Reaction mass of butyl diphenyl phosphate and dibutyl phenyl phosphate and tributyl phosphate

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:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study is performed according to the most recent guidelines (OECD & GLP).

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

Test substance:
- Direct weighings were prepared to give the different test item concentrations. The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 16 hours.
- Test item concentrations were: 10, 100 and 1000 mg/L
- Test item concentration in physico-chemical oxygen consumption control was: 1000 mg/L

Reference substance:
- For the reference compound a stock solution at a concentration of 500 mg/L was prepared by dissolving 250 mg 3,5-Dichlorophenol in 5 mL of 1 N NaOH and diluting to 0.5 litre with deionised water. The pH was adjusted to pH 7 ± 0.5 with HCl.
- Concentrations of reference compound 3,5-Dichlorophenol were: 2.5, 5, 10, 20 and 40 mg/L

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Type: mixed population of aquatic microorganisms (activated sludge)
- Origin: aeration tank of a domestic sewage treatment plant (Municipal STP Cologne-Stammheim)
- Date of collection: 2012-08-13
- Microbial inoculum: The sludge was settled and the supernatant was decanted. After centrifuging the sludge (15 min at 4500 rpm and 20°C) the supernatant was decanted again. Approximately 1 g of the wet sludge was dried in order to calculate the amount of wet sludge to achieve a concentration of activated sludge of 3 g/L (dry weight) suspended solids. The calculated amount of sludge was dissolved in synthetic medium and then filled up to a defined end volume with deionised water.
- Storage of sludge: aeration of the activated sludge at 20 ± 2 °C, daily fed with synthetic medium
- pH of the suspension before application: 7.6
- Synthetic sewage feed: A synthetic waste water feed was prepared by dissolving the following amounts of substance per 1 litre of water. (16.0 g peptone, 11.0 g meat extract, 3.0 g urea, 0.7 g NaCl, 0.4 g CaCl2 x 2H2O, 0.2 g MgSO4 x 7H2O, 2.8 g K2HPO4)
- pH of the synthetic sewage feed: 7.5 ± 0.5

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

with permanant aeration

Test temperature:

20 ± 2°C

pH:

6 to 8

Nominal and measured concentrations:

Nominal concentrations test substance: 10, 100 and 1000 mg/L
Nominal concentrations reference substance: 2.5, 5, 10, 20 and 40 mg/L

Details on test conditions:

- Before use the wet weight/dry weight relationship of the activated sludge was determined by drying 10 mL of sludge suspension. Subsequently, a sludge suspension of 2 g (dry weight)/L was prepared. The pH of this suspension was measured and adjusted to 6-8.
- 8 mL of the synthetic medium and 100 mL of activated sludge were added to the dissolved test item. The mixture was filled up with deionised water to 250 mL and aerated at 20 ± 2 °C.
- The exposure medium with the reference substance was prepared by adding 8 mL of the synthetic medium, 100 mL of activated sludge and a defined amount of the stock solution to achieve the test concentrations, and was filled up with deionised water to 250 mL and aerated at 20 ± 2°C.
- Control vessels (inoculated sample without test item) were prepared the same way.
- Additional vessels to determine the physico-chemical oxygen consumption were prepared containing the test item, and the synthetic medium but no activated sludge.
- Oxygen consumption was measured and recorded after an aeration time of 3 hours in all these vessels starting with control 1. Thereafter, temperature and pH were measured as well. Then the other test vessels were measured. Control 2 terminated the measurements.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: oxygen concentration

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: by probit analysis

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

402 mg/L

Nominal / measured:

nominal

Conc. based on:

other: oxygen concentration

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: by probit analysis, 95% CL could not be determined

Details on results:

Dibutyl phenyl phosphate showed 12.73% respiration inhibition of activated sludge at the highest concentration of 1000 mg/L.

Results with reference substance (positive control):

The EC50 value of the refernce substance is 8.248 mg/L (95% CL: 5.062 - 13.396)

Reported statistics and error estimates:

Results were generated via probit analysis. The selected effective concentrations (ECx) of the test item and their 95%- and 99%-confidence limits (according to Fieller`s theorem).
- For test substance: Computation of variances and confidence limits was adjusted to metric data (Christensen & Nyholm 1984). The p(F) is greater than 0.05; i.e. the slope was not significantly different from zero. The effect parameters and confidence limits can be meaningless. Slope function after Litchfield and Wilcoxon: 201.280.
- For reference substance: Computation of variances and confidence limits was adjusted to metric data (Christensen & Nyholm 1984). Slope function after Litchfield and Wilcoxon: 2.140.

