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Ecotoxicological information

Toxicity to soil microorganisms

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Administrative data

Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June - October 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: in accordance with guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Ascophyllum nodosum, ext.
EC Number:
283-907-6
EC Name:
Ascophyllum nodosum, ext.
Cas Number:
84775-78-0
Molecular formula:
Not applicable; a generic molecular formula cannot be provided for this UVCB substance.
IUPAC Name:
(2R,3R,4R,5R)-hexane-1,2,3,4,5,6-hexol; (2S,3S,4S,5S,6R)-6-{[(2S,3S,4R,5S,6R)-2-carboxy-4,5,6-trihydroxyoxan-3-yl]oxy}-3,4,5-trihydroxyoxane-2-carboxylic acid; [(2S,3R,4R,5S,6S)-3-hydroxy-6-{[(2S,3R,4R,5S,6S)-3-hydroxy-6-{[(2S,3R,4R,5S,6S)-6-{[(2S,3R,4R,5S,6S)-3-hydroxy-6-{[(2S,3R,4R,5S,6R)-6-hydroxy-2-methyl-4,5-bis(sulfooxy)oxan-3-yl]oxy}-2-methyl-5-(sulfooxy)oxan-4-yl]oxy}-2-methyl-4,5-bis(sulfooxy)oxan-3-yl]oxy}-2-methyl-5-(sulfooxy)oxan-4-yl]oxy}-2-methyl-5-(sulfooxy)oxan-4-yl]oxidanesulfonic acid
Details on test material:
Name /Code: Algeafert Solid K+
Batch number: 00680
Active ingredient: Ascophyllum nodosum (algal extract)
Certificate of analysis (date): 25th May 2010
Indication: Agriculture fertilizer
Expiry date: 09th March 2012
Storage conditions: dark, room temperature
Appearance/Colour: Black micro-flakes
Aggregate state at room temperature: solid
Solubility: 500 g/l (25°C) in water

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Nitrification of lucerne meal – Determination of the nitrates :

On each sampling day, aliquots equivalent to almost 10 g of dry soil from the incubated soil treatment were transferred into 250 ml glass flasks. 50 ml of KCl solution 0.01 M, stored at 20°C, was added to each flask. Flasks were closed with stoppers and vigorously shaken by hand for 1 minute. Flasks were then transferred in a shaker incubator and agitated at 200 rpm (run per minute) for 60 minutes, at 20°C.
After the incubation, the content of each flask was transferred in a centrifuge plastic tube and centrifuged for 12 minutes at 3500 rpm at 20°C. Supernatant of each tube was separated and transferred into a microtube (tube Eppendorf®). Aliquots of the cleaned supernatants were centrifuged at 14.000 rpm for 10 minutes using a bench centrifuge.
Nitrates in the soil extracts were determined by using the Ionic Chromatograph. Before analysis, the chromatographic system was calibrated using 5 standard solutions of NO3 that cover samples’ extracts range. At the end of the analysis the 5 standard solutions of NO3 for system calibration were injected again to test the system repeatability.

At each sampling point, 0, 7, 14 and 28 days after treatments administration, the following parameters were determined:
1. Soil dry weight (one representative sample was made by mixing an aliquot of each replicate per sampling date).
Dry weight determined on 10 g soil sample.
2. pH (one representative sample was made by mixing an aliquot of each replicate per sampling date).
pH calculated on at least 5 ml soil sample according to the method ANNEX 3 (“Determination of soil pH”) of the OECD 222 “Earthworm Reproduction Test (Eisenia fetida/andrei) “ adopted on 13th April 2004.
3. Nitrogen turn-over and short-term respiration (all three replicates per treatments and sampling date).
The nitrate content was determined on 10 g of soil dry weight using Ion Chromatography technique according to the Sop BT 215.
The short-term respiration was determined in 200 g of moist soil sample, after adding the glucose, by the use of OxiTop System®.


Test substrate

Vehicle:
no
Details on preparation and application of test substrate:
The soil used in the present study was an agricultural soil type. The loamy sand soil was taken from an agricultural field in Offenbach (Rheinland-Pfalz, Germany) “Im Bildegarten” Nr. 508/2 on April the 15th, 2010. The sampling site was treated neither with pesticides for at least 5 years nor with organic fertilizer or fertilizers that affect the soil microflora (i.e. calcium cyanamide) within one year before the sampling date as requested in OECD Guidelines 216 and 217. The sampling depth was about 20 cm.

The soil was placed in plastic box with a loose lid in order to have a sufficient gas exchange and was pre-incubated at (18-19.5) °C at 40 % about of its WHCmax, in the dark, for 16/17 days before starting the experimental phase of the carbon transformation test and the nitrogen turnover test respectively.

