Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Aug 2001 to 10 Oct 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Version / remarks:

January 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)

Version / remarks:

January 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: agricultural soil, a sandy loam

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: A high dose stock solution was prepared by dissolving 26.078 mg of the test item in 3500 µL acetone. For the low dose stock solution, 500 µL of the high dose stock solution were diluted to 2000 µL with acetone. For the preparation of the high and low dose application mixtures, aliquots of 1220 µL and 1000 µL of the corresponding high and low dose stock solutions were added uniformly to 30 g aliquots of quartz sand. After complete evaporation of the acetone, under a stream of nitrogen gas, each application mixture was mixed homogeneously using a mixer. The application mixtures were stored in a refrigerator (about 4 °C) until application for one day. Each soil sample (150 g dry weight) received 1.5 g of the application mixtures from the low and high dose application mixtures, corresponding to concentrations of 0.62 mg and 3.03 mg test item/kg dry soil, respectively. During application, the soil was thoroughly mixed by carefully swivelling the flasks. For the nitrification experiments, the soil samples were amended with 0.7 g Iucerne meal (nitrogen content approximately 3%) after application. Finally, the soil moisture content of all samples was adjusted to 40% of the MWC by adding purified water.

VEHICLE:
- Chemical name of vehicle: organic solvent, acetone
- Evaporation of vehicle before use: Yes

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20 ± 2 °C

Moisture:

40 %

Organic carbon content (% dry weight):

1.32

Nitrogen content (% dry weight):

0.09

Details on test conditions:

TEST SYSTEM
- Test container: 1 L incubation flasks
- Amount of soil: 150 g dw
- No. of replicates per concentration: 3
- No. of replicates per control: 3

SOIL INCUBATION
- Method: Series of individual subsamples

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- History of site: The freshly sampled soil (sieved through a 2-mm sieve by the supplier) was obtained from
the supplier on August 15, 2001. At the testing facility the soil was stored at 4 °C. The soil has not been subjected to any pesticide or organic treatment for at least four years. In 2000, Phacelia was
grown. The soil was treated with inorganic fertiliser in 1996, 1998, 1999 and 2000
- % sand: 57.8
- % silt: 33.3
- % clay: 9.0
- Soil taxonomic classification: USDA
- pH (in water): 7.5
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 13.3
- Maximum water holding capacity (in % dry weigth): 38 ± 1
- Cation exchange capacity (mvaI/100 g soil): 10
- Storage: 4 ˚C
- Initial microbial biomass as % of total organic C: 1.1

