Ascophyllum nodosum, ext.

Toxicity to soil microorganisms

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

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:

Ascophillum nodosum, extract

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 21^thJanuary 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).