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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:
02 Mar 2017 to 03 Apr 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Version / remarks:
January, 2000
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material (as cited in study report): ZDEC
- Batch No.: 70100804
- Purity: 99.2%
- Zn. content: 18.1%
- Date of manufacture: January 20, 2017
- Expiry date: January 20, 2019
- Appearance: Solid white to off white powder or Granules
- Solubility (water): 1.06 mg/L
- Storage conditions: at 20 ± 5 °C, in the dark
Analytical monitoring:
no
Vehicle:
yes
Remarks:
quartz sand and lucerne green grass meal
Details on preparation and application of test substrate:
Appropriate amounts of the test item were mixed with the quartz sand. Afterwards, the treated quartz sand and additionally 0.5% lucerne meal (related to soil dry weight) were mixed to the soil using a laboratory mixer. Throughout the application the soil was ventilated and the soil water content was adjusted between 54% and 55% WHC. For the control, quartz sand and additionally 0.5% lucerne meal (based on soil dry weight) was added to the control. The soil water content was adjusted to 55% WHC. The test item was applied to the total soil amount for each treatment and soil from each treatment was divided into three replicates after application. The applications were conducted in the following order: control and subsequently increasing test substance concentrations.
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
18 - 22 °C
Moisture:
54% to 56% of maximum water holding capacity.
Details on test conditions:
TEST SYSTEM
- Test container: Disposable plastic boxes; 400 g soil (dry weight), box size approximately 0.5 L, filled up to 6 cm (dimensions: 0.10 m width x 0.10 m depth x 0.065 m height).
- No. of replicates per concentration: 3
- Filling of containers: The soil was filled loosely into the boxes, which were covered by perforated lids to allow air exchange to ensure aerobic incubation conditions.
- Light regime: In the dark

SOURCE AND PROPERTIES OF SUBSTRATE
- Origin: The soil batch used in this study was according to the guidelines and was taken from fallow grassland
- District authority: Darmstadt-Dieburg
- Municipality: 64380 Rossdorf, Germany
- Geographical position: longitude 8° 44' 38.70''; latitude 49° 51' 59.59'' N
- Field history: No pesticides or organic or mineral fertilizer had been used on the soil for at least four years prior to test initiation. The field has been rented since 2004.
- Soil texture: Silty sand
- pH: 6.5
- (NH4+)N (mg/kg dry weight): 0.337
- (NO2-)N (mg/kg dry weight): 0.024
- (NO3-)N (mg/kg dry weight): 5.511
- Nmin-N (mg/kg dry weight): 5.872
- CEC (mmol Ba/kg dry weight): 43
- WHC (%): 35.5
- Clay (%): 2.6
- Silt (%): 35.0
- Sand (%): 62.4
- Soil dry weight at test start(%): 84.1
- Microbial biomass (mg C/kg dry weight), calculated from respiration activity (based on CO2 production): 214.45
- Microbial biomass (% of total organic carbon) (based on CO2 production): 2.58
- (NO3-)N (mg/kg dry weight): 15.208

SOIL PREPARATION
The soil was sampled (according DIN 10381-6, sampling depth 0.05 to 0.2 m) and brought to the laboratory. In the laboratory it was air dried and sieved (mesh 2 mm) at room temperature.

SOIL STORAGE UNTIL USE (PRE-INCUBATION)
The soil was stored at 20°C ± 2°C with appropriate ventilation and periodical moisture adjustment. The soil dry weight prior to test start was determined to be 84.1%.
- Date of soil sampling: February 06, 2017
- Date of sieving, start of preincubation: February 21, 2017
- End of pre-incubation, date of application of the test item onto soil: March 02, 2017
- Pre-incubation temperature: 20 ± 2°C
- Total duration of pre-incubation: 9 days

WATER CONTENT AND PH
- Determination of Soil-pH, Dry Weightand Water Content: Dry weight and water content were determined at test start and at day 28 gravimetrically according to ISO 11465; pH was determined at test start and day 28 according to ISO 10390 (CaCl2-method); in each case one replicate per treatment group was used.
- Control of Water Content: The water content of one replicate of each treatment group was determined at each sampling date. Water losses were compensated weekly by adding pure water.
- Soil pH: The pH was determined at test start and on day 28 in one replicate of each treatment group. Over the course of the study, the pH value ranged between 7.0 and 7.2.

EFFECT PARAMETERS MEASURED: nitrate
For the determination of nitrate-N content, soil samples were taken within 6 hours after application and at test end at day 28. The nitrate content was determined in each sample of treated and control soils. For extraction, 24 g to 25 g soil were suspended in 100 mL 0.1 M KCl-solution and agitated for one hour. The suspension was centrifuged (Multifuge 3s+, 4350 rpm) and the extracts were stored deep frozen. Amounts of 74.0 mg, and 72.2 sodium nitrite and potassium nitrate, respectively, were diluted in 1000 mL (sodium nitrite) and 100 ml (potassium nitrate) 0.1 M KCl to prepare the standard stock solutions for nitrite-N and nitrate-N + nitrite-N determination. Appropriate aliquots of the stock solutions were automatically diluted by the dilution unity with 0.1 M KCl to prepare 6 standard solutions at a range of 0.5 mg/L to 3.0 mg/L for nitrite-N and 7 standard solutions at a range of 1.0 mg/L to 12.0 mg/L for nitrate-N + nitrite-N determination. Before photometric determination, frozen soil extracts were thawed. For nitrite-N and nitrate-N + nitrite-N determination undiluted extracts were used. The concentrations of (NO3-)-N in the soil were calculated from the measured values by subtracting the (NO2-)-N concentrations from the (NO3-)-N+(NO2-)-N concentrations. The nitrogen content was determined using a AA3 Continuous Flow Analyzer and equipment (see 'Any other information on materials and methods incl. tables' for details).

