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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 12 May 2008 and 19 June 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect 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 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

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 reconstituted 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:21/08/2007 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
- Stability under test conditions: Stable
- 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.
The test samples were analysed directly without further treatment.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Based on the result of the range-finding test a "limit test" was conducted. The test material solution for the definitive test was prepared by stirring an excess (100 mg/l) of the test material in reconstituted water for a period of time and then removing any undissolved test material by filtration to give a saturated solution.

An amount of test material (1100 mg) was dispersed in 11 litres of reconstituted water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for 48 hours. After 48 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. Aliquots (50, 90, 160 and 280 ml) of this saturated solution were each added separately to a final volume of 500 ml of reconstituted water to give the remaining test concentrations of 10, 18, 32 and 56 % v/v saturated solutions respectively.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 (fresh media) and 48 hours (old media)


- Eluate: Not applicable

- Controls: A positive control (Safepharm Laboratories Project No: 0039/0977) conducted approximately every six months used potassium dichromate as the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.

An amount of reference material (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/l stock solution. An aliquot (50 ml) of this stock solution was diluted in reconstituted water and the volume adjusted to 500 ml to give a 10 mg/l stock solution. Aliquots (16, 28, 50, 90 and 160 ml) of the 10 mg/l stock solution were each separately dispersed in a final volume of 500 ml of reconstituted water to give the test series of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.

Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Exposure conditions for the positive control were similar to those used in the definitive test.

The temperature was maintained at 21°C.


- Chemical name of vehicle : Not applicable

- Concentration of vehicle in test medium: Not applicable

- Evidence of undissolved material : After the stirring period any undissolved test material was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a saturated solution of the test material.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea

- Source: Derived from in-house laboratory cultures.

- Age at study initiation: The test was carried out using 1st instar Daphnia Magna.

- Feeding during test: Received no food during exposure

ACCLIMATION
- Acclimation period: Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.

These young were removed from the cultures and used for testing.

- Acclimation conditions: Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20 degC.

The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

- Type and amount of food:Each culture was fed daily with a suspension of algae (Chlorella sp.).

The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

- Health during acclimation: No mortality observed


Test Water:
The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

Reconstituted Water

i)Stock Solutions

a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l

ii)Preparation

An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable.

Test conditions

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

Temperature was maintained at 21°C to 22°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Temperatures in the control and test vessels were maintained at 21°C to 22°C throughout the test which were slightly in excess of the 20 ± 1°C given in the protocol. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification.

pH:

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.

Dissolved oxygen:

The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.

Salinity:

freshwater used.

Nominal and measured concentrations:

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10, and 100 % v/v saturated solution. The test material was prepared as a saturated solution.
Main test: Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test material at concentrations of 10, 18, 32, 56 and 100 % v/v saturated solution.
Test results based on nominal concentrations only.

Details on test conditions:

TEST SYSTEM
As in the range-finding tests 250 ml glass jars containing approximately 200 ml of test preparation were used. At the start of the study 10 daphnids were placed in each test and control vessel at random, in the test preparations. Duplicate test vessels were used for each test and control group. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 21C to 22C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test material.
The test preparations were not renewed during the exposure period. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water with a conductivity of <5 µS cm-1.

- Total organic carbon: Not recorded

- Particulate matter: Not recorded

- Metals: Not recorded

- Pesticides: Not recorded

- Chlorine: Not recorded

- Alkalinity: pH of 7.8 ± 0.2

- Ca/mg ratio: The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

- Conductivity: Not recorded

- Culture medium different from test medium: The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

- Intervals of water quality measurement: Not recorded

Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration was measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter and the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
The results of the physico-chemical measurements are given in Appendix 3.

OTHER TEST CONDITIONS
- Adjustment of pH: Adjusted (if necessary) with NaOH or HCl.

- Photoperiod: Photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.

- Light intensity: Not recorded


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

The test media preparations were observed to be clear colourless solutions throughout the duration of the test.







TEST CONCENTRATIONS
- Spacing factor for test concentrations: Water samples were taken from the control (replicates R1 – R2 pooled) and the 10, 18, 32, 56 and 100 % v/v saturated solution test groups (replicates R1 – R2 pooled) at 0 and 48 hours for quantitative analysis.

Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

The method of analysis, stability, recovery and test preparation analyses are described in Appendix 2 (attachment 1).


- Justification for using less concentrations than requested by guideline: Not applicable

- Range finding study
- Test concentrations:
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 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: Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in Table 1.
No immobilisation was observed at the test concentrations of 0.10, 1.0 and 10 % v/v saturated solution. However, immobilisation was observed at 100 % v/v saturated solution.
Based on this information test concentrations of 10, 18, 32, 56 and 100 % v/v saturated solution were selected for the definitive test

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

100 other: %v/v

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL of 85 - 140% v/v saturated solution

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

32 other: %v/v

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL not stated

Details on results:

- Behavioural abnormalities: Not recorded

- Other biological observations: None recorded

- Immobilisation of control:
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 2. The relationship between percentage immobilisation and concentration at 48 hours is given in Figure 1 (please see attached background materila).

