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

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 27 May 2008 and 21 June 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

yes

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test material in cases where the test material is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (100 mg/l) of test material in dechlorinated tap water for a period of 48 hours prior to removing any undissolved test material present by filtration (0.2 µm Sartorius Sartopore, first approximate 2 litres discarded in order to pre-condition the filter) to give a saturated solution of the test material.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 21/07/2008 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: The test material concentration in the test samples was determined by high performance liquid chromatography (HPLC) using an external standard. The test material gave a chromatographic profile consisting of a single peak.
- Sampling method: The test samples were analysed directly without further treatment.


Water samples were taken from the control and each replicate test vessel at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 hours (old media) hours for quantitative analysis.
Duplicate samples were taken and stored at approximately -20degC for further analysis if necessary.
The method of analysis, stability, recovery and test preparation analyses are described in Appendix 2 (see attached background material).

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:

An amount of test material (2250 mg) was dispersed in 22.5 litres of dechlorinated tap water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 14°C for a period of 48 hours. After 48 hours the stirring was stopped and the undissolved test material removed by filtration (0.2 micro.m Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to give the 100% v/v saturated solution.

This method of preparation was conducted in duplicate to give replicates R1 and R2.
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 hours (old media) (see Appendix 2 - attached background material).

- 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 stated
- Evidence of undissolved material (e.g. precipitate, surface film, etc):None recorded

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Strain: Not available
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Age at study initiation (mean and range, SD): Not available
- Length at study initiation (length definition, mean, range and SD): the fish had a mean standard length of 4.5 cm (sd = 0.1)
- Weight at study initiation (mean and range, SD): mean weight of 1.32 g (sd = 0.07) at the end of the definitive test.
- Method of breeding: Not available
- Feeding during test: None.
- Food type: . The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test.



ACCLIMATION
- Acclimation period:. Fish were acclimatised to test conditions from 5 June 2008 to 17 June 2008
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test.
- Feeding frequency: Not stated
- Health during acclimation (any mortality observed):There was zero mortality in the 7 days prior to the start of the test.


QUARANTINE (wild caught)
- Duration: Not applicable
- Health/mortality: Not applicable

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure.

Test conditions

Hardness:

The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
Laboratory tap water was 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 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 (see attached background material).

Test temperature:

Temperature was recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. Temperature was measured using a Hanna Instruments HI93510 digital thermometer.
Temperature was maintained at approximately 14°C throughout the test.

pH:

pH was recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The pH was measured using a WTW pH/Oxi 340I pH meter.
There were no treatment related differences for pH.

Dissolved oxygen:

Dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. Dissolved oxygen concentration was measured using a dissolved oxygen meter.
Dissolved oxygen content of greater than or equal to 9.7 mg O2/l.
There were no treatment related differences for oxygen concentration.

Salinity:

Not applicable

Nominal and measured concentrations:

In the range-finding test fish were exposed to a series of nominal test concentrations of 10 and 100% v/v saturated solution. The test material was prepared as a saturated solution.
Definitive test - 100% v/v saturated solution

The test material solutions for the definitive test were prepared by stirring an excess (100 mg/l) of test material in dechlorinated tap water for a period of time and then removing any undissolved test material by filtration to give a saturated solution.

Details on test conditions:

TEST SYSTEM
- Test vessel:glass exposure vessels
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 20 litre glass exposure vessels were used for each test concentration.
- Aeration: The test vessels were aerated via narrow bore glass tubes
- Type of flow-through (e.g. peristaltic or proportional diluter): Not recorded
- Renewal rate of test solution (frequency/flow rate):A semi-static test regime was employed in the test involving a daily renewal of the test preparations to ensure that the concentrations of the test material remained near nominal and to prevent the build up of nitrogenous waste products.
- No. of organisms per vessel:7
- No. of vessels per concentration (replicates):7
- No. of vessels per control (replicates):7
- No. of vessels per vehicle control (replicates): 7
- Biomass loading rate: Not recorded


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
Laboratory tap water was 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.
- Total organic carbon:1.041 mg/l

- Particulate matter: Not recorded

- Metals: Not recorded

- Pesticides: 0.000 µg/l

- Chlorine: 0.297 mg/l

- Alkalinity: 7.719 pH value

- Ca/mg ratio:Not recorded

- Conductivity: 421.385 µS/cm at 20°C

- Culture medium different from test medium: No

- Intervals of water quality measurement: Not stated


OTHER TEST CONDITIONS
- Adjustment of pH: No treatment related changes to pH.
- Photoperiod:photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours.
- Light intensity:Not recorded


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: In the range-finding test fish were exposed to a series of nominal test concentrations of 10 and 100% v/v saturated solution. The test material was prepared as a saturated solution.
- 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. Based on the results of the range-finding test a "Limit test" was conducted. The test material solutions for the definitive test were prepared by stirring an excess (100 mg/l) of test material in dechlorinated tap water for a period of time and then removing any undissolved test material by filtration to give a saturated solution.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 other: %v/v Saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL not stated

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 other: %v/v Saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL not stated

Details on results:

Range-finding Test:
Cumulative mortality data from the exposure of rainbow trout to the test material during the range-finding test are given in Table 1. There were no sub-lethal effects of exposure during the range-finding test.
The results showed no mortalities at the test concentrations of 10 and 100% v/v saturated solution.
Based on this information, the test material solution for the definitive test was prepared by stirring an excess (100 mg/l) of test material in dechlorinated tap water for a period of time and then removing any undissolved test material by filtration to give the 100% v/v saturated solution. This experimental design conforms to a "Limit test" to confirm that no immobilisation or adverse reactions to exposure were observed.