VEHICLE CONTROL PERFORMED: yes. For the control, quartz sand and additionally 0.5% lucerne meal (based on soil dry weight) was added to the control. The lucerne meal used was fine powdered lucerne green grass meal; the analysed carbon and nitrogen content was 40.9% and 2.7%, respectively. The ratio of carbon to nitrogen was 15.0 / 1.
Nominal and measured concentrations:
- Nominal test concentrations: 0 (control), 5, 20, 80, 320 and 1000 mg/kg soil dw
Reference substance (positive control):
yes
Remarks:
sodium chloride
Key result
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:
NITRATE CONTENT
No adverse effects of the test item on nitrate content in soil were observed at day 28. At day 28 differences to the control were 1.66%, 4.29%, 8.01%, 13.12% and 11.14% for the lowest test concentration (5 mg test item / kg soil dry weight) up to the highest test concentration (1000 mg test item/kg soil dry weight). At day 28, the differences were statistically not significant compared to the control for all test rates. See 'Any other information on results incl. tables'.

NITRATE FORMATION RATE
The soil nitrate formation rates did not exceed the trigger range of ± 25% set by OECD guideline 216 at the 0 - 28 day determination. Differences to the control were -0.33%, 4.62%, 11.77%, 19.03% and 17.05% for the lowest test concentration (5 mg test item / kg soil dry weight) up to the highest test concentration (1000 mg test item/kg soil dry weight). The differences were statistically not significant at day 28 for all test rates. See 'Any other information on results incl. tables'.
Results with reference substance (positive control):
Sodium chloride was tested at 16 g/kg soil dry weight.The variation of replicate control samples was less than 15%. The reference item had a retarding effect of more than ± 25% compared to the control at days 28 and 97 after application. The results of the study proved sensitivity of the test system and provided assurance that the laboratory test conditions are adequate.
Reported statistics and error estimates:
Calculation of mean values per treatment, standard deviation and coefficient of variation. Normality and homogeneity of variances were tested using the Shapiro-Wilk`s Test (α = 0.01) and Levene´s test (α = 0.01), respectively and pair-wise comparisons of treated and control values according to Student t-test (α = 0.05) were conducted. For NOEC determination, the multiple sequentially-rejective Median (2x2-Table) test after Bonferroni-Holm (nitrate content day 0) or the Williams Multiple Sequential t-test procedure (nitrate content day 28, nitrate rate) was conducted. The software used to conduct the statistical analysis was ToxRat Professional, Version 3.2.1, ® ToxRat Solutions GmbH.

Table: Effects of the test item on Nitrate-Content and Nitrate Rate (Mean Values)

Test concentration

[mg/kg soil dw]

Day 0

Day 28

Interval day 0-28

NO3-N

mg/kg dry weight

Dev. %(1)

sig.(2)

NO3-N

mg/kg dry weight

Dev. %(1)

sig.(2)

NO3-N

mg/d/kg dry weight

Dev. %(1)

sig.(2)

0 (control)

14.268

--

--

39.722

---

--

0.909

---

--

5

15.007

5.18

n.s.

40.381

1.66

n.s.

0.906

-0.33

n.s.

20

14.784

3.62

n.s.

41.425

4.29

n.s.

0.951

4.62

n.s.

80

14.474

1.44

n.s.

42.905

8.01

n.s.

1.016

11.77

n.s.

320

14.638

2.59

n.s.

44.935

13.12

n.s.

1.082

19.03

n.s.

1000

14.359

0.64

n.s.

44.146

11.14

n.s.

1.064

17.05

n.s.

(1) Dev., deviation from control

(2) sig.: significance according to Student-t-test procedure, one sided, α = 0.05;

n.s.: not significant

Validity criteria fulfilled:
yes
Remarks:
see 'Any other information on materials and methods incl. tables'.

Description of key information

The 28-d NOEC value was determined to be 1000 mg/kg soil dw, as the test item had no impact on nitrogen transformation (nitrate content and nitrate formation rate) of soil microorganisms.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:
1 000 mg/kg soil dw

Additional information

The toxicity to soil microorganisms was determined in a study according to OECD 216 and in compliance with GLP criteria (Ibacon GmbH, 2017). In this study, soil (silty sand) was incubated for 28 days with test substance concentrations of 0 (control), 5, 20, 80, 320 and 1000 mg/kg soil dw. The test substance was mixed with quartz sand and lucerne meal (0.5%) and subsequently mixed with the soil. The soil contained approximately 2.6% clay, 35% silt and 62.4% sand. For the determination of nitrate-N content, soil samples were taken within 6 hours after application and at test end at day 28. Nitrate content and the nitrate formation rate were subsequently compared to that of the controls. No adverse effects of the test item on nitrate content in soil were observed. The soil nitrate formation rates did not exceed the trigger range of ± 25% set by OECD guideline 216 at the 0 - 28 day determination. Differences to the control were -0.33%, 4.62%, 11.77%, 19.03% and 17.05% for the lowest test concentration (5 mg test item / kg soil dry weight) up to the highest test concentration (1000 mg test item/kg soil dry weight). The differences were statistically not significant at day 28 for all test rates. Based on these results, the 28-d NOEC value (based on the nitrate formation rate) is determined to be 1000 mg/kg soil dw.