Analysis of the immobilisation data by the probit method (Finney 1971) at 48 hours based on the nominal test concentrations gave the following results:


Time (h) EC50 (mg/l) 95% Confidence limits (mg/l)

24 >100 -
48 100 85 - 140


The No Observed Effect Concentrations after 24 and 48 hours exposure were 56 and 32 mg/l respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

The slopes and their standard errors of the response curves at 48 hours were 6.6 (SE = 2.1). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of response curve at 24 hours.

- Other adverse effects control: No other effects observed.

- Abnormal responses: None recorded

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test media preparations were observed to be clear colourless solutions throughout the duration of the test.

Results with reference substance (positive control):

- Results with reference substance

Cumulative immobilisation data from the exposure of Daphnia magna to the reference material (Safepharm Laboratories Project No: 0039/0977) during the positive control are given in Table 3. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 2 and 3 (please see attached background material).

Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the trimmed Spearman-Karber method (Hamilton et al 1977) at 24 hours and the probit method (Finney 1971) at 48 hours based on the nominal test concentrations gave the following results:

Time (h)
EC50 (mg/l) 95% Confidence limits
(mg/l)
3 > 3.2* -
24 0.97 0.85 - 1.1
48 0.70 0.62 - 0.80

The No Observed Effect Concentration after 24 and 48 hours were 0.56 and 0.32 mg/l respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

The slope and standard error of the response curve at 48 hours was 11 (SE = 2.3). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of the response curve at 24 hours.

The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/l (sd = 0.22).

Reported statistics and error estimates:

The slopes and their standard errors of the response curves at 48 hours were 6.6 (SE = 2.1). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of response curve at 24 hours.

Any other information on results incl. tables

Table1               Cumulative Immobilisation Data in the Range-finding Test

Nominal Concentration (% v/v saturated solution)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
0.10	0	0
1.0	0	0
10	0	0
100	1	4

 

Table2               Cumulative Immobilisation Data in the Definitive Test

Nominal Concentration (% v/v saturated solution)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
10	0	0	0	0	0	0	0	0
18	0	0	0	0	0	0	0	0
32	0	0	0	0	0	0	0	0
56	0	0	0	0	1	0	1	5
100	1	0	1	5	6	4	10	50

 

 

Table3               Cumulative Immobilisation Data in the Positive Control

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	3 Hours				24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0	0	0	0	0
0.56	0	0	0	0	0	0	0	0	1	2	3	15
1.0	0	0	0	0	6	5	11	55	9	10	19	95
1.8	0	0	0	0	10	10	20	100	10	10	20	100
3.2	2	1	3	15	10	10	20	100	10	10	20	100

 

R₁– R₂= Replicates 1 and 2

Appendix3         Physico-Chemical Measurements

Nominal Concentration (% v/v saturated solution)		0 Hours				24 Hours	48 Hours
		pH	mg O2/1	%*	T°C	TºC	pH	mg O2/1	%*	T°C
Control	R1	8.0	8.6	97	21	22	8.1	8.4	94	21
	R2	8.0	8.5	96	21	22	8.1	8.4	94	21
10	R1	8.0	8.6	97	21	22	8.1	8.3	93	21
	R2	8.0	8.6	97	21	22	8.1	8.3	93	21
18	R1	8.0	8.6	97	21	22	8.1	8.3	93	21
	R2	8.0	8.6	97	21	22	8.1	8.3	93	21
32	R1	8.0	8.6	99	22	22	8.0	8.4	94	21
	R2	8.0	8.6	99	22	22	8.0	8.4	94	21
56	R1	8.0	8.5	98	22	22	8.0	8.4	94	21
	R2	8.0	8.6	99	22	22	8.0	8.3	93	21
100	R1	7.9	8.5	98	22	22	7.9	8.4	94	21
	R2	7.9	8.5	98	22	22	7.9	8.4	97	22

 

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

R₁- 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 invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of 100 % v/v saturated solution with 95% confidence limits of 85 - 140 % v/v saturated solution. The No Observed Effect Concentration at 48 hours was 32 % v/v saturated solution.

Executive summary:

Introduction. A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Directive 92/69/(which constitutes Annex V of Council Directive 67/548/).

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, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test material at concentrations of 10, 18, 32, 56 and 100 % v/v saturated solution for 48 hours at a temperature of 21°C to 22°C under static test conditions. The test material solution was prepared by stirring an excess (100 mg/l) of test material in reconstituted water using a propeller stirrer at approximately 1500 rpm at a temperature of approximately 21°C for 48 hours. After the stirring period any undissolved test material was removed by filtration (0.2 µm 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 immobilised Daphnia were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of 21°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results.

The 48-Hour EC₅₀for the test material to Daphnia magna based on nominal test concentrations was 100 % v/v saturated solution with 95% confidence limits of 85 - 140 % v/v saturated solution. The No Observed Effect Concentration was 32 % v/v saturated solution.

Analysis of the test preparations at 0 (fresh media) and 48 (old media) hours 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 iron 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.

The 48-Hour EC₅₀for the reference material to Daphnia magna based on nominal concentrations was 0.70 mg/l with 95% confidence limits of 0.62 – 0.80 mg/l. The No Observed Effect Concentration was 0.32 mg/l.