Barium, 1,4-bis (C13-rich C11-14-isoalkyl) 2-sulfobutanedioate phosphate complex

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 29 February 2008 and 28 April 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 21/08/2009 Date of Signature: 15/10/2007

Test material

Test material information

Constituent 1

Reference substance name:

dibarium(2+) bis(1,5-dioxo-1,5-bis(tridecyloxy)pentane-3-sulfonate) hydrogen phosphate

EC Number:

931-037-9

Molecular formula:

Molecular formula for each combination of chain length. C13,13: (C30H57O7S)2 Ba @ BaHOP4 C11,12: (C27H51O7S)2 Ba @ BaHOP4 C12,12: (C28H53O7S)2 Ba @ BaHOP4 C12,13: (C29H55O7S)2 Ba @ BaHOP4 C13,14: (C31H59O7S)2 Ba @ BaHOP4 C14,14: (C32H61O7S)2 Ba @ BaHOP4

IUPAC Name:

dibarium(2+) bis(1,5-dioxo-1,5-bis(tridecyloxy)pentane-3-sulfonate) hydrogen phosphate

Details on test material:

- Name of test material (as cited in study report): Barium di(bistridecylsulfosuccinate) in mixture with Barium hydrogen phosphate
- Substance type: pale yellow solid
- Physical state: Solid
- Lot/batch No.: Y-T-1
- Storage condition of test material: room temperature in the dark
- Other:

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Analysis of the concentration, homogeneity and stability of the test material in the test preparations was not appropriate to the Test Guideline.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the purpose of the definitive test, the test material was dispersed directly in water.
An amount of test material (500 mg) was dispersed in approximately 250 ml of water and subjected to ultrasonication for approximately 30 minutes. Synthetic sewage (16 ml), activated sewage sludge (200 ml) and water were added to a final volume of 500 ml to give the required concentration of 1000 mg/l.
This method of preparation was performed for the three separate replicate test vessels.
The control group was maintained under identical conditions but not exposed to the test material.
Analysis of the concentration, homogeneity and stability of the test material in the test preparations was not appropriate to the Test Guideline.

- Eluate: Not applicable
- Differential loading: Not applicable
- Controls: The control group was maintained under identical conditions but not exposed to the test material.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Observations made throughout the test period (see Table 5) showed that at the test concentration of 1000 mg/l, large lumps of undissolved test material were visible resting on the bottom of the flask at 0 hours, 30 minutes and 3 hours contact time. This was considered to be due to the insoluble nature of the test material in the test media. However the test material was dispersed with the aid of ultrasonication in the test diluent for approximately 15 minutes prior to the addition of the synthetic sewage, activated sewage sludge and water and each vessel was aerated with compressed air at a rate of approximately 0.5-1 litre per minute to ensure that there was maximum contact between the test material and activated sewage sludge in the test medium.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: A mixed population of activated sewage sludge micro-organisms was obtained on 29 February 2008 for the range-finding test and on 28 April 2008 for the definitive test from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
- Method of cultivation: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and was used on the day of collection. The pH of the sample was 7.9 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter.
- Preparation of inoculum for exposure:Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 ml of deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 4.2 g/l prior to use.

A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:
16 g Peptone
11 g Meat extract
3 g Urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
dissolved in 1 litre of water with the aid of ultrasonication.

- Pretreatment: Not applicable
- Initial biomass concentration:4.2 g/l

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

The test water used for both the range-finding and definitive tests was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener) giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

Test temperature:

The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1°C.

pH:

The pH of the sample was 7.9 measured using a WTW pH/Oxi 340I pH meter

Dissolved oxygen:

Measured using a dissolved oxygen meter.

Salinity:

Not applicable.

Nominal and measured concentrations:

In the range-finding test activated sewage sludge micro-organisms were exposed to nominal test concentrations of 100 and 1000 mg/l.
Based on the results of the range-finding test a "limit test" was conducted at a concentration of 1000 mg/l for the definitive test.

Details on test conditions:

TEST SYSTEM
-At time "0" 16 ml of synthetic sewage was diluted to 300 ml with water and 200 ml of inoculum added in a 500 ml conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 – 1 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference material being added. Finally a second control was prepared.

As each vessel reached 30 minutes contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 ml darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 6.5 mg O2/l and 2.5 mg O2/l). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period. After measurement the contents of the BOD bottle were returned to the test vessel.
This procedure was repeated after 3 hours contact time. The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1°C.

Observations were made on the test preparations throughout the test period, and the pH of the control, reference material and test material preparations were measured using a WTW pH 320 pH meter and a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:

The test water used for both the range-finding and definitive tests was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener) giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Appendix 1.

- Total organic carbon:1.429 mg/l
- Metals: <0.021 mg/l Copper
- Chlorine:0.274mg/l
- Ca/mg ratio: Not stated
- Conductivity: 403.577 µS/cm at 20°C



OTHER TEST CONDITIONS
- Adjustment of pH: Not available
- Photoperiod:The test was conducted under normal laboratory lighting
- Light intensity:Not stated


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
In order to calculate the inhibitory effect of the test and reference materials the respiration rate was expressed as a percentage of the two control respiration rates.