Definitive Test:

Mortality data

Cumulative mortality data from the exposure of rainbow trout to the test material during the definitive test are given in Table 2.
There were no mortalities in 14 fish exposed to a test concentration of 100% v/v saturated solution for a period of 96 hours. Inspection of the mortality data gave the following results:
Time (h) LC50 (% v/v saturated solution) 95% Confidence limits
(% v/v saturated solution)
3 >100 -
6 >100 -
24 >100 -
48 >100 -
72 >100 -
96 >100 -
The results of the definitive test showed the highest test concentration resulting in 0% mortality to be greater than or equal to 100% v/v saturated solution, the lowest test concentration resulting in 100% mortality to be greater than 100% v/v saturated solution and the No Observed Effect Concentration (NOEC) to be 100% v/v saturated solution. The No Observed Effect Concentration is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this concentration

Sub-lethal effects:
There were no sub-lethal effects of exposure observed in 14 fish exposed to a test concentration of 100% v/v saturated solution for a period of 96 hours.

Observations on test material solubility:
The test material preparation was observed to be a clear, colourless solution throughout the duration of the test.

Physico-chemical measurements:
The results of the physico-chemical measurements are given in Appendix 3. Temperature was maintained at approximately 14°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Verification of test concentrations:
Analysis of the test preparations (see Appendix 2) at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 hours (old media) showed measured concentrations in the test preparations to be less than the limit of quantitation (LOQ) of the analytical method which was assessed down to 0.21 mg/l.
This does not infer that no test material was in solution but that the dissolved concentration (ie bioavailable to the test organisms) was below the limit of quantitation of the analytical method. As such the test results are based on nominal concentrations only.
This study showed that there were no toxic effects at saturation.

Results with reference substance (positive control):

- Results with reference substance valid? Not applicable

Reported statistics and error estimates:

No Data

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table1               Cumulative Mortality Data in the Range-findingTest

Nominal Concentration (% v/v saturated solution)	Cumulative Mortality (Initial Population = 3)
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours
Control	0	0	0	0	0	0
10	0	0	0	0	0	0
100	0	0	0	0	0	0

 

 Table2              Cumulative Mortality Data in the DefinitiveTest

Nominal Concentration (% v/v saturated solution)	Cumulative Mortality (Initial Population =7)						% Mortality
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	0	0	0	0	0	0	0
100 R1	0	0	0	0	0	0	0
100 R2	0	0	0	0	0	0	0

Appendix 3 - Physico-Chemical Measurements

Nominal Concentration (% v/v saturated solution)	Time (Hours)
	0 Hours (Fresh Media)				24 Hours (Old Media)				24 Hours (Fresh Media)
	pH	mg O2/l	%ASV*	TºC	pH	mg O2/l	%ASV*	T°C	pH	mg O2/l	%ASV*	T°C
Control	7.9	10.1	98	14	8.1	9.9	98	15	7.8	10.0	97	14
100 R1	7.8	9.9	98	15	7.8	9.5	94	15	8.0	10.0	99	15
100 R2	7.7	9.9	98	15	7.8	9.2	91	15	7.9	9.9	98	15

 

Nominal Concentration (% v/v saturated solution)	Time (Hours)
	48 Hours (Old Media)				48 Hours (Fresh Media)				72 Hours (Old Media)
	pH	mg O2/l	%ASV*	TºC	pH	mg O2/l	%ASV*	T°C	pH	mg O2/l	%ASV*	T°C
Control	8.1	9.9	98	15	7.9	9.8	95	14	8.1	10.5	104	15
100 R1	7.9	9.2	91	15	7.9	10.0	99	15	7.8	9.9	98	15
100 R2	7.9	9.2	91	15	7.8	10.0	99	15	7.8	9.9	98	15

 

Nominal Concentration (% v/v saturated solution)	Time (Hours)
	72 Hours (Fresh Media)				96 Hours (Old Media)
	pH	mg O2/l	%ASV*	TºC	pH	mg O2/l	%ASV*	T°C
Control	7.7	10.0	97	14	8.1	10.2	99	14
100 R1	7.9	10.6	105	15	7.9	10.0	97	14
100 R2	7.8	10.5	104	15	7.9	10.0	97	14

*ASV= Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

R₁and R₂= Replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 of greater than 100% v/v saturated solution. Correspondingly the No Observed Effect Concentration was 100% v/v saturated solution.

Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test material to rainbow trout (Oncorhynchus mykiss). The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods.

Information provided by the Sponsor indicated that the water solubility of the test material was very low. Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the material using traditional methods of preparation e.g. ultrasonication and high shear mixing. As the test material is a preparation it was considered appropriate to prepare the test material using a saturated solution method of preparation.

Following a preliminary range-finding test fish were exposed, in two groups of seven, to an aqueous solution of the test material, at a single concentration of 100% v/v saturated solution for a period of 96 hours at a temperature of approximately 14ºC under semi-static test conditions. The test material solution was prepared by stirring an excess (100 mg/l) of test material in dechlorinated tap water using a propeller stirrer at approximately 1500 rpm at a temperature of approximately 14°C for 48 hours. After the stirring period any undissolved test material was removed by filtration (0.2 micro.m Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a saturated solution of the test material.

The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results.

The 96-Hour LC₅₀based on nominal test concentrations was greater than 100% v/v saturated solution and correspondingly the No Observed Effect Concentration was 100% v/v saturated solution.

Analysis of the test preparations at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 hours (old media) showed measured concentrations in the test preparations to be less than the limit of quantitation (LOQ) of the analytical method which was assessed down to 0.21 mg/l.

This does not infer that no test material was in solution but that the dissolved concentration (ie bioavailable to the test organisms) was below the limit of quantitation of the analytical method. As such the test results are based on nominal concentrations only.

This study showed that there were no toxic effects at saturation.