1,4-Benzenedicarboxylic acid, 1,4-diisononyl ester

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:

2010-02-10 to 2011-03-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

(1992)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

Commission Regulation (EC) No. 440/2008

Deviations:

no

Principles of method if other than guideline:

Pre-study solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. Based on this information the test item was categorised as being a 'difficult substance' as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,4-Benzenedicarboxylic acid, 1,4-diisononyl ester

EC Number:

700-453-0

Cas Number:

59802-05-0

Molecular formula:

C26H42O4

IUPAC Name:

1,4-Benzenedicarboxylic acid, 1,4-diisononyl ester

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: "Limit test" was conducted at a concentration of 100% v/v saturated solution
- Sampling method: The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 (fresh media), 24 and 96 hours (old media). Water samples were taken from the control and each replicate test vessel at 0 (fresh media), 24 and 96 hours (old media) for quantitative analysis. Duplicate samples and samples at 24 (fresh media), 48 and 72 hours (fresh and old media) were taken and stored at approximately -20°C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Pre-study solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. Based on this information the test item was categorised as being a 'difficult substance' as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.
An amount of test item (200 mg) was dispensed in duplicate, on to the surface of 2 litres of reconstituted water with the aid of magnetic stirring at a rate such that a dimple was formed at the water surface for periods of 24 or 48 hours. After stirring the aqueous phase was removed from the bottom of the mixing vessel and samples taken for chemical analysis. The pre-study media preparation trials indicated that a saturated solution method of preparation utilising a very slow stir rate was most appropriate for this test item due to its inherent tendency to form micro micelles.
For the definitive test the saturated solution was prepared using a very slow stir method of preparation with a 24 hour stir. An amount of test item (2100 mg) was dispensed on to the surface of 21 litres of dechlorinated tap water prior to stirring slowly via magnetic stirrer for 24 hours. After 24 hours the stirring was stopped and the aqueous phase removed by mid-depth siphoning to give a 100% v/v saturated solution of test item. This method of preparation was conducted in duplicate. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 (fresh media), 24 and 96 hours (old media).

- Controls: test medium without test substance. The control group was maintained under identical conditions but not exposed to the test item.
- Vehicle: Auxiliary solvents to aid dissolution were not employed.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Microscopic inspection of the saturated solution showed no micro-dispersions or undissolved test item to be present in the range finding test. The test preparations were observed to be clear, colourless solutions throughout the duration of the definitive test.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout (Oncorhynchus mykiss)
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Age at study initiation: juvenile
- Length at study initiation: mean standard length of 5.6 em (sd = 0.5)

ACCLIMATION
- Acclimation period: Fish were acclimatised to test conditions for 12 days.
- Acclimation conditions: 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 during acclimation: The stock fish were fed commercial trout pellets which was discontinued 24 hours prior to the start of the definitive test.
- Health during acclimation: There was 0% mortality in the 7 days prior to the start of the test.

FEEDING DURING TEST
- The fish received no food during exposure.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Hardness:

140 mg/l as CaC03

Test temperature:

14 °C

Dissolved oxygen:

9.83 mg O2/l

Nominal and measured concentrations:

nominal: 100 % (v/v) saturated solution (initnital loading rate 100 mg test substance per litre, slow stir method). The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 (fresh media), 24 and 96 hours (old media).

Details on test conditions:

TEST SYSTEM
- Test vessel: 20 litre glass exposure vessels for each test concentration
- Type: covered to reduce evaporation
- Aeration: aerated via narrow bore glass tubes
- Renewal rate of test solution: daily renewal of the test preparations
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 1
- Biomass loading rate: Loading rate of 0.87 g bodyweight/litre (static volume).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated by passage through an activated carbon filter and partly softened. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Ca/mg ratio: 140 mg/L CaCO3
- Culture medium different from test medium: The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.

OTHER TEST CONDITIONS
-pH: The pH was measured using a Hach hq30d pH.
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Limit test
- Justification for using less concentrations than requested by guideline: Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration, no mortalities or sub-lethal effects of exposure were observed.
- Range finding study
- Test concentrations: In the range-finding test fish were exposed to a single nominal concentration of 100% v/v saturated solution, as the Acute Toxicity to Daphnia magna test indicated that toxicity was not expected at this concentration.
- Results used to determine the conditions for the definitive study: Based on initial preliminary work the test item was prepared as a saturated solution, using a column elution method. The results from the range-finding test for this study showed no effects at 100% v/v saturated solution. The range-finding and definitive tests for the Acute Toxicity to Daphnia magna test showed no toxic effects to exposure using the slow-stirring method of preparation. Therefore, for ethical reasons, it was considered unnecessary to conduct a further range-finding test for this study using the slow-stirring method of preparation given that toxic effects were not expected. Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration, no mortalities or sub-lethal effects of exposure were observed.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

2 h

Dose descriptor:

LC50

Effect conc.:

> 0.007 mg/L

Effect concentrations 2

Duration:

6 h

Dose descriptor:

LC50

Effect conc.:

> 0.007 mg/L

Effect concentrations 3

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 0.007 mg/L

Effect concentrations 4

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 0.007 mg/L

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 0.007 mg/L

Effect concentrations 6

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.007 mg/L

Details on results:

Analysis of the test preparations at 0, 24 and 96 hours showed measured test concentrations to range from 0.00108 to 0.0220 mg/L and so it was considered justifiable to estimate the LC50 values in terms of the nominal test concentrations only.

There were no mortalities in 14 fish exposed to a mean measured test concentration of 0.0066 mg/L for a period of 96 hours. The results of the definitive test showed the highest test concentration resulting in 0% mortality to be greater than or equal to 0.0066 mg/L and the No Observed Effect Concentration (NOEC) to be 0.0066 mg/L. The No Observed Effect Concentration is
based upon zero mortalities and the absence of any sub-lethal effects of exposure at this concentration. There were no sub-lethal effects of exposure observed in the test. The test preparations were observed to be clear, colourless solutions throughout the
duration of the test.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Cumulative Mortality Data in the Definitive Test

Mean Measured Test Concentration (mg/L)	Cumulative Mortality (Initial Population=7)						% Mortality
Control	0	0	0	0	0	0	0
100R1	0	0	0	0	0	0	0
100R2	0	0	0	0	0	0	0

Concentrations in Aqueous Test Media Samples Determined Using HPLC Analysis

Sample	Nominal Concentration (% v/v Saturated Solution)	Concentration Found (mg/L)
0 hours	Control 100 R1 100 R2	<LOQ 0.00108 0.00810
24 hours	Control 100 R1 100 R2	<LOQ 0.00114 0.00578
96 hours	Control	<LOQ 0.0220 0.00121

Applicant's summary and conclusion

Conclusions:

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated No mortalities were observed within the water solubility limits of the test item. The 96-Hour LC50 is therefore considered to be greater than the maximum achievable water solubility. This corresponds to greater than 0.0066 mg/L based on the mean measured test concentration. The No Observed Effect Concentration was greater than or equal to the water solubility (0.0066 mg/L based on the mean measured test concentration). This study showed that there were no toxic effects at saturation.