Dibutyl [[bis[(2-ethylhexyl)oxy]phosphinothioyl]thio]succinate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 October 2017 to ****

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

-Purity: 84%, based on P-31 NMR data
-Description: Pale yellow, clear liquid
-Storage: room temperature, in the dark

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Each treatment vessel contained a synthetic sewage feed, laboratory well water, test or reference substance (blank controls had neither) and microbial inoculum (except for abiotic control) to achieve total test volumes of 500 mL.

Since the test substance was insoluble in water at the concentrations to be used in this study, a primary stock solution was not prepared. The test substance, which was in a liquid state, was added directly to the test vessels by volume. The volume of test substance to be added to the test vessels was adjusted for the purity and relative density of the test substance.

The reference substance, 3,5-dichlorophenol (3,5-DCP), was added the test vessels as a prepared aqueous stock solution (500 mg/L) in 5.0 mL of 1 N sodium hydroxide that had been adjusted to a final pH of 7.43 with 1 N sulfuric acid to achieve nominal concentrations of 1.0, 3.0, 10, and 30 mg/L (single vessel for each).

At time zero, 16 mL of synthetic sewage feed was added to a 1-L beaker and then brought to a volume of 250 mL with laboratory well water. An aliquot (250 mL) of the microbial inoculum was added until the solution was at full volume (500 mL). This mixture was identified as Control 1. The reference substance (3,5 dichlorophenol) flasks (1.0, 3.0, 10, and 30 mg/L) were then prepared by adding appropriate volumes of the 3,5-dichlorophenol stock solution to the sludge mixture prepared in the same manner as in Control 1. Control 2 was prepared in the same manner as Control 1, 15 minutes after the last reference substance test system was prepared. The abiotic control was prepared by adding the appropriate amount of the test substance to the vessel with synthetic sewage feed and dilution water; this flask did not contain inoculum. The test substance flasks were initiated following the same procedure as Control 1 and Control 2 with the exception that the appropriate amount of the test substance was added to the sludge mixture. All test and reference substance concentrations were based on nominal levels.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Wareham Waste Water Treatment Plant (WWTP), Wareham, Massachusetts, which receives primarily domestic sewage
- Method of cultivation: The inoculum received 3.0 mL of an undiluted synthetic sewage feed and was aerated overnight at a temperature of 20 ± 2 ºC.
- Initial biomass concentration: 3.18 g/L
- Preparation of inoculum for exposure: sludge was passed through a 2-mm stainless steel sieve, concentrated by centrifuging for 10 minutes at 1000 rpm and washed with laboratory well water a total of four times, with the supernatant being discarded each time, then resuspended in water to obtain an activated sludge inoculum containing a solids level of 3 g/L (+/- 10%). The pH value of the sludge inoculum was 6.46 and was adjusted to a final pH of 7.29 using 10 M sodium bicarbonate solution.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20.4 to 20.6 °C (environmental chamber); 20.0 to 20.9 °C (respirator water bath)

pH:

7.45 in the abiotic control; the remaining pH values ranged from 7.59 to 7.81

Dissolved oxygen:

Aerated; see tabulated oxygen consumption data

Nominal and measured concentrations:

- test substance: 1, 10, 100 and 1000 mg/L (nominal)
- reference substance: 1.0, 3.0, 10, and 30 mg/L (nominal)

Details on test conditions:

The study was conducted as a combined range finding / limit test. A definitive test was not conducted in this study because as no significant respiration inhibition was observed in the presence of test substance during the combined range finding / limit test.
TEST SYSTEM
- Test vessel: 1-L glass beakers (containing a total volume of 500 mL)
- No. of vessels per concentration (replicates):
- Test substance: 10, 100, and 1000 mg/L, with the highest concentration tested in triplicate
- Reference substance: 1.0, 3.0, 10, and 30 mg/L (single vessel for each)
- No. of vessels per control (replicates): two
- No. of vessels per abiotic control: 1000 mg/L, in triplicate

- Aeration: yes (0.612 to 0.998 L/minute)
- Nutrients provided for bacteria: synthetic sewage feed prepared according to OECD guideline 209
- Nitrification inhibitor used: not applicable; nitrification inhibition not assessed.
- Biomass loading rate: Following inoculation, each vessel contained a nominal suspended solids concentration of ca.1.5 g/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: a mixture of unadulterated water from a 100-meter bedrock well and dechlorinated Town of Wareham well water.
- Particulate matter: not specified

OTHER TEST CONDITIONS
Following 3 hours of incubation with aeration, a 23-mL aliquot of each sludge mixture was transferred to a 50 mL cylinder placed inside a 20 +/- 1˚C water bath placed over a stir plate. The pH of the sludge mixture was measured before the respiration measurement. The cylinders received a stir bar and were fitted with a dissolved oxygen probe and attached to a Strathkelvin Instruments (SI) Model 928 oxygen system (Strathkelvin software version 4.0.6.2), which began monitoring the depletion of oxygen of the samples (six vessels at a time) rates immediately. The sludge mixtures were stirred continuously while the oxygen measurements were taken. The oxygen measurements were terminated after 10 minutes.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Details on results:

The oxygen consumption rate was calculated within the Strathkelvin instrument for each control, test substance, and reference substance concentration by using the dissolved oxygen concentration versus time curves over the linear portions of each curve (2.00 to 8.00 minutes for the reference substance and 4.00 to 8.00 minutes for the test substance).