Test organisms

Test organisms (inoculum):
soil

Study design

Total exposure duration:
30 d

Test conditions

Test temperature:
Room Temperature.
Soil Storage temperature: 4 ± 2 °C
Pre-incubation temperature: (18-19.5) °C
Moisture:
41.0% of the WHCmax
Details on test conditions:
Characteristics of the experimental analytical conditions were:
Eluent Solution: NaHCO3 1.0 mM/Na2CO3 3.2 mM
Flux: 0.70 ml/min
Injected volume: 20 µl
Temperature: Room Temperature
Nominal and measured concentrations:
116.28 mg/Kg soil dry weight and 232.56 mg/Kg soil dry weight.
Reference substance (positive control):
yes
Remarks:
Dinoseb acetate

Results and discussion

Effect concentrations
Remarks on result:
other: Please refer to details on results
Details on results:
According to the results obtained and according to the OECD Guidelines 216 and 217, the Test item Algeafert Solid K+ had no adverse effects on soil microbial nitrogen turn-over and soil short-term respiration at the two doses tested of 116.28 mg/Kg soil dry weight and 232.56 mg/Kg soil dry weight.
Therefore no inhibitory effect on carbon and nitrogen cycle of microflora in the soil can be forecasted for the recommended use.
Results with reference substance (positive control):
The Toxic reference item (Dinoseb acetate) is routinely tested using the same soil batch used in this study and it was tested for this soil batch in a previous trial to confirm the normal reaction of the soil towards herbicides.
Reported statistics and error estimates:
The statistical analysis on results of the nitrogen turn-over and the short-term respiration test consisted as a first step in determining the homogeneity of the variance of the groups by F-test (α=0.05) followed by statistical analysis to compare the treated groups to the control by t-test (two sides, α=0.05) for significant or not-significant differences.

Any other information on results incl. tables

Table 5: Results of the Short-term respiration test

Sample

CO2produced mg/Kg

dry soil*h Average

Deviation from

the control (%)

                  0 days after treatment (3 hours)

 

Untreated Control

21.88

--------

Soil treated withAlgeafert Solid K+low dose

21.19

- 3.2

Soil treated withAlgeafert Solid K+high dose

20.96

- 4.2

              7 days after treatment

 

Untreated Control

26.71

--------

Soil treated withAlgeafert Solid K+low dose

26.95

+ 0.9

Soil treated withAlgeafert Solid K+high dose

25.79

- 3.4

              14 days after treatment

 

Untreated Control

26.71

--------

Soil treated withAlgeafert Solid K+low dose

26.71

0.0

Soil treated withAlgeafert Solid K+high dose

29.71

+ 11.2

              28 days after treatment

 

Untreated Control

26.95

--------

Soil treated with Algeafert Solid K+low dose

27.18

+ 0.9

Soil treated with Algeafert Solid K+high dose

27.18

+ 0.9

Table 6: Results of the Nitrogen turn-over test

Sample

mg NO3/Kg dry soil

Average

Deviation from

the control (%)

                  0 days after treatment (3 hours)

 

Untreated Control

90.41

--------

Soil treated withAlgeafert Solid K+low dose

90.71

+ 0.3

Soil treated withAlgeafert Solid K+high dose

95.91

+ 6.1

             7 days after treatment

 

Untreated Control

63.97

--------

Soil treated withAlgeafert Solid K+low dose

43.24

- 32.4

Soil treated withAlgeafert Solid K+high dose

63.26

- 1.1

              14 days after treatment

 

Untreated Control

81.29

--------

Soil treated withAlgeafert Solid K+low dose

49.94

 - 38.6

Soil treated withAlgeafert Solid K+high dose

64.11

- 21.1

             28 days after treatment

 

Untreated Control

111.19

--------

Soil treated withAlgeafert Solid K+low dose

108.47

- 2.4

Soil treated withAlgeafert Solid K+high dose

124.17

+ 11.7

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Following the OECD Guidelines 216 and 217 (2000), the results of the study were considered valid because of the variation among replicates of untreated samples is less than ± 15%.
Conclusions:
According to the results obtained and according to the OECD Guidelines 216 and 217, the Test item Algeafert Solid K+ had no adverse effects on soil microbial nitrogen turn-over and soil short-term respiration at the two doses tested of 116.28 mg/Kg soil dry weight and 232.56 mg/Kg soil dry weight.
Therefore no inhibitory effect on carbon and nitrogen cycle of microflora in the soil can be forecasted for the recommended use.
Executive summary:

The effects of the Test item Algeafert Solid K+on soil microbial nitrification (nitrogen turn-over) and carbon transformation (short-term respiration) processes were studied according to the OECD Guidelines 216 and 217, adopted 21thJanuary 2000.Test item was mixed into a sandy loam agricultural soil, at the application rates provided by the Sponsor: 116.28 mg of Algeafert Solid K+/Kg soil dryweight (low dose)and 232.56 mg of Algeafert Solid K+/Kg soil dry weight (high dose). The control consisted of soil treated with tap water and incubated at the same condition of the treated soil in the dark at 20±for 28 days. The Toxic reference item (Dinoseb acetate) was tested, for this soil batch, within a previous study to confirm the normal reaction of the soil against herbicides.

The influence of the Test item on the nitrification of lucerne meal was investigated and the results were compared to untreated samples.

The CO2produced by microbial soil microflora during short-term respiration trials, after addition of glucose, was measured and the results were compared to untreated samples.

The Test item Algeafert Solid K+, at the lowest dose applied and at the highest dose applied did not affect the microbial short-term respiration in soil since the treated samples deviated less than 25% from the control after 28 days (+0.9%for both low and high dose of the Test item).

The Test item,Algeafert Solid K+, at the lowest dose applied and at the highest dose applied did not affect the microbial nitrogen turn-over in soil since the treated samples deviated less than 25% from the control after 28 days 

(- 2.4%and+ 11.7%for the low and the high dose respectively).

According to the results of the present study, the Test item Algeafert Solid K+has no effects neither on carbon transformation nor on soil nitrification at the rates: 116.28 mg of Algeafert Solid K+/Kg soil dryweight (low dose) and 232.56 mg of Algeafert Solid K+/Kg soil dry weight (high dose).