stock solution was prepared and the material was added directly to each test vessel by way of glass microscope cover slips. The nominal test material concentrations were 1, 10, 100, 1000 and 10000 mg/L. A vessel with no test material was also prepared as a test control replicate.

After three hours of exposure, or three hours within the test vessel, the sludge mixture was transferred into a BOD bottle containing a Teflon coated magnetic stir bar. The bottle was capped with the BOD bottle oxygen probe to eliminate air space and placed on a magnetic stirring plate. The sludge mixture was stirred at a constant rate and the consumption of oxygen over time was recorded on a strip chart recorder. The oxygen readings were recorded for ten minutes or until a linear trace covering a sufficient range of oxygen concentrations was obtained.

Number of Vessels and Concentrations (Definitive Study):
Controls – two controls
Reference – 10, 20 and 40 ppm 3,5-dichlorophenol, no replicates
Test Material – 1, 10, 100, 1000, 10000 mg/L nominal, no replicates

-Test Water: Reagent grade water was used for preparing the test and reference materials. The dilution water was from the City of Franklin Tennessee water supply and was softened and dechlorinated prior to use.

- Adjustment of pH: No.
- Photoperiod: No data.

During testing, each mixture was aerated with low pressure oil free air controlled by brass needle valves.

EQUIPMENT:-Measuring Vessel: BOD bottles
-Oxygen Meter: Yellow Springs Instruments Model 51 DO meter

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Change in O2 mg/L per change in time and percent inhibition

Test concentrations: 0, 1, 10, 100, 1000, 10000 mg/L nominal

Reference substance (positive control):

Results and discussion

Effect concentrations
3 h
Dose descriptor:
Effect conc.:
> 10 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
The average respiration rates for the two controls was 77.2 mg O2/L*hr.

The resultant consumption rates for the inoculum exposed to the reference toxicant were used to calculate the EC50 of the reference toxicant in the sludge used to test the experimental material. The EC50 was calculated to be 18.5 mg/L 3,5-dichlorophenol.

Five nominal concentrations of OS#13961BA were tested with the activated sludge inoculum at 1, 10, 100, 1000 and 10000 mg/L. The resultant respiration rates were respectively: 67.0, 58.2, 67.2, 72.0 and 54.0 mg O2/L*hr. These data translate into percent inhibitions of 13.2%, 24.6%, 13.0%, 6.7% and 30.1%. Because none of the test concentrations produced an inhibition greater than 50%, the EC50 was estimated to be > 10,000 mg/L.
Results with reference substance (positive control):
- Results with reference substance valid? Yes.

To assure quality control, the reference toxicant 3,5-dichlorophenol needs to be between 5 and 30 mg/L. The EC50 for the reference toxicant in this test was calculated to be 18.5 mg 3,5-dichlorophenol per liter.

Any other information on results incl. tables

Test Material Data

Test Concentration (mg/L)

Change in O2 (mg/L)

Change in Time





Percent Inhibition



3 4/6





5 4/6










3 3/6





8 2/6



Justification for Read Across from Analogue EC 283-392-8

Common Manufacturing Process: The submission substance (EC 273-527-9) 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 an alcohol or a mixture of two alcohols of a similar class - branched alcohol containing C8, C5 and C4 carbons (submission 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 – 10.3 % 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 isopropyl and 1,3-dimethylbutyl alcohols in the analogue).

Same Chemical Category:The submission substance (EC 273-527-9) 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 submission substance (EC 273-527-9) 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 submission substance and the analogue are related to the alkyl (R) groups of the alkyldithiophosphate ligands.

The analogue/data source (EC 283-392-8) is a multicomponent mixture of ZDDP monomers and dimers containing isopropyl dithiophosphate ligands, 1,3-dimethylbutyl dithiophosphate ligands and mixtures of isopropyl and 1,3-dimethylbutyl dithiophosphate ligands with a molecular weight range of 492 – 660 (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:
The effect of the test material on the respiration of activated sewage sludge micro-organisms using standard guidelines gave an EC50 of >10,000 mg/L
Executive summary:

Introduction.A study was performed to assess the effect of the OS#13961BA test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984), No. 209 "Activated Sludge, Respiration Inhibition Test".

Methods.Activated sewage sludge was exposed to directly added test material at concentrations of 1, 10, 100, 1000 and 10000 ppm, a duplicate control group, and an assessment of the sensitivity of the inoculum used in the test to a reference toxicant 3,5-dichlorophenol. The test duration was a 3 hour exposure period at 20 degrees C to the test material followed by measurement of oxygen consumption.


Results.The effect of the test material on the respiration of activated sewage sludge gave an EC50 of >10,000 mg/L. The reference material gave an EC50 value of 18.5 mg/L. The activated sludge respiration test passed the quality control criteria for an acceptable test. The two control replicates produced oxygen consumption rates within the required 15% of each other