EFFECT PARAMETERS MEASURED
- Sampling interval: Respiration, nitrification and ammonification were determined for all treatments at the intervals of 0-3 hours, 7, 14 and 28 days after treatment.
- Respiration: The microbial biomass was determined according to Anderson and Domsch (1978). For the short-term respiration experiments the glucose concentration which exerted a maximum respiration response was added.
- Principle of measurement: The CO2 evolved after the addition of glucose was measured semicontinuously with an infrared (IR) gas analyser for approximately 24 hours. After addition of glucose/talc mixtures, soil aliquots were packed into glass columns and maintained at 20 °C. Each column was to a trapping system for water and CO2 at the inlet and to the IR gas analyser at the outlet. After an accumulation time (AT), one column was flushed for 25 seconds (measuring time, MT) with air (flow rate F, eg. 300 mL per MT) and the concentration air) was measured. After a waiting period (7 seconds), the next column was flushed. The volume of evolved per hour (VCO2 in mL/h) per kg dry soil equivalents was calculated based on the concentration, the soil sample dry weight equivalents and the air flow rate.
- Microbial biomass: Bulk samples of untreated soil were prepared for microbial biomass analysis by sieving and adjusting moisture content to 40% of its MW. Seven subsamples of 40 g wet soil each (corresponding to 34.7 g dry soil) were mixed with different concentrations of glucose and talc. The talc served as an inert ingredient to improve the homogeneous distribution of the glucose in the soil. For the biomass determination, only those glucose concentrations providing the three highest initial and constant CO2 production rates (substrate saturation conditions) were considered. The CO2 production rates were calculated as an average value from the rate determinations during the constant initial respiration period (lag phase). The microbial biomass was then calculated according to Anderson & Domsch (1978).
- Glucose Induced Short-Term Respiration (Carbon Mineralisation Test): For the short-term respiration experiment the glucose concentration, which exerted a maximum respiration response was added to the soil samples after sampling. Triplicate aliquots of 40 g wet soil (corresponding to 34.7 g dry soil) were amended with the glucose-talc mixtures, namely 1.15 g glucose/kg dry soil. After amending the soil samples with glucose, the short-term respiration rates were measured by analysing evolved CO2 for about 22 hours. In order to evaluate the influence of the test item on carbon mineralisation in soil, the respiration rates of treated and control soil during the first 12 consecutive hours were compared. The dinoseb acetate treated samples were compared to the control samples in order to show the sensitivity of the test system and method.
- Nitrification and Ammonificatlon: The concentrations of nitrite and nitrate were determined for each sampling interval in a 2M KCl extract of the soil sample.
- Nitrification of Lucerne Meal - Determination of Nitrite and Nitrate: After adjustment to the target moisture, triplicate aliquots of about 46 to 47 g wet soil (corresponding to 40 g dry soil) of the sample were taken at each sampling day. These aliquots were extracted for 15 minutes with 2M KCI (30 mL) by shaking them at room temperature on a horizontal shaker. After centrifugation at about 2000 x for 10 minutes, the supernatant liquid was decanted through fluted filter paper. The extraction process was repeated using 25 mL 2M KCI. The combined aqueous extracts were adjusted to a final volume of 50 mL with 2M KCI. The concentrations of nitrite- and nitrate-nitrogen were then determined in triplicate using a Flow Injection Analyser, equipped with a controller and a sampler and a PC data-processing system. The system was calibrated using standard solutions. The initial nitrogen content (nitrate and nitrite) of the soil was measured in unamended and untreated soil samples at Day 0.
- Nitrite-nitrogen Analysis: The determination of nitrite-nitrogen is based on a colorimetric method. In this method, the nitrite ion reacts with sulfanilamides under acidic conditions to yield a diazo compound which is coupled with N-1-naphthylethylene-diamine dihydrochloride to form a soluble dye. The concentration is measured colorimetrically at a wavelength of 540 nm. The limit of quantitation is 0.05 mg/L extract, corresponding to 0.06 mg/kg dry soil.
- Nitrate-nitrogen Analysis: Nitrate is reduced to nitrite in a cadmium reductor. The nitrite is then determined as described above. The method determines the sum of free nitrite and nitrate simultaneously. Hence, the Nitrate-nitrogen concentrations are calculated by subtracting Nitrite-nitrogen as measured above from the free nitrite and nitrate mixture concentration. The limit of quantitation is 0.5 mg/L extract, corresponding to 0.6 mg/kg dry soil.

VEHICLE CONTROL PERFORMED: no

Nominal and measured concentrations:

Low dose: 0.60 mg/kg soil dw
High dose: 3 mg/kg soil dw

Reference substance (positive control):

yes

Remarks:

Dinoseb Acetate

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 3 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

respiration rate

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 3 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

An overview of the result sis provided in Table1 - Table 2 in 'Any other information on results incl. tables'.
- Glucose-induced short-term respiration: The rate of respiration at test initiation was 7.76 mL CO2/h per kg dry soil for the control, 7.35 mL CO2/h for the low dose and 7.46 mL CO2/h for the high dose treated samples. In comparison to the control, decreases in respiration of - 5.3 and - 3.9% were observed for the low and high dose treated samples, respectively. On Day 7 mean increases of 4.2 and 12.7% were observed for the two dose rates in comparison to the control. By Day 14 mean respiration was between - 11.9 and - 13.6% below the mean control respiration. After 28 days of incubation, the rate of respiration was 5.49 mL CO2/h for the control, 5.41 mL CO2/h for the low and 5.38 mL for the high dose treated soil samples, respectively. This resulted in a deviation of - 1.6 and - 2.1% from the control for the low and high dose treated samples. respectively. Thus, no significant influence of the test substance on microbial respiration in soil was observed for the two treatments. According to the Malkomes scheme, rates up to ten times the maximum expected field rate of the test item caused negligible effects on nitrate formation.

- Nitrification: Initially, no nitrite and 13.3 mg nitrate per kg dry soil were detected in untreated and unamended soil, respectively. On Day 0, 0.1 mg nitrite dry soil was detected for the control and the treated samples. On the following sampling dates, the nitrite concentration was below the limit of determination (< 0.1 mg/kg dry soil) in all samples. Thus, the treatment with the test item had no influence on the nitrite formation and transformation.
The mean initial concentration of nitrate-nitrogen was 14.8 mg for the control, and 13.9 and 13.7 mg for the low and high dose treated samples, respectively. Mean nitrate levels decreased until Day 7 and increased thereafter in all sample types, reaching 23.7, 20.9 and 21.8 mg, respectively, on Day 28. The calculated deviation to control was - 11.8 and - 8.0%, for low and high dose treated samples, respectively. Thus, no significant influence of the test item on nitrate formation was observed up to 10 times the maximum expected single field rate.
According to the Malkomes scheme, rates up to ten times the maximum expected field rate of the test item caused negligible effects on nitrate formation.

