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

Toxicity to soil microorganisms

Administrative data

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-01-19 to 2010-12-01

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:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Version / remarks:

(2000)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.21 (Soil Microorganisms: Nitrogen Transformation Test)

Version / remarks:

Commission Regulation (EC) No. 440/2008

Deviations:

no

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:

no

Details on sampling:

- Concentrations:
- Sampling method: On days 0, 7 and 28 three control and solvent control vessels and six replicate test item vessels were sacrificed for nitrate analysis. The contents of the test vessel were transferred to 500 ml polyethylene bottles and an aliquot (250 mL) of potassium chloride (0.1 M) added. The mixtures were then shaken (150 rpm, 60 minutes) prior to removal of the aqueous phase by centrifugation (3500 rpm, 15 minutes). 5 mL of the soil filtrate was added to an aliquot (35 ml) of acid mixture followed by 5 mL of 2,6-dimethylphenol solution. The mixture was thoroughly mixed and allowed to stand for between 24 and 36 minutes prior to determination of the absorbance at 324 nm.

Test substrate

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: The soil for the definitive tests was obtained from LUFA Speyer, Speyer, Germany. The sampling site had not been treated with crop protection products or organic fertiliser for at least 3 years prior to sampling (data from supplier).The moisture content of the soil was determined prior to the start of the definitive test by drying an amount of soil (1 00.00 g) at approximately 105°C until a constant weight was obtained.

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: An amount of test item (5000 mg) was dissolved in acetone and the volume adjusted to 10 mL to give a 5000 mg/ 10 mL solvent stock solution. An aliquot (2 mL) of the 5000 mg/ 10 mL solvent stock solution was added dropwise onto dry quartz sand (10 g) and the solvent allowed to evaporate to dryness in a fume cupboard. The test item/sand combination and an amount of powdered Lucerne-green-grass-meal (5 g) was then added to a final bulk soil weight of 1 kg (dry weight) and thoroughly mixed prior to the addition of an aliquot (98.2 mL) of water to give the required test concentration of 1000 mg/kg with a nominal moisture content of 40% of the Water Holding Capacity (WHC). The soil for each test concentration was then mixed again to ensure homogeneity prior to splitting into 18 replicates for incubation.

VEHICLE:
- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium (stock solution and final test solution): The solvent control soil was prepared by dispensing an aliquot (1.0 mL) of acetone dropwise onto dry quartz sand (5 g) and allowing the solvent to evaporate to dryness in a fume cupboard.
- Evaporation of vehicle before use: yes

Test organisms

Test organisms (inoculum):

soil

Study design

Total exposure duration:

28 d

Test conditions

Test temperature:

21 °C

Moisture:

The moisture content of the soil was maintained to 40% ± 5% of the WHC of the soil.

Details on test conditions:

TEST SYSTEM
- Test container (type, material, size): glass beakers
- Amount of soil: 50 g of soil (dry weight)
- No. of replicates per concentration: 6 (overall 18 replicates for analysis after 0, 7, and 28 days)
- No. of replicates per control: 3 (overall 9)
- No. of replicates per vehicle control: 3 (overall 9)

SOIL INCUBATION
- Method: The test vessels were covered with loosely fitting lids in order to minimise moisture loss by evaporation and maintained in a temperature controlled room at approximately 21°C, in darkness.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): The field-fresh soil for the range-finding and definitive tests was obtained from LUFA Speyer, Speyer, Germany.
- History of site: The sampling site had not been treated with crop protection products or organic fertiliser for at least 3 years prior to sampling (data from supplier).
- Soil type: silty sand/ sandy loam
- Sand content: 60.1 %
- pH (in water): 7.4 (data from supplier)
- Initial microbial biomass as % of total organic C: 0.99 ± 0.08%
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 98 mg/100 g (data from supplier)
- Maximum water holding capacity (in % dry weigth): 35.6 ±3.0 g/100g
- Pretreatment of soil: The moisture content of the soil was determined prior to the start of the definitive test by drying an amount of soil (100.00 g) at approximately 105°C until a constant weight was obtained. The moisture content of the soil, expressed as a percentage of the dry weight, was determined to be 4%. The Water Holding Capacity (WHC) of the soil was 35.6 g/100g and hence 98.2 mL of deionised reverse osmosis water per 1 kg (dry weight) of soil was added. This gave a final water content of 13.4 g/100 g (wet weight), i.e. 40% of the WHC as recommended by the Test Guidelines.
- Storage (condition, duration): stored at approximately 4°C prior to use.
- Initial microbial biomass as % of total organic C: The microbial biomass of the soil was shown to be 108 µg C/g, equivalent to 1.1% of the total soil organic carbon content.

DETAILS OF PREINCUBATION OF SOIL:
Prior to the start of the range-finding and definitive tests, an amount of soil (approximately 2.5 kg) was removed and stored at approximately 21°C for 2 days for the range-finding test and for 3 days for the definitive test and shielded from light.

EFFECT PARAMETERS MEASURED:
Room temperature was recorded daily throughout the test. Each individual test vessel was weighed on Day 0 and deionised reverse osmosis water added on a weight basis on days 7, 14, 21 and 28 where necessary in order to maintain the moisture content within ±5% of the initial moisture content. On days 0, 7 and 28 the control and solvent control vessels and the test item vessels were sacrificed for nitrate analysis.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: nominal test concentrations of 10, 100 and 1000 mg/kg
- Results used to determine the conditions for the definitive study: The results showed no significant effect on nitrogen transformation activity at all concentrations tested. Based on this information a single test concentration of 1000 mg/kg soil was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the maximum recommended concentration given in the Test Guidelines no effect on nitrogen transformation was observed.

Nominal and measured concentrations:

1000 mg/kg

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Effect concentrations 2

Key result

Duration:

28 d

Dose descriptor:

EC25

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Effect concentrations 3

Key result

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Effect concentrations 4

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

Absorbance values and nitrate concentrations determined on each sampling day. The nitrate formation rates and percentage inhibition of nitrogen transformation activity on days 7 and 28 were calculated.
The microbial biomass of the soil was shown to be 108 µg C/g. This was equivalent to 1.1% of the total soil organic carbon content. The variation between replicate control nitrification rates was less than 15% and therefore satisfied the validation criterion given in the Test Guidelines. The "No Observed Effect Concentration" (NOEC) was 1000 mg/kg, this was the maximum concentration tested.

Reported statistics and error estimates:

Statistical analysis of the nitrate concentration values was carried out for the solvent control and test item group using a Students t-test. The test item group was shown to have similar nitrate concentrations as the solvent control group (P≥0.05). It was therefore considered that the test item exhibited no adverse effect on the nitrogen transformation rate. The "No Observed Effect Concentration" (NOEC) was 1000 mg/kg, this was the maximum concentration tested. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg /kg.

Any other information on results incl. tables

Inhibition of Nitrogen Transformation Activity from the Definitive Test

Nominal Concentration (mg/kg soil)	Day 7		Day 28
	Nitrate Formation (mg N03/kg/d)	% Inhibition	Nitrate Formation (mg N03/kg/d)	% Inhibition
Control	2.16	-	3.60	-
Solvent Control	2.58	-	3.73	-
1000	3.96	[53]*	4.52	[21]*

*[Increasing nitrate formation rate as compared to controls]

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the nitrogen transformation activity of soil microorganisms has been investigated over a 28-Day period and gave an EC50 value of greater than 1000 mg/kg. Correspondingly the No Observed Effect Concentration was 1000 mg/kg.