At test termination, the respiration rate for the two control vessels was determined to be 30.4 and 29.8 mg O2/L/hr, which met the acceptability criterion (i.e., within 30% of each other during the test). Respiration rate in the test substance test vessels (10, 100, 1000, 1000, and 1000 mg/L) was 29.0, 26.6, 34.1, 25.9, and 28.8 mg O2/L/hr, respectively. Compared to the controls, respiratory inhibition for the test substance test vessels was 3.7, 11.6, -13.3, 14.0, and 4.3%, respectively.

Based on these results, the EC10 and EC50 values were estimated to be >1000 mg/L. The no observed effect concentration (NOEC) for total respiration was determined to be 1000 mg/L.

Results with reference substance (positive control):

Respiration rate in the 3,5-dichlorophenol test vessels (1.0, 3.0, 10, and 30 mg/L) was 20.4, 22.0, 10.2, and 4.6 mg O2/L/hr, respectively. Compared to the controls, respiratory inhibition by 3,5 dichlorophenol in test vessels was 32.2, 26.9, 66.1, and 84.7%, respectively. Based on these results, the EC50 value for 3,5 dichlorophenol was calculated to be 4.7 mg/L, which was within the acceptable limits as specified in the OECD 209 Guideline (i.e., 2 to 25 mg/L).

Reported statistics and error estimates:

The percent inhibition was plotted against the log of the test concentration and the EC50 and EC10 values derived by linear regression using the Comprehensive Environmental Toxicity Information System (CETIS). No statistically significant effect was observed between the controls and the highest sample concentration (1000 mg/L), based on a one-tail, two-sample Student’s Paired t-Test, assuming unequal variances (p > 0.05).

Sample Type	pH	Respiration Rate (mg O2/L/hr.)	% Inhibition*
Abiotic	7.45	0.7	NA
Control 1	7.60	30.4	NA
Control 2	7.71	29.8	NA
	Control Mean:	30.1
3,5 dichlorophenol (mg/L)
1.0	7.79	20.4	32.2
3.0	7.81	22.0	26.9
10	7.76	10.2	66.1
30	7.73	4.6	84.7
EC50: 4.7 mg/L
Test Substance (mg/L)
10	7.70	29.0	3.7
100	7.67	26.6	11.6
1000	7.64	34.1	-13.3
1000	7.60	25.9	14.0
1000	7.59	28.8	4.3
EC10 >1000 mg/L
EC50 >1000 mg/L

NA: Not Applicable

* 3,5-Dichlorophenol and butanedioic acid, 2-[[bis[(2-ethylhexyl)oxy]phosphinothioyl]thio]-, 1,4-dibutyl ester compared to control mean (n = 2).

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the activated sludge respiration inhibition study, it was concluded that the test substance does not inhibit the respiration of the sludge microbial population in a domestic waste water treatment plant up to a concentration of 1000 mg/L.

Executive summary:

The study was conducted to determine the median effect concentration (EC50) and, where possible, EC10 and NOEC for activated sludge microorganisms exposed to the test substance in accordance with OECD Guideline 209. The activated sludge inoculum was collected from the Wareham Waste Water Treatment Plant (WWTP), Wareham, Massachusetts, which receives primarily domestic sewage.

A combined range finding / limit test, employing nominal test concentrations of 1, 10, 100 and 1000 mg/L, was undertaken to determine the appropriate concentration levels for a definitive test. A definitive test was not conducted in this study because no statistically significant respiration inhibition was observed during the combined range finding / limit test following a 3-hour exposure to the test substance.

At test termination, the respiration rate for the two control vessels was determined to be 30.4 and 29.8 mg O2/L/hr, which met the acceptability criterion (i.e., within 30% of each other during the test). Respiration rate in the test substance test vessels (10, 100, 1000, 1000, and 1000 mg/L) was 29.0, 26.6, 34.1, 25.9, and 28.8 mg O2/L/hr, respectively. Compared to the controls, respiratory inhibition for the test substance vessels was 3.7, 11.6, -13.3, 14.0, and 4.3%, respectively. Based on these results, the EC10 and EC50 values were estimated to be >1000 mg/L. The no observed effect concentration (NOEC) for total respiration was determined to be 1000 mg/L.

Respiration rate in the 3,5-dichlorophenol test vessels (1.0, 3.0, 10, and 30 mg/L) was 20.4, 22.0, 10.2, and 4.6 mg O2/L/hr, respectively. Compared to the controls, respiratory inhibition for the 3,5‑dichlorophenol test vessels was 32.2, 26.9, 66.1, and 84.7%, respectively. Based on these results, the EC50 value for 3,5 dichlorophenol was calculated to be 4.7 mg/L, which was within the acceptable limits as specified in the OECD 209 Guideline (i.e., 2 to 25 mg/L).

Description of key information

In an OECD 209 study, conducted according to GLP, no significantrespirationinhibition was observed following a 3-hour exposure of activated sewage sludge to the test substance, resulting in an EC50 value of greater than 1000 mg/L.  The No Observed Effect Concentration (NOEC) following the 3-hour exposure was 1000 mg/L (Smithers Viscient, ****).