Results with reference substance (positive control):

- Glucose-induced short-term respiration: The dinoseb acetate treated samples showed an increasing inhibiting effect when compared to the non-treated samples. The deviation from the control was - 35.8% on Day 0, - 4.6% (Day 7), - 51.1% (Day 14) and - 62.2%. Thus, the reference item showed a clear inhibitory effect. According to the Malkomes scheme, rates up to ten times the maximum concentration of the test item expected under field conditions result in negligible effects on soil respiration. Dinoseb acetate resulted in critical effects on soil respiration.

- Nitrification: In the dinoseb acetate treated samples the amount of nitrite per kg dry soil was between 0.1 mg and 0.7 mg nitrite dry soil. Thus, dinoseb acetate showed an effect on the turn-over of nitrite. The nitrate concentration on Day 28 for the dinoseb acetate treated samples was 40.0 mg nitrate dry soil. This resulted in a deviation of 68.8% to control. Thus, the reference item dinoseb acetate showed a strong stimulating effect. According to the Malkomes scheme, rates up to ten times the maximum expected field rate of the test item caused negligible effects on nitrate formation. Treatment with dinoseb acetate resulted in critical effects.

Reported statistics and error estimates:

The Dixon-test, as reported by Sachs (1999) or Dixon (1953) was used to eliminate outliers in the respiration and nitrification experiments, if needed. The test was performed with three values (n = 3) and a confidence interval of 95%.
In the respiration and nitrification experiments, the mean of individual values at the end of their respective incubation period were statistically evaluated on a 5%-significance level = 0.05, two-sided) by the Dunnett-test (Days 0 to 28) to find significant differences between control and treated samples (Dunnett, 1955 and 1964; Sachs, 1999).
For the calculation of the microbial biomass the initial and constant production rate per kg dry soil and Anderson and Domsch (1978) equation were used.

Table 1. Effect of the test substance on the respiration rate in a sandy loam soil

Treatment Rate (mg a.s./kg soil d.w.)	% deviation from control on day:	% deviation from control on day:	% deviation from control on day:	% deviation from control on day
	0	7	14	28
0.60	-5.3	+4.2	-11.9	-1.6
3.0	-3.9	+12.7	-13.6	-2.1
Dinoseb acetate	-35.8	-4.6	-51.1	-62.2

 

 Table 2. Effect of the test substance on nitrification in a sandy loam soil

Treatment Rate (mg a.s./kg soil d.w.)	Nitrate levels (mg/kg) on day:	Nitrate levels (mg/kg) on day:	Nitrate levels (mg/kg) on day:	Nitrate levels (mg/kg) on day:
	0	7	14	28
0 (control)	14.8	5.1	9.0	23.7
0.60	13.9*	4.0*	8.1*	20.9*
3.0	13.7*	5.4	9.5	21.8*
Dinoseb acetate	13.6*	9.6*	20.0*	40.0*
Treatment Rate (mg a.s./kg soil d.w.)	% deviation (nitrate levels) from control on day:	% deviation (nitrate levels) from control on day:	% deviation (nitrate levels) from control on day:	% deviation (nitrate levels) from control on day:
	0	7	14	28
0.60	-6.1	-21.6	-10.0	-11.8
3.0	-7.4	+5.9	+5.6	-8.0
Dinoseb acetate	-8.1	+88.2	+122.2	+68.8
Treatment Rate (mg a.s./kg soil d.w.)	% deviation (nitrate formation rate) from control on day:	% deviation (nitrate formation rate) from control on day:	% deviation (nitrate formation rate) from control on day:	% deviation (nitrate formation rate) from control on day:
	0	7	14	28
0.60	n.a.[KHm2]	2.1	5.1	-12.9
3.0	n.a.	-14.1	5.1	-16.3
Dinoseb acetate	n.a.	-58.8	166.7	36.1

 

* Significantly different from control at 5% level

Validity criteria fulfilled:

yes

Conclusions:

In a toxicity test to soil microorganisms, performed in accordance with OECD TG 216 and 217, the test substance caused no effects on soil respiration (measured as CO2 production) and on nitrogen transformation (measured as NO3-N formulation) by the end of the 28-day incubation period at the highest treatment tested (3.0 mg/kg dry soil).