Table 1: Oxygen content, temperature and pH values during exposure phase (test item)

	Test item concentration [mg/L]	O2start [mg O2/L]	O2end [mg O2/L]	Time (start-end) [minutes]	Temp. [°C]	pH
Test item	10	4.3	2.8	3	20.7	7.9
Test item	100	4.6	2.5	5	20.7	7.8
Test item	1000	4.5	2.9	4	20.8	7.9
Control 1		5.3	2.6	6	20.6	7.9
Control 2		5.4	2.6	6	21.2	7.9
Physico-chemical oxygen consumption control	1000	7.8	7.8	9	20.8	7.4

 

Table 2: Oxygen content, temperature and pH values during exposure phase (reference compound)

	Reference compound concentration [mg/L]	O2start [mg O2/L]	O2end [mg O2/L]	Time (start-end) [minutes]	Temp. [°C]	pH
3,5-Dichloro-phenol	2.5	5.3	2.6	7	20.6	7.9
3,5 -Dichloro-phenol	5	4.5	2.6	5	20.6	8.0
3,5 -Dichloro-phenol	10	6.5	5.2	8	20.7	7.8
3,5 -Dichlorophenol	20	6.9	6.5	7	20.8	7.8
3,5 -Dichlorophenol	40	7.1	6.9	8	20.8	7.9

 

Table 3: Results of test item Dibutyl phenyl phosphate

Test item concentration (nominal)	Respiratory rate test item	Phys.-chem. O2consumption	Respiratory rate - phys.-chem. O2consumption	Inhibition
[mg/L]	[mg/L×h]	[mg/L×h]	[mg/L×h]	[%]
10	30.0	0.0	30.0	0.0
100	25.2	0.0	25.2	8.364
1000	24.0	0.0*	24.0	12.727
Control, mean	27.5
Control 1	27.0
Control 2	28.0

Comments: Concentrations are given as nominal concentrations and were not confirmed by analytical methods.
* The physico-chemical oxygen consumption has been determined at 1000 mg/L test item concentration. As no physico-chemical oxygen consumption was observed at that test item concentration this observation also holds true for the lower test item concentrations.

Table 4: Results of reference compound 3,5-Dichlorophenol

 

Reference compound concentration (nominal)	Respiratory rate reference compound	Inhibition
[mg/L]	[mg/L×h]	[%]
2.5	23.14	15.84
5	22.80	17.09
10	9.75	64.55
20	3.43	87.53
40	1.5	94.55
Control, mean	27.5
Control 1	27.0
Control 2	28.0

 

Comments: Concentrations are given as nominal concentrations and were not confirmed by analytical methods.

Validity criteria fulfilled:

yes

Conclusions:

A study was performed to assess the toxicity of Dibutyl phenyl phosphate to bacteria. The study was conducted in accordance with Council Regulation (EC) No 440/2008, Method C.11 ”Biodegradation: Activated Sludge Respiration Inhibition Test” (2008). This test method is equal to OECD Guideline 209 (1984). The activated sludge was exposed to Dibutyl phenyl phosphate at different concentrations. The respiration rate of each mixture was determined after aeration periods of 3 hours. Dibutyl phenyl phosphate showed 12.73 % respiration inhibition of activated sludge at a test item concentration of 1000 mg/L. Consequently, the EC50 is higher than 1000 mg/L. The effect value relates to a nominal concentration, since no analytical monitoring was performed.

Executive summary:

With the used method the effect of DBPP on microorganisms was assessed by measuring the respiration rate under defined conditions in the presence of different concentrations of the test item. The study was conducted in accordance with Council Regulation (EC) No 440/2008, Method C.11 ”Biodegradation: Activated Sludge Respiration Inhibition Test” (2008). This test method is equal to OECD Guideline 209 (1984).

 

To measure the oxygen consumption, 250 mL of sludge with the test item (or control or reference compound) was incubated for 3 h in 300 mL closed Erlenmeyer flasks and aerated through a glass tube at 50-100 L/h with clean oil-free air. For the measurement, the content of the Erlenmeyer flasks was completely transferred to 250 mL BOD bottles and oxygen content was measured with an oxygen meter (redox electrode) with writer.

 

Two controls without the test item were included in the test design, one at the start and the other at the end of the test series. Each batch of activated sludge was checked using 3,5-Dichlorophenol as a reference compound. Since some substances may consume oxygen by chemical reactivity, a physico-chemical oxygen consumption control was carried out additionally. In order to be able to differentiate between physico-chemical oxygen consumption and biological oxygen consumption (respiration), at least the maximum concentration of the test item was tested without activated sludge.

 

The respiration rate for each concentration was determined graphically from the linear part of the curve of the oxygen content versus time. The inhibitory effect of the test item at a particular concentration is expressed as a percentage of the mean of the respiration rates of two controls. An EC50 value was calculated from the respiration rates at different test item concentrations and for the reference compound the EC50 value was calculated using the statistics programme ToxRatPro Version 2.10. The EC50 of DBPP was > 1000 mg/L in the current test.  

Description of key information

The key study was a respiration inhibition study (Richter, 2012) performed according to the current guidelines and GLP criteria. The determined EC10 value was 402 mg/L, this was used as starting value for the risk analysis.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

402 mg/L

Additional information

The study was performed to assess the toxicity of Dibutyl phenyl phosphate to bacteria. The study was conducted in accordance with Council Regulation (EC) No 440/2008, Method C.11 ”Biodegradation: Activated Sludge Respiration Inhibition Test” (2008). This test method is equal to OECD Guideline 209 (1984). The activated sludge was exposed to Dibutyl phenyl phosphate at different concentrations. The respiration rate of each mixture was determined after aeration periods of 3 hours. Dibutyl phenyl phosphate showed 12.73 % respiration inhibition of activated sludge at a test item concentration of 1000 mg/L. Consequently, the EC50 is higher than 1000 mg/L. The calculated EC10 was 402 mg/L. The effect value relates to a nominal concentration, since no analytical monitoring was performed.