%inhibition = 1 -[(2RS/(RC1 + RC2)] x 100
where
RS                  =         oxygen consumption rate for test or reference sample
RC1+ RC2     =         oxygen consumption rates for controls 1 and 2

The percentage inhibition values were plotted against concentration for the reference material only, a line fitted using the Xlfit software package (IDBS) and the EC20, EC50 and EC80 values determined from the equation for the fitted line.
The EC20, EC50 and EC80 values for the test material were determined by inspection of the inhibition of respiration rate data.
95% confidence limits for the reference material only, were calculated for the EC50 values using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon 1949).
The No Observed Effect Concentration (NOEC) was determined by inspection of the inhibition of respiration rate data.
The results of the study are considered valid if (i) the two control respiration rates are within 15% of each other and (ii) the EC50 (3-Hour contact time) for 3,5-dichlorophenol lies within the range 5 to 30 mg/l.


TEST CONCENTRATIONS

- Range finding study
- Test concentrations: In the range-finding test activated sewage sludge micro-organisms were exposed to nominal test concentrations of 100 and 1000 mg/l. The test material was dispersed directly in water.
- Results used to determine the conditions for the definitive study:The test concentration to be used in the definitive test was determined by a preliminary range-finding test.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL not stated

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
Range-finding Test

Oxygen consumption rates and percentage inhibition values for the control, test and reference materials in the range-finding test are given in Table 1.
No significant effect on respiration was observed at either test concentration employed. Based on this information, a single test concentration, in triplicate, of 1000 mg/l was selected for the definitive test.
Data supplied by the Sponsor indicated that the test material contained Barium di(bistridecylsulfosuccinate) in mixture with Barium hydrogen phosphate. A study to determine the General Physico-Chemical Properties of the test material (SafePharm Laboratories Limited Project Number 2086/0018) gave a water solubility of equivalent to or less than 2.2 x 10-5 g/l of solution at 20.0 ±0.5°C for Bistridecylsulfosuccinate and a water solubility of 2.15 x 10-2 g/l of solution at 20.0 ±0.5°C for Barium di(bistridecylsulfosuccinate) in mixture with Barium hydrogen phosphate (expressed as total soluble barium).
Although the test material was not in solution in the range-finding test, no significant inhibition was observed at the test concentrations employed and given the extremely low water solubility of the test material it was considered that no inhibition would occur at these levels of water solubility. As the test material was also a mixture of two components it was considered justifiable to conduct the definitive test at a single nominal test concentration of 1000 mg/l in excess of the water solubility level in order to ensure that the toxicity of the test material as a whole was determined.
Using this approach the effect of an excess amount of undissolved test material can be assessed in order to determine whether the test material has any effect on the activated sewage sludge micro-organisms exoenzymes, or whether the uptake of undissolved test material by processes such as phagocytosis has any adverse effect on the activated sewage sludge.

Definitive test

Observations made throughout the test period (see Table 5) showed that at the test concentration of 1000 mg/l, large lumps of undissolved test material were visible resting on the bottom of the flask at 0 hours, 30 minutes and 3 hours contact time. This was considered to be due to the insoluble nature of the test material in the test media. However the test material was dispersed with the aid of ultrasonication in the test diluent for approximately 15 minutes prior to the addition of the synthetic sewage, activated sewage sludge and water and each vessel was aerated with compressed air at a rate of approximately 0.5-1 litre per minute to ensure that there was maximum contact between the test material and activated sewage sludge in the test medium.

- Effect concentrations exceeding solubility of substance in test medium:

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: Please see results section.
- Other:

Reported statistics and error estimates:

No Data

Any other information on results incl. tables

 DefinitiveTest

Oxygen consumption rates and percentage inhibition values for the control, test and reference materials in the definitive test are given in Tables 2 and 3. The pH values of the test preparations at the start and end of the exposure period are given in Table 4, and observations made on the test preparations throughout the study are given in Table 5.

Percentage inhibition is plotted against concentration for the reference material, 3,5‑dichlorophenol only (Figures 1 and 2 - see attached background material).

The following results were derived:

Bariumdi(bistridecylsulfosuccinate) in mixture with Barium hydrogen phosphate:

	ECx(30 Minutes) (mg/l)	95% Confidence Limits (mg/l)	ECx(3 Hours) (mg/l)	95% Confidence Limits (mg/l)
EC20	>1000	-	>1000	-
EC50	>1000	-	>1000	-
EC80	>1000	-	>1000	-
NOEC	1000	-	1000	-

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/l.

3,5-dichlorophenol:

	ECx(30 Minutes) (mg/l)	95% Confidence Limits (mg/l)	ECx(3 Hours) (mg/l)	95% Confidence Limits (mg/l)
EC20	6.0	-	3.5	-
EC50	14	12 - 17	9.8	7.8 - 12
EC80	33[1]	-	28	-

Variation in respiration rates of controls 1 and 2 after 30 minutes was ± 0%, and ± 2% after 3 hours contact time.