Executive summary:

The influence of the test substance on soil microorganisms was determined by measuring the soil respiration and nitrification processes according to the OECD Guidelines 216 and 217 (2000). The study was conducted in compliance with GLP criteria. The test substance was added to quartz sand as a solution in acetone. After evaporation of the acetone the treated quartz sand was mixed into batches of a sandy loam soil (pH (CaCl2) 6.5, 1.32% OC, microbial biomass 142 mg C/kg (i.e. 1.1% of the total soil organic carbon)). The treatment rates were 0.60 and 3.0 mg/kg soil d.w. Control soil was treated in the same way, but without the addition of test substance. A positive control (Dinoseb acetate, 25 mg/kg soil d.w.) was included. Aliquots (150 g d.w.) were dispensed into 1 L incubation flasks closed with cotton wool plugs (1 replicate per sampling point), which were incubated at 20 ± 2°C. Soil moisture was maintained at 40% MWHC. For nitrification, soil was amended with lucerne meal (0.5%). On day 0, 7, 14 and 28, three aliquots of 40 g d.w. were removed from each flask for nitrite and nitrate measurements. For respiration, on day 0, 7, 14 and 28, three aliquots of 34.7 g d.w. were removed from each flask, amended with glucose, and CO2 production was measured for 12 hours.

The respiration rate in the control was 7.76 mL CO2/hour/kg soil d.w. on day 0. This rate decreased to 5.49 mL CO2/hour/kg soil d.w. on day 28. Respiration of treated sandy loam soil differed from untreated soil by ≤ 14% at all sampling points. The positive control gave an adequate response (as low as 62.2% inhibition on the respiration on day 28 compare to the control group). The variation (CV) between nitrate levels of control replicates was ≤ 5% at any sampling. After 28 days, at 0.60 and 3.0 mg/kg, respectively, absolute nitrate-N values differed from untreated soil by 12% and 8%, and nitrate formation rates by 13% and 16%. The positive control gave an adequate response (68% deviation from control for nitrate formulation on day 28). Based on the findings, it is concluded that the NOEC value for the respiration and nitrogen transformation is ≥ 3.0 mg/kg soil dw.

Description of key information

All available data was assessed and the study representing the worst-case effect is included here as key. The result can be considered worst-case and is selected for the CSA.

28-d NOEC = 3 mg/kg dry soil (sandy loam), respiration and nitrification, OECD TG 216 and 217, Volkel 2001

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

3 mg/kg soil dw

Additional information

OECD TG 216 and 217, Volkel 2001

The influence of the test substance on soil microorganisms was determined by measuring the soil respiration and nitrification processes according to the OECD Guidelines 216 and 217 (2000). The study was conducted in compliance with GLP criteria. The test substance was added to quartz sand as a solution in acetone. After evaporation of the acetone the treated quartz sand was mixed into batches of a sandy loam soil (pH 6.5 (CaCl2), 1.32% OC, microbial biomass 142 mg C/kg (i.e. 1.1% of the total soil organic carbon)). The treatment rates were 0.60 and 3.0 mg/kg soil d.w. Control soil was treated in the same way, but without the addition of test substance. A positive control (Dinoseb acetate, 25 mg/kg soil d.w.) was included. Aliquots (150 g d.w.) were dispensed into 1 L incubation flasks closed with cotton wool plugs (1 replicate per sampling point), which were incubated at 20 ± 2°C. Soil moisture was maintained at 40% MWHC. For nitrification, soil was amended with lucerne meal (0.5%). On day 0, 7, 14 and 28, three aliquots of 40 g d.w. were removed from each flask for nitrite and nitrate measurements. For respiration, on day 0, 7, 14 and 28, three aliquots of 34.7 g d.w. were removed from each flask, amended with glucose, and CO2 production was measured for 12 hours.

The mean respiration rate in the control was 7.76 mL CO2/hour/kg soil d.w. on day 0. This rate decreased to 5.49 mL CO2/hour/kg soil d.w. on day 28. Respiration of treated sandy loam soil differed from untreated soil by ≤ 14% at all sampling points. The positive control gave an adequate response (as low as 62.2% inhibition on the respiration on day 28 compared to the control group). The variation (CV) between nitrate levels of control replicates was≤5% at any sampling. After 28 days, at 0.60 and 3.0 mg/kg, respectively, absolute nitrate-N values differed from untreated soil by 12% and 8%, and nitrate formation rates by 13% and 16%. The positive control gave an adequate response (69% deviation from control for nitrate formulation on day 28). Based on the findings, it is concluded that the NOEC value for the respiration and nitrogen transformation is ≥3.0 mg/kg soil dw.