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Toxicity to soil microorganisms

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Reference
Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 May 2016 to 19 August 2016
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Version / remarks:
Industrial Chemical Guidance adhered to with regard to sampling times and endpoints.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identity: Di-isodecyl azelate
CAS Registry Number: 28472-97-1
Batch Number: 0001090238
Date of Manufacture: 04 February 2016
Retest Date: 07 February 2018
Storage policy: The test item was stored at ambient temperature.
Analytical monitoring:
not required
Remarks:
I No Test Substance monitoring, transformation analytics only.
Details on sampling:
Days 0 and 28
Vehicle:
yes
Remarks:
Acetone evaporated onto fine quartz sand
Details on preparation and application of test substrate:
SOIL
The study was carried out with a single, common agricultural soil as described in OECD Guideline 216 (2000). The soil used was LUFA standard soil type 2.3 (Batch number F2.32116) and was supplied by LUFA-Speyer, Obere Langgasse 40, 67346 Speyer, Germany.

The soil sampling was carried out according to ISO 10381-6 (1993) as recommended in OECD 216 (2000). Details of the soil characterisation data and soil collection, site history and soil treatment (Appendices 2 and 3) were provided by LUFA-Speyer. Soil was received at CEMAS on the 07 June 2016 and was stored in the dark in a refrigerator (nominally at 4oC) until required.

Sand content of 59.6 %
pH of 7.1 (measured in water)
Organic carbon content of 0.67 %
Microbial biomass (257 mg Cmic/kg) of not less than 1 % of the total organic carbon content (actual value 3.83 %).

In most cases, a soil with these characteristics represents a worst-case situation, since adsorption of the test chemical to soil is minimal and consequently its availability to the microflora is maximal.

Acclimation of the soil used for the study was started on 13 July 2016, when the soil was brought to a moisture content of 35 % (±5 %) of the maximum water holding capacity (MWHC) of the soil, and left to acclimate in a test cabinet in the dark at 20 ± 2oC for six days before treatment.

REPLICATION
Each replicate was contained within a 2.0 L plastic container (internal dimensions 16.7 cm x 16.7 cm x 9 cm deep) with the lid perforated with a single 3 mm diameter hole to ensure aerobic conditions.

Three replicates were prepared for each treatment in the bioassay. Each replicate contained 500 g (dry weight equivalent) of soil.

DOSE APPLICATION
A solution was prepared at a nominal concentration of 50 mg/mL by direct addition of di-isodecyl azelate (5.0006 g) to 100 mL of acetone. This solution was mixed thoroughly and serially diluted to prepare solutions to dose the soil at 455, 207, 94 and 43 mg di-isodecyl azelate/kg dry soil treatment levels.
15g fine quartz sand was dosed with 30 mL of the relevant solution and then evaporated to dryness. Solvent control =15g sand dosed with 30mL acetone; water control =15g sand (undosed).

The treatment solutions (20 mL dose volume/kg dry soil) were added to fine quartz sand carrier (at a ratio of 10 g/kg dry weight soil). The dosed sand was left for ~2-3 hours so the acetone could evaporate prior to mixing into the soil. Treated sand was added to the pre-acclimated soil along with a volume of deionised water required to achieve the test moisture of 40 %
(5 %) of the MWHC.

The deionised water control soil was treated with the equivalent amount of deionised water and undosed quartz sand. Solvent control soil was treated with the equivalent amount of deionised water and sand dosed with acetone (evaporated prior to mixing).

NITROGEN SOURCE AMENDMENT
The test soil was amended with powdered dried ground lucerne. The lucerne was added at a rate of 2.5 g of lucerne/500 g of soil (dry weight equivalent) to the control and treatment groups on Day 0. This was mixed into the soil by hand prior to addition of the sand and deionised water. The lucerne had a C:N ratio of 14.4:1.

The soil was bulk dosed, mixed in a Crypto Peerless mixer and then split equally into replicate test vessels (3 x ca. 500g). Treatments were mixed in the following order: deionised water control, solvent control then test item in ascending rates.

SAMPLING
Soil was taken from the bulk sample from each treatment for pH (in water) determination at the start and from one replicate on Day 28. Soil pH was determined using SOP CEM-3373: Determination of the pH of water, soil and sediment samples in water and/or salt solutions (0.01M CaCl2, 0.1M KCl, 1.0M KCl).

Soil was taken from one replicate from each treatment for moisture and dry matter content determination on Day 28, using SOP CEM-3088: Determination of the Dry Matter and Moisture Content of Soils and Plant Materials.

Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20oC ± 2oC
Moisture:
42 % ± 5% MWHC
Details on test conditions:
The vessels were maintained in the dark at 20oC ± 2oC. Temperature was monitored throughout the study using IceSpy (Skye Instruments, Powys, UK) electronic environmental data loggers.
A satellite moisture control vessel was included in the test for the sole purpose of monitoring moisture content. The moisture content of the soil was maintained at 42 % ± 5% MWHC and on a weekly basis, the weight of the moisture control vessel was determined as a guide to test vessel water loss. Weight loss since the previous sampling occasion was restored by the addition of deionised water to all replicates in all treatments, based on the assumption that all vessels had experienced similar moisture change.
Nominal and measured concentrations:
Achieved concentration was not determined analytically as it was not a requirement of the OECD TG 216.
Reference substance (positive control):
no
Remarks:
| A positive reference substance is not a requirement of OECD TG 216
Key result
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
729.7 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Key result
Duration:
28 d
Dose descriptor:
other: NOAEL
Effect conc.:
455 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
EC25
Effect conc.:
592.4 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
509.9 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Details on results:
Day 0-28 Nitrate production rate (with 95 % confidence limits)
NOEL: 43 mg/kg
NOAEL: 455 mg/kg
EC10: 509.9 mg/kg (509.4 – 510.8)
EC25: 592.4 mg/kg (591.0 – 594.6)
EC50: 729.7 mg/kg (727.1 – 734.1)


Day 28 Nitrate concentration (with 95 % confidence limits)
NOEL: 43 mg/kg
NOAEL: 455 mg/kg
EC10: 530.1 mg/kg (529.0 – 531.7)
EC25: 642.8 mg/kg (640.0 – 646.6)
EC50: 830.6 mg/kg (825.0 – 838.3)

NOEL No-Observed Effect Level (Dunnett’s Test, two-tail, p ≤ 0.05)
NOAEL No-Observed Adverse Effect Level (empirically derived)
EC Effect Concentration (Linear interpolation)
Reported statistics and error estimates:
The data generated were subject to statistical analysis to determine significant treatment related effects compared to the solvent control using Dunnett’s two-tailed t test, p ≤ 0.05, ToxCalc (1999).

The EC10, EC25, EC50 (with 95 % confidence limits) and the NOEL were determined by linear interpolation, ToxCalc (1999).

Treatment

Day 28 data

Day 0 to Day 28 rate data

Mean Ammonium

(mg/kg dry soil)

% Variation (ammonium production) from solvent control

Mean Nitrate (mg/kg dry soil)

% Variation (Nitrate) from solvent control

Mean Nitrate Production

(mg/kg/day)

% Variation (from solvent control)

Water Control

1.64

27.6

194.7

-6.8

4.68

-9.8

Solvent Control

1.28

N/A

208.8

N/A

5.19

N/A

43 mg/kg

1.28

0.0

210.8

0.9

5.25

1.2

94 mg/kg

1.28

0.0

244.4*

17.0

6.36*

22.7

207 mg/kg

1.20

-6.9

351.7*

68.4

10.20*

96.5

455 mg/kg

20.86

1524

263.2*

26.1

7.04*

35.6

1000 mg/kg

79.37

6079

70.2*

-66.4

0.05*

-99.1

All concentrations on a dry soil basis.

* Statistically different to solvent control (Dunnett’s Test, two-tail, p ≤ 0.05).

Validity criteria fulfilled:
yes
Conclusions:
Di-isodecyl azelate showed a statistically significant effect on nitrate production at Day 28 compared to the solvent control treatment at tested at 94, 207, 455 and 1000 mg product/kg dry soil (Dunnett’s two-tail, p≤ 0.05, ToxCalc version 5.0.23, 1999).

At Day 28 the EC10, EC25 and EC50 for nitrate production were estimated at 530.1, 642.8 and 830.6 mg di-isodecyl azelate/kg dry soil, respectively. For the Day 0-28 period the EC10, EC25 and EC50 for the nitrate production rate were estimated at 509.9, 592.4 and 729.7 mg di-isodecyl azelate/kg dry soil, respectively (linear interpolation, ToxCalc version 5.0.23, 1999).

The overall NOAEL was determined to be 455 mg di-isodecyl azelate/kg dry soil.

Description of key information

EC50 (28 d) = 729.7 mg test item/kg dry soil (soil microorganism, OECD 216)

NOEC (28 d) = 455 mg test item/kg dry soil (soil microorganism, OECD 216)

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:
729.7 mg/kg soil dw
Long-term EC10 or NOEC for soil microorganisms:
455 mg/kg soil dw

Additional information

One study is available assessing the toxicity to soil microorganism of Diisodecyl azelate (CAS 28472-97-1, CEMAS 2016). The study was performed under GLP conditions according to the OECD Guideline 216. The test was conducted with the following concentrations 43, 94, 207, 455 and 1000 mg/kg soil dry weight. Statistically significant effect on nitrate production were observed at Day 28 compared to the solvent control treatment at 94, 207, 455 and 1000 mg /kg dry soil (Dunnett’s two-tail, p≤ 0.05, ToxCalc version 5.0.23, 1999). For the Day 0-28 period the EC10, EC25 and EC50 for the nitrate production rate were estimated at 509.9, 592.4 and 729.7 mg/kg dry soil, respectively (linear interpolation, ToxCalc version 5.0.23, 1999). The overall NOEC was determined to be 455 mg/kg dry soil.