The validation criteria for the control respiration rates and reference material EC₅₀values were therefore satisfied.

In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O₂/l and 2.5 mg O₂/l respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

Relatively low initial oxygen readings were observed in all vessels (see Tables 2 and 3). This was considered to be due to the response of the activated sewage sludge on the day and was considered not to have had a significant effect on the results of the test given that the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

[1]Value obtained by calculation using the the ‘best fit’ line for the concentration response curve (Xlfit4 software package)

Table1               Oxygen Consumption Rates and Percentage Inhibition Values in the Range-findingTest

Nominal Concentration (mg/l)		Initial O2 Reading (mg O2/l)	Measurement Period (minutes)	Final O2Reading (mg O2/l)	O2Consumption Rates (mg O2/l/min)	% Inhibition
Control	R1	3.8	3	2.2	0.53	-
	R2	3.8	3	2.1	0.57	-
Test Material	100	5.3	3	3.4	0.63	[15]
	1000	4.4	4	1.9	0.63	[15]
3,5-dichlorophenol	3.2	6.1	8	2.3	0.48	13
	32	8.4	10	7.4	0.10	82

 

Table2               Oxygen Consumption Rates and Percentage Inhibition Values in the
Definitive Test after 30 Minutes Contact Time

Nominal Concentration (mg/l)		Initial O2 Reading (mg O2/l)	Measurement Period (minutes)	Final O2Reading (mg O2/l)	O2Consumption Rates (mg O2/l/min)	% Inhibition
Control	R1	4.6	3	2.2	0.80	-
	R2	3.3	2	1.7	0.80	-
Test Material	1000 R1	2.1	1	1.4	0.70	13
	1000 R2	2.5	1	1.7	0.80	0
	1000 R3	1.6	1	0.9	0.70	13
3,5-dichlorophenol	3.2	3.8	2	2.2	0.80	0
	10	4.8	5	2.1	0.54	33
	32	6.5	10	5.0	0.15	81

 

Table3               Oxygen Consumption Rates and Percentage Inhibition Values in the
Definitive Test after 3 Hours Contact Time

Nominal Concentration (mg/l)		Initial O2 Reading (mg O2/l)	Measurement Period (minutes)	Final O2Reading (mg O2/l)	O2Consumption Rates (mg O2/l/min)	% Inhibition
Control	R1	4.9	4	2.0	0.73	-
	R2	3.3	2	1.9	0.70	-
Test Material	1000 R1	3.3	2	1.8	0.75	[5]
	1000 R2	3.7	2	2.2	0.75	[5]
	1000 R3	3.9	2	2.3	0.80	[12]
3,5-dichlorophenol	3.2	5.4	5	2.4	0.60	16
	10	6.4	10	3.1	0.33	54
	32	7.5	10	6.2	0.13	82

 

Table4               pH Values of the Test Preparations at the Start and End of the Exposure Period

Nominal Concentration (mg/l)		pH
		0 Hours	3 Hours
Control	R1	7.4	8.1
	R2	7.8	8.0
Test Material	1000 R1	7.8	7.9
	1000 R2	7.9	7.9
	1000 R3	7.9	7.9
3,5-dichlorophenol	3.2	7.4	8.2
	10	7.8	8.3
	32	7.8	8.4

 

Table5               Observations on the Test Preparations Throughout the Test Period

Nominal Concentration (mg/l)		Observations on Test Preparations
		0 Hours	30 Minutes Contact Time	3 Hours Contact Time
Control	R1	Dark brown dispersion	Dark brown dispersion	Dark brown dispersion
	R2	Dark brown dispersion	Dark brown dispersion	Dark brown dispersion
Test Material	1000 R1	Dark brown dispersion with large lumps of test material resting on the bottom of the flask	Dark brown dispersion with large lumps of test material resting on the bottom of the flask	Dark brown dispersion with large lumps of test material resting on the bottom of the flask
	1000 R2	Dark brown dispersion with large lumps of test material resting on the bottom of the flask	Dark brown dispersion with large lumps of test material resting on the bottom of the flask	Dark brown dispersion with large lumps of test material resting on the bottom of the flask
	1000 R3	Dark brown dispersion with large lumps of test material resting on the bottom of the flask	Dark brown dispersion with large lumps of test material resting on the bottom of the flask	Dark brown dispersion with large lumps of test material resting on the bottom of the flask
3,5-dichlorophenol	3.2	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible
	10	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible
	32	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/l.

Executive summary:

Introduction.

A study was performed to assess the effect of the 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", Method C.11 of EEC Commission Directive 88/302/EEC and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Methods.

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test material at a concentration of 1000 mg/l (three replicate flasks) for a period of 3 hours at a temperature of approximately 21°C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 30 minutes and 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

Results.

The effect of the test material on the respiration of activated sewage sludge gave a 3‑Hour EC₅₀of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was1000mg/l.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/l.

The reference material gave a 3-Hour EC₅₀value of 9.8 mg/l, 95% confidence limits 7.8 - 12